BMSCs通过线粒体途径修复镉致大鼠睾丸损伤的研究

发布时间：2018-03-18 12:30

本文选题：镉　切入点：睾丸损伤　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:观察骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)静脉移植是否对镉致睾丸损伤具有修复作用,并初步探讨其线粒体途径作用机制。方法:采用贴壁培养法纯化、扩增获取Wistar大鼠乳鼠BMSCs。利用流式细胞仪检测第三代BMSCs细胞周期及表面标志物CD45和CD90。21只成年雄性Wistar大鼠,随机分为对照组、模型组和细胞治疗组,每组7只。分别通过腹腔注射给予0、0.4、0.4 mgkg-1体重的Cd Cl2生理盐水溶液,每周连续5次,持续5周。染毒结束后,将1×107氯甲基苯甲酰胺(CM-Dil)标记的BMSCs分两次于24 h和48 h经球后静脉移植到细胞治疗组大鼠体内,对照组及模型组给予同等体积的磷酸盐缓冲液。2周后取大鼠睾丸组织称重并计算脏器系数;HE染色进行病理组织学观察;睾丸组织冰冻切片后,采用激光共聚焦显微镜观察BMSCs在细胞治疗组大鼠睾丸组织内的定植情况;原子吸收法测定睾丸组织内镉含量;TUNEL法检测睾丸组织细胞凋亡;免疫组织化学法及Western blot法检测线粒体凋亡相关蛋白Bim、Bax、Bcl-2、Cytochrome C、Caspase-3、active-Caspase-3和AIF,线粒体自噬相关蛋白Beclin1、PINK1、Parkin、p-Parkin、LC3B,线粒体生物发生相关蛋白SIRT1、PGC-1α的表达;实时定量PCR检测线粒体基因组(mt DNA)拷贝数。结果:流式细胞仪检测结果显示,第三代BMSCs大部分处于相对不活跃的分裂间期,细胞基本不表达CD45,高表达CD90。细胞移植2周后,模型组大鼠明显精神萎靡且少动,细胞治疗组大鼠精神行为状态明显好于模型组,且细胞治疗组大鼠体质量显著高于模型组(P0.05)。模型组大鼠睾丸组织质量与脏器系数与对照组相比无显著性差异。HE染色结果显示,模型组大鼠睾丸组织曲细精管萎缩,小管内细胞排列紊乱,细胞层次与成熟生精细胞减少;细胞治疗组病变明显改善。激光共聚焦显微镜可在细胞治疗组大鼠睾丸组织内观察到CM-Dil红色荧光标记的BMSCs。原子吸收法检测结果显示,模型组与细胞治疗组大鼠睾丸组织内镉蓄积量显著高于对照组,二者间比较无明显差别。TUNEL法检测结果显示,细胞治疗组大鼠睾丸组织细胞凋亡率显著低于模型组(P0.05)。免疫组织化学法和Western blot检测结果显示,模型组大鼠睾丸组织线粒体途径凋亡相关蛋白Bim、Bax、Cytochrome C、Caspase-3、active-Caspase-3和AIF表达比其他两组显著增多,Bcl-2表达最少(P0.05);模型组线粒体自噬相关蛋白Beclin1、PINK1、Parkin、p-Parkin、LC3B蛋白表达水平显著高于另外两组(P0.05);线粒体生物发生相关蛋白SIRT1在模型组大鼠睾丸组织表达减少、PGC-1α去乙酰化水平减少(P0.05)。mt DNA拷贝数结果显示,模型组显著低于对照和细胞治疗组(P0.05)。结论:镉可以进入大鼠体内,在睾丸组织蓄积并导致睾丸组织损伤;静脉移植的BMSCs可以定位在镉致损伤的睾丸组织,并且修复局部组织损伤;其修复机制可能是通过抑制睾丸组织线粒体途径细胞凋亡,线粒体途径细胞自噬和促进线粒体生物发生实现的。
[Abstract]:Objective: To observe the effect of bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BMSCs) intravenous transplantation on whether cadmium induced testicular injury repair, and to investigate the mechanism of mitochondrial pathway. Methods: the adherent culture method of purification, amplification of Wistar rat BMSCs. obtained by using the third generation BMSCs cell cycle and detection the surface markers of CD45 and CD90.21 in adult male Wistar rats by flow cytometry, were randomly divided into control group, model group and treated group, 7 rats in each group were given by intraperitoneal injection. 0,0.4,0.4 mg? Kg-1 Cd Cl2 weight of normal saline solution for 5 times a week, for 5 weeks. After the end of the exposure. 1 x 107 chloromethyl benzamide (CM-Dil) labeled BMSCs two to 24 h and 48 h after the ball after intravenous transplantation to cell therapy in vivo rat group, control group and model group were given equal volume of phosphate buffer solution after.2 weeks Take the rat testicular tissue and weighed to calculate the organ coefficient; HE staining was performed for histopathological observation; testicular tissue sections, using laser confocal microscope BMSCs treatment group rat testicular tissue colonization in the cell; Determination of cadmium content in testis cell apoptosis was detected by atomic absorption spectrometry; testis TUNEL; immunohistochemistry method and Western blot method to detect mitochondrial apoptosis related proteins Bim, Bax, Bcl-2, Cytochrome, C, Caspase-3, active-Caspase-3 and AIF, mitochondrial autophagy related protein Beclin1, PINK1, Parkin, p-Parkin, LC3B, SIRT1 associated protein of mitochondrial biology, expression of PGC-1; real-time quantitative PCR detection of mitochondrial genome copy number (MT DNA). Results: flow cytometry showed that the third generation of BMSCs, mostly in the relatively inactive interphase cells, the expression of CD45, high expression of CD90. cells 2 weeks after transplantation, the rats in model group was significantly depressed and less dynamic, cell therapy group rats behavior and mental status is significantly better than the model group, and cell therapy weight group rats were significantly higher than those in the model group (P0.05). The testicular tissue of rats in model group and organ coefficient compared with the control group no significant difference the difference of.HE staining showed that the atrophy of model group rats testis seminiferous tubule cells, disordered cell level and mature spermatogenic cells decreased; cells in the treatment group were significantly improved. The lesions were detected by confocal microscope CM-Dil red fluorescence labeled BMSCs. atomic absorption assay in testis cell therapy group, model group and treatment group rat testis cells in Cd accumulation was significantly higher than the control group, two patients had no significant difference in the.TUNEL assay showed that the cell treatment of testicular tissue in rats The apoptosis rate was significantly lower than that of model group (P0.05). Immunohistochemistry and Western blot assay showed that the mitochondrial pathway of apoptosis in testis of rats in the model group related protein Bim, Bax, Cytochrome, C, Caspase-3, active-Caspase-3 and AIF expression increased significantly than the other two groups, Bcl-2 was the least (P0.05); model group, mitochondrial autophagy PINK1 related protein Beclin1, Parkin, p-Parkin, LC3B protein expression was significantly higher than that of the other two groups (P0.05); mitochondrial biogenesis related protein SIRT1 expression decreased in the testis of rats in the model group, PGC-1 alpha acetylation level reduction (P0.05).Mt DNA copy number showed that the model group was significantly lower than that of control and cell therapy group (P0.05). Conclusion: cadmium can enter the rats, in the testis tissue accumulation and lead to testicular tissue injury; intravenous transplantation of BMSCs can locate the lesions caused by testicular tissue in cadmium, and repair The mechanism of repair may be by inhibiting the apoptosis of mitochondria in testicular tissue, mitochondrial autophagy and promoting mitochondrial biogenesis.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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