间充质干细胞修复PBC胆管上皮细胞功能的机制研究
发布时间：2019-06-24 14:23
【摘要】：背景:原发性胆汁性胆管炎(primary biliary cholangitis,PBC)是一种慢性胆汁淤积性自身免疫疾病。早期主要累及小叶间胆管及间隔胆管,肝内胆管上皮细胞(intrahepatic biliary epithelial cells,IBEC)在 PBC 的发病中起着重要的作用。间充质干细胞(mesenchymal stem cells,MSCs)有向肝细胞分化的潜能,也可以通过分泌生长因子修复损伤的组织。有研究证明MSCs移植治疗PBC是安全有效的。然而具体机制尚不清楚。目的:探讨MSCs对甘氨酸鹅脱氧胆酸(glycochenodeoxycholic acid,GCDC)造成胆管上皮细胞损伤的修复作用及机制,为MSCs治疗PBC提供依据。方法:BEC体外培养后,经不同浓度的GCDC作用不同时间后,采用流式细胞术检测BEC Annexin V/7-ADD阳性凋亡细胞的百分数。在此基础上,通过MSCs与损伤的BEC共培养,通过流式细胞术检测Annexin V/7-ADD凋亡百分数、线粒体膜电位JC-1的表达及活性氧自由基(reactive oxygen species,ROS)表达量。CCK-8法(cell counting kit8)检测BEC的细胞活力,蛋白印迹法(Western Blotting)检测各组PBC特异性自身抗原丙酮酸脱氢酶复合体E2亚基(E2 components of the pyruvate dehydrogenase complex,PDC-E2)、凋亡相关蛋白天冬氨酸蛋白水解酶 3/8/9(cysteinyl aspartate specific proteinase,Caspases)、自噬相关蛋白微管相关蛋白轻链(microtubule-associated protein-light chain 3,LC3)的表达。免疫组织化学法染色各组BEC特异性标记角蛋白19(cytokeratin-19,CK-19)。免疫荧光法检测自噬相关蛋白LC3B的表达。酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)检测患者血清及细胞培养上清血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达。结果:1.GCDC 可诱导 BEC 凋亡,1,000μM GCDC 作用 BEC 2h、6h、24h 后,与对照组相比凋亡细胞比例显著增加,(37.6±5.5、74.2±4.0、62.6±7.7)%vs(6.0 ± 0.5、16.0±4.3、16.4±4.0)%,p0.001。2.MSCs 与 BEC 共培养可以抑制 GCDC 引起的凋亡,(19.3±4.8)%vs(38.9±4.7)%,p0.05。MSCs 可以部分缓解GCDC引起的BEC细胞线粒体膜电位的损伤,但差异无统计学意义;MSCs可部分抑制GCDC引起的BEC凋亡水解相关蛋白活化的Caspase 8、Caspase 9的表达。3.GCDC作用BEC后胆管上皮细胞特异性标志物CK19表达降低,而MSCs共培养后,CK-19的表达增加。作为PBC特异性自身抗原的PDC-E2在GCDC处理后,与对照组相比,相对表达量显著增加,(1.4 ±0.2)vs(0.7±0.1),(p0.05),与MSCs共培养后,PDC-E2的相对表达量下降,(0.9±0.1)vs(1.4±0.2),(p0.05)。4.MSCs 可抑制 GCDC 诱导的 BEC 自噬的激活。MSCs可以减少GCDC引起的LC3的表达。5.MSCs部分缓解GCDC对BEC细胞活力的影响。GCDC作用2h和6h可明显降低BEC的细胞活力,而MSCs共培养之后BEC的增殖能力有部分提高,但差异无统计学意义。6.MSCs共培养有缓解GCDC诱导的细胞内的活性氧自由基表达的趋势,但差异无统计学意义。7.MSCs减少BEC上清VEGF的表达。患者血清VEGF的水平明显高于健康对照组,(1128.0 ± 650.4)pg/ml vs(92.6 ± 39.8)pg/ml,(p0.05)。且在体外实验中MSCs共培养可显著降低GCDC作用后BEC培养上清的VEGF表达水平,(296.5±38.8)pg/mlvs(712.7±90.2)pg/ml,(p0.001)。结论:GCDC可在体外对BEC造成损伤。而MSCs可通过减少BEC的凋亡,抑制其自噬的激活,减少PDC-E2表达,增加CK-19表达,从而对BEC产生修复作用,其机制可能与下调BEC表达VEGF有关。
[Abstract]:BACKGROUND: Primary biliary cholangitis (PBC) is a kind of autoimmune disease of chronic cholestasis. In the early stage, the interlobular bile duct and the interlobular bile duct and the intrahepatic biliary epithelial cells (IBEC) play an important role in the pathogenesis of PBC. Mesenchymal stem cells (MSCs) have the potential to differentiate into hepatocytes, and can also be used to repair the damaged tissue by secreting growth factors. It is proved that the transplantation of MSCs is safe and effective. However, that specific mechanism is not clear. Objective: To study the effect and mechanism of MSCs on the damage of bile duct epithelial cells caused by glycinodeoxycholic acid (GCDC), and to provide the basis for the treatment of PBC. Methods: After the in vitro culture of BEC, the percentage of BEC Annexin V/7-ADD positive apoptotic cells was detected by flow cytometry after different concentrations of GCDC. On this basis, the expression of Annexin V/7-ADD, the expression of mitochondrial membrane potential JC-1 and reactive oxygen species (ROS) expression were detected by flow cytometry. The cell activity of BEC was detected by the CCK-8 method, and the E2 components of the pyruvate dehydroxygenase complex (PDC-E2) and the apoptosis-related protein aspartate hydrolase 3/8/9 (cysteinyl-specific protein, Casastes) were detected by Western Blotting. The expression of microtubule-associated protein-light chain 3 (LC3) in the self-autophagy-related protein. BEC-specific marker keratin 19 (cytokeratin-19, CK-19) was stained by immunocytochemical method. The expression of autophagy-related protein LC3B was detected by immunofluorescence. The expression of vascular endothelial growth factor (VEGF) was detected by enzyme-linked immunosorbent assay (ELISA). Results:1. The apoptosis of BEC induced by GCDC could be induced by 1,000. m u.M GCDC. After 24 h, the percentage of apoptotic cells increased significantly (37.6% 5.5, 74.2% 4.0, 62.6% 7.7)% vs (6.0% 0.5, 16.0% 4.3, 16.4% 4.0)%, and p0.2. 1.2. The co-culture with BEC could inhibit the apoptosis induced by GCDC (19.3% 4.8)% vs (38.9% 4.7)%. p0.05. MSCs can partially alleviate the damage of the mitochondrial membrane potential of the BEC cells induced by the GCDC, but the difference is not significant; the MSCs can partially inhibit the expression of the Caspase 8 and the Caspase 9 which are activated by the BEC induced by the GCDC, and the expression of the specific marker CK19 of the bile duct epithelial cell after the GCDC acting BEC is reduced, The expression of CK-19 increased after the co-culture of MSCs. Compared with the control group, the relative expression of pPDC-E2 as the PBC-specific autoantigen increased significantly (1.4-0.2) vs (0.7-0.1), (p0.05), and the relative expression of PDC-E2 decreased after co-culture with MSCs (0.9-0.1) vs (1.4-0.2). (p0.05).4. MSCs can inhibit the activation of the autophagy of BEC induced by GCDC. MSCs can reduce the expression of LC3 induced by GCDC. The effects of GCDC on the cell viability of BEC could be significantly reduced by 2 h and 6 h, and the proliferation ability of BEC was partly improved after the co-culture of MSCs, but the difference was not significant.6. The co-culture of MSCs was a trend to alleviate the expression of reactive oxygen free radicals in the cells induced by GCDC. 7.MSCs decreased the expression of VEGF in BEC. The level of VEGF in patients was significantly higher than that in healthy control group (1128.0-650.4) pg/ ml vs (92.6-39.8) pg/ ml (p0.05). In vitro, the expression of VEGF in the culture supernatant of BEC after GCDC was significantly reduced (296.5-38.8) pg/ ml vs (712.7-90.2) pg/ ml (p0.001). Conclusion: The GCDC can damage BEC in vitro. In addition, MSCs can reduce the apoptosis of BEC, inhibit the activation of autophagy, decrease the expression of PDC-E2, increase the expression of CK-19, and thus produce a repair effect on BEC, and the mechanism may be related to down-regulation of BEC expression of VEGF.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.7
本文编号：2505131
[Abstract]:BACKGROUND: Primary biliary cholangitis (PBC) is a kind of autoimmune disease of chronic cholestasis. In the early stage, the interlobular bile duct and the interlobular bile duct and the intrahepatic biliary epithelial cells (IBEC) play an important role in the pathogenesis of PBC. Mesenchymal stem cells (MSCs) have the potential to differentiate into hepatocytes, and can also be used to repair the damaged tissue by secreting growth factors. It is proved that the transplantation of MSCs is safe and effective. However, that specific mechanism is not clear. Objective: To study the effect and mechanism of MSCs on the damage of bile duct epithelial cells caused by glycinodeoxycholic acid (GCDC), and to provide the basis for the treatment of PBC. Methods: After the in vitro culture of BEC, the percentage of BEC Annexin V/7-ADD positive apoptotic cells was detected by flow cytometry after different concentrations of GCDC. On this basis, the expression of Annexin V/7-ADD, the expression of mitochondrial membrane potential JC-1 and reactive oxygen species (ROS) expression were detected by flow cytometry. The cell activity of BEC was detected by the CCK-8 method, and the E2 components of the pyruvate dehydroxygenase complex (PDC-E2) and the apoptosis-related protein aspartate hydrolase 3/8/9 (cysteinyl-specific protein, Casastes) were detected by Western Blotting. The expression of microtubule-associated protein-light chain 3 (LC3) in the self-autophagy-related protein. BEC-specific marker keratin 19 (cytokeratin-19, CK-19) was stained by immunocytochemical method. The expression of autophagy-related protein LC3B was detected by immunofluorescence. The expression of vascular endothelial growth factor (VEGF) was detected by enzyme-linked immunosorbent assay (ELISA). Results:1. The apoptosis of BEC induced by GCDC could be induced by 1,000. m u.M GCDC. After 24 h, the percentage of apoptotic cells increased significantly (37.6% 5.5, 74.2% 4.0, 62.6% 7.7)% vs (6.0% 0.5, 16.0% 4.3, 16.4% 4.0)%, and p0.2. 1.2. The co-culture with BEC could inhibit the apoptosis induced by GCDC (19.3% 4.8)% vs (38.9% 4.7)%. p0.05. MSCs can partially alleviate the damage of the mitochondrial membrane potential of the BEC cells induced by the GCDC, but the difference is not significant; the MSCs can partially inhibit the expression of the Caspase 8 and the Caspase 9 which are activated by the BEC induced by the GCDC, and the expression of the specific marker CK19 of the bile duct epithelial cell after the GCDC acting BEC is reduced, The expression of CK-19 increased after the co-culture of MSCs. Compared with the control group, the relative expression of pPDC-E2 as the PBC-specific autoantigen increased significantly (1.4-0.2) vs (0.7-0.1), (p0.05), and the relative expression of PDC-E2 decreased after co-culture with MSCs (0.9-0.1) vs (1.4-0.2). (p0.05).4. MSCs can inhibit the activation of the autophagy of BEC induced by GCDC. MSCs can reduce the expression of LC3 induced by GCDC. The effects of GCDC on the cell viability of BEC could be significantly reduced by 2 h and 6 h, and the proliferation ability of BEC was partly improved after the co-culture of MSCs, but the difference was not significant.6. The co-culture of MSCs was a trend to alleviate the expression of reactive oxygen free radicals in the cells induced by GCDC. 7.MSCs decreased the expression of VEGF in BEC. The level of VEGF in patients was significantly higher than that in healthy control group (1128.0-650.4) pg/ ml vs (92.6-39.8) pg/ ml (p0.05). In vitro, the expression of VEGF in the culture supernatant of BEC after GCDC was significantly reduced (296.5-38.8) pg/ ml vs (712.7-90.2) pg/ ml (p0.001). Conclusion: The GCDC can damage BEC in vitro. In addition, MSCs can reduce the apoptosis of BEC, inhibit the activation of autophagy, decrease the expression of PDC-E2, increase the expression of CK-19, and thus produce a repair effect on BEC, and the mechanism may be related to down-regulation of BEC expression of VEGF.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.7
【参考文献】
相关期刊论文 前1条
1 Ying-Qiu Huang;;Recent advances in the diagnosis and treatment of primary biliary cholangitis[J];World Journal of Hepatology;2016年33期
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