本文選題：骨髓間充質干細胞 + 肝星狀細胞��； 參考：《廣西醫(yī)科大學》2012年碩士論文

【摘要】：目的：觀察大鼠骨髓間充質干細胞(BMSCs)對肝星狀細胞(HSCs)死亡受體5和Caspase-3蛋白表達的影響,探討B(tài)MSCs誘導HSCs凋亡及其機制。 方法：貼壁篩選法培養(yǎng)、純化SD大鼠BMSCs,傳代至第3代使用,大鼠肝纖維原細胞系和原代HSCs細胞凍融后傳至第4代使用。在半透膜(transwell insert)上分別接種肝纖維原細胞和BMSCs細胞,在6孔培養(yǎng)板上接種大鼠HSCs細胞,建立上下雙層共培養(yǎng)體系。實驗分4組：①空白對照組：BMSCs和HSCs分別單獨培養(yǎng)；②陰性對照組：HSCs與肝纖維原細胞共培養(yǎng)(模擬肝星狀細胞體內生長環(huán)境)；③共培養(yǎng)組：HSCs與BMSCs共培養(yǎng)；④實驗組：用腫瘤壞死因子相關凋亡誘導配體(TRAIL)多克隆抗體先與BMSCs作用6h后,不換液,再與HSCs共培養(yǎng)。在倒置相差顯微鏡下,于24h,48h,72h各時間段動態(tài)觀察HSCs和BMSCs細胞形態(tài)學改變；MTT法檢測BMSCs對HSCs增殖的抑制率；應用流式細胞儀Annexin-V-FITC/PI雙染法檢測HSCs凋亡率；Western Blot法分別檢測HSCs細胞上Caspase-3蛋白和死亡受體5蛋白的表達；用SYBR熒光實時定量RT-PCR法檢測HSCs死亡受體5mRNA的表達；收集各組上清液,ELISA法檢測各組上清液中TRAIL的濃度。 結果：1BMSCs對HSCs的增殖具有抑制作用,在共培養(yǎng)組中,培養(yǎng)24后,肝星狀細胞增殖抑制率升高,72h后明顯升高,且呈現(xiàn)時間依賴性,在共培養(yǎng)的各個時間段與空白組、陰性對照組和實驗組比較差異均有顯著性統(tǒng)計學意義(P0.01)。2.Annexin-V-FITC/PI雙染法檢測BMSCs和HSCs共培養(yǎng)24,48,72h后HSCs的凋亡率分別為10.17%、15.15%和30.18%,與其他各組比較差異有顯著性統(tǒng)計學意義(P0.01)。3ELISA法檢測各組上清液中TRAIL的濃度：各組在培養(yǎng)48h時,TRAIL的分泌到達生理的增殖期高峰,72h后有所下降；共培養(yǎng)組的TRAIL濃度與BMSCs單獨培養(yǎng)組比較差異有顯著性統(tǒng)計學意義,在共培養(yǎng)各個時間段低于BMSCs單獨培養(yǎng)組。4.共培養(yǎng)24h后,共培養(yǎng)組Caspase-3蛋白的表達顯著升高,與空白組和陰性對照組比較差異有顯著性統(tǒng)計學意義(P0.01),且隨時間延長呈現(xiàn)遞增趨勢；實驗組Caspase-3蛋白與共培養(yǎng)組比較差異有顯著性統(tǒng)計學意義(P0.01),在共培養(yǎng)的各個時間段均低于共培養(yǎng)組。5.共培養(yǎng)組死亡受體5蛋白的表達隨共培養(yǎng)時間的延長呈遞增趨勢,與空白組和陰性對照組比較差異有顯著性統(tǒng)計學意義(P0.01),在共培養(yǎng)的各個時間段均高于以上兩組；實驗組死亡受體5蛋白在共培養(yǎng)的各個時間段均低于共培養(yǎng)組,二者比較差異有顯著性統(tǒng)計學意義(P0.01)；空白組死亡受體5蛋白的表達和陰性組在共培養(yǎng)的各個時間段比較無差異(P0.05)6.共培養(yǎng)組BMSCs能明顯上調HSCs中死亡受體5mRNA的表達,在共培養(yǎng)的各個時間段與其他各組比較差異有顯著性統(tǒng)計學意義(P0.01)且表達高于以上各組；而空白組和陰性對照組死亡受體5mRNA的表達在共培養(yǎng)的各個時間段比較無差異(P0.05) 結論：BMSCs與HSCs共培養(yǎng)能抑制HSCs的增殖,促進HSCs的凋亡。其機制可能為BMSCs旁分泌的TRAIL通過上調Caspase-3和DR5蛋白的表達實現(xiàn)的。
[Abstract]:Objective: To observe the effect of rat bone marrow mesenchymal stem cells (BMSCs) on the expression of death receptor 5 and Caspase-3 protein in hepatic stellate cells (HSCs) and explore the apoptosis and its mechanism induced by BMSCs in HSCs.
Methods: the SD rat BMSCs was purified by the adherent wall screening method and passed to the third generation. The rat liver fibroblast cell line and the original HSCs cell were transmitted to the fourth generation after freezing and thawing. The liver fibroblasts and BMSCs cells were inoculated on the semi permeable membrane (Transwell insert), and the rat HSCs cells were inoculated on the 6 Hole culture plate, and the upper and lower bilayer co culture bodies were established. The experiment was divided into 4 groups: (1) blank control group: BMSCs and HSCs were cultured separately; (2) negative control group: co culture of HSCs and hepatic fibroblast (simulating the growth environment of hepatic stellate cells); (3) co culture group: co culture of HSCs and BMSCs; (4) experimental group: polyclonal antibody with tumor necrosis factor related apoptosis inducing ligand (TRAIL) After 6h was first acted with BMSCs, the liquid was not changed and then co cultured with HSCs. Under the inverted phase contrast microscope, the morphological changes of HSCs and BMSCs cells were observed dynamically at all time segments of 24h, 48h and 72h, and the inhibition rate of BMSCs to HSCs proliferation was detected by MTT method, and the apoptosis rate was detected by the method of flow cytometry. The expression of Caspase-3 protein and death receptor 5 protein on s cells. The expression of HSCs death receptor 5mRNA was detected by SYBR fluorescence real-time quantitative RT-PCR method, the supernatant of each group was collected, and the concentration of TRAIL in the supernatant of each group was detected by ELISA method.
Results: 1BMSCs had a inhibitory effect on the proliferation of HSCs. In the co culture group, the proliferation inhibition rate of hepatic stellate cells increased after 24, and 72h was significantly increased and showed time dependence. There was significant statistical significance (P0.01).2.Annexin-V-FITC/ between the negative control group and the experimental group in each time period of co culture. The apoptosis rate of HSCs was 10.17%, 15.15% and 30.18% respectively after BMSCs and HSCs were co cultured for 24,48,72h. There was significant statistical significance (P0.01).3ELISA method to detect the concentration of TRAIL in the supernatant of each group with the other groups (P0.01), and the.3ELISA method was used to detect the concentration of TRAIL in each group: each group reached the peak of the physiological proliferation period of the TRAIL, and decreased after 72h when the 48h was cultured. The concentration of TRAIL in the co culture group was significantly different from that in the BMSCs group, and the expression of Caspase-3 protein in the co culture group was significantly higher than that of the BMSCs single culture group.4. in the co culture group. The difference was statistically significant (P0.01) with the blank group and the negative control group (P0.01). The extension of Caspase-3 protein in the experimental group was significantly higher than that of the co culture group (P0.01). The expression of death receptor 5 protein in the co culture group was lower than that of the co culture group, and the expression of the 5 protein in the co culture group increased with the prolongation of the co culture time, and there was a significant difference from the blank group and the negative control group. The statistical significance (P0.01) was higher than that of the above two groups in all the time periods of co culture; the 5 protein of the death receptor in the experimental group was lower than that of the co culture group, and the difference between the two groups was significant (P0.01); the expression of the death receptor 5 egg white in the blank group and the negative group were in each time period of co culture. The P0.05 6. co culture group BMSCs can obviously increase the expression of death receptor 5mRNA in HSCs, and there is significant statistical significance (P0.01) in each time period of co culture with the other groups (P0.01), and the expression of 5mRNA in the blank group and negative control group is in each time period of co culture. No difference (P0.05)
Conclusion: the co culture of BMSCs and HSCs can inhibit the proliferation of HSCs and promote the apoptosis of HSCs. The mechanism may be that the TRAIL paracrine secreted by BMSCs can be realized by up regulation of the expression of Caspase-3 and DR5 protein.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

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