本文選題：肝硬化 + 骨髓間充質(zhì)干細(xì)胞��； 參考：《蘭州大學(xué)》2017年碩士論文

【摘要】：背景:干細(xì)胞移植治療肝硬化在動(dòng)物實(shí)驗(yàn)、臨床研究已被廣泛研究,堿性成纖維細(xì)胞生長(zhǎng)因子體外條件下可以促進(jìn)干細(xì)胞增殖,利用bFGF培養(yǎng)后的干細(xì)胞治療肝硬化的體內(nèi)療效目前尚不確定。目的:探索bFGF對(duì)BMSC生長(zhǎng)和增殖的影響。加入0.1%bFGF培養(yǎng)后的骨髓間充質(zhì)干細(xì)胞通過(guò)小鼠尾靜脈注射后肝功能ALT、AST、ALB水平、組織病理學(xué)改變及膠原沉積變化情況。方法:全骨髓貼壁法提取雄性SD大鼠骨髓間充質(zhì)干細(xì)胞;流式細(xì)胞檢測(cè)BMSC。雌性昆明小鼠46只隨機(jī)分為分為5組:A組(CCl4+PBS)、B組(CCl4+bFGF)、C組(CCl4+bFGF-BMSC)、D組(CCl4+bFGF+BMSC)、E組(CCl4+bFGF+BMSC3次移植)。小鼠CCl4造模10周后分別尾靜脈注射bFGF+或bFGF-培養(yǎng)的BMSC,酶聯(lián)免疫法檢測(cè)肝功能,HE染色檢測(cè)組織病理學(xué)改變情況;Masson三色染色法檢測(cè)肝組織膠原沉積情況。結(jié)果:(1)BMSC生長(zhǎng)情況:觀察各組傳代細(xì)胞的生長(zhǎng)情況,bFGF-、bFGF+組細(xì)胞24h完全貼壁,貼壁細(xì)胞初期呈梭形或不規(guī)則形,細(xì)胞形態(tài)無(wú)明顯異常,主要以梭形為主,bFGF+組細(xì)胞增殖較快,于第4天鋪滿瓶底。bFGF-組較bFGF+細(xì)胞生長(zhǎng)相對(duì)緩慢,第6-7天時(shí)基本完全鋪滿細(xì)胞培養(yǎng)瓶。(2)細(xì)胞生長(zhǎng):10ng/ml bFGF體外培養(yǎng)條件下,BMSC生長(zhǎng)速率顯著高于無(wú)bFGF培養(yǎng)。(3)流式檢測(cè):CD29、CD44陽(yáng)性表達(dá),CD45呈陰性表達(dá)。(4)化學(xué)法檢測(cè)肝臟功能:ALT水平在模型對(duì)照組(A組)與B組比較無(wú)顯著性差異(P0.05),C組、D組、E組ALT水平與A組、B組比較均有下降(兩兩比較具有顯著性差異,P0.05);ALT水平在D組、E組均較C組下降(P0.05);E組較D組下降(P0.05)。AST水平在A組與B組間無(wú)顯著性差異(P0.05);C組、D組、E組兩兩之間無(wú)顯著性差異(P0.05);而C組、D組、E組較A組或B組具有明顯下降(P0.05)。(5)HE染色結(jié)果:A組、B組HE染色見肝細(xì)胞明顯水腫、脂肪變性、橋接壞死,肝索排列紊亂,假小葉廣泛形成。C組肝細(xì)胞水腫、脂肪變性,炎性細(xì)胞浸潤(rùn),但程度較模型組有所減輕,匯管區(qū)纖維沉積,可見假小葉結(jié)構(gòu);D組、E組肝細(xì)胞水腫情況、炎性細(xì)胞浸潤(rùn)程度較BMSC組明顯改善,假小葉結(jié)構(gòu)可見,匯管區(qū)可見纖維沉積。(6)Masson結(jié)果顯示,A組、B組肝門脈區(qū)大量綠染纖維沉積,肝實(shí)質(zhì)內(nèi)綠色纖維浸潤(rùn),纖細(xì)的纖維間隔從匯管區(qū)延伸至肝實(shí)質(zhì),分隔肝小葉形成假小葉。A組纖維面積較B組無(wú)顯著性差異(P0.05);與A組、B組相比,C組、D組、E組纖維量減少,較A組、B組明顯改善,差異有統(tǒng)計(jì)學(xué)意義(p值均0.05);C組、D組匯管區(qū)、肝實(shí)質(zhì)有可見纖維沉積,但兩組纖維面積無(wú)顯著性差異(P0.05);E組較C組、D組面積均有顯著降低(P0.05)。結(jié)論:體外培養(yǎng)加入bFGF后BMSC生長(zhǎng)速度較無(wú)bFGF時(shí)顯著提高,細(xì)胞形態(tài)大致相同,流式檢測(cè)細(xì)胞表面標(biāo)志物CD29、CD44、CD45表達(dá)與無(wú)bFGF時(shí)相同。BMSC移植能夠改善肝硬化小鼠肝功能、肝硬化程度、膠原纖維沉積,培養(yǎng)中加入bFGF的BMSC更為顯著地改善肝損傷程度和纖維化程度,重復(fù)細(xì)胞移植后肝纖維化程度改善更為顯著。
[Abstract]:Background: stem cell transplantation in the treatment of liver cirrhosis in animal experiments, clinical research has been widely studied, basic fibroblast growth factor can promote stem cell proliferation in vitro. The in vivo efficacy of stem cells cultured with bFGF in the treatment of liver cirrhosis is uncertain. Objective: to explore the effect of bFGF on the growth and proliferation of BMSC. Bone marrow mesenchymal stem cells (BMSCs) cultured with 0.1GF were injected through caudal vein in mice. The level of ALB, histopathological changes and collagen deposition in liver function were observed. Methods: bone marrow mesenchymal stem cells from male SD rats were extracted by whole bone marrow adherent method, and BMSCs were detected by flow cytometry. 46 female Kunming mice were randomly divided into 5 groups: group B: CCl4 bFGF group B group CCl4 bFGF group B group (group C): CCl4 bFGF group B group (group D): CCl4 bFGF group B group (group E): CCl4 bFGF BMSC3 transplants. After 10 weeks of mouse CCl4 model, bFGF or bFGF- cultured BMSCs were injected into tail vein respectively. Liver function was detected by enzyme-linked immunosorbent assay (Elisa) to detect histopathological changes. Masson trichrome staining was used to detect collagen deposition in liver tissue. Results the growth of BMSC: the growth of BMSCs in each group was observed. The cells in the bFGF-bFGF group were completely adhered to the wall for 24 hours. The adherent cells were fusiform or irregular at the initial stage, and the morphology of the cells was not obviously abnormal. The proliferation of the cells in the main fusiform group was faster than that in the bFGF group. On the 4th day, the growth of bFGF cells was slower than that of BFGF- group. After 6-7 days, the cell growth rate of bFGF was significantly higher than that of bFGF free culture.) flow cytometry showed negative expression of CD44 + CD44 and negative expression of CD45. 4) the liver function was detected by chemical method. There was no significant difference between group A and group B (P 0.05). The level of ALT in group D was significantly lower than that in group A (P 0.05) and the level of alt in group B (P 0.05) was significantly lower than that in group C (P 0.05). There was no significant difference between group A and group B in the level of P0.05A. There was no significant difference between group D and group E, while group C had a significant decrease in P0.05. 5HE staining results showed that the liver cells were edema in group A and group B compared with those in group A or group B, while in group C, there was no significant difference between group A and group B in the level of P0.05A, and there was no significant difference between group D and group B in the levels of P0.05A and P0.05.The results showed that there was no significant difference between group D and group B. Fatty degeneration, bridging necrosis, disordered arrangement of hepatic cord, edema of hepatocytes, steatosis and infiltration of inflammatory cells were widely formed in group .C, but the degree was less than that in the model group. The edema of hepatocytes, the degree of inflammatory cell infiltration and the structure of pseudolobules were obviously improved in group D and group E, and the fibrous deposition was observed in portal area of group A and B. The results showed that a large number of green stained fibers were deposited in portal vein of group A and B. Green fiber infiltration in hepatic parenchyma, fine fibrous septum extending from catchment area to hepatic parenchyma, no significant difference in fiber area between group A and group B in the formation of pseudolobules, and decrease in fiber content in group C and group D in comparison with group A and group B. Compared with group A, group B, the difference was statistically significant (P < 0.05). There was significant difference between group C and group C (P < 0.05), but there was no significant difference in fiber area between group A and group C (P 0.05), but the area of fiber in group E was significantly lower than that in group C (P 0.05), but there was no significant difference in fiber area between group C and group C (P 0.05). Conclusion: the growth rate of BMSC in vitro was significantly higher than that without bFGF, and the cell morphology was approximately the same. The expression of CD29, CD4, CD4, CD45, a marker on cell surface, could improve liver function and degree of liver cirrhosis in cirrhotic mice as compared with those without bFGF. The degree of liver injury and fibrosis was significantly improved by adding bFGF to BMSC and the degree of liver fibrosis was improved after repeated cell transplantation.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575.2

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