【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)干細胞(mesenchymal stem cells,MSCs)有向肝細胞分化的潛能,也可以通過分泌生長因子修復(fù)損傷的組織。有研究證明MSCs移植治療PBC是安全有效的。然而具體機制尚不清楚。目的:探討MSCs對甘氨酸鵝脫氧膽酸(glycochenodeoxycholic acid,GCDC)造成膽管上皮細胞損傷的修復(fù)作用及機制,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作用不同時間后,采用流式細胞術(shù)檢測BEC Annexin V/7-ADD陽性凋亡細胞的百分數(shù)。在此基礎(chǔ)上,通過MSCs與損傷的BEC共培養(yǎng),通過流式細胞術(shù)檢測Annexin V/7-ADD凋亡百分數(shù)、線粒體膜電位JC-1的表達及活性氧自由基(reactive oxygen species,ROS)表達量。CCK-8法(cell counting kit8)檢測BEC的細胞活力,蛋白印跡法(Western Blotting)檢測各組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。免疫組織化學(xué)法染色各組BEC特異性標記角蛋白19(cytokeratin-19,CK-19)。免疫熒光法檢測自噬相關(guān)蛋白LC3B的表達。酶聯(lián)免疫吸附(enzyme linked immunosorbent assay,ELISA)檢測患者血清及細胞培養(yǎng)上清血管內(nèi)皮生長因子(vascular endothelial growth factor,VEGF)的表達。結(jié)果:1.GCDC 可誘導(dǎo) BEC 凋亡,1,000μM GCDC 作用 BEC 2h、6h、24h 后,與對照組相比凋亡細胞比例顯著增加,(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細胞線粒體膜電位的損傷,但差異無統(tǒng)計學(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。3.GCDC作用BEC后膽管上皮細胞特異性標志物CK19表達降低,而MSCs共培養(yǎng)后,CK-19的表達增加。作為PBC特異性自身抗原的PDC-E2在GCDC處理后,與對照組相比,相對表達量顯著增加,(1.4 ±0.2)vs(0.7±0.1),(p0.05),與MSCs共培養(yǎng)后,PDC-E2的相對表達量下降,(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。5.MSCs部分緩解GCDC對BEC細胞活力的影響。GCDC作用2h和6h可明顯降低BEC的細胞活力,而MSCs共培養(yǎng)之后BEC的增殖能力有部分提高,但差異無統(tǒng)計學(xué)意義。6.MSCs共培養(yǎng)有緩解GCDC誘導(dǎo)的細胞內(nèi)的活性氧自由基表達的趨勢,但差異無統(tǒng)計學(xué)意義。7.MSCs減少BEC上清VEGF的表達�；颊哐錠EGF的水平明顯高于健康對照組,(1128.0 ± 650.4)pg/ml vs(92.6 ± 39.8)pg/ml,(p0.05)。且在體外實驗中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BEC造成損傷。而MSCs可通過減少BEC的凋亡,抑制其自噬的激活,減少PDC-E2表達,增加CK-19表達,從而對BEC產(chǎn)生修復(fù)作用,其機制可能與下調(diào)BEC表達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)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R575.7

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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：2505131