【摘要】： 目的研究大鼠骨髓間充質(zhì)干細(xì)胞(MSCs)體外誘導(dǎo)大鼠肝星狀細(xì)胞(HSCs)凋亡及其機制。 方法MSCs的分離：從SD雄性大鼠的股骨骨髓中獲得,經(jīng)分離,培養(yǎng),傳代至第4代使用；大鼠肝星狀細(xì)胞系(HSC-T6)及纖維原細(xì)胞系復(fù)蘇后傳代使用。用6孔塑料培養(yǎng)板,在半透膜(transwell insert)上層接種MSCs(2×105cells／well),在下層接種HSC-T6細(xì)胞(2X105cells/well),建立上下雙層細(xì)胞共培養(yǎng)體系,常規(guī)培養(yǎng)。實驗分組：①空白對照組：HSCs單獨培養(yǎng)；②陰性對照組：纖維原細(xì)胞(Fiberoblasts)與HSCs共培養(yǎng)；③MSCs組：MSCs與HSCs共培養(yǎng)。以上體系培養(yǎng)觀察24h,48h和72h,于倒置相差顯微鏡下動態(tài)觀察HSCs細(xì)胞形態(tài)；免疫組化法檢測HSCs a-SMA表達(dá)；WST-8法檢測HSCs增殖抑制率；流式細(xì)胞儀Annexin-V-FITC/PI雙染法和DNA凝膠電泳(DNA Ladder)檢測HSCs細(xì)胞凋亡；RT-PCR檢測HSCs Caspase-3, Bax基因mRNA表達(dá)；Western blot檢測HSCs Caspase-3, Bax蛋白表達(dá)。 結(jié)果(1)MSCs與HSCs共培養(yǎng)24h,48h和72h, HSCs表現(xiàn)明顯增殖抑制(P0.01),且呈現(xiàn)時間依賴性,MSCs組與空白對照組、陰性對照組比較均有顯著性差異(P0.01；P0.01)。(2)流式細(xì)胞儀Annexin-V-FITC/PI雙染法檢測MSCs與HSCs共培養(yǎng)后HSCs的凋亡率：共培養(yǎng)24h后HSCs凋亡率呈現(xiàn)時間依賴性,與空白對照組和陰性對照組比較有統(tǒng)計學(xué)差異(P0.01；P0.01)。(3)DNA凝膠電泳(DNA Ladder測定)MSCs組中出現(xiàn)了明顯的DNA Ladder,空白對照組和陰性對照組中無DNA Ladder。(4)RT-PCR檢測共培養(yǎng)后各組HSCs的Caspase-3, Bax mRNA表達(dá)：共培養(yǎng)24h后,MSCs組Caspase-3, Bax mRNA表達(dá)呈現(xiàn)時間依賴性,與空白對照組、陰性對照組比較均有統(tǒng)計學(xué)差異(P0.01；P0.01)。(5)Western blot檢測共培養(yǎng)后各組HSCs的Caspase-3, Bax蛋白質(zhì)表達(dá)：MSCs組24h后Caspase-3, Bax表達(dá)呈現(xiàn)時間依賴性,與空白對照組,陰性對照組比較均有統(tǒng)計學(xué)差異(P0.01；P0.01)。 結(jié)論(1)MSCs可在體外抑制HSCs增殖和誘導(dǎo)其凋亡。(2)MSCs可能通過旁分泌途徑誘導(dǎo)HSCs凋亡。(3)MSCs誘導(dǎo)HSCs凋亡發(fā)生是通過上調(diào)Caspase-3, Bax表達(dá)發(fā)揮作用。
[Abstract]:Objective to investigate the apoptosis of rat hepatic stellate cell (HSCs) induced by rat bone marrow mesenchymal stem cell (MSCs) in vitro and its mechanism. Methods MSCs was isolated from the femur bone marrow of SD male rats. It was isolated, cultured and subcultured to the 4th passage. The rat hepatic stellate cell line (HSC-T6) and fibroblast cell line were resuscitated and then subcultured. MSCs (2 脳 105cells/well) was inoculated on the semi-permeable membrane (transwell insert) and HSC-T6 cell (2X105cells/well) was inoculated in the lower layer with a 6-well plastic culture plate. The co-culture system of upper and lower double layer cells was established and cultured routinely. The control group was divided into two groups: the control group was isolated and the control group was isolated from the control group: the fibroblast (Fiberoblasts) and HSCs co-cultured in the control group, and the control group was co-cultured with HSCs. The morphology of HSCs cells was observed by inverted phase contrast microscope for 48h and 72h respectively, and the expression of HSCs a-SMA was detected by immunohistochemistry and the inhibitory rate of HSCs proliferation was detected by WST-8 method. Flow cytometry Annexin-V-FITC/PI double staining and DNA gel electrophoresis (DNA Ladder) were used to detect the apoptosis of HSCs cells. RT-PCR was used to detect the mRNA expression of HSCs Caspase-3, Bax gene. Western blot was used to detect the expression of HSCs Caspase-3, Bax protein. Results (1) MSCs and HSCs co-cultured for 48h and 72h showed obvious proliferation inhibition (P0.01), and there were significant differences between the control group and the control group (P0.01). P0.01). (2) flow cytometry Annexin-V-FITC/PI double staining method was used to detect the apoptosis rate of HSCs after co-culture of MSCs and HSCs. The apoptosis rate of HSCs was time-dependent after 24 hours of co-culture, which was significantly different from that of blank control group and negative control group (P0.01). P0.01). (3) DNA gel electrophoresis (DNA Ladder assay) in MSCs group, there were no DNA Ladder. (4 in DNA Ladder, blank control group and no DNA Ladder. (4 in negative control group. Caspase-3, Bax mRNA expression of HSCs in each group after co-culture was detected. Caspase-3, Bax mRNA expression in MSCs group was time-dependent after 24 hours of co-culture. Compared with the control group and the negative control group, there were significant differences between the control group and the negative control group (P0.01P0.01). (5) Western blot was used to detect the Caspase-3, Bax protein expression of HSCs in each group in a time-dependent manner after 24 hours, compared with the blank control group. There was statistical difference in negative control group (P 0.01, P 0.01). Conclusion (1) MSCs can inhibit the proliferation and induce apoptosis of HSCs in vitro. (2) MSCs may induce HSCs apoptosis through paracrine pathway. (3) MSCs induces HSCs apoptosis through up-regulation of Caspase-3, Bax expression.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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本文編號：2211317