一氧化氮联合大鼠骨髓间充质干细胞对小鼠肝硬化的治疗效应

发布时间：2018-04-03 10:14

  本文选题：全骨髓贴壁法　切入点：肝硬化　出处：《兰州大学》2016年硕士论文

【摘要】：目的:建立稳定的大鼠骨髓间充质干细胞(Bone Marrow Mesenchymal Stem Cells,BMSCs)分离、培养及鉴定体系,并研究一氧化氮(Nitric Oxide,NO)联合BMSCs对小鼠肝硬化的治疗效果。方法:(1)体外大鼠BMSCs的分离、培养及鉴定:全骨髓贴壁法分离大鼠BMSCs,连续传代培养。取第三代细胞,进行流式细胞表型鉴定,MTT比色法测定细胞增殖曲线,并进行成骨诱导培养鉴定其分化能力,检测NO对BMSCs增殖能力的影响;(2)体内检测NO联合BMSCs对肝硬化的治疗效果:68只雌性KM小鼠随机分为空白组(10只)、模型组(10只)、硝普纳(Sodium Nitroprusside,SNP)组(12只)、BMSCs组(12只)、BMSCs+SNP(一次)组(12只)、BMSCs+SNP(多次)组(12只)。45%CCL4腹腔注射造肝硬化模型,10周后BMSCs组、BMSCs+SNP(一次)组、BMSCs+SNP(多次)组采用性别交叉的移植方法,通过尾静脉移植BMSCs,SNP组、BMSCs+SNP(一次)组、BMSCs+SNP(多次)组通过腹腔注射SNP,空白组和模型组注射PBS。4周后处死全部小鼠,采血测血清中AST、ALT、ALB;RT-PCR测性别决定区(SRY)mRNA的含量;HE染色检测肝纤维化程度。结果:(1)体外实验:成功分离、培养大鼠BMSCs,并绘制其生长曲线,流式细胞鉴定符合间充质干细胞表型,通过茜素红染色检测成骨诱导成功;不同浓度梯度的NO及不同加药次数对BMSCs增殖无明显影响(P0.05);(2)体内实验:10周后肝硬化模型成功建立;BMSCs组、BMSCs+SNP(一次)组、BMSCs+SNP(多次)组相对于模型组和SNP组ALT、AST水平下降,ALB升高(P0.05),模型组和SNP组ALT、AST、ALB差异无统计学意义(P0.05);BMSCs+SNP(多次)组较BMSCs组、BMSCs+SNP(一次)组ALT、AST下降显著(P0.05),ALB差异无统计学意义(P0.05);而BMSCs组、BMSCs+SNP(一次)组ALT、AST、ALB差异无统计学意义(P0.05)。空白组、模型组、SNP组无SRY mRNA的表达,BMSCs组、BMSCs+SNP(一次)组和BMSCs+SNP(多次)组可检测到SRY mRNA;其中BMSCs+SNP(多次)组较BMSCs+SNP(一次)组、BMSCs组SRY mRNA升高(P0.05);BMSCs组较BMSCs+SNP(一次)组SRYmRNA差异无统计学意义(P0.05)。HE染色可观察到模型组、SNP组存在肝硬化,BMSCs组、BMSCs+SNP(一次)组、BMSCs+SNP(多次)组肝硬化明显改善,其中BMSCs+SNP(多次)组改善最为明显。结论:全骨髓贴壁法可分离大鼠骨髓间充质干细胞,并稳定传代、增殖、分化;NO对其增殖无明显影响;移植的BMSCs可改善小鼠肝硬化,联合NO可增强BMSCs治疗肝硬化的疗效。
[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.妫，

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