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Objective Through observing the effect of low-dose T-2 toxin on chondrocyte,to study the molecular mechanism of cartilage damage.Methods The primary chondrocytes were isolated from articular cartilage of d 1-2 Wistar neonate rats through enzymatic digestion.Different doses (0.005,0.010,0.100 μg/L) of T-2 toxin were added after 24 h in vitro culture.The survival rate of chondrocytes was detected with Trypan blue staining.Echylosis (matrix metalloproteinase,MMP1) was analyzed by immunohistochemistry.The damage of articular chondrocyte was observed by transmission electron microscope.Results ①Cell morphology of in vitro cultured chondrocyte:the newly isolated chondrocytes were spherical.After 24 hours,the adherent cells gradually began to stretch the triangle or polygon;the nucleus was large and round;the cell was clear and transparent,containing secretory granules.②Cell proliferation:T-2 toxin had a significant inhibitory effect on chondrocyte proliferation,the higher the concentration of T-2 toxin,the significant the inhibitory effects [0.000 μg/L (control) group:3.45 × 108/L,0.005 μg/L T-2 toxin group:3.45 × 108/L,0.010 μg/L T-2 toxin group:2.06 × 108/L,x2 =9.554,P ＜ 0.05].③Immunohistochemical observation:dysplasia,nucleus condensation and membrane rupture were observed in T-2 toxin treated group,brown staining was observed in all groups at varying degrees.The deepest staining was in 0.005 μg/L T-2 toxin group,with the strongest secretion of MMP1;with increasing doses of the toxin,the damage to cartilage cells was severe,MMP1 secretion was less,staining was weak,and the weakest staining was in the 0.100 μg/L T-toxin group.④Under transmission electron microscopy:in control group,cytoplasm was rich in rough endoplasmic reticulum,nuclear membrane and cell membrane were clear;in 0.005 μg/L T-2 toxin group,the cell nucleus showed pyknosis,organelles were decreased in cytoplasm;in 0.100 μg/L T-2 toxin group,the microvilli was dropped out of cartilage surface,nuclear changes were obvious,and mitochondria was myeloid degeneration;rough endoplasmic reticulum was degranulation and expansion into cystiform,chondrocytes were apoptosis occasionally,the cell nucleus showed pyknosis,and the formation of high-density plaque.Conclusion Low dose of T-2 toxin could damage the primary cultured articular chondrocyte in vitro.The results have showed that there are damaged cytostasis,chondrocyte degeneration,necrosis and apoptosis.
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Background Previous study showed that both hydroxycamptothecin (HCPT) and etoposide (VP-16) can induce the apoptosis of human Tenon capsule fibroblasts (HTFs).However,whether the combination of HCPT with VP-16 enhance the efficacy of drugs is unknown.Olbjeetive This study was to investigate the synergistic effect and its mechanism of HCPT combined with VP-16 on apoptosis of HTFs.Methods Human Tenon capsule tissue was obtained from the eye bank of Jiangsu Province People's Hospital.HTFs were cultured in vitro using explant method and identified by immunofluorescence with vimentin.The fourth generation of cells were incubated in 96-well plate,and different concentrations of HCPT (1,5,10,50,100 mg/L),VP-16 (0.6,2.5,5.0,25.0,50.0 mg/L) and HCPT+VP-16 (2:1,final concentrations 0.80,3.75,7.50,37.50,75.00 mg/L) were added for 24 hours.The inhibiting rate of drugs to HTFs growth was detected using CCK-8 kit.The HTFs were divided into blank control group,HCPT (50 ng/L) treated group,VP-16 (25 mg/L) treated group and HCPT+ VP-16 (37.5 mg/L) treated group,and the apoptosis rates of HTFs in various groups were assayed by flow cytometry.The expressions of caspase-3,cleaved caspase-3,bax,bcl-2,JNK,p-JNK,Akt,p-Akt in the cells were detected by Western blot assay.Results Cultured cells grew well with the polygon shape and positive response for vimentin.The inhibiting rate was elevated with the increase of drug dosage 24 hours after addition of drugs (HCPT:F=41.34,P=0.00 ; VP-16:F =62.60,P =0.00 ; HCPT+VP-16:F =46.77,P =0.00).The half maximal inhibitory concentrations (IC50) of HCPT,VP-16,HCPT+VP-16 were 80.99,27.93,19.81 mg/L,respectively,and the combined index (CI) of HCPT with VP-16 was 0.399,showing a stronger synergistic action.The apoptotic rates of HTFs were (4.87±0.78) ％,(11.20± 1.94)％,(12.67±1.51)％ and (19.77±2.01)％ in the blank control group,HCPT treated group,VP-16 treated group and HCPT+VP-16 treated group,respectively,with a significant difference among them (F=18.23,P ＜ 0.01),and the apoptotic rate was significantly raised in the HCPT + VP-16 treated group,HCPT treated group and VP-16 treated group compared with the blank control group (q'=15.67,16.32,26.88,all at P＜0.01).Compared with the blank control group,the grey scale values of cleaved caspase-3,bax,p-JNK in the cells of HCPT+VP-16 treated group,HCPT treated group and VP-16 treated group were significantly increased (all at P＜0.01),and those in the HCPT+VP-16 treated group significant ascent in comparison with the HCPT treated group and VP-16 treated group (all at P＜0.01).However,the changes of caspase-3,JNK and Akt expression were insignificant.The grey scale values of bcl-2 and p-Akt in the HTFs of the HCPT,VP-16 and HCPT+VP-16 treated groups were significantly lower than those of the blank control group,with a dominant reducing in the HCPT+VP-16 treated group (all at P＜0.01).Conclusions HCPT and VP-16 induce the apoptosis of HTFs in vitro at a dose-dependent manner.The combination of HCPT with VP-16 has a stronger synergistic efficacy.The up-regulation of p-JNK and bax as well as the down-regulation of p-Akt and bcl-2 in HTFs are involved in the coaction of HCPT and VP-16.
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Background When limbal stem cell deficiency (LSCD) occurs,not only the limbal stem cells (LSCs) were damaged,but also the LSCs matrix microenvironment was under destruction.The treatment of LSCD include both replenishing of stem cells and restoration of microenvironment.So far,the method to improve the microenvironment of LSCD still exist limitation and urgently need to establish more appropriate microenvironment for the LSCs growth in vitro.Objective This study was to investigate whether the human bone marrow-derived mesenchymal stem cells (BMSCs) can be used as the ideal cells to repair limbal microenvironment and its possible mechanism of repairing limbal microenvironment during the human LSCs amplification in vitro as feeder cells.Methods BMSCs were cultured and passaged in vitro,and flow cytometry was used to assay the expressions of CD45,CD71,CD90,CD105 and HLA-DR and directionally induced BMSCs to the osteoblasts and adipocytes.BMSCs were treated using mitomycin C (MMC) to use as the feeder cells.LSCs were separately co-cultured with BMSCs,Swiss-3T3 feeder cells and free-feeder cells,and colony-forming efficiency (CFE) of the LSCs was compared among different co-cultured groups.LSCs were then cultured sequentially and identified by flow cytometry.Expression of cytokines in BMSCs was confirmed by reverse transcriptional polymerase chain reaction (RT-PCR).Results Cultured BMSCs showed a good homogeneity,with a lot of expressions of interstitial cell markers such as CD71,CD90,CD105 and less expressions of hematopoietic cell markers including CD45 and HLA-DR.After separately cocultured with feeder cells for 12 days,the CFE of the LSCs co-cultured with BMSCs,Swiss-3T3 and no feeder cells was 3.67％ ±0.58％,4.30％ ± 1.54％ and 0.20％ ±0.10％,showing a statistical significant difference among the three groups(F =15.420,P =0.040).There was no statistically significant difference in the C FE of the LSCs between the BMSCs feeder group and the Swiss-3T3 feeder cells group(P =0.456),between the BMSCs feeder group and the free-feeder cells group or the Swiss-3T3 co-culture group and the free-feeder cells group (P =0.005,0.002).LSCs presented with a positive response for ABCG2 antigen in the co-cultured with BMSCs group.Basic fibroblast growth factor(bFGF),stem cell factor (SCF) and N-cadherin(N-cad) were positively expressed in the BMSCs as feeder cells.Conclusions Human BMSCs-derived feeder cells can improve the growth of the stromal microenvironment of the LSCs and enhance their proliferation ability.Human BMSCs are suitable for engineering of epithelial sheets.
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Background Primary open angle glaucoma (POAG) is a major blindness-causing disease,characterized by elevated intraocular pressure due to an insufficient outflow of aqueous humor. The trabecular meshwork lining the aqueous outflow pathway modulates the aqueous outflow facility. To study the biological characteristics of the trabecular meshwork cells has important significance. Objective This study was to culture the trabecular cells from primary open-angle glaucomatous eye (POAG) and study the biologic characteristics of passaged cells. Methods The deep scleral tissue with trabecular meshwork was obtained during the trabeculectomy from 8 eyes with POAG. The trabecular cells were primarily cultured and passaged in vitro. The generation 3 cells were identified by immunochemistry with the laminin (LM), fibronectin (FN) and neuron specific endolase (NSE)monoclonal antibodies. The ultrastructure was examined to observe the biological characteristics of the cells under the transmission electronic microscope. The experimental results were compared among POAG group, normal control group and blank control group. Results The primarily cultured POAG trabecular cells migrated from the edge of tissue mass about 10 days. The cells of generation 3 presented the logarithmic phase in the first 4 days and fused in the 7th day. FN,LM and NSE were positively expressed in the generated cells in POAG group and normal control group rather than blank control group. The MOD values of the generation 3 cells for FN in POAG group and normal control group were 0. 35 ± 0.06 and 0. 26 ± 0. 01, and those for LM were 0. 34 ± 0. 03 and 0. 25 ± 0. 02 respectively, showing statistically significant difference between these two groups ( FN: t = 14. 446, P＜0.001; LM: t = 9. 346, P＜0. 001 ). The microvilli, cytolysosome and phagocytic vesicle were obviously decreased in the trabcular cells of POAG group compared with normal control group under the transmission electron microscope. Conclusion The trabecular meshwork cells from POAG can be successfully cultured and passaged in vitro. It provides a cytology basis for further glaucoma research.
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0.05),but 5-Aza at the concentration of 15?mol/L inhibited the growth of MSCs(P
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Objective To establish a method for the in-vitro culture of astrocytes from cerebral cortex of neonatal rats.Methods Astrocytes were isolated from cerebral cortex of the neonatal SD rats aged 24 hours,and then were cultured in the high-glucose DMEM medium with 10% fetal bovine serum added.The morphological features of astrocytes were examined under inverted microscope.The growth of the third generation of astrocytes was observed with counting plate method and methyl thiazolyl tetrazolium (MTT) assay.Results The astrocytes from cerebral cortex of neonatal rats grew well,the astrocytes showed typical morphological features,and no other kinds of cells was found.Conclusion A simple and practical in-vitro culture method for rats astrocytes is established.