D-Hanks Solution Research Articles

PRIMARY CULTURE AND IDENTIFICATION OF MOUSE BRAIN MICROVASCULAR ENDOTHELIAL CELLS

Brain microvascular endothelial cells are the basic components of the blood-brain barrier. Many neurological diseases are related to the loss of blood brain barrier (BBB) function. Isolating and culturing primary BMVEC is an important means to study the function and regulation of BBB in vitro. To establish a method for isolation and culture of primary mouse brain microvascular endothelial cells (BMECs). The 7-10-days mice were sacrificed by neck removal, the cranial cavity was opened, the brain was aseptically removed and the cerebral cortex was retained. To extract brain microvascular segments, the brain underwent three D-Hank solution rinses. It was then homogenized, twice digested by enzyme, and centrifuged using a Bovine Serum Albumin (BSA) gradient. For primary culture, the brain microvascular segments were injected into gelatin-coated culture plates using DMED complete media. When the cell density reaches 90 %, the media is removed, and the cells are then given two PBS washes. A new medium was introduced after adding 1ml of the trypsin-EDTA solution to digest for 2–5 minutes for passage. Cell culture plates were rinsed and pre-cooled 95 % ethanol was added for 20 minutes after passaged cells had grown to 80–90 % of their original size. After a third wash, 1 ml of mouse factor VIII antibody was added to the culture wells, where it was left for 4 hours at 37 °C. FITC-labeled rabbit anti-mouse antibody in a volume of 1 mL was added. Under a confocal laser microscope, the plates were examined and taken pictures of. The outcome demonstrates positive expression of the marker factor VIII associated antigen. To create a cell suspension, monolayer-growing microvascular endothelial cells were chosen. 100 L of cell suspension was used to inoculate each well in 96-well plates, and the cells were then grown. On days 1, 2, 3, 4, 5, 6, and 7, the original medium was removed, and then 180 μL of DMEM and 20 μL of MTT were simultaneously added to each well for a 4-hour culture. After adding dimethyl sulfoxide (DMSO), each well's absorbance value (A value) was measured at 492 nm. The outcome demonstrates that the growth peak is attained between 6 and 8 days. This method can successfully isolate and culture primary BMECs, which lay a foundation for the study of BMEC in vitro.

The protective effects of Astragaloside Ⅳ on diastolic function of rat thoracic aortic rings impaired by microvesicles

To investigate the effects of Astragaloside IV (AST) on diastolic function of rat thoracic aorta rings which was injured by microvesicles derived from hypoxia/reoxygenation (H/R)-treated human umbilical vein endothelial cells (HUVECs), and the mechanism of AST. H/R-induced endothelial microvesicles (H/R-EMVs) were generated from cultured HUVECs in vitro under the condition of hypoxia for 12 hour/Reoxygenation for 4 hour, H/R-EMVs were stored in D-Hank's solution. Male Wistar rats were underwent thoracotomy, the thoracic aorta with intact endothelium were carefully removed and cut into 3~4 mm rings. The experiment was divided into six groups. H/R-EMVs group:thoracic aortic rings of rats were incubated in culture medium and treated with H/R-EMVs in a final concentration of 10μg/ml; different doses of AST groups:thoracic aortic rings of rats were treated with 10, 20, 40, 60 mg/L AST co-incubated with 10μg/ml H/R-EMVs respectively; control group were treated with the same volume of D-Hank's solution. Duration of incubation was 4 h, each group was tested in five replicate aortic rings. Effects of AST on endothelium-dependent relaxation were detected. The production of nitric oxide (NO) and the level of endothelial NO synthase (eNOS), phosphorylated eNOS (p-eNOS, Ser-1177), serine/threonine kinase (Akt), phosphorylated Akt (p-Akt, Ser-473), extracellular regulated protein kinases (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2, Thr202/Tyr204) of rat thoracic aortic rings were detected. Tenμg/ml H/R-EMVs could impaire the relaxation of rat thoracic aortic rings significantly (P<0.01). Compared with H/R-EMVs group, relaxation of rat thoracic aortic rings was increased by 20, 40 and 60 mg/L AST in a concentration-dependent manner (P<0.01), the level of NO production was also enhanced (P<0.05, P<0.01). The level of t-eNOS, t-Akt and ERK1/2 was not changed, but the level of p-eNOS, p-Akt and p-ERK1/2 increased by the treatment with AST (P<0.01). AST could effectively ameliorate endotheliumdependent relaxation of rat thoracic aortic rings impaired by H/R-EMVs in a concentration-dependent manner, the mechanism might involve the increase in production of NO, and the protein level of p-eNOS, p-Akt and p-ERK1/2.

Preparation and investigation of novel SrCl2/DCMC-modified (via DOPA) decellularized arteries with excellent physicochemical properties and cytocompatibility for vascular scaffolds.

A new method of fabricating vascular scaffolds was designed in this article by crosslinking the porcine arteries using dialdehyde carboxymethyl (DCMC) and further introducing the Sr element on the surface of modified arteries using DOPA. DCMC had been selected as an ideal crosslinking reagent for its excellent cytobiocompatibility and suitable chemical reactivity. Unfortunately, the endothelialization of biological vascular scaffolds fixed by DCMC was unsatisfactory. To overcome this deficiency, the Sr element was introduced onto arteries to improve the endothelialization of fixed arteries due to the Sr element being able to promote the expression of vascular endothelial growth factor (VEGF) being crucial for growth and proliferation of HUVECs. After modifying and crosslinking, their chemical structures, mechanical properties, stability, and cytocompatibility were examined. Our findings demonstrated that DCMC could improve the mechanical properties of animal-derived materials successfully and possess suitable biocompatibility compared with glutaraldehyde (GA). The Sr element can easily be introduced onto the surface of DCMC modified arteries by DOPA. Compared with purely DCMC-crosslinked ones, SrCl2/DCMC modification has no significant effect on the mechanical strength of fixed arteries, but a slight tendancy to improve the stability of fixed samples in D-Hanks solution. MTT assay and fluorescence tests implied that SrCl2/DCMC modification could effectively stimulate HUVECs' adhesion and proliferation, and thus promote the endothelialization process of fixed arteries. SrCl2/DCMC-modified arteries with excellent physicochemical properties and appealing HUVEC-cytocompatibility should be promising materials for fabricating vascular scaffolds.

Role of HIF-1.ALPHA./HKII signaling pathway in inhibition of hypoxia/reoxygenation-induced apoptosis in rat cardiomyocytes by hypoxic preconditioning

Objective To investigate the role of hypoxia-inducible factor-1α/ hexokinase Ⅱ (HIF-1α/HKⅡ) signaling pathway in the inhibition of hypoxia/reoxygenation (H/R)-induced apoptosis in the rat cardiomyocytes by hypoxic preconditioning (HPC). Methods Primarily cultured cardiomyocytes obtained from the neonatal rats were seeded in culture dishes at the density of 5×105 cells/ml.The cardiomyocytes were attached to the wall for 72 h and then randomly divided into 6 groups (n=15 each) using a random number table: control group (group C); group H/R; group HPC; HIF-1α inhibitor YC-1 group (group YC-1); group HPC + YC-1; dimethyl sulfoxide (DMSO) group.The cells were exposed to D-Hank solution saturated with 95% N2 and 5% CO2 for 4 h, and then cultured in the normal culture atmosphere for 2 h. The cells in YC-1 and DMSO groups were incubated in the culture medium containing 10 μmol/l YC-1 and 0.1% DMSO (100 μl) for 24 h, respectively.HPC was induced by 3 cycles of 10 min hypoxia followed by 30 min reoxygenation, and H/R injury model was then established in group HPC.In group HPC+ YC-1, the cells were incubated for 5 min in the M199 culture medium supplemented with 10 μmol/L YC-1 and 10% fetal bovine serum, and the other treatments were similar to those previously described in group HPC.After the end of treatments, the apoptosis in cardiomyocytes was detected by TUNEL, the expression of HIF-1α, HKⅡ and cytochrome c was detected by Western blot, and the mitochondrial membrane potential was quantitatively measured using fluorescence.Apoptotic rate was calculated. Results Compared with group C, the apoptotic rate was significantly increased, the mitochondrial membrane potential was significantly decreased, and the expression of HIF-1α, HKⅡ and cytochrome c was significantly up-regulated in group H/R(P<0.05=. Compared with group H/R, the apoptotic rate was significantly decreased, and the mitochondrial membrane potential was significantly increased, and the expression of HIF-1α and HKⅡ was significantly up-regulated, and the expression of cytochrome c was significantly down-regulated in group HPC(P<0.05=. Compared with group HPC, the apoptotic rate was significantly increased, the mitochondrial membrane potential was significantly decreased, the expression of HIF-1α and HKⅡ was significantly down-regulated, and the expression of cytochrome c was significantly up-regulated in group HPC+ YC-1 (P<0.05=. Conclusion The mechanism by which HPC inhibits H/R-induced apoptosis in rat cardiomyocytes is associated with activation of HIF-1α/HKⅡ signaling pathway. Key words: Anoxias; Ischemic preconditioning; Myocardial reperfusion injury; Apoptosis; Hypoxia-inducible factor 1, alpha subunit; Hexokinase

In vitro study of strontium doped calcium polyphosphate-modified arteries fixed by dialdehyde carboxymethyl cellulose for vascular scaffolds

Dialdehyde carboxymethyl (DCMC) was select as an optimal crosslinking reagent for its excellent biocompatibility, appropriate degradable property and suitable chemical reactivity in our previous research. However, similar to other traditional crosslinking reagent, the endothelialization of biological vascular scaffolds fixed by DCMC was not sufficient enough. To overcome this limitation presented in DCMC-fixed arteries, a novel strategy was developed by introducing SCPP into arteries to investigate effects of strontium doped calcium polyphosphate (SCPP)-doping on the properties of DCMC-fixed arteries, especially on the endothelialization of fixed arteries. After modifying and crosslinking, their chemical structures, mechanical properties, stability, and cytocompatibility were examined. The result obtained from EDS analysis indicated SCPP was successfully introduced into DCMC-fixed arteries. Compared with purely DCMC-crosslinked ones, DCMC/SCPP modification has no significant effect on mechanical strength of fixed arteries, but a slight tend to improve the stability of fixed samples in D-Hanks solution. Moreover, MTT assay, SEM observation and live/dead assay implied that DCMC/SCPP modification could effectively stimulate HUVECs’ adhesion and proliferation, and thus promote endothelialization process of fixed arteries. In this study, micron-sized SCPP particles were firstly applied to modify DCMC-crosslinked porcine arteries, and the results demonstrated this method could improve the endothelialization of fixed arteries. Therefore, DCMC/SCPP-modified arteries with excellent physicochemical properties and biocompatibility should be a promising materials for fabricating vascular scaffolds.

Crosslinking effect of dialdehyde starch (DAS) on decellularized porcine aortas for tissue engineering

Biological tissue-derived biomaterials must be chemically modified to avoid immediate degradation and immune response before being implanted in human body to replace malfunctioning organs. DAS with active aldehyde groups was employed to replace glutaraldehyde (GA), a most common synthetic crosslinking reagent in clinical practice, to fix bioprostheses for lower cytotoxicity. The aim of this research was to evaluate fixation effect of DAS. The tensile strength, crosslinking stability, cytotoxicity especially the anti-calcification capability of DAS-fixed tissues were investigated. The tensile strength and resistance to enzymatic degradation of samples were increased after DAS fixation, the values maintained stably in D-Hanks solution for several days. Meanwhile, ultrastructure of samples preserved well and the anti-calcification capability of samples were improved, the amount of positive staining points in the whole visual field of 15% DAS-fixed samples was only 0.576 times to GA-fixed ones. Moreover, both unreacted DAS and its hydrolytic products were nontoxic in cytotoxicity study. The results demonstrated DAS might be an effective crosslinking reagent to fix biological tissue-derived biomaterials in tissue engineering.

Effects of histamine on ciliary beat frequency of ciliated cells from guinea pigs nasal mucosa

We aimed to investigate the effect of histamine on ciliary beat frequency (CBF) through combining high-speed digital microscopy and patch-clamp technology. Ciliated cells were obtained from septum and turbinate of 90-120-day-old healthy male guinea pigs. Tight seal was formed by applying negative pressure on the glass electrode after the drawing and pushing progress. Then, we enrolled high-speed digital microscopy to measure CBF before and after treatment with histamine of different concentrations ranging from 10(-6) to 10(-1) mol/L in Hank's solution and D-Hank's solution as well as after administrating adenosine triphosphate. One-way ANOVA, Student's t test or Kruskal-Wallis test was used for statistical comparisons. Glass electrode fix up ciliated cell is available at tip diameter of 2-5 μm and negative pressure of 10-20 cmH2O column. The baseline CBF in Hank's solution was higher than in D-Hank's solution. Treatment with 10(-6)-l0(-3) mol/L histamine of concentrations can stimulate a rise of CBF. Nevertheless, CBF in all groups decreased to baseline CBF within 20 min. Generally, 10(-2) mol/L histamine can stimulate a rise of CBF; meanwhile, the high concentration of histamine killed 50% ciliated cell. Histamine at 10(-1) mol/L killed all ciliated cells. Ciliary beating activity decreased in Ca(2+)-free solution. Moreover, adenosine triphosphate could increase CBF effectively after the stimulation effect of histamine. We construct an effective technology integrating patch-clamp technique with CBF measurements on ciliated cells. Extracellular histamine stimulation could increase CBF effectively.

Pathological and MR-DWI study of the acute hepatic injury model after stem cell transplantation.

To investigate apparent diffusion coefficient (ADC) values as an indication of reconditioning of acute hepatic injury (AHI) after allogeneic mononuclear bone marrow cell (MBMC) transplantation. Three groups were used in our study: a cell transplantation group (n = 21), transplantation control group (n = 21) and normal control group (n = 10). AHI model rabbits in the cell transplantation group were injected with 5 mL of MBMC suspension at multiple sites in the liver and the transplantation controls were injected with 5 mL D-Hanks solution. At the end of the 1st, 2nd and 4th wk, 7 rabbits were randomly selected from the cell transplantation group and transplantation control group for magnetic resonance diffusion-weighted imaging (MR-DWI) and measurement of the mean ADC values of injured livers. After MR-DWI examination, the rabbits were sacrificed and the livers subjected to pathological examination. Ten healthy rabbits from the normal control group were used for MR-DWI examination and measurement of the mean ADC value of normal liver. At all time points, the liver pathological scores from the cell transplantation group were significantly lower than those in the transplantation control group (27.14 ± 1.46 vs 69.29 ± 6.16, 22.29 ± 2.29 vs 57.00 ± 1.53, 19.00 ± 2.31 vs 51.86 ± 6.04, P = 0.000). The mean ADC values of the cell transplantation group were significantly higher than the transplantation control group ((1.07 ± 0.07) × 10⁻³ mm²/s vs (0.69 ± 0.05) × 10⁻³ mm²/s, (1.41 ± 0.04) × 10⁻³ mm²/s vs (0.84 ± 0.06) × 10⁻³ mm²/s, (1.68 ± 0.04) × 10⁻³ mm²/s vs (0.86 ± 0.04) × 10⁻³ mm²/s, P = 0.000). The pathological scores of the cell transplantation group and transplantation control group gradually decreased. However, their mean ADC values gradually increased to near that of the normal control. At the end of the 1st wk, the mean ADC values of the cell transplantation group and transplantation control group were significantly lower than those of the normal control group [(1.07 ± 0.07) × 10⁻³ mm²/s vs (1.76 ± 0.03) × 10⁻³ mm²/s, (0.69 ± 0.05) × 10⁻³ mm²/s vs (1.76 ± 0.03) × 10⁻³ mm²/s, P = 0.000]. At any 2 time points, the pathological scores and the mean ADC values of the cell transplantation group were significantly different (P = 0.000). At the end of the 1st wk, the pathological scores and the mean ADC values of the transplantation control group were significantly different from those at the end of the 2nd and 4th wk (P = 0.000). However, there was no significant difference between the 2nd and 4th wk (P = 0.073 and 0.473, respectively). The coefficient of correlation between the pathological score and the mean ADC value in the cell transplantation group was -0.883 (P = 0.000) and -0.762 (P = 0.000) in the transplantation control group. Tracking the longitudinally dynamic change in the mean ADC value of the AHI liver may reflect hepatic injury reconditioning after allogeneic MBMC transplantation.

The experimental study of magnetic resonance diffusion-weighted imaging to the treatment of acute hepatic injury with allogeneic MBMCs transplantation into liver

To investigate the therapeutic effect of mononuclear bone marrow cells (MBMCs) transplantation to rabbit liver with acute hepatic injury (AHI) and the feasibility that evaluated the functional recovery of acute hepatic injury model with MR-DWI. 10 healthy rabbits were chosen to be normal control group which was only carried out MR-DWI scan of normal liver. 42 model rabbits of acute liver injury were randomly selected and divided into 2 groups:transplanted group (n = 21) and control group(n = 21). Each rabbit of the transplanted group was infused 5 ml MBMCs suspension (about containing 2 × 10⁷ MBMCs) into its liver at multiple sites. All manipulations to each rabbit in the control group were as same as those in the transplanted group except that 5 ml of D-Hanks solution was injected instead of MBMCs suspension.7 model rabbits respectively chosen from the transplanted group and the control group were carried out MR-DWI scan and calculated the mean ADC value of the injury liver and then were killed on day 7, day 14 and day 28 of AHI establishment after transplantation. Other comparative assays were performed including: functional assay of liver, pathological examination of liver sections. Before MBMCs transplantation, the difference of liver function was not significant between the transplanted group and the control group. But after MBMCs transplantation, the liver functions of the transplanted group were significantly better than those of the control group at all time points tested (P < 0.05). On day 7 of AHI establishment after MBMCs transplantation, the mean ADC values of the transplanted group and the control group were significant lower than those of the normal control group (P < 0.05). The mean ADC values of the transplanted group and the control group increased to the mean ADC values of the normal control group over time. At the same time point, the mean ADC values of the transplanted group were significantly higher than those of the control group. In the transplanted group, the difference of average ADC values between any two time points were significantly (P < 0.05). In the control group, the mean ADC values on day 7 were lower than those on day 14 and day 28 (P < 0.05), the mean ADC values on day 14 were lower than those on day 28 (P = 0.417). The correlation between the average ADC value and the ALT or AST was negative (P < 0.05), the correlation between the average ADC value and the ALB was positive (P < 0.05). Along with the increase of the average ADC value, the liver function of the AHI model rabbit gradually got better. Transplantation of MBMCs promoted the recovery of liver function of AHI model rabbit. The recovery of the injury liver could be detected with observing dynamic change of its mean ADC value.

Study on the mechanisms of chitosan and its derivatives used as transdermal penetration enhancers

The efficacy of chitosan (CS) and its derivatives used as transdermal penetration enhancers has been confirmed in our previous research. This study investigated the mechanisms of penetration enhancement by CS and its derivatives, i.e., N-trimethyl chitosan (TMC) with different degree of quaternization (DQ) and mono-N-carboxylmethyl chitosan (MCC). After treatment with CS, TMCs or MCC, the secondary structure changes of keratin in stratum corneum (SC) from mice were examined by an Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) combined with the application of the second-order derivative, deconvolution and curve-fitting. The water content in the SC was also studied by ATR-FTIR. HaCaT cell lines were employed as the cell models in the study. HaCaT cells were first treated with blank D-Hanks solution, CS or its derivatives, and were then fluorescent labeled with DiBAC 4 (3). The change of membrane potential was measured by a flow cytometer (FCM). Alternatively, the treated HaCaT cells were labeled with NBD-C 6-HPC and the change of membrane fluidity was examined under a Confocal Laser Scanning Microscope (CLSM). It was found that CS, TMCs and MCC could significantly affect the secondary structure of keratin in SC in different ways. Although the amide II absorption peak of keratin moved to a lower wave number following treatment with CS, TMCs, or MCC, the β-turning structure of keratin was converted to β-sheeting and random coiling after treatment with TMCs and was converted to β-sheeting and α-helix following treatment with MCC and CS. At the same time, CS and its derivatives all could increase the water content of SC, decrease HaCaT cells membrane potentials and enhance HaCaT cells membrane fluidity significantly. The effect of TMCs appeared to be independent of their DQ. The results suggest that the mechanisms of transdermal enhancement of CS, TMCs and MCC are closely related to their effects on the secondary structure of keratin and water content in SC, cell membrane potential and fluidity.

Quercetin-crosslinked porcine heart valve matrix: Mechanical properties, stability, anticalcification and cytocompatibility

Bioprosthetic heart valves, prepared by glutaraldehyde (GA) crosslinking, have some limitations due to poor durability, calcification and immunogenic reactions. The aim of this study was to evaluate the crosslinking effect of a natural product, quercetin, on decellularized porcine heart valve extracellular matrix (ECM). After crosslinking, the mechanical properties, stability, anticalcification and cytocompatibility were examined. The results showed that the tensile strength of quercetin-crosslinked ECM was higher than that of GA-crosslinked ECM. After crosslinking with quercetin, the thermal denaturation temperature of ECM was clearly increased. Quercetin-crosslinked ECM could be stored in D-Hanks solution for at least 30 days without any loss of ultimate tensile strength and elasticity. After soaking in D-Hanks solution for 36 days, there was only 11.55% non-crosslinked excess quercetin released and no further release thereafter. Cell culture study shows that no inhibition on proliferation of vascular endothelial cells occurred when the quercetin concentration was lower than 1 μg ml −1. This non-cytotoxic concentration was 100 times higher than that of GA. The resistibility of quercetin-crosslinked ECM to in vitro enzymatic hydrolysis was comparable to that of GA-crosslinked ECM. An in vitro anticalcification experiment showed that quercetin crosslinking could protect ECM from deposition of minerals in simulated body fluid. The present study demonstrated that quercetin can crosslink porcine heart valve ECM effectively, which suggests that quercetin might be a new crosslinking reagent for the preparation of bioprosthetic heart valve xenografts and scaffolds for heart valve tissue engineering.

Effects of propofol on cytotoxicity of bupivacaine: intracellular Ca2+ concentration and nitric oxide synthase activity in PC12 cells exposed to bupivacaine

Objective To investigate the effects of propofol on intracellular free Ca2+ concentration and nitric oxide synthase (NOS) activity in PC12 cells exposed to bupivacaine. Methods The PC12 cells were provided by Shanghai Cell Biology Research Institute, Chinese Academy of Sciences and cultured in DMEM liquid culture medium. The cultured PC12 cells were seeded in 36 well plates and randomly assigned to one of 4 groups (n=9 wells each): group Ⅰ control (C);group Ⅱ propofol (P);group Ⅲ bupivacaine (B) and group Ⅳ propofol + bupivacaine (PB). In control group D-Hank solution was added. The cells were exposed to propofol 2 mmol/L and bupivacaine 0.09 mmol/L in group P and B respectively. In group PB the cells were incubated with propofol 2 mmol/L and bupivacaine 0.09 mmol/L simultaneously. After being incubated for 6 and 24 h the apoptosis in BC12 cells was assessed by flow cytometry. Apoptotic rate was calculated. NOS activity and intracellular free Ca2+ coneentration in PC12 cells were determined. Results Bupivacaine significantly increased the apoptotic rate of PC12 cells, the intracellular free Ca2+ concentration and NOS activity in PC12 cells in group B as compared with control group. Propofol significantly decreased the toxic effects of bupivacaine on PC12 cells in group PB compared with group B. Conclusion Bupivacaine is toxic to PC12 cells by increasing apoptosis, intracellular Ca+ concentration and NOS activity in the cells. The toxic effects can be prevented to some extent by concomitant administration of propofol. Key words: Propofol; Buoivacaine; Calcium channels; Nitric-oxide synthase; Apoptosis

Culture of rat adipose-diverted stem cells by serial explant after enzymatic predigestion

Objective To isolate and culture adipose-diverted stem cells (ADSCs) in SD rats using serial explant after enzymatic predigestion, and compare, the results with that from Zuk's method. Methods Adipose tissue from groin area of one-month-old SD rats was divided into two groups. In group A, the adipose tissue was snipped into 8mm3 blocks after washed with D-Hank's solution. The tissue blocks were digested by 0.25% trypsin for 5 minutes and 0.1% collagenase type 1 for 20 minutes, and put on 1mm grid nylon net for 3 to 4 days of cultivation. The tissues were then removed to another culture flask. They were repeatedly cultured by serial explant method for three or four times. In group B, the adipose tissue was treated by Zuk's method. MTT was used to measure bioactivity of the culture and flow cytometry was used to detect surface markers of the stem cells. Results In both groups, the cells had typical morphological characteristics of stem cells and expressed adipose stem cell surface markers CD105 and CD44. Conclusion The adipose-diverted stem cells cultured by technique of enzymatic digestion and serial explant in vitro have the same characteristics of those cultured with Zuk's method. This technique can yield plenty of ADSCs samples during a short period of time and thus enables its practical use in ADSCs cultivation. Key words: Stem cells; Cell culture techniques; Adipose; Explant method

Treatment of stroke in rats with bone marrow mesenchymal stem cells

To investigate the effects of treatment of stroke in rats with bone marrow mesenchymal stem cells (BMSCs) and mechanism thereof. Bone marrow of a healthy volunteer was collected and the BMSCs were separated with density gradient centrifugation. The hBMSC were cultivated and harvested until the third passage. A number of adult male Sprague-Dawley rats received corresponding behavioral training before surgery and underwent transient middle cerebral arterial occlusion (MCAO) for 2 hours. Sixty of them showing the scores of 6 approximately 12 according to the modified neurological severity score system were randomly divided into 2 groups: treatment group (n = 48, injected into the cortex around the ischemic areas with hBMSCs 3x10(5)/15 microl) and control group (n = 12, injected with D-Hanks solution 15 microl 24 hours after the establishment of MCAO models. Morris water maze test, Rotarod test and adhesive-removal test were performed since the 4th day to the 32 day after transplantation once every 3 days. 1, 2, 3, and 4 weeks after the transplantation 12 rats from each group were killed randomly to take out their brains. Immunofluorescence was used to identify the migration, survival and differentiation of the hBMSC. A large number of hBMSC could be seen within 2 weeks after transplantation. The number of hBMSC decreased since the 21st day after transplantation and few cells could be found at the end of 1 month after. No definite evidence supported the differentiation of neural cells derived from the hBMSCs during the whole process. Morris water maze test showed that the mean escape time 1 week after transplantation of the treatment group was (69 +/- 10) s, significantly shorter than that of the control group [(120 +/- 0) s, P < 0.05] The significant difference persisted until the 4(th) week (P > 0.05). Rotarod test with the speed of 10 r/min showed that the mean latency period 10 days after transplantation of the treatment group was (167 +/- 18) s, significantly longer than that of the control group [(37 +/- 19) s, P < 0.05]. The significant difference persisted until the experimental terminal. The adhesive-removal test showed that the mean latency period 13 days after transplantation of the treatment group was (33 +/- 8) s, significant shorter than that of the control group [(84 +/- 13) s, P < 0.05]. The significant difference persisted until the experimental terminal. Injection of hBMSCs into brain cortex improves neurological functional recovery after stroke. The transplanted cells can migrate and survive for a certain period, but no hBMSC express proteins phenotype of neural cells.

Crosslinking of decellularized porcine heart valve matrix by procyanidins

Heart valve diseases have a significant high mortality, and the valve replacement using glutaraldehyde crosslinked porcine heart valves is one of the main curing techniques. But its application is limited due to poor durability, calcification of the valves and immunogenic reactions. The aim of this study was to evaluate the crosslinking effect of procyanidins on porcine heart valve matrix. After crosslinking of the decellularized porcine aortic heart valves by procyanidins, the tensile strength, the in vitro enzymatic degradation resistance, procyanidins release from the crosslinked materials and the cytotoxicity of procyanidins to heart valvular interstitial cells were examined. The results showed that the tensile strength of procyanidins crosslinked valve matrix was higher than that of glutaraldehyde crosslinked valve matrix. Valve matrix crosslinked by 10 mg/ml procyanidins could be stored in D-Hanks solution for at least 45 days without any decline in ultimate tensile strength and maintained the elasticity as the fresh valves. Furthermore, procyanidins was found to release when the crosslinked tissue stored in D-Hanks solution. The release rate was high during the first 4 days and then dramatically decreased thereafter. During releasing phase, the concentration of procyanidins was no toxicity to heart valve interstitial cells. In vitro enzymatic degradation revealed that crosslinked matrix could resist the enzymatic hydrolysis, and the resistant capacity was approximately the same as glutaraldehyde crosslinked valve matrix. This study shows that procyanidins can crosslink porcine heart valves effectively without toxicity. Our results suggested that this method might be a useful approach for preparation of bioprosthetic heart valve.

104 DEVELOPMENTAL POTENTIAL OF CLONED EMBRYOS FROM ADULT AND FETAL OVINE SOMATIC CELLS

This study reconstructed embryos using adult and fetal skin fibroblast cells as donor karyoplasts and ovine enucleated oocytes as recipient cytoplasts for comparing the developmental potential of the reconstructed embryos. Ovine ovaries were collected at a local slaughterhouse and the cumulus–oocyte complexes (COCs) were extracted from antral follicles 2 to 5 mm in diameter. A group of 20 to 30 COCs were matured in a 50-�L microdrop of maturation medium that was composed of TCM-199 supplemented with 20% FBS, 10 �g mL-1 FSH, 20 �g mL-1 LH, and 1.5 �g mL-1 17β-estradiol under mineral oil in a 35-mm petri dish in humidified atmosphere of 5% CO2 in air at 38.5�C for 18–22 h. Then oocytes with extruded first polar body (MII) were selected and enucleated for use as recipient cytoplasm. Adult and fetal ovine skin tissues were cut into small pieces (1 mm3), transferred to a 25-mL culture flask containing 2 mL DMEM-F12 medium supplemented with 10% FBS, and then incubated by using explant tissue culture in humidified atmosphere of 5% CO2 in air at 37�C for 5 to 7 days. The medium and unattached epithelial cells were discarded. The attached fibroblast cells were digested by 0.25% trypsin in D-Hanks solution at 37�C for 5 min and dispersed by pipetting. The cell suspensions were transferred to a centrifuge tube and centrifuged at 100g for 10 min. Subsequently, the recovered cells were subcultured for 4–6 passages and then frozen in DMEM-F12 medium containing 10% dimethyl sulfoxide (DMSO) and 20% FBS in liquid nitrogen. The fibroblast cells were serum-starved in DMEM-F12 supplemented with 0.5% FBS for 3 to 5 days and transferred into a micromanipulation drop consisting of H-M199 supplemented with 10% FBS and 5 �g mL-1 cytochalasin B for use. The adult and fetal skin fibroblast cells were injected into the recipient cytoplasm. The fusion of fibroblast cells into the recipient cytoplasm was induced by electrofusion (1500 V cm-1 for 40 �s two times with an interval of 0.125 s). The fused oocytes were activated by 5 mM mL-1 ionomycin with 2 mM mL-1 6-dimethylaminopurine (6-DMAP). A group of 6–10 of the activated reconstructed embryos were co-cultured with ovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% FBS in a 20-�L microdrop for 168 h. The results indicated that 76.0% (596/784) and 75.5% (249/330) of the nuclear transfer couplets were successfully fused from adult fibroblasts and fetal fibroblasts, respectively; 76.2% (454/596) and 79.5% (198/249) of the fused oocytes cleaved within 48 h after activation for adult and fetal, respectively; 26.9% (122/454) and 28.3% (56/198) of the cleaved oocytes developed to morula or/and blastocyst embryo stages, respectively. This study demonstrated that the ovine somatic cell transferred embryos were initiated for cell cycle of mitosis and underwent subsequent development to morula/blastocyst embryo stage in vitro, and that there were no statistical differences (P &amp;gt; 0.05) in developmental capacity between the cloned embryos from adult and fetal skin fibroblast cells.

Quantitative study on the mobility of Demodex folliculorum (Acari: Demodicidae) and inhibitive effects of metronidazole combined with bezoar

Abstract The mobility of human follicle mites may play an important role in the infestation and transmission of demodicidosis. In order to better understand the mobility of follicle mites and to test some miticides which may be responsible for the alteration of mobility of the mite, a Computer-Aided Microimage Processing and Analysis System (CAMPAS) for assaying the mobility of Demodex folliculorum was developed. The crawling velocity, as an index of mite mobility, showed a bimodal distribution with peaks from 0 to 40 µm/min and from 200 to 260 µm/min. Scarcely any mites with crawling velocity ranging from 80 to 160 µm/min were detected in these experiments. Adults of D. folliculorum showed two different mobility patterns: inactive and active. All mites with crawling velocity more than 200 µm/min were randomly exposed to (1) a mixture of 1% metronidazole plus 1% bezoar, (2) a mixture of 2% metronidazole plus 2% bezoar and (3) D-Hank's solution (as control). Based on the sequential data on the inhibitive e...