Collagenase Research Articles

Isolation and identification of muscle-derived stem cells in the rat penile corpus cavernosum

Objective To investigate the methods for isolation and identification of muscle-derived stem cells (MDSCs) in vitro.Methods The penile corpus cavemosums,from 2-month-old Sprague-Dawley rats,were digested and separated with0.5％ type Ⅰ collagenase and 0.1％ trypsin.Stem cells were isolated by the modified Preplate technique.The cells were cultured for 1 h,and the adherent cells were named PP1.The non-adherent cells were cultured in the other flask for 2 h,and adherent cells named PP2.Non-adherent cells were cultured again in the other flask for 18 h,and the adherent cells named PP3.Thereafter,non-adherent cells were cultured at intervals of 24 h in order to obtain the adherent cells PP4,PP5,PP6.The morphological changes of the cells were observed.The expression of stem cell marker (Sca-1) and desmin was detected by flow cytometry in PP3,PP6 cells; The positive expression of Sca-1 and desmin was detected by Western blotting in PP1-PP6 cells ; The expression of Sca-1,Desmin,Sca-1/ Desmin was detected by immunofluorescence cytochemistry in PP6 cells.Results The adherence of PP1- PP6 cells was decreased gradually.PP6 cells were smaller and round floating on the culture medium.Adherend cells could be seen and shaped round or spindly from day two to three.The positive rate of Sca-1 ( ± ) in PP3 cells was (0.3 ± 0.2) ％ ( P ＞ 0.05 ),and that of Desmin ( + ) was ( 15.3 ± 1.5 ) ％ ( P ＜0.05 ) in comparison to the controls.The positive rate of Sca-1 ( + ) in PP6 cells was (5.7 ±0.7)％,and that of Desmin ( + ) was (41.1 ± 1.6)％ (P＜0.05) in comparison to PP3 cells.Western blotting showed that there was no obvious expression of Sca-1 protein in PP1 -PP5 cells,but there was in pp6 cells.Desmin protein was detected in pp1-pp6 cells and increased gradially.Positive immunofluorescence outcomes were found with the stem cell markers and myoblast markers.The cells which expressed Sca-1 scattered in microscopic field of vision.Positive staining of Sca-1 mainly distributed in the cytoplasm.The cells expressing Desmin aggregated,and the positive staining of Desmin distributed in the cytoplasm.It was also found that very few cells were double-positive staining of Sca-1 and Desmin,which were stained in the cytoplasm simultaneously.Conclusion We separated the cells with phenotype of stem cells and myoblasts from rat penile corpus cavernosum. Key words: Penile corpus cavernosum; Muscle-derived stem cells; Stem cell marker; Desmin

Endogenous neurogenesis after intracerebral hemorrhage.

Currently, it is accepted that brain injury promotes endogenous neurogenesis in mammals, primarily in the subventricular zone (SVZ), and newborn cells can migrate to the injured area. We examined the pattern of endogenous neurogenesis in adult rats after intracerebral hemorrhage (ICH) that was caused by intrastrial administration of collagenase type IV. Our results showed that ICH induced strong endogenous neurogenesis between 72 hours and 7 days after injury, but that the majority of newborn cells did not survive longer than 3 weeks due to apoptosis-mediated cell death. Furthermore, endogenous neurogenesis remained into a small extent at least 1 year after ICH. Because of the growing interest in new strategies for brain regeneration, these data suggest endogenous neurogenesis and inhibiting apoptosis of newborn neuroblasts as potential strategies to improve the consequences of hemorrhagic stroke in humans.

The study of the insulin secretory function of the bioartificial pancreas in vitro

Objective To explore the impact of insulin secretory function of adult islet cells from barium-alginate microencapsulation in vitro. Methods After weighting the pancreas,human pancreatic islets were isolated with type V collagenase and purified by Ficoll's discontinuous density gradient centrifugation.The islet cell yield and purity were evaluated with microscope by DTZ staining.Human islets were coated by barium-alginate microencapsulation,and the viability was assessed by insulin release assay in vitro.Results After isolation,the average number of islet was about 3600 ±447 per gram pancreas.It was about 2140±207 after purification with more than 70％ purity.At day 2,4 and 6 after islet cell culture in vitro,basal insulin concentrations from the culture medium was measured,and the mean insulin concentration (mU/L) in media of the microencapsulated islet group at 2nd,4th and 6th day were 3.302±1.63、3.504±1.10 and 2.921±1.13 respectively,and those non-microencapsulated islet group were 3.814 ± 1.49、4.175 ±1.60、3.617± 1.34.There were no significant differences between these two groups (P＞0.05).Conclusions The bioartificial pancreas has effective insulin secretory function in vitro without being affected by barium-alginate microencapsulation. Key words: Bioartificial pancreas; Islet; Insulin,secretory

Cross-linking of human amniotic membrane by ultraviolet A-riboflavin

Background Studies confirmed that ultraviolet A (UVA)- riboflavin photodynamic therapy can control keratoconus progresses by altering the physicochemical property of cornea.The collagen components of amniotic membrane transplantation is similar to that of cornea and amniotic membrane transplantation has been widely used to ocular surface reconstruction.However,the study on UVA riboflavin-induced-collagen crosslinking for amniotic tissue is less now. Objective This study was to investigate the role of UVA-riboflavin on frozen-preserved human amniotic membrane. Methods Human amnions were obtained in informed consent and prepared into 2 mm×15 mm pieces and were then divided into 4 groups using lottery method and 6 pieces for each group.The first 3 groups were treated with the photosensitizer riboflavin and UVA-irradiation ( wavelength:370 nm ; irradiation energy:1,2 or 3 mW/cm2,distance:10 mm) for 30 minutes,and the untreated fourth group was as control group.Biomechanical stress-strain test was performed using a microcomputer-controlled biomaterial tester and the stress(mN) was recorded when the strains were set to 5％,10％ and 15％.The 7 mm diameter of human amniotic membrane pieces were trephined and divided into 4 groups(5 pieces for each group) with the treated method as mentioned above,and then the buttons were exposed to 0.1％ collagenase Ⅰ solution.The transparency was scored and the complete dissolving time was record.In histological evaluation,three groups (3 pieces for each group) of human amniotic membranes were treated using UVAriboflavin(3 mW/cm2),0.1％ riboflavin,normal saline for 30 minutes respectively and examined under the transmission electron microscopy.This study was performed under the permission of the Ethic Commission of Beijing Tongren Hospital. Results When the strain was 5％,10％,15％,the stress of control group and 1,2,3 mW/cm2UVA group were statistically signifcantly different ( F =3.411,P =0.037; F =9.927,P =0.001;F=11.118,P=0.000).The tensile strength of human amniotic membrane cross-linked with UVA-riboflavin was statistically significantly increased in comparison to the control group (P＜0.05 ),and the tensile strength of human amniotic membrane became stronger as UVA power increased.The complete dissolve time was (8.6± 1.8 ) hours for the control group,(39.6± 2.3 ) hours for 1 mW/cm2 UVA group,(71.4±0.9 ) hours for 2 mW/cm2 UVA group,(78.8± 1.8 ) hours for 3 mW/cm2 UVA group,showing the enhanced anti-enzyme ability of human amniotic membrane after cross-linking(P＜0.01 ).The collagen density in the UVA-riboflavin treated group was increased,the connection among the collagen fibers as well as between the stroma and the epithelium became tighter than those of control group.Conclusions Collagen cross-linking with UVA-riboflavin make the biomechanical strength and enzymatic resistance of human amniotic membrane enhance and ultrastructure change of human amniotic membrane. Key words: Amniotic membrane; Ultraviolet A; Riboflavin; Collagen cross-linking

Dispase rapidly and effectively purifies Schwann cells from newborn mice and adult rats.

In the present study, Schwann cells were isolated from the sciatic nerve of neonatal mice and purified using dispase and collagenase. Results showed that after the first round of purification with dispase, most of the Schwann cells appeared round in shape and floated in culture solution after 15 minutes. In addition, cell yield and cell purity were higher when compared to the collagenase group. After the second round of purification, the final cell yield for the dispase group was higher than that for the collagenase group, but no significant difference was found in cell purity. Moreover, similar results in cell quantity and purity were observed in adult Sprague-Dawley rats. These findings indicate that purification with dispase can result in the rapid isolation of Schwann cells with a high yield and purity.

Influence of lipopolysaccharide on collagen metabolism of normal skin fibroblasts of human

Objective To observe the influence of lipopolysaccharide (LPS) on collagen metabolism of normal human skin fibroblasts and its biological role in the formation of hypertrophic scar.Methods Fibroblasts were isolated and cultured in vitro,and then exposed to different doses of LPS (0.005,0.01,0.05,0.1,0.5,1.0 μg/ml) from E.coli.055:B5 respectively.The expression of proccllagen type Ⅰ,Ⅲand collagenase mRNAs was tested by RT -PCR.Fibroblasts from hypertrophic scar tissue obtained from the same patients in the same culture passage were used as control.Results Compared with control group,the expression of procollagen type Ⅰ,Ⅲ mRNAs in normal skin fibroblasts increased (0.323 ± 0.041,0.303 ± 0.063,0.391 ± 0.071,0.344 ± 0.086,0.488 ± 0.059,0.401 ± 0.087,0.616 ± 0.107,0.434 ±0.084,0.823 ±0.092,0.542 ± 0.082),while the expression of collagenase mRNAs of normal skin fibroblasts depressed(0.598 ± 0.068,0.556 ± 0.049,0.441 ± 0.043,0.372 ± 0.083,0.260 ± 0.027 ).When LPS was set to the concentration of 0.005 μg/ml,it showed a concentration dependent manner.However,when the concentration of LPS was set to 0.5 μg/ml,the expression of procollagen type Ⅰ,Ⅲ and collagenase mRNAs of normal skin fibroblasts began to decrease (0.451 ± 0.063,0.374 ± 0.072,0.360 ± 0.062).When the concentration of LPS was set to 1.0 μg/ml,the expression of procollagen type Ⅰ,Ⅲ mRNAs (0.162 ± 0.025,0.171 ± 0.061 )were inhibited and the expression of collagenase mRNAs began to increase (0.444 ±0.114).When the concentration of LPS was set to 0.1 μg/ml,the expression of procollagen type Ⅰ,Ⅲ and collagenase mRNAs of normal skin fibroblasts(0.823 ±0.092,0.542 ±0.082,0.260 ±0.027)was similar to that of hypertrophic scar tissue fibroblasts(0.829 ±0.049,0.569 ±0.038,0.277 ±0.059).Conclusions This result supported that LPS may be an important factor in collagen metabolism of normal skin fibroblasts and it plays an important role in hypertrophic scar formation. Key words: Lipopolysaccharides/PD ; Collagen/ME ; Skin/CY/DE; Fibroblasts/ME/DE

Effect of the emodin thermosensitive hydrogel on coilagenase in gingival crevicular fluid of patients with chronic periodontitis

Objective To observe the effect of the emodin thermosensitive hydrogel on collagenase in gingival crevicular fluid of patients with chronic periodontitis and to evaluate the clinical efficacy.Methods Forty patients with chronic periodontitis were selected.For each patient after scaring and root planning,the one teeth on one side in a person was assigned at the test group and treated with the emodin thermosensitive hydrogel in the pocket once a week for four weeks.The teeth on the other side were assigned as the control group,just with the primary periodontal treatment.The PLI、SBI、PD、AL、MD and content of collagenase in gingival crevicular fluid were examined at baseline,before administration and after administration four weeks.The COL-II level in GCF was detected using ELISA method.Results There were no significant differences in PLI、SBI、PD、AL、MD and content of collagenase in gingival crevicular fluid before the treatment(P ＞ 0.05).While there are some evidences that periodontal indexes dropped down and the type Ⅱ collagenase level in gingival crevicular fluid also declined after using the emodin thermosensitive hydrogel than the control group(t =3.46,4.02,4.18,3.03,2.79,4.29,all P ＜ 0.05).Conclusion Ultrasonic scaling using the emodin thermosensitive hydrogelas was more effective in reducing the type Ⅱ collagenase level in gingival crevicular fluid.And improving clinical parameters associated with periodontal health in patients with chronic periodontitis. Key words: Collagen type Ⅱ ; Emodin; Silicone gels; Periodontitis; Fluids and secretions

Hair follicle regeneration by injection of follicular cells

To explore the mechanisms of hair follicle regeneration by injection of follicular cells isolated from murine skin. Epidermis was peeled off from the dermis of 3-5 d C57BL/6J mouse by 0.2% Dispase digestion at 37 degrees C for 2 hours. Dermis was cut into small pieces and digested in 0.2% collagenase at 37 degrees C for 30 minute with low speed stirring to isolate hair follicles from dermis. Hair follicles were collected through filtration, low-speed centrifugation and density gradient centrifugation. Collagenase and trypsin were added to digest hair follicles into dissociated cells which were marked by Dio and injected into the nude mouse skin. 2 d after intradermal injection of hair follicle cells, a cyst was formed containing lots of round and elliptical cells and homogeneous eosin stained cell-free tissues. The cyst wall was composed of many spindle shaped fibroblast cells and showed sparsely localized green fluorescence. The contents of the cyst showed bright green fluorescence. 4 d after injection, the skin became slightly thicken with grey appearance, a lots of hair follicles formed with black bulb. 1 weeks after injection, the injection site became black and evaluated with a lots of black hair follicles and hyperproliferation of capillary blood. Newly formed hair follicles showed bright green fluorescence. 3 weeks after injection, a cyst containing lots of black hairs formed in the injection site. Newly formed hair follicles showed positive for Dio. Sebaceous gland can be seen accompanied with hair follicles. 6 weeks after injection, the cyst contained lots of sheded club hair shafts and hair follicles on the stage of anagen. Cultured follicular cells and injection below 1 x 10(5) failed to regenerate hairs. While the regenerated hair follicle was few when the hair follicle cells were injected subcutaneously. Follicular cells can aggregate spontaneously and develop synergistically into hair follicles with normal growth cycle after implantation. The regeneration depends on the interactions between follicular cells, as well as on the recipient sites and cell numbers.

In vitro osteogenic potential of canine adipose derived stem cells.

Stem cells based tissue engineering is a promising approach for the regenerative treatment of various tissue disorders. Adipose tissue is an abundant source of cells which are competent of multipotential differentiation, called adipose-derived stem cells (ADSCs). The present study was contemplated with the objective of assessing the osteogenic differentiation potential of the canine ADSCs in vitro. The canine ADSCs were isolated from adipose tissue around falciform ligament and abdominal subcutaneous fat. Yield of viable ADSCs from both the tissue sources was found to be nearly equivalent. Tissue subjected to trypsinization yielded more viable, but lesser number of cells as compared to collagenase treatment. The stemness of ADSCs was affirmed by reverse transcriptase PCR which exhibited the expression of stem cell specific genes, OCT4 and NANOG.The monolayer of ADSCs was subjected to differentiation into adipogenic and osteogenic lineages. Assessment of the osteogenic potential of ADSCs in vitro opens a new therapeutic horizon for development of in vivo strategies employing autologous stem cell based tissue regeneration in orthopedics.

The progress in minimally invasive treatment of cervical disc herniation

Background Cervical disc herniation is a clinical syndrome,which developed from cervical intervertebral disc degeneration.Due to external force or unclear reason,an intervertebral disc bulges out compressing spinal cord and nerve root and leads to corresponding symptom.As the accelerated pace of life,the number of patients suffering from cervical disc herniation has grown continuously.There are a number of treatments at present,which can be roughly classified into conservative treatment,operative treatment and minimally invasive treatment.Objective To illustrate the advantages of the interventional treatment,including less trauma,multilevel segments treatment at a time and less complications.Content This paper summarizes the advantages and disadvantages of different minimally invasive treatments,including collagenase chemical dissolution technique,percutaneous laser disc decompression vaporization,endoscopic posterior laser disc decompression,coblation nucleoplasty and ozone ablation.Trend Minimally invasive treatment of cervical disc herniation will be accepted by more and more patients owing to its multiple advantages and less trauma. Key words: Cervical disc herniation; Interventional treatment; Collagenase chemonucleolysis percutaneous

Effectiveness of tilapia islets transplantation in diabetic mice

Objective To observe the effect of tilapia islets transplantation through intraperitoneal injection in diabetic mice.Methods Tilapia islets were harvested,and isolated with the collagenase enzyme.The diabetic models in BALB/C mice were made by intraperitoneal injection of staptozocine (STP).Diabetic mice were divided randomly into 2 groups:islets group ( n =14),NS group ( n =10).The survival rate of all groups was observed.The blood glucose levels were determined and compared after transplantation.The samples were evaluated and examined histologically in Hematoxylin and Eosin (HE) and Immunohistochemistrical staining.Results The 3-week survival rate in islets group was higher than in NS group (P＜0.05).In islets group,tilapia islets could maintain normoglycemia for (3.70 ±0.95) days in diabetic mice.At the 5th day after transplantation,the grafts showed a globally necrosis in islets group.No deposition of IgM and IgG was detected around the grafts at every period in immunohistpchemical staining.Conclusion Transplantation of tilapia islets intraperitoneally in diabetic mice could maintain blood glucose levels in a short time.Humoral immunity is not the reason to cause islet early death,but the cell-mediated immune rejection is the reason to cause graft rejection. Key words: Xenotransplantation; Islets; Tilapia

Isolation and identification of cancer stem cells from primary human ovarian cancer tissues

To isolate and identify the cancer stem cells from primary human ovarian cancer tissues. Fresh tumor tissues from five cases of pathologically diagnosed ovarian cancers were taken, minced and then digested with collagenase and hyaluronidase to obtain single cell suspension. The erythrocytes were removed with ACK Lysis buffer. The suspensions were sorted by magnetic activated cell sorting (MACS) using CD133-binding microbeads. Then the sorted CD133(+) cells were verified by flow cytometry. The cells were cultured in serum-free medium supplemented with EGF, bFGF, insulin and BSA, and grew into spheroids. Immunofluorescence, differentiation and tumor formation tests of the cells were performed to characterize the properties of cancer stem cells. The ovarian cancer stem cells were successfully isolated from primary human ovarian tumors, which formed typical spheroids in serum-free medium and had stronger ability of tumorigenesis. The results of related experiments verified that CD133 positive cells owned the properties of cancer stem cells. The ovarian cancer stem cells presenting the characteristics of stemness in vitro and in vivo, have been successfully isolated from primary human ovarian tumor tissues by MACS. The isolated ovarian cancer stem cells could be used in future researches of their biological functions.

Change of voltage-gate potassium channel in pulmonary arterial smooth muscle cells of pulmonary hypertension induced by left-to-right shunt in rats

To investigate the electrophysiological changes of voltage-gate potassium channel (Kv) of pulmonary arterial smooth muscle cells of pulmonary arterial hypertension in rats induced by left to right shunt, and to analyze the role of Kv during the progress of pulmonary arterial hypertension. Forty male SD rats were randomly divided into three groups, group A (control, n = 10), group B (sham operated only group, n = 10), and group C (PAH model group, n = 20). Mean pulmonary artery pressure (mPAP) and right ventricular hypertrophy index (RVHI) of each rat were measured, single pulmonary artery smooth muscle cell (PASMC) was obtained by acute enzyme separation method (collagenase I plus papain) and the conventional whole-cell patch clamp technique was used to record resting membrane potential (Em), potassium ion current of voltage-gated potassium channel, the I-V curve between each 2 groups was compared, and correlation of each parameter was analyzed. (1) The mPAP and RVHI of group C were significantly higher than those of group A and group B (P < 0.01, respectively). (2) The Em of group C [(-33.00 ± 4.09) mV] was significantly higher than that of group A [(-48.10 ± 4.54) mV] and group B [(-51.11 ± 3.66) mV], P < 0.01. (3) The peak current at +50 mV of voltage-gated potassium channel: in group C [(64.80 ± 8.40) pA/pF], it was significantly lower than that of group A [(120.85 ± 11.66) pA/pF] and group B [(118.03 ± 10.18) pA/pF] (P < 0.01, respectively). None of the parameters showed any significant difference between group A and group B (P > 0.05 for all comparisons). (4) Compared with group A and group B, the I-V curve of group C significantly downward shifted (P < 0.01, respectively). The difference in I-V curve between group A and group B was not significant, P > 0.05. (5) The correlation of resting membrane potential and mPAP and RVHI had significantly positive correlation (P < 0.001, respectively); but the correlation of membrane current, membrane current density and mPAP, RVHI and resting membrane potential had significantly negative correlation (P < 0.001, respectively). During the formation process of left-to-right shunt induced pulmonary arterial hypertension, function of Kv channel was inhibited, suggesting that Kv channel may be the mechanism of pulmonary arterial hypertension induced by left-to-right shunting.

Study of the effects of mild hypothermia on improvement of cardiomyocyte contractility after ischemia-reperfusion in rats

Objective To study the effects of mild hypothermia on cardiomyocyte contractility improvement after ischemia-repeffusion injury and on the preservation of well-functioning mitochondrial respiratory capability.Methods A total of 50 newborn SD rats 1 ～ 2 days after delivery were sacrificed and their hearts taken to preserved in 4 ℃ cold D-hanks buffer solution with 0.12％ pancreatic proteinase and collagenase and then processed with 37 ℃ water bath to collect the cardiomyocytes cultured in DMEM medium with 10％ FBS for 5 days.The cardiomyocytes of rats were subjected to ischemia/reperfusion,in vitro,by oxygen and glucose deprivation(OGD)/oxygen and glucose restoration(OGR).The cardiomyocytes of rats after ischemia/reperfusion were divided into three groups:control group,hypothemia group and normothermia group.Contractile frequency and velosity were determined before OGD and 0 h,0.5h,1 h,1.5 h and 2 h after OGR.Ultrastructure changes of cardiomyocytes and mitochondrion were observed under transmission electron microscope(TEM)0 h and 2 h after OGR as well as assessment ot respiratory rate and respiratory control rate(RCR)with Clark oxygen electrode in each group.All data were analyzed with statistical software of SPSS 13.0.Results Contractile function of cardiomyocytes in hypothermia group and normothermia group declined to nadir at 0 h after OGR(P =0.000)and the contractile function of cardiomyocytes in hypothermia group was improved one hour later,compared with the normothermia group(P =0.000).Obvious swelling of mitochondrion was observed under TEM in normothermia group with little alteration after OGR.The RCR assessments indicated respiratory function in normothermia group was impaired after OGR(P =0.000)and this may be responsible for contractility dysfunction.Conclusions Mild hypothemia used after ischaemia can optimize the contractility of cardiomyocytes after a normothermia OGR,and the well-functioning respiratory capability of mitochondrion may be preserved in this process. Key words: Oxygen and glucose deprivation; Cultured cardiomocytes; Mitochondrion; Mild hypothermia; Ultrastructure; Respiratory function

Construction of CTGF shRNA expression vector and its effect on the expression of CTGF in rat MDSCs

Objective To observe the influence of the short hairpin RNA (shRNA) on the expression of connective tissue growth factor (CTGF) by transforming growth factor β1 (TGF-β1) induced rat muscle-derived stem cells (MDSCs).Methods CTGF-targeted mRNA sequences were designed to synthesize shRNA expressing DNA plasmid pGenesil-3-shRNA in vitro.Then the sequences were analyzed.Rat MDSCs were obtained from newborn rat skeletal muscles with the two-step method of collagenase and trypsin digestion and dissociation.The cells were identified with cellular immunochemical staining.The MDSCs were cultured with TGF-β1 in the control group,and were transfected with pGenesil-3-shRNA plasmid and then cultured with TGF-β1in the experimental group.The levels of CTGF mRNA and protein were measured by Real-Tune PCR and Western Blot,respectively.Results The plasmid vector with target CTGF gene was designed correctly.The CTGF mRNA and protein expression in the experimental group were significantly lower than the control group at 48,72and 96 hours afier transfection and culture ( P ＜ 0.05 ).Conclusion CTGF-targeted pG,enesil-3-shRNA plasmid can transfect MDSCs and reduce TGF-β1-induced expression of CTGF by MDSCs. Key words: Transforming growth factor beta; Animal experimentation; Gene interference; Short hairpin RNA; Muscle-derived stem cells

Culture and identification of microvascular endothelial cells from human endometriosis

To establish the methods of isolating and culturing human ovarian endometriosis-derived microvascular endothelial cells (OEMEC). The tissues of human endometriotic cyst of ovary were finely minced with scissors, then digested by collagenase I, II and trypsin-ethylene diamine tetraacetic acid (EDTA). The cells were purified by using centrifugation of 2000 r/min speed. OEMEC were identified by light microscope and transmission electron microscope observing CD(34), FVIII-Rag and Weibel-Palade in microvascular endothelial cells. The OEMEC grew as confluent monolayer like cobblestones under light microscope. CD(34) and FVIII-Rag were expressed strongly, and the percentages of CD(34) and FVIII-Rag positive cells were 91.4% and 92.5%. Weibel-Palade bodies could be observed under transmission electron microscope. The time of cell doubling proliferation was 4.5 days. The established system of isolating OEMEC would provide lab base for studying the mechanisms of angiogenesis in endometriosis lesions.

Efficacy of plasma radiofrequency ablation at low temperature in disc combined with collagenase injection out of disc in patients with cervical intervertebral disc herniation

Objective To evaluate the efficacy of plasma radiofrequency ablation at low temperature in disc combined with collagenase injection out of disc in patients with cervical intervertebral disc herniation.Methods Fifty-six patients suffering from cervical intervertebral disc herniation with headache,dizziness,and pain in the neck and in the shoulder were randomly divided into 2 groups ( n =28 each):collagenase injection out of disc group (group C) and plasma radiofrequency ablation at low temperature in disc combined with collagenase injection out of disc group (group R).All operations were carried out under CT guidance.Results At the sixth month of follow up after treatment,the remission rates of headache,dizziness and pain in the neck and in the shoulder were 86％,79％,and 93％ in group C and 96％,93％,and 100％ in group R,.respectively,with significant difference between the two groups ( P ＜ 0.05 ) Conclusion The efficacy of plasma radiofrequency ablation at low temperature in disc combined with collagenase injection out of disc is much better than collagenase single in patients with cervical intervertebral disc herniation. Key words: Catheter ablation; Collagenases; CERVICAL SPONDYLOSIS

Regulation of extracellular matrix mRNA expression by mechanical stretch in human uterosacral ligament fibroblasts

Objective To investigate the effects of mechanical stretch on the pelvic floor dysfunction and study the regulation of mechanical stretch on the morphology and extracellular matrix mRNA expression of fibroblasts derived from human uterosacral ligament.Methods 13 patients who underwent hysterectomy surgery due to uterine benign diseases in the Obstetric and Gynecological Department of the Second Hospital of Hebei Medical University from October 2009 to September 2010 and had no pelvic organ prolapse were recruited in this study.Fibroblasts of uterosacral ligament were cultured by collagenase digestion and identified by morphology and immunocytochemical methods.Fibroblasts of 3 ～ 4 generations were stretched by flexcell-4000 tension system for 0,4,15,24 hours.Total RNA was extracted from the fibroblasts and mRNA of Collagen Ⅰ,Collagen Ⅲ,matrix metalloproteinase-1 and tissue inhibitor of metalloprot einase-1were measured with real-time fluorescence quantitative PCR.Results In response to mechanical stretch,the morphology and orientation of the uterosacral ligament fibroblasts were changed as well as their synthetic function.Collagen Ⅰ mRNA for 4 or 15 hours' stretch (0.777 ± 0.251,0.730 ± 0.248)were statistically down regulated compared with 0 hour ( 1.000 ±0.284),( q =4.5786,4.6458,p ＜0.05).But for 24 hours'stretch,the mRNA expression ( 1.102 ±0.308) of collagen Ⅰ restored to the level of 0 hours.The expressions of collagen Ⅲ mRNA for 4,15,24 hours' stretch (4.771 ± 0.879,6.146 ± 1.109,5.022 ± 0.907 )were statistically up regulated compared with 0 hour (1.001 ± 0.284),(q =15.9481,21.7647,17.0099,P ＜ 0.01 ).MMP-1mRNA for 4,15,24 hours' stretch (6.420 ± 2.143,23.695 ± 2.221,20.169 ±2.794) were statistically up regulated compared with 0 hour ( 1.000 ±0.414),( q =9.3421,39.1178,33.0403,P ＜ 0.01 ).TIMP-1 mRNA for 4,15 hours' stretch (0.455 ± 0.370,0.481 ± 0.386)were statistically down regulated compared with 0 hour( 1.000 ± 0.425 ),( q =6.5265,6.3106,P ＜0.05).But for 24 hours'stretch,the mRNA expression( 0.883 ± 0.538 ) restored to the level of 0 hours.Conclusions The morphology,orientation and the synthesis function of the uterosacral ligament fibroblasts can be regulated by mechanical stress.Extracellular matrix remodeling may be adaptive changes due to the mechanical stress. Key words: Mechanics; Sacrum; Ligaments/CY ; Fibroblasts/CY/ME; Extracellular matrix/ME

Viscoelastic properties of human normal nucleus pulposus cells

Objective To study the viscoelastic properties of nucleus pulposus (NP) cells from human in vitro. Methods NP was obtained from discarded NP tissue of 3 scoliosis patients aged from 13 to 16 years. Pancreatin and collagenase type Ⅱ were used to digest NP and cells were isolated from NP. Type Ⅱ collagen immunofluorescence and Fan seaing were used to identify NP cells. The micropipette aspiration test was used in combination with a three-parameter viscoelastic solid model to measure the mechanical properties of NP cells. Results The mean diameter of the digested NP cells was ( 15.40 ± 1.83) μm. In response to a prescribed pressure, the NP cells exhibited viscolastic solid creep behavior, which was characterized initially by a jump in displacement followed by a monotony decreasing rate of deformation that generally reached an equilibrium. NP cells were deformed to a length as much as 2 times the radius of the micropipette without completely entering the micropipette. The viscolastic parameters were k1 (0. 101 ±0. 052) kPa, k2 (0. 353 ± 0. 199) kPa, and μ ( 3. 034 ± 1. 843 ) kPa· s, respectively. Only the k1 was positively correlated to the cell diameter (r =-0. 389, P ＜ 0. 05 ). Conclusion Human normal NP cells behave as a typical viscolastic solid creep. Micropipette aspiration technique is a valid method for the study on biomechanics of NP cells. Key words: Nucleus pulposus cells; Biomechanics; Viscoelastic properties

Matrix metalloproteinases (with accent to collagenases)

Matrix metalloproteinases (MMPs) are a major group of enzymes that regulate cell–matrix composition. The MMPs are zinc-dependent endopeptidases known for their ability to cleave one or several extracellular matrix (ECM) constituents, as well as nonmatrix proteins. This review focuses on structural and functional elements of MMPs, and their roles in physiological and pathological processes, in which it is believed the MMPs play an important, or even indispensable role. According to their structural and functional characteristics, MMP family members have been classified into six different but closely related subgroups with fairly characteristic but often overlapping substrate specificities. MMP synthesis and functions are regulated by transcriptional activation, post-transcriptional processing (release of pro-domain, cell surface shedding), and control of activity by a family of endogenous inhibitors collectively known as tissue inhibitors of metalloproteinases (TIMPs). The balance of MMPs to TIMPs therefore determines matrix turnover, where either an excess of MMPs or a deficit of TIMPs may result in excess ECM degradation. Moreover, the recent development of selective and nonselective inhibitors of MMPs would also provide new insights in the knowledge of the relationship between activation of inflammatory cells and tissue remodeling and propose new therapeutic possibilities to the treatment of inflammatory disease.