發(fā)布時(shí)間：2018-06-21 03:01

  本文選題：BMSCs + 基質(zhì)金屬蛋白酶1�。� 參考：《第三軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：肝纖維化發(fā)病率和死亡率高,是能夠?qū)θ祟惤】翟斐鼍薮笸{的一種全球流行的疾病,它的終末階段是肝硬化,肝硬化以假小葉形成和肝功能進(jìn)行性改變?yōu)橹饕憩F(xiàn),然而,肝纖維化是一個(gè)動(dòng)態(tài)過(guò)程,并對(duì)肝細(xì)胞損傷有著復(fù)雜的調(diào)節(jié)機(jī)制,大量證據(jù)表明,肝纖維化是一個(gè)可逆的疾病,因此,采取有效的治療可以防止甚至逆轉(zhuǎn)肝纖維化。骨髓間充質(zhì)干細(xì)胞(bone marrow stem cells, BMSCs)由于其多向分化和自我更新的潛能,被認(rèn)為是一種行之有效的方法,BMSCs在體外可分化為肝細(xì)胞,更有研究證實(shí)BMSCs移植到大鼠肝臟后可以轉(zhuǎn)化為肝樣細(xì)胞。然而大量實(shí)驗(yàn)已經(jīng)證實(shí)單獨(dú)使用BMSCs作用有限,而基因修飾的BMSCs是提高其治療能力的一種可行的方法,基質(zhì)金屬蛋白酶(MMPs)是一組能夠降解細(xì)胞外基質(zhì)(ECM)的酶,它們?cè)谡５纳磉^(guò)程,例如生長(zhǎng)、組織重塑、炎癥、血管生成、損傷修復(fù)和細(xì)胞遷移中起著重要的作用。這其中的基質(zhì)金屬蛋白酶-1(matrix metalloproteinase1,MMP-1)能夠降解纖維膠原Ⅰ,Ⅱ,Ⅲ,X型,明膠和蛋白聚糖類,在肝臟的基質(zhì)降解中發(fā)揮著重要的作用。 目的： 通過(guò)將人基質(zhì)金屬蛋白酶-1(hMMP-1)修飾的BMSCs注入肝纖維化大鼠體內(nèi),觀察其可否增強(qiáng)BMSCs對(duì)肝纖維化的修復(fù)作用。 方法： 貼壁法培養(yǎng)BMSCs,體外用重組腺病毒Ad-hMMP-1-增強(qiáng)綠色熒光蛋白(enhanced green fluorescent protein, EGFP)轉(zhuǎn)染大鼠BMSCs,熒光顯微鏡觀察綠色熒光蛋白(green fluorescent protein, GFP)表達(dá),、Vestern blot法檢測(cè)各組細(xì)胞內(nèi)hMMP-1蛋白表達(dá)情況,細(xì)胞增殖-毒性檢測(cè)試劑盒Cell Couning Kit-8(CCK-8)法檢測(cè)轉(zhuǎn)染后細(xì)胞增殖情況。 使用50%四氯化碳(Carbon tetrachloride, CCl4)+植物油溶液0.3ml/100g皮下注射造肝纖維化模型,10周后驗(yàn)證肝纖維化模型,測(cè)量各組大鼠體重和肝臟重量,下腔靜脈取血檢測(cè)肝纖維化指標(biāo),蘇木素-伊紅染色(hematoxylin-eosin, HE)染色觀察病理組織學(xué)改變并行病理組織學(xué)評(píng)分,確認(rèn)建模成功后,尾靜脈注射轉(zhuǎn)染后的BMSCs。 轉(zhuǎn)染后3周,處死各組大鼠,酶聯(lián)免疫吸附劑測(cè)定(enzyme linked immunosorbentassay, ELISA)法檢測(cè)血清中hMMP-1表達(dá)情況,hMMP-1酶活性熒光定量試劑盒檢測(cè)組織中酶活性,測(cè)量第3周各組大鼠的體重及大鼠肝臟的重量,下腔靜脈取血檢測(cè)肝功及肝纖維化的指標(biāo),冰凍切片于熒光顯微鏡下觀察肝臟組織中的綠色熒光的表達(dá)情況,通過(guò)HE染色觀察病理組織學(xué)的改變并行肝臟組織病理組織學(xué)評(píng)分,糖原染色或者稱為PAS染色法(Periodic Acid-Schiff staining)觀察肝臟組織糖原合成情況,膠原染色或者稱為MASSON染色法(masson staining)觀察膠原合成情況,熒光定量PCR、Westeron blot和肝臟組織免疫組織化學(xué)(immunohistochemistry, IHC)檢測(cè)MMP-1、組織金屬蛋白酶組織抑制劑—1(tissue inhibitor of metalloproteinase1, TIMP-1).甲胎蛋白(a-fetoprotein, AFP).白蛋白(Albumin, ALB).細(xì)胞角蛋白-18(Cytokeratin18, CK-18)的基因和蛋白表達(dá)情況。 結(jié)果： BMSCs用貼壁法成功培養(yǎng),在體外成功將重組腺病毒Ad-hMMP-1-eGFP導(dǎo)入大鼠BMSCs中,轉(zhuǎn)染后對(duì)細(xì)胞增殖無(wú)明顯影響；10周后造模成功,尾靜脈注射后轉(zhuǎn)染的BMSCs成功在大鼠肝臟定植,行冰凍切片在肝組織中可見GFP標(biāo)記的細(xì)胞,ELISA檢查血清中僅有轉(zhuǎn)染hMMP-1組有hMMP-1表達(dá),酶活性為1.438×10-3mmol/(gmin); hMMP-1組較肝纖維化對(duì)照組體重明顯增加,較空載體組、肝纖維化對(duì)照組兩組肝臟重量明顯增加,肝功能較肝纖維化對(duì)照組明顯改善；HE染色可見hMMP-1組平均視野下假小葉的數(shù)量最少,細(xì)胞變性及脂肪變性的程度也是三者中最輕；MASSON染色顯示hMMP-1組大鼠膠原含量較空載體組、肝纖維化對(duì)照組兩組均低；PAS染色顯示hMMP-1組大鼠糖原合成能力較空載體組、肝纖維化對(duì)照組兩組強(qiáng)；免疫組化與熒光定量PCR結(jié)果顯示hMMP-1組ALB. CK-18. AFP. MMP-1明顯增高,TIMP-1降低。證實(shí)轉(zhuǎn)染hMMP1能夠顯著提高BMSCs對(duì)肝纖維化的修復(fù)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:
By injecting BMSCs modified by human matrix metalloproteinase -1 (hMMP-1) into liver fibrosis rats, we can see whether it can enhance the effect of BMSCs on liver fibrosis.
Method錛，

本文編號(hào)：2046897