MMP1基因修饰的骨髓间充质干细胞对肝纤维化修复作用的实验研究

发布时间：2018-06-21 03:01

  本文选题：BMSCs + 基质金属蛋白酶1　； 参考：《第三军医大学》2014年硕士论文

【摘要】：肝纤维化发病率和死亡率高,是能够对人类健康造出巨大威胁的一种全球流行的疾病,它的终末阶段是肝硬化,肝硬化以假小叶形成和肝功能进行性改变为主要表现,然而,肝纤维化是一个动态过程,并对肝细胞损伤有着复杂的调节机制,大量证据表明,肝纤维化是一个可逆的疾病,因此,采取有效的治疗可以防止甚至逆转肝纤维化。骨髓间充质干细胞(bone marrow stem cells, BMSCs)由于其多向分化和自我更新的潜能,被认为是一种行之有效的方法,BMSCs在体外可分化为肝细胞,更有研究证实BMSCs移植到大鼠肝脏后可以转化为肝样细胞。然而大量实验已经证实单独使用BMSCs作用有限,而基因修饰的BMSCs是提高其治疗能力的一种可行的方法,基质金属蛋白酶(MMPs)是一组能够降解细胞外基质(ECM)的酶,它们在正常的生理过程,例如生长、组织重塑、炎症、血管生成、损伤修复和细胞迁移中起着重要的作用。这其中的基质金属蛋白酶-1(matrix metalloproteinase1,MMP-1)能够降解纤维胶原Ⅰ,Ⅱ,Ⅲ,X型,明胶和蛋白聚糖类,在肝脏的基质降解中发挥着重要的作用。 目的： 通过将人基质金属蛋白酶-1(hMMP-1)修饰的BMSCs注入肝纤维化大鼠体内,观察其可否增强BMSCs对肝纤维化的修复作用。 方法： 贴壁法培养BMSCs,体外用重组腺病毒Ad-hMMP-1-增强绿色荧光蛋白(enhanced green fluorescent protein, EGFP)转染大鼠BMSCs,荧光显微镜观察绿色荧光蛋白(green fluorescent protein, GFP)表达,、Vestern blot法检测各组细胞内hMMP-1蛋白表达情况,细胞增殖-毒性检测试剂盒Cell Couning Kit-8(CCK-8)法检测转染后细胞增殖情况。 使用50%四氯化碳(Carbon tetrachloride, CCl4)+植物油溶液0.3ml/100g皮下注射造肝纤维化模型,10周后验证肝纤维化模型,测量各组大鼠体重和肝脏重量,下腔静脉取血检测肝纤维化指标,苏木素-伊红染色(hematoxylin-eosin, HE)染色观察病理组织学改变并行病理组织学评分,确认建模成功后,尾静脉注射转染后的BMSCs。 转染后3周,处死各组大鼠,酶联免疫吸附剂测定(enzyme linked immunosorbentassay, ELISA)法检测血清中hMMP-1表达情况,hMMP-1酶活性荧光定量试剂盒检测组织中酶活性,测量第3周各组大鼠的体重及大鼠肝脏的重量,下腔静脉取血检测肝功及肝纤维化的指标,冰冻切片于荧光显微镜下观察肝脏组织中的绿色荧光的表达情况,通过HE染色观察病理组织学的改变并行肝脏组织病理组织学评分,糖原染色或者称为PAS染色法(Periodic Acid-Schiff staining)观察肝脏组织糖原合成情况,胶原染色或者称为MASSON染色法(masson staining)观察胶原合成情况,荧光定量PCR、Westeron blot和肝脏组织免疫组织化学(immunohistochemistry, IHC)检测MMP-1、组织金属蛋白酶组织抑制剂—1(tissue inhibitor of metalloproteinase1, TIMP-1).甲胎蛋白(a-fetoprotein, AFP).白蛋白(Albumin, ALB).细胞角蛋白-18(Cytokeratin18, CK-18)的基因和蛋白表达情况。 结果： BMSCs用贴壁法成功培养,在体外成功将重组腺病毒Ad-hMMP-1-eGFP导入大鼠BMSCs中,转染后对细胞增殖无明显影响；10周后造模成功,尾静脉注射后转染的BMSCs成功在大鼠肝脏定植,行冰冻切片在肝组织中可见GFP标记的细胞,ELISA检查血清中仅有转染hMMP-1组有hMMP-1表达,酶活性为1.438×10-3mmol/(gmin); hMMP-1组较肝纤维化对照组体重明显增加,较空载体组、肝纤维化对照组两组肝脏重量明显增加,肝功能较肝纤维化对照组明显改善；HE染色可见hMMP-1组平均视野下假小叶的数量最少,细胞变性及脂肪变性的程度也是三者中最轻；MASSON染色显示hMMP-1组大鼠胶原含量较空载体组、肝纤维化对照组两组均低；PAS染色显示hMMP-1组大鼠糖原合成能力较空载体组、肝纤维化对照组两组强；免疫组化与荧光定量PCR结果显示hMMP-1组ALB. CK-18. AFP. MMP-1明显增高,TIMP-1降低。证实转染hMMP1能够显著提高BMSCs对肝纤维化的修复能力。
[Abstract]:Hepatic fibrosis with high morbidity and mortality is a global epidemic disease can produce a great threat to human health, it is the end stage of liver cirrhosis, liver cirrhosis to form pseudolobule and liver function changes as the main performance, however, liver fibrosis is a dynamic process, and the liver cell injury with the conditioner complex System, substantial evidence that hepatic fibrosis is a reversible disease, therefore, to take effective treatment can prevent or reverse hepatic fibrosis. Bone marrow mesenchymal stem cells (bone marrow stem cells, BMSCs) because of its differentiation and self-renewal potential, is considered to be a kind of effective method, BMSCs can be differentiated for liver cells in vitro More cells, the study confirmed that BMSCs transplanted into rat liver could transform into hepatocyte like cells. However, a large number of experiments have confirmed that BMSCs alone is limited, and the modified BMSCs is a feasible method to improve the treatment ability, matrix metalloproteinase (MMPs) is a group capable of degrading extracellular matrix (ECM) the enzyme, they are in Often the physiological process, such as growth, tissue remodeling, inflammation, angiogenesis, and plays an important role in repair and cell migration. Matrix metalloproteinases of the -1 (matrix metalloproteinase1 MMP-1) to the degradation of collagen I, II, III, X, gelatin and proteoglycans, in matrix degradation the liver plays an important role Use.
Objective:
The matrix metalloproteinase -1 (hMMP-1) modified BMSCs injection in hepatic fibrosis rats, to observe the possibility of strengthening the repair effect of BMSCs on liver fibrosis.
Method锛，

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