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BACKGROUND: It is important to study the methods of culturing bone marrow-derived mesenchymal stem cells (MSCs) to obtain a great amount of high purity MSCs for applying ocular tissues constructed by tissue engineering technique to treat eye diseases.OBJECTTVE: To separate and culture in vitro MSCs from bone marrow of the adult rats by density gradient centrifugation combined with adherence culture, and observe the growing characteristics and the possibility of mass multiplication.DESIGN: A completely randomized grouping design/repetitive measuring experiment.SETTING: Pathological laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University.MATERIALS: Four six-week-old SD rats about 250 g, grade Ⅱ of cleaning, were provided by the Animal Center of Sun Yat-sen University [certificate number: SCSK(Yue)2004/0011], about 250 g each rat and there was no limit to the sex. The main reagents and instruments included low sugar Dulbecco modified Eagle culture medium (DMEM/F12, American Gibco Corporation), trypsin (fetal bovine serum (FBS, Hangzhou Sijiqing Bio-Engineering Material Research Institute), American Gibco Corporation), disodium edetate, lymphocyte separating medium, fibronectin, CD44, CD34, CD31 monoclonal antibodies, two-step-method kit for immunohistochemistry (Beijing Zhong shan Biotechnology Corporation).METHODS: This experiment was conducted at the Key laboratory of Ophthalmology (Sun Yat-sen University), Ministry of ethanol (750 g/L) for 10 minutes. Under aseptic condition, the medullaris cavitas was exposed, the syringe containing application m edium was directly punctured into the femoral cavity, the cells in the medullaris cavitas were washed out with the culture medium containing heparin and taken as the cell suspension. The bone marrow-derived MSCs were separated and purified by density gradient centrifugation combined with adherence culture, and the growing conditions of the wells. When the cells had generally connected with each other, they were fixed with methanol or dimethy ketone in situ for 10 minutes, and then hematoxylin eosin (HE) staining or immunohistochemical staining. Antibodies against fiinoculated to 96-well culture plate by a cell density of 4.25×107/L, with 200 μL every well; then put the culture plate into culture box. Then from the next day to the sixth day, 5 g/L MTT solution was added into two rows (20 μL every well) every day, continuously cultured for 4 hours, then the supernatant was removed, and 200 μL DMSO was added to each well, agitated for 5 minutes, then detected the absorbance (A) values at 570 nm wave length, and a growth curve was drawn.branes were clear and the cell bodies were lucent. Being cultured for 2 days, there appeared adherent cells. 3 days later,most of the adherent cells extended and appeared to be in polygon, star or long-shuttle shapes. 4 days later, the cells showed to be in division growth stage; and about 12 days later, the cell clones were connected to each other, appearing curve of subcultured MSCs was in S shape. Cells began growing fast on the 2nd day after being passaged, and they entered the growth period for 3-4 days followed by saturation period, and then cells stopped growing.CONCLUSION: It is a simple and practical method to separate MSCs from the bone marrow of adult rats by means of density gradient centrifugation and adherence. MSCs can greatly proliferate in vitro and offer seed cells for the application of tissue engineering technique to treat eye diseases.
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Objective:To investigate the effects of the β-casomorphin-7 on in vitro and in vivo mice splenic lymphocyte and macrophage nitric oxide production.Methods:Splenocytes proliferation assay and nitrite determination were employed for the studies of effects of β-casomorphin-7 on mice splenic lymphocyte and macrophage following in vitro stimulation of β-casomorphin-7 and in vivo intrapetitoneal administration and drinking β-casomorphin-7 solution administration.Results:In vitro study, β-casomorphin-7 showed a stimulation as well as a suppression of lymphocyte proliferation and significantly(P＜0.01) suppressed the production of nitric oxide. In vivo study, β-casomorphin-7 actions on lymphocytes and macrophages were accordant in intraperitoneal administration and drinking β-casomorphin-7 solution administration. β-casomorphin-7 significantly(P＜0.01) increased in splenic lymphocyte proliferative response and suppressed nitric oxide production in peritoneal macrophages.Conclusion:The present study indicates that the β-casomorphin-7 has the immunomodulatory effects and β-casomorphin-7 may be permeable into peripheral blood intact in mice of 2-3 weeks of age.
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·AIM: To investigate the preparation of endostatin protein and its biologic activity on vascular endothelial cell.· METHODS: pBlast-hEndostatin and pBlast-Mcs were identified by digesting with Nhe Ⅰ and Sal Ⅰ, by PCR reaction, by sequencing, and by Alignments of PCR products with gene bank using NCBIBLAST software. The identified pBlast-hEndostatin as well as pBlast-Mcs were then purified with QIAGEN Endofree plasmid maxi kit.The purified plasmids transfected human fibroblasts. The expression of endostatin was detected by RT-PCR, Westem-Blot and immunohistochemistry. The endostatin prorein produced by transfected fibroblasts was purified by ultrafiltration and affinity chromatography. The inhibitory action of endostatin on human umbilical vein endothelium was measured by MTT assay.· RESULTS: pBlast-hEndostatin was found to contain human endostatin gene. Endostatin protein was produced by transfected fibroblasts. The inhibitory ratio of 2.5,5,10,20,40,80mg/L endostatin on human umbilical vein endothelium for 48h were 8.5%,13.1%,27.7%,38.1%,56.7%,63.8% respectively. IC50 value was 34.5mg/L.No inhibition action was found on fibroblasts.·CONCLUSIONS: Endostatin protein can be produced by the transfected fibroblasts. The produced endostatin has inhibitory action on human umbilical vein endothelium and has no inhibition action on fibroblasts.
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OBJECTIVE:To study the effect of dexamethasone (Dex) on expression of aquaporin-1(AQP1) in cultured human trabecular meshwork(HTM) cells METHODS:Reverse transcription combined with polymerase chain reaction(RT-PCR) was used to detect the expression of AQP1 in cultured HTM cells and those treated by Dex RESULTS:The mRNA of AQP1 expressed in normal HTM cells was 1 643?0 354,while 1 577?0 405,1 117?0 443,0 458?0 301,0 267?0 243 in those treated with Dex for 3 days in concentrations of 10-8mol,5?10-8 mol,10-7 mol,5?10-7mol As the concentrations of Dex increased to≥5?10-8mol,the expression of AQP1 was inhibited(P
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Objective To culture astrocytes of human optic nerve and establish the cell lines for further study of healing process after optic nerve trauma. [WT5”HZ]Methods [WT5”BZ]Astrocytes of infantile optic nerve were cultured by tissue inoculation or tissue digestion with 0.25% trypsin and 0.06% EDTA. The second and fourth passage cells were stained with HE and anti GFAP, S 100 protein, vimentin, and CD34 antibodies. [WT5”HZ]Results The trypsinized astrocytes of infantile optic nerve reached confluence in 7 days. The cultured cells were in polygonal shape with processes and the cytoplasm was abundant. These cells were positive in GFAP, S 100 protein and vimentin staining, and negative in CD34 staining. Conclusions Astrocytes of human optic nerve can be successfully cultured by trypsinization rather than tissue inoculation.