Microvilli Research Articles

Protective effects of recombinant trichinella spiralis-53 000 protein combining with imipenem on polymicrobial septic mice

To observe the protective effects of recombinant trichinella spiralis-53 000 protein (rTsP53 protein) combining with imipenem (IMP) on septic mice and their underlying mechanisms. . Male BALB/c mice were divided into five groups randomly. Cecal ligature and puncture (CLP) operation was used for building polymicrobial septic model (CLP group).Mice in sham group were only subjected to laparoromy and abdominal closure without cecum ligation. At 6 hours post CLP, mice in CLP+IMP,CLP+rTsP53,and CLP+IMP+rTsP53 groups were injected intraperitoneally with IMP (20 mg/kg) + 0.1 mL albumin,rTsP53 protein (6 mg/kg) + 0.1 mL normal saline (NS),IMP (20 mg/kg) + rTsP53 protein (6 mg/kg) respectively, mice in sham group and CLP group were injected intraperitoneally with 0.1 mL albumin + 0.1 mL NS, then these therapies were repeated every 12 hours until the experiment ended. Twenty mice were extracted randomly from each group for surveying 72-hour survival rate.At 0,6,12,24,36,48,72 hours post CLP,3 mice in each group were collected and cytokines in serum were tested by enzyme-linked immunosorbent assay (ELISA).Whole blood was cultured, then the numbers of bacteria colony-forming units (CFU) were counted. Mice were executed at 24 hours, then the epithelial cells ultrastructures of the mice small intestinal mucosa were observed by transmission electron microscope (TEM). ① Compared with CLP,CLP+IMP or CLP+rTsP53 group,72-hour survival rate of the mice in CLP+IMP+rTsP53 group was significantly elevated (85％ vs.20％,55％,25％,all P ＜ 0.05).② No bacteria was found in cultured whole blood of mice in sham group at all time-points. At 6 hours post CLP operation, the number of bacterial clone of all experimental groups was rose significantly. The changed trend of bacterial number in CLP group was rising at the beginning, then declining, and the bacterial number reached the peak at 24 hours (× 106 cfu/L:12.74± 2.33).From 12 hours, the bacterial numbers of CLP+rTsP53 group were higher than those of CLP group, and reached the peak at 36 hours (× 106 cfu/L:22.13 ± 4.28),then declined gradually. The bacterial numbers of CLP+IMP and CLP+IMP+rTsP53 groups reached the peak at 6 hours (× 106 cfu/L:5.72 ± 0.50,5.49 ± 0.59),then declined. They were significantly less than those of CLP group from 12 hours.③ From 6 hours after CLP,the cytokines levels of mice in all experimental groups were higher than those in sham group. The tumor necrosis factor-α (TNF-α) levels in CLP group showed a trend of elevation in the beginning, and decrease thereafter. It reached the peak at 36 hours (ng/L:1 422.67 ±72.19).The TNF-α level peak time of CLP+IMP group,CLP+rTsP53 group,CLP+IMP+rTsP53 group was advanced to 12 hours post CLP (ng/L:1376.29±44.67,929.36±40.42,809.61±22.61).At 24 hours post CLP, the interleukin-6 (IL-6) level of CLP group and CLP+IMP group reached the peak (ng/L:215.39 ± 16.05,191.63 ± 8.99).The peak time of CLP+rTsP53 group and CLP+IMP+rTsP53 was advanced to 12 hours post CLP (ng/L:113.01 ± 12.11,92.43±6.11).The level of IL-4,IL-10 in CLP group raised gradually to the highest at 72 hours (ng/L:366.25 ±24.25,923.14±30.36).The IL-4 and IL-10 levels of CLP+IMP group raised to their maximum value at 12 hours and 24 hours respectively (ng/L:281.47±16.33,555.67±13.57),then declined. The IL-4 and IL-10 levels of CLP+rTsP53 group and CLP+IMP+rTsP53 group gradually ascended their peak value at 72 hours [IL-4 (ng/L) was 453.14±18.53,410.43 ± 15.75,IL-10 (ng/L) was 1 185.61 ± 16.74,1 006.77 ± 36.91,respectively].From 12 hours, the pro-inflammatory cytokines levels of CLP+IMP+rTsP53 group were significantly less than those of CLP+IMP group and CLP+rTsP53 group.④ At 24 hours post CLP, compared with mice in CLP,CLP+IMP, or CLP+rTsP53 group, mice in CLP+IMP+rTsP53 group had slighter ultra structure injuries in the microvilli, cell junction and mitochondria of small intestinal mucosa epithelial cells. The levels of pro-inflammatory cytokines were reduced and the levels of anti-inflammatory eytokines were escalated by intervention of rTsP53 protein combining with IMP boosted in polymierobial septic mice serum, and enhanced the survival rate of the mice. The injection of rTsP53 protein alone had no protective effects on polymicrobial septic mice,because the amount of bacteria in mice blood was augmented

Feasibility of bacterial cellulose membrane as biological scaffold for construction of tissue engineering corneal epithelium

Background Corneal transplantation is a primary method for the treatment of serious corneal diseases, but its application is limited because of the shortage of corneal donor. The study on tissue engineering corneal epithelium provides a new approach to corneal transplantation, and the biological scaffold materials for tissue engineering corneal epithelium is an issue of increasing concern. Bacterial cellulose membrane has been used in medical field, but its application in tissue engineering corneal epithelium deserves more researching. Objective This study was to evaluate the biocompatibility of bacterial cellulose membrane as a biological scaffold of tissue engineering corneal epithelium. Methods Corneal epithelium was isolated from 1 month-old New Zealand White rabbit. Corneal epithelial cells were cultured using explant method and identified by detecting the CK-3 expression using immunofluorescence technique. The second generation cells were inoculated on bacterial cellulose membrane and culture plate, respectively, and the growth status of the cells were examined and compared under the optical microscope. The cell activity/toxicity test was performed by LIVE/DEAD cell staining kit at the third day after inoculation to evaluate the survival rate. The ultrastructure of the cell surface was examined under the scanning electron microscope. The study was performed in accordance with the ARVO Statement. Results Rabbit corneal epithelial cells grew well 1 week after primarily cultured with a cobblestone-like appearance and positive response for CK3 antibody. The cells on the bacterial cellulose membrane presented a round shape and regular arrangement and showed the green fluorescence for LIVE/DEAD test, with the survival rate 100%. Abundant leafy protrusion, microvilli and intercellular junction were seen under the scanning electron microscope. In addition, mitosis phase of cells and many filopodia between the cells and bacterial cellulose membrane were also exhibited. Conclusions Rabbit corneal epithelial cells can grow well in bacterial cellulose membrane. Bacterial cellulose membrane has good biocompatibility, indicating that bacterial cellulose membrane can be used as new biological material for tissue engineering corneal epithelium. Key words: [Key words] Epithelium, corneal/cytology; Tissue engineering; Tissue scaffolds/microbiology; Biocompatible materials; Cells, cultured; Rabbits; Bacterial cellulose

Dysfunction of tear film and ultrastructural changes of corneal epithelial cells in castrated male mice

Background Studies indicate that the decline of androgen level affects the secretion function of epithelial cells of meibomian glands and lacrimal glands, which results in the abnormality of tear quality and quantity. However, whether androgen level regulates the mucin adhesion to ocular surface is still not elucidated. Objective This study was to investigate the tear functional changes and corneal ultrastructural changes in castrated male mice. Methods Fifty-six SPF male BALB/c mice were devided into normal control group, sham group, and orchectomy 1-week group, orchectomy 2-week group, orchectomy 4-week group, orchectomy 6-week group, orchectomy 8-week group according to random number table. Orchectomy was performed in the mice of the orchectomy 1-, 2-, 4-, 6-and 8-week group, and only incision rather than enucleation of testis was carried out in the mice of the sham group. Peripheral venous blood samples were collected during eyeball enucleation in all the mice and serum androgen concentration was detected by radioimmunoassay. Tear secretion level was measured with phenol red thread. Break-up time of tear film (BUT) was tested, and corneal inflorescence staining was scored based on the criterion of Toshida. The corneas of all the mice were collected for ultrastructural examination under the transmission electron microscope. The study complied with the Regulations for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Commission. Results Mouse serum androgen concentration was (573.87±6.74)ng/μl in the normal control group and (579.74±8.24)ng/μl in the sham group, and there was no significant difference between these two groups (t=1.432, P=0.251), however, the serum androgen concentration was 0 ng/μl in all castrated mice after orchectomy. The tear secretion levels were (5.26±0.99), (4.89±0.56), (4.62±0.83), (4.28±0.63), (3.36±0.56), (2.60±0.27) and (2.08±0.35)mm/5 minutes in the normal control group, sham group and orchectomy 1-, 2-, 4-, 6-and 8-week groups, respectively, and the tear secretion levels were significantly lower in the orchectomy 2-, 4-, 6-and 8-week groups than those in the normal control group (all at P<0.05). The BUT were (68.33±12.86), (62.47±3.75), (58.67±10.03), (47.17±7.64), (39.51±3.39), (32.67±3.88) and (31.00±2.36) seconds in the normal control group, sham group and orchectomy 1-, 2-, 4-, 6-and 8-week groups, respectively, and the BUTs were significantly shorter in the orchectomy 2-, 4-, 6-and 8-week groups than those in the normal control group (all at P<0.05). The fluorescein stain score of corneal epithelium was gradually increased with the lapse of castrated time. The decrease of number, shortening and edema of microvilli as well as separation of desmosomes of corneal epithelial cells were found under the transmission electron microscope in castrated mice. Conclusions Decreasing of serum androgen level, declining of aqueous tears, instability of tear film and damage of corneal epithelial cell ultrastructures occur in castrated male mice, and these manifestations are similar to dry eyes. Key words: [Key words] Androgen; Epithelial cells, corneal; Dry eye; Tear; Ultrastructure; Orchectomy; Disease models, animal; Mice, BALB/c

Gastrospheres of human gastric mucosa cells: an in vitro model of stromal and epithelial stem cell niche reconstruction.

The molecular characterization of mechanisms involved in the gastrointestinal tract disorders needs an in vitro 3D culture model able to mimic the in vivo gastric microenvironment. Herein, we propose a 3D coculture system where gastric epithelial and stromal cells are grown together building spherical and solid structures using the NASA bioreactor - cell culture system (RCCS), a bioreactor. Epithelial and stromal cells from human antral gastric mucosa were isolated from endoscopic gastric biopsies. Thereafter, these cells were mechanically and enzymatically dispersed by treatment with dispase and collagenase, respectively. Using specific culture procedures, these cells formed 3D structures by using a RCCS, named "gastrospheres". Briefly, gastrospheres were obtained by initial seeding of 2.5x10⁴ cells/well in 96 well culture plates. At 24 h after their formation, they were transferred into RCCS, and maintained for 7, 14, 21, and 28 days. The gastrospheres were morphologically characterized by immunocytochemisty to evaluate extracellular matrix (ECM), and by electron microscopy. These analysis of gastrospheres revealed that the epithelial cells were cytokeratin (CK) and lectin reactive and were arranged in the outer layer; stromal cells presented long cytoplasmic processes and were localized inside the gastrosphere. They were vimentin (VIM) and α-smooth muscle actin (α-SMA) positive and expressed ECM components such as laminin (LN), fibronectin (FN), and type IV collagen (CIV). Electron microscopy revealed groups of cohesive gastric cells surrounded by complex stromal structures, with multiple microvilli, and tight cellular junctions interspersed with extracellular matrix fibrils and fibers. The presence of some nestin-positive cells was observed in the inner region of the gastrospheres, suggesting an intermediary localization between epithelial and stromal cells. Altogether, our data suggest that in vitro gastrospheres recapitulate the in vivo gastric niche microenvironment.

Effect of a novel photopesticide on some biological aspects of milkweed bug Spilostethus pandurus (Scopoli), with reference to ultrastructural changes in the midgut

The Hemipteran bug Spilostethus pandurus is a serious pest infesting plant seeds. Haematoporhyrin (HPF) was successful as a novel trend for S. pandurus control in the field due to its accumulation inside their organs after incubation and exposure to sunlight in the summer season. The survival rate of nymphs decreased with increasing HPF concentration. A concentration of 1× 10-3 M/l HPF increased the mortality of the first nymphal instar by 6.06- and 3.9-fold after 2 and 3 days as compared to the control, it produced the highest mortality that remained nearly constant throughout most of the 16 days of development. All biological parameters were affected, showing increased nymphal mortality, decreased number of deposited eggs by females and decreased percentage of egg hatchability. Light and electron microscopic studies on midgut regions (mg1-mg4) of adults resulting from treated nymphs revealed severe disorganisation and disintegration of cells and the presence of a great number of vacuoles. The basement membrane was detached. The microvilli were destroyed in the apical part and the food discharged. In addition, cell lysis appeared from mg1 to mg4 and many of cell organelles disappeared. The appearance of autophagic lysosomes was evident together with looped, vacuolated or swollen mitochondria. It seems likely that the nuclear membrane was ruptured or detached and clumping of the chromatin occurred. The muscle fibre and trachea were malformed. The obtained results might reflect the potency of HPF as a novel photoinsecticide in milkweed bug control in an integrated pest management (IPM) programme.

Morphological assessment of sulfur mustard-induced acute lung injury in rats through different routes

To establish an animal model of sulfur mustard (SM)-induced acute lung injury in rats through different routes and compare the morphological changes in lung tissue and cells. One hundred and thirty-six male rats were selected and randomly divided into 5 groups, namely peritoneal cavity SM group (n=32), trachea SM group (n=32), peritoneal cavity propylene glycol group (n=32), trachea propylene glycol group (n=32), and normal control group (n=8). The rats in peritoneal cavity SM group were injected intraperitoneally with diluted SM (0.1 ml, 8 mg/kg), and the rats in trachea SM group were injected intratracheally with diluted SM (0.1 ml, 2 mg/kg). Once the rats were sacrificed at 6, 24, 48, and 72 h after SM treatment, morphological changes in lung tissue and cells were observed by light and electron microscopy. In the peritoneal cavity SM group, the epithelial cells of bronchioles maintained intact with increased exudate and bleeding in alveolar cavity and large areas of pulmonary consolidation under the light microscope. In the tracheal SM group, focal ulcer formed in the epithelial cells of bronchioles with increased exudate and bleeding in alveolar cavity, partial pulmonary consolidation, and compensatory emphysema in peripheral alveolar space under the light microscope. The alveolar interval areas were widened obviously in both groups in a time-dependent manner. Under the electron microscope, we observed local loss of cellular membrane in type I alveolar epithelium, broken or lost microvilli in cells of typeⅡalveolar epithelium and fuzzy mitochondrial crista as well as the appearance of ribosome detached from rough endoplasmic reticulum in both two groups. Compared with those in the trachea SM group and the control group, the ratio of the alveolar septum average area to the visual field area in the peritoneal cavity SM group at 6, 24, 48, and 72 h was significantly higher (P<0.05). The lung tissue injury through the intraperitoneal route is more severe than that through the tracheal route, while focal ulceration of bronchioles epithelial cells appears in the case of tracheal route. The degree of injury increases over time in both groups, and the cellular damage is approximately the same in both groups.

Histology and scanning electron microscopy of the tubal tonsil of goats.

Aim:To observe the light and scanning electron microscopy (SEM) of the caprine tubal tonsil.Materials and Methods:The study was conducted on six crossbred male goats of 6 months of age. From the median sections of the head, tissue pieces from the nasopharynx around the auditory tube were collected and fixed for histology and SEM.Results:Tonsillar lymphoid tissue was located in the nasopharynx ventral to the auditory tube opening in the lateral wall of the pharynx. The height of the surface epithelium of the tubal tonsil measured 80.17±1.08 µm and was a pseudostratified ciliated columnar type with basal, supporting, and goblet cells. Above the dome of lymphoid nodules, the epithelium was modified into a follicle associated epithelium (FAE), also called lympho-epithelium or reticular epithelium and was characterized by the absence of goblet cells and cilia, reduced number of cell layers, and a large number of lymphoid cells due to interrupted basement membrane. The height of FAE was smaller than that of the surface epithelium and measured 34.33±0.92 µm. The surface of tubal tonsil showed folds and invaginations, which formed crypts. The lamina propria-submucosa underneath the epithelium was formed by the meshwork of reticular and, thin and loose collagen fibers with dome-like accumulation of lymphoid nodules. In the secondary lymphoid nodules, a corona, parafollicular area, and interfnodular area were observed. The average number of lymphoid nodules counted per field under low power magnification of microscope was 1.17±0.17, and the internodular distance was 34.00±4.37 µm. The mean diameter of lymphoid nodules was 566.67±11.45 µm and the lymphocyte count per nodule was 14741.67±174.36. The number of plasma cells counted per field under low power was 44.38±2.90 below the surface epithelium. The tubal tonsil was not encapsulated. In SEM, the surface epithelium of the tubal tonsils presented ciliated cells, microvillus (MV) cells, and goblet cells. The region of FAE possessed Type-I and Type-II MV cells and microfold (M) cells in between.Conclusion:It was concluded that the tubal tonsils were well developed in goats, which might serve as a means of protection against the spread of infection to the middle ear cavity.

Ameliorative effect of androgen therapy tear on film stability in castrate female rats

Background Prevalence of dry eye is significantly inceasing in postmenopausal women than that in men, suggesting that sex hormone plays a role in the pathogenesis of dry eye. In addition, dry eye might become worse following estrogen therapy in postmenopausal women. However, whether application of androgen can ameliorate dry eye is being concerned. Objective This study was to investigate the effect of androgen on tear film of ovariectomized female rats. Methods Forty-eight 3-month-old sexually mature female Wistar rats were randomized into the normal control group, sham group, ovariectomy (OVX) model group and testosterone-injected group. OVX models were established by bilateral ovaries enucleation in the rats of the model group and testosterone-injected group, and then androgen (3.75 mg/kg) was intramuscularly injected since 5 months after OVX at 3-day interval for 6 weeks. Only intraperitoneal fat was cut off in the sham group. In 6 weeks after injection of androgen, serum androgen concentration detected and Schirmer Ⅰtest (SⅠt), tear film break-up time (BUT) were performed. The rats were sacrificed to prepare the corneal and conjunctival samples. The expression of MUC5AC in conjunctival tissue was examined by immunofluorescence staining, and the microstructure of corneal cellular surface was observed under the scanning electron microscope before and 6 weeks after application of androgen. Animals in this study were treated in accordance with Animal Experimentation Ethic Committee Guidelines of Southern Medical University and the study protocol was approved by Ethic Committee of this University. Results The mean serum testosterone concentration was (1.83±0.12)ng/ml, and SⅠt or BUT was (3.63±0.26)mm/5 minutes or (3.73±0.38)seconds, respectively, in the OVX model group, which was significantly declined in comparison with (2.56±0.14)ng/ml, (7.47±0.66)mm/5 minutes or (9.57±0.76)seconds in the normal control group (all at P=0.000). However, the serum testosterone concentration was (3.38±0.24)ng/ml, SⅠt was (6.37±0.45)mm/5 minutes and BUT was (7.54±0.55)seconds in the testosterone-injected group, which was significantly higher than that in the OVX model group (all at P=0.000). The positive staining of MUC5AC in rat conjunctival tissue weakened in the OVX model group compared with the normal control group, and the fluorescence intensity in the testosterone-injected group was stronger than that in the OVX model group. Regularly arranged microvilli and cell metabolism hiatus on the surface of corneal cells were seen in the normal control group and the sham group; while the microvilli were shorter and irregularly arranged, and the cell metabolism hiatus were disappeared in the OVX model group. However, microvilli and cell metabolism hiatus were close to normal ones in the testosterone-injected group. Conclusions Deterioration of tear secretion and instability of tear film are are probably associated with the lower serum androgen levels in castrated female rats. Systemic supplement of androgen can promote tear secretion, improve tear film stability and alleviate ocular surface damage. Key words: Dry eye/therapy; Tears/secretion; Ovariectomy; Androgens/drug therapy; Disease models, animal; Rats, Wistar; Female

ADF/COF Have Different Effects on Intestinal Brush Border Structure and NHE3 Activity

The Na‐H exchanger 3 (NHE3) protein complex accounts for most Na absorption at the intestinal brush border. NHE3 expression/activity is acutely regulated by endocytosis/exocytosis. Cofilin‐1 (COF) and actin depolymerizing factor (ADF) are highly conserved isoforms of the cofilin family of actin severing proteins. COF/ADF are ubiquitously expressed though ADF has a stronger presence in intestinal tissue. We hypothesized that COF and/or ADF are necessary for basal and regulated NHE3 activity by remodeling the terminal web actin structure to allow endocytosis/exocytosis. COF and ADF knockdown (KD) cells were engineered with lentivirus/puromycin in Caco‐2 cells, a polarized intestinal Na‐absorptive cell line. COF‐KD reduced COF by 67±5% (p蠄0.01) without significantly altering ADF, while ADF‐KD cells reduced ADF by 60±7% (p蠄0.01) without altering COF. By transmission electron microscopy, COF‐KD cells had straighter and a greater number of microvilli (MV) than control and ADF‐KD cells had more bent MV. By immunofluorescence, neither COF nor ADF had a predominantly MV distribution. NHE3 activity was quantified by measuring pH change (BCECF/fluorometry) under basal and inhibited (forskolin (FSK), 2.5 uM) conditions. COF‐KD had 35±12% less activity than control cells (p蠄.05). ADF‐KD had 34±12% greater NHE3 activity than control cells (p蠄.05). FSK inhibited NHE3 similarly in all three conditions (approximately 49%).Conclusions1. Using KD studies we conclude that COF normally increases and ADF decreases basal NHE3 activity, but neither are involved in cAMP induced inhibition of NHE3. 2. COF normally reduces apical microvillar number while ADF is necessary for microvillar rigidity. The lack of ADF/COF in MV suggest that they affect the MV through intermediate proteins.

Corneal epithelial changes of soft contact lens wearers under a transmission electron microscope

To investigate the difference in corneal epithelium between patients with and without soft contact lens (SCL) wearing, and to analyze corneal epithelial changes of the eyes with long-time SCL wearing. In this cross sectional study, the subjects were divided into two groups: 13 patients of daily SCL wearers and 11 control subjects who had never worn contact lenses. The flap of corneal epithelium was observed by transmission electron microscopy. The corneal epithelial microvillus density was compared between the two groups. Transmission electron micrographs of the control group showed a tight connection between cells, regularly aligned basal cells, and continuous basement membrane. Compared with the control group, SCL wearers showed incomplete basement membrane, swollen epithelial cells, swollen mitochondria, and widened intercellular interstices. The density of corneal epithelium microvilli [(0.071466 +/- 0.015889)/microm2 vs. (0.139851 +/- 0.024171)/micro2] was lower (t = 8.312, P < 0.05). Long-term SCL wearing can induce remarkable changes of corneal epithelial tissue, and the density of corneal epithelial microvilli decreases.

Effects of the pesticide Lindane on granulosa cell ultrastructure

The excessive exposure to pesticides in the Aral Sea area was correlated to the increased reproductive pathologies in those regions [1]. One of the principal chemical employed was the gamma-hexachlorocycloexane herbicide Lindane (L), a persistent organochlorine that may induces alterations in granulosa cell (GCs) survival [2, 3]. However, a comprehensive experimental study on the L-induced dose-effect morpho - logical alterations, has not yet addressed. Therefore, we studied by means of trans- mission and scanning electron microscopy, the morphological changes of mouse GCs, matured in vitro with increasing concentrations of L [4-6]. GCs showed several dose-dependent changes, in respect to controls. In particular, we observed significant reduction of GC microvilli and decrease of cytoplasmic pro- cesses between adjacent GCs. In addition, peripheral aggregation of chromatin under the nuclear membrane, extensive plasma membrane blebbing, abundant GC remnants and cellular debris were also present. Mitochondria, endoplasmic reticula and Golgi apparatuses did not show significant changes. In conclusion, our results showed a dose-dependent toxicity of L on GCs, associated to morphological signs of apoptosis. Alterations of GCs may be associated to impaired oocyte competence and sterility [7].

Formulation and evaluation of matrix tablets of miglitol using different grades of HPMC

Miglitol is a second‐generation ‐glycosidase inhibitor with a chemical structure of 1‐desoxynojiromycin. It acts as a potent competitive inhibitor of the alfa glycosidase in the microvilli of the intestinal brush border. Miglitol has a short biological half‐life (2 h) and its bioavailability is >90%. Moreover, the site of absorption of miglitol is in the intestine.Therefore, the objective of the present work is to develop a sustained release matrix drug delivery systemfor the drug miglitol for the better management of the disease, to minimize side‐effects as well as to improve patient compliance using different grades of HPMC K‐4, HPMC K‐15 and HPMC K‐100 in different proportions and combinations by the direct compression technique. Key words: Bioavailability, controlled release, direct compression technique, hydrophilic polymer, miglitol

Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells.

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin-Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization.

Effects of GnRHa active immunity on FSHR expression and uterine development in prepuberty and non cyclic ewes

Summary This study explored the effects of GnRH agonist (GnRHa) immunity on expression of FSHR mRNA in pituitary and FSHR protein in uteri, and also confirmed its efficacy on uterine development. 42 prepuberty and non cyclic ewes were assigned to 6 experimental groups (EG) during anestrus season. Animals in EG-I, EG-II and EG-III were subcutaneously injected with 200, 300 and 400 µg alarelin antigens (day 0 and 14). Animals in EG-IV and EG-V were subcutaneously injected with 200 and 300 µg alarelin antigen (day 0, 7, 14 and 21). Uterine FSHR protein expression was detected using Western-blotting. RT-PCR was implemented to measure expression of pituitary FSHR mRNA on day 70 after the immunity. Compared to the CG, pituitary FSHR mRNA levels reduced by 58%, 88% and 91% (P<0.01) in EG-I, EG-II and EG-III. FSHR mRNA in EG-IV and EG-V reduced by 64.29% (P<0.05) and 10.11% compared to EG-I and EG-II. Expression levels of uterine FSHR protein in EG-III and EG-V were higher than that in CG. The uterine glands, mitochondria and mitochondrial crista decreased, and the glandular cavity reduced, also the microvilli shortened. Uterine horn weights in EG-I, EG-II and EG-III reduced by 4.66%, 10.20% and 16.63%, respectively. Uterine wall thickness (UWT) in EG-I, EG-II and EG-III reduced by 1.41%, 5.84% and 8.75% (P<0.05). The endometrial epithelium thickness (EET) in EG-I, EG-II and EG-III decreased 2.70%, 8.87% and 19.15%. The positive correlations between uterine horn weights and UWT, EET and FSHR were calculated.

In vitro induction of human breast adipose-derived stem cells into epithelial-like cells by co-culturing

To explore the feasibility of the transdiferentiation of human breast adipose-derived stem cells (hbASCs) into mammary epithelial-like cells after co-culturing in Transwell in vitro. The third passage hbASC and the HBL-100 cell line were co-cultured in a Transwell culture system for 15 days. The hbASCs were observed and identified by inverted phase contrast microscope and transmission electron microscopy, and immunocytochemistry staining in the induced and control groups. Both the third passage hbASCs and the HBL-100 cell line cells could adhere and grow rapidly after co-culture in the Transwell system. After co-culture for 15 days, the morphology of some induced hbASCs changed into epithelial-like cells. Some induced hbASCs showed positive expression of CK18, CK19 by immunocytochemistry staining, and typical epithelium cells with microvilli, desmosomes and tonofilaments observed under TEM. The positive rate of CK18 and CK19 was (24.4 +/- 12.0)% and (21.6 +/- 16.4)% in experimental group, and (1.8 +/- 1.7)% and (1.1 +/- 0.6)% in control group. The data suggests that hbASCs may have the potential to transdifferentiate into human mammary epithelial-like cells after co-culturing in Transwell in vitro.

Establishment of CPPs-BDI-1-loading MMC conjugated with albumin nanoparticles microspheres and its transfecting to human bladder cancer cell in vitro

Objective To explore and identify its penetrating membrane activity of cell penetrating peptide CPPs-monoclonal antibody BDI-1-MMC-loaded albumin nanoparticle triad,in order to construct new targeted therapy drug delivery system.Methods From February 2011 to October 2013,the TAT-EGFP,MMC,BDI-1-NS were prepared and SPDP decoupling league building nanometer microspheres triplet CPPs-BDI-1-MMC-NS was used.Triplet nanometer microspheres was built to in vitro transfect human bladder cancer cells,and laser confocal,scanning electron microscope and transmission electron microscopy were used to detect the possibility to transfect human bladder cancer cells by albumin nanoparticles microspheres triplet and its therapeutic effect.Results Bifunctional coupling agent SPDP was used to couple membrane peptides,BDI-1,MMC albumin nanoparticles microspheres in order to produce triplet CPPs-BDI-1-MMC-NS with 300 nm in diameter of nanometer microsphere,which is portable of chemotherapy drugs.Triplet experiments have been established in which microspheres strongly targeted tumor cells as well as had membrane activity,and it can effectively transfect human bladder cancer cells.Conclusions The albumin triplet nanometer microspheres can be used as a new targeted drug delivery system of bladder cancer.The microvilli on the surface of tumor cell membrane was associated with cell membrane peptide internalization nanometer microspheres loaded mitomycin albumin triplet internalizing the ball into the cells,and microvilli disappeared after micro ball internalized into tumor cells. Key words: Bladder cancer; Nanoparticles; Cell-penetrating peptides; Penetrating membrane activity

Culture of human nasal polyp epithelial cells at an air-liquid surface

To study the human nasal ciliated epithelial cells at an air-liquid surface (ALI) so as to establish a reliable cell culture model for nasal mucociliary transport study. The human nasal ciliated epithelial cells were cultured by low-temperature enzymatic digestion method at an air-liquid surface, the cell growth behavior was observed under the inverted phase-contrast microscope, the proliferation, confluence and differentiation of cultured cells were examined by scanning electron microscope and immunocytochemistry, the basal and stimulated ciliary beat frequency (CBF) of cultured epithelial cells were measured by using high-speed digital microscopic imaging system. Prism 4.0 software was used to analyze the data. (1) Under microscope, the cells on transwell membrane adhered well at 24 h and locked tightly to display with a cobblestone-like appearance; the monolayer cells got confluence to 80%-90% after one-week submersion culture, and thereafter exposed to air-liquid interface. (2) Under scanning electron microscope, the cilia and also the small microvilli could be observed to protrude from the cell's surface at ALI day 7; the ciliated cells differentiated well and distributed in cluster; goblet cells and nonciliated columnar cells distributed between ciliated cells. (3) Immunocytochemistry of β-tubulin IV and zona occludens-1 showed a good confluence and differentiation of cilia in cultured epithelial cells at ALI day 14, and the percentage of ciliated epithelial cells was 50%-60 %. (4) The basal CBF of cultured epithelial cells was (8.42 ± 1.24), (8.71 ± 1.11), (9.17 ± 1.11), (8.89 ± 0.91), (8.99 ± 0.91) Hz at ALI day 7, 14 , 21, 28, 35, respectively, no significant difference was found among them(F = 1.451, P > 0.05). (5) At the concentration of 100 µmol/L ATP, an exogenous stimulating agent, significantly increased the CBF of cultured epithelial cells. Air-liquid interface cultured human nasal epithelial cells by enzymatic digestion method manifest with good confluence and differentiation status; the cells could maintain differentiated morphology and physiological function close to in vivo epithelium for a long term, therefore, it may serve as an ideal cell model for mucociliary transport study.

Protection effect of sodium hyaluronate on ocular surface toxicity induced by Benzalkonium chloride-preserved brimonidine in rabbits

To investigate the protective effects of sodium hyaluronate on ocular surface toxicity induced by a prolonged use of benzalkonium chloride-preserved Brimonidine eye drops. Experimental study. Thirty adult female New Zealand rabbits were divided into three groups with randomized numbers design. Ten rabbits were treated with 0.2% Brimonidine eye drops and PBS (PBS group), the other ten rabbits with 0.2% Brimonidine combined with sodium hyaluronate eye drops (SH group), and control group received no treatment for 60 days. Schirmer test, fluorescein (FL) and Rose Bengal (RB) staining, conjunctival impression cytology specimens collecting were performed on day 0, 31, and 61. Apoptosis of conjunctival epithelium was detected by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay on day 61. Conjunctival inflammation was observed by hematoxylin eosin staining. Histomorphological changes of cornea and conjunctiva were observed by light microscopy, and scanning and transmission electron microscopy at day 61. Fluorescein and Rose Bengal scores were analysed by Kruskal-Wallis test. Schirmer scores, goblet cell density and inflammatory cells infiltration were analysed by repeated measures analysis of variance. There were significant differences in fluorescein and Rose bengal (H = 22.031, 15.303, P < 0.01) staining among the groups on day 61. Compared with the control group (FL: 0, 0-1, RB: 0, 0-1), fluorescein and Rose Bengal scores were significantly (P < 0.001) increased in PBS group (FL: 1.5, 1-2, RB: 1, 1-2), whereas was significantly (P < 0.001) decreased in SH group (FL:0, 0-1 RB:1, 0-1) when compared to PBS group. There were significant differences in aqueous tear production and goblet cell density (F = 7.980, 14.545, both P < 0.01) among the groups on day 61. Compared with the control group [(9.43 ± 0.57) mm, (87.73 ± 2.34/HP)], Schirmer scores and goblet cell density were significantly (P < 0.01) reduced in PBS-treated group [(6.61 ± 0.38) mm, (68.06 ± 3.61)/HP], but significantly (P < 0.05) increased in SH-treated group [(8.75 ± 0.57) mm, (82.31 ± 1.64)/HP] compared with PBS-treated group. The number of inflammatory cells was significant difference (F = 56.306, P < 0.001) among the groups on day 61. Compared with the control group [(39.89 ± 2.03)/HP], inflammatory cells infiltration was significantly (P < 0.01) increased in both PBS [(73.18 ± 2.17)/HP] and SH groups [(48.79 ± 2.64)/HP], however, SH-treated group was significantly lowered when compared with PBS-treated group. In addition, decrease in apoptosis, complete microvilli and cell organelles were found in the corneal and conjunctival epithelial cells in SH-treated group. Our results demonstrate that topical application of SH reduces the ocular toxicity and protect the ocular surface in the long term anti-glaucomatous medical therapies and may be considered as a vehicles or neutralizing material for future ocular application.

Biocompatibility of chitosan carrier with rabbit corneal endothelium

Background There are a lot of studies about the carrier of corneal endothelial transplantation,but the best carrier has not been defined.Objective This study was to investigate the biocompatibility of chitosan carrier with rabbit corneal endothelium in vivo.Methods Fresh eye-balls were obtained from 10 New Zealand white rabbits.Rabbit corneal endothelial cells (CECs) were isolated and cultured on chitosan carrier in vitro.The morphology and density of rabbits CECs were observed every day,and the expressions of fibronectin (FN),collagen-1 (Coil-I) and Zonula occludens 1 (ZO-1) were detected by immunoinfluorescence.The morphology and ultrastructure of CECs were observed under the scanning and transmission electron microscope.Chitosan carrier with CECs was implanted into the anterior chamber of the left eyes in ten healthy New Zealand white rabbits,and only paracentesis of anterior chamber was performed in the right eyes as controls.The inflammation of ocular anterior segment was examined under the slit lamp microscope,and corneal thickness was measured 1 week,4 and 8 weeks after operation.Corneal endothelium cell density and morphology were examined under the corneal endothelial microscope at postoperative 2 weeks.Corneal samples were collected for the regular histopathological examination to observe the inflammatory reaction at postoperative 1 month and 3 months.Paired t test was used for statistical analyses between the control group (left eyes) and the experimental group (right eyes).The use and care of the animals followed the Statement of ARVO.Results CECs formed an intact monolayer of cells with the uniform shape and size on the chitosan membrane after incubated for 5 days.The cells reached confluence of 90％ 7 days after cultured with the 40％ hexagon cells.Under the scanning electron nicroscope,rabbit CECs showed the round or polygon in the shape with the microvillus on the cell surface.The cells connected closely by desmosome.The processes,pseudopodiums and microvillus on the cellular surface,vacuole in the cytoplasm,expanded endoplasmic reticulum with ribosome and abundant chromatin were exhibited under the transmission electron microscope.The immunofluorescence examination revealed the positive expressions of FN,Coll-Ⅰ and ZO-1 in the CECs on the chitosan carrier.In the in vivo experiment,the exudation in the anterior chamber and corneal edema were seen under the slit lamp microscope 3 days after implantation of chitosan carrier with CECs.However,the inflammation was gone 14 days after operation.The differences of the corneal thickness were no significant between the experimental group and the control group 1 week and 4,8 weeks after operation (t =1.377,P=0.265;t =1.795,P=0.165 ; t =0.390,P =0.760).In addition,no significant differences were found in the CECs density and the hexagon cells rate between the two groups(P =0.365,0.062).The histopathological examination showed that the inflammatory cells around the chitosan membrane were disappeared 3 months after operation and showed a good corneal structure.Conclusions Chitosan carrier has a good biocompatibility with rabbit CECs and anterior chamber,and it may be a potentially good carrier for CECs transplantation. Key words: Chitosan; Cornea; Endothelial cell; Biocompatibility; Biotoxity; Rabbit

Dulbecco's modified eagle's medium and minimum essential medium--which one is more preferred for establishment of Caco-2 cell monolayer model used in evaluation of drug absorption?

The Caco-2 cell monolayer model is widely used in drug absorption studies. Dulbecco's Modified Eagle's Medium (DMEM) and Minimum Essential Medium (MEM) have been used alternatively in the development of this model, although they are different in composition which may affect the differentiation and junction formation of the Caco-2 cell monolayer. Two Caco-2 cell monolayers cultured in both media were compared herein in order to underlay the standardization of this model. These two monolayers were comparatively evaluated regarding reliability and stability by morphology, transepithelial electrical resistance (TEER), alkaline phosphatase (AKPase) activity and transport experiments. Although the results showed that characteristic microvilli were present at the apical side of both monolayers, the dynamic change of TEER of the monolayer cultured in DMEM was more stable than that cultured in MEM, and AKPase activity of the former was stronger than that of the latter. Furthermore, the quantity of atenolol, a key indicator usually used for assessment of this model, across the monolayer cultured in MEM was significantly more than that cultured in DMEM. Therefore, the Caco-2 monolayer cultured in DMEM was more reliable and stable than that cultured in MEM, and thus the former was preferred for drug absorption investigation in vitro.