本文選題：全骨髓貼壁法　切入點：肝硬化　出處：《蘭州大學(xué)》2016年碩士論文

【摘要】：目的:建立穩(wěn)定的大鼠骨髓間充質(zhì)干細胞(Bone Marrow Mesenchymal Stem Cells,BMSCs)分離、培養(yǎng)及鑒定體系,并研究一氧化氮(Nitric Oxide,NO)聯(lián)合BMSCs對小鼠肝硬化的治療效果。方法:(1)體外大鼠BMSCs的分離、培養(yǎng)及鑒定:全骨髓貼壁法分離大鼠BMSCs,連續(xù)傳代培養(yǎng)。取第三代細胞,進行流式細胞表型鑒定,MTT比色法測定細胞增殖曲線,并進行成骨誘導(dǎo)培養(yǎng)鑒定其分化能力,檢測NO對BMSCs增殖能力的影響;(2)體內(nèi)檢測NO聯(lián)合BMSCs對肝硬化的治療效果:68只雌性KM小鼠隨機分為空白組(10只)、模型組(10只)、硝普納(Sodium Nitroprusside,SNP)組(12只)、BMSCs組(12只)、BMSCs+SNP(一次)組(12只)、BMSCs+SNP(多次)組(12只)。45%CCL4腹腔注射造肝硬化模型,10周后BMSCs組、BMSCs+SNP(一次)組、BMSCs+SNP(多次)組采用性別交叉的移植方法,通過尾靜脈移植BMSCs,SNP組、BMSCs+SNP(一次)組、BMSCs+SNP(多次)組通過腹腔注射SNP,空白組和模型組注射PBS。4周后處死全部小鼠,采血測血清中AST、ALT、ALB;RT-PCR測性別決定區(qū)(SRY)mRNA的含量;HE染色檢測肝纖維化程度。結(jié)果:(1)體外實驗:成功分離、培養(yǎng)大鼠BMSCs,并繪制其生長曲線,流式細胞鑒定符合間充質(zhì)干細胞表型,通過茜素紅染色檢測成骨誘導(dǎo)成功;不同濃度梯度的NO及不同加藥次數(shù)對BMSCs增殖無明顯影響(P0.05);(2)體內(nèi)實驗:10周后肝硬化模型成功建立;BMSCs組、BMSCs+SNP(一次)組、BMSCs+SNP(多次)組相對于模型組和SNP組ALT、AST水平下降,ALB升高(P0.05),模型組和SNP組ALT、AST、ALB差異無統(tǒng)計學(xué)意義(P0.05);BMSCs+SNP(多次)組較BMSCs組、BMSCs+SNP(一次)組ALT、AST下降顯著(P0.05),ALB差異無統(tǒng)計學(xué)意義(P0.05);而BMSCs組、BMSCs+SNP(一次)組ALT、AST、ALB差異無統(tǒng)計學(xué)意義(P0.05)�？瞻捉M、模型組、SNP組無SRY mRNA的表達,BMSCs組、BMSCs+SNP(一次)組和BMSCs+SNP(多次)組可檢測到SRY mRNA;其中BMSCs+SNP(多次)組較BMSCs+SNP(一次)組、BMSCs組SRY mRNA升高(P0.05);BMSCs組較BMSCs+SNP(一次)組SRYmRNA差異無統(tǒng)計學(xué)意義(P0.05)。HE染色可觀察到模型組、SNP組存在肝硬化,BMSCs組、BMSCs+SNP(一次)組、BMSCs+SNP(多次)組肝硬化明顯改善,其中BMSCs+SNP(多次)組改善最為明顯。結(jié)論:全骨髓貼壁法可分離大鼠骨髓間充質(zhì)干細胞,并穩(wěn)定傳代、增殖、分化;NO對其增殖無明顯影響;移植的BMSCs可改善小鼠肝硬化,聯(lián)合NO可增強BMSCs治療肝硬化的療效。
[Abstract]:Aim: to establish a stable bone Marrow Mesenchymal Stem BMSCs system for the isolation, culture and identification of rat bone marrow mesenchymal stem cells (BMSCs), and to study the therapeutic effects of nitric oxide nitric oxide (no) combined with BMSCs on liver cirrhosis in mice.Methods: isolated, cultured and identified rat BMSCs in vitro: BMSCs were isolated by whole bone marrow adherent method and cultured continuously.The third generation of cells were identified by flow cytometry and the proliferation curve was determined by MTT colorimetry, and the differentiation ability was evaluated by osteogenic induction culture.媯，

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