【摘要】：背景:原發(fā)性膽汁性膽管炎(primary biliary cholangitis,PBC)是一種慢性膽汁淤積性自身免疫疾病。早期主要累及小葉間膽管及間隔膽管,肝內(nèi)膽管上皮細(xì)胞(intrahepatic biliary epithelial cells,IBEC)在 PBC 的發(fā)病中起著重要的作用。間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)有向肝細(xì)胞分化的潛能,也可以通過(guò)分泌生長(zhǎng)因子修復(fù)損傷的組織。有研究證明MSCs移植治療PBC是安全有效的。然而具體機(jī)制尚不清楚。目的:探討MSCs對(duì)甘氨酸鵝脫氧膽酸(glycochenodeoxycholic acid,GCDC)造成膽管上皮細(xì)胞損傷的修復(fù)作用及機(jī)制,為MSCs治療PBC提供依據(jù)。方法:BEC體外培養(yǎng)后,經(jīng)不同濃度的GCDC作用不同時(shí)間后,采用流式細(xì)胞術(shù)檢測(cè)BEC Annexin V/7-ADD陽(yáng)性凋亡細(xì)胞的百分?jǐn)?shù)。在此基礎(chǔ)上,通過(guò)MSCs與損傷的BEC共培養(yǎng),通過(guò)流式細(xì)胞術(shù)檢測(cè)Annexin V/7-ADD凋亡百分?jǐn)?shù)、線粒體膜電位JC-1的表達(dá)及活性氧自由基(reactive oxygen species,ROS)表達(dá)量。CCK-8法(cell counting kit8)檢測(cè)BEC的細(xì)胞活力,蛋白印跡法(Western Blotting)檢測(cè)各組PBC特異性自身抗原丙酮酸脫氫酶復(fù)合體E2亞基(E2 components of the pyruvate dehydrogenase complex,PDC-E2)、凋亡相關(guān)蛋白天冬氨酸蛋白水解酶 3/8/9(cysteinyl aspartate specific proteinase,Caspases)、自噬相關(guān)蛋白微管相關(guān)蛋白輕鏈(microtubule-associated protein-light chain 3,LC3)的表達(dá)。免疫組織化學(xué)法染色各組BEC特異性標(biāo)記角蛋白19(cytokeratin-19,CK-19)。免疫熒光法檢測(cè)自噬相關(guān)蛋白LC3B的表達(dá)。酶聯(lián)免疫吸附(enzyme linked immunosorbent assay,ELISA)檢測(cè)患者血清及細(xì)胞培養(yǎng)上清血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor,VEGF)的表達(dá)。結(jié)果:1.GCDC 可誘導(dǎo) BEC 凋亡,1,000μM GCDC 作用 BEC 2h、6h、24h 后,與對(duì)照組相比凋亡細(xì)胞比例顯著增加,(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 與 BEC 共培養(yǎng)可以抑制 GCDC 引起的凋亡,(19.3±4.8)%vs(38.9±4.7)%,p0.05。MSCs 可以部分緩解GCDC引起的BEC細(xì)胞線粒體膜電位的損傷,但差異無(wú)統(tǒng)計(jì)學(xué)意義;MSCs可部分抑制GCDC引起的BEC凋亡水解相關(guān)蛋白活化的Caspase 8、Caspase 9的表達(dá)。3.GCDC作用BEC后膽管上皮細(xì)胞特異性標(biāo)志物CK19表達(dá)降低,而MSCs共培養(yǎng)后,CK-19的表達(dá)增加。作為PBC特異性自身抗原的PDC-E2在GCDC處理后,與對(duì)照組相比,相對(duì)表達(dá)量顯著增加,(1.4 ±0.2)vs(0.7±0.1),(p0.05),與MSCs共培養(yǎng)后,PDC-E2的相對(duì)表達(dá)量下降,(0.9±0.1)vs(1.4±0.2),(p0.05)。4.MSCs 可抑制 GCDC 誘導(dǎo)的 BEC 自噬的激活。MSCs可以減少GCDC引起的LC3的表達(dá)。5.MSCs部分緩解GCDC對(duì)BEC細(xì)胞活力的影響。GCDC作用2h和6h可明顯降低BEC的細(xì)胞活力,而MSCs共培養(yǎng)之后BEC的增殖能力有部分提高,但差異無(wú)統(tǒng)計(jì)學(xué)意義。6.MSCs共培養(yǎng)有緩解GCDC誘導(dǎo)的細(xì)胞內(nèi)的活性氧自由基表達(dá)的趨勢(shì),但差異無(wú)統(tǒng)計(jì)學(xué)意義。7.MSCs減少BEC上清VEGF的表達(dá)�；颊哐錠EGF的水平明顯高于健康對(duì)照組,(1128.0 ± 650.4)pg/ml vs(92.6 ± 39.8)pg/ml,(p0.05)。且在體外實(shí)驗(yàn)中MSCs共培養(yǎng)可顯著降低GCDC作用后BEC培養(yǎng)上清的VEGF表達(dá)水平,(296.5±38.8)pg/mlvs(712.7±90.2)pg/ml,(p0.001)。結(jié)論:GCDC可在體外對(duì)BEC造成損傷。而MSCs可通過(guò)減少BEC的凋亡,抑制其自噬的激活,減少PDC-E2表達(dá),增加CK-19表達(dá),從而對(duì)BEC產(chǎn)生修復(fù)作用,其機(jī)制可能與下調(diào)BEC表達(dá)VEGF有關(guān)。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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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本文編號(hào)：2505131