本文選題：鎘　切入點(diǎn)：睪丸損傷　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:觀察骨髓間充質(zhì)干細(xì)胞(Bone marrow mesenchymal stem cells,BMSCs)靜脈移植是否對鎘致睪丸損傷具有修復(fù)作用,并初步探討其線粒體途徑作用機(jī)制。方法:采用貼壁培養(yǎng)法純化、擴(kuò)增獲取Wistar大鼠乳鼠BMSCs。利用流式細(xì)胞儀檢測第三代BMSCs細(xì)胞周期及表面標(biāo)志物CD45和CD90。21只成年雄性Wistar大鼠,隨機(jī)分為對照組、模型組和細(xì)胞治療組,每組7只。分別通過腹腔注射給予0、0.4、0.4 mgkg-1體重的Cd Cl2生理鹽水溶液,每周連續(xù)5次,持續(xù)5周。染毒結(jié)束后,將1×107氯甲基苯甲酰胺(CM-Dil)標(biāo)記的BMSCs分兩次于24 h和48 h經(jīng)球后靜脈移植到細(xì)胞治療組大鼠體內(nèi),對照組及模型組給予同等體積的磷酸鹽緩沖液。2周后取大鼠睪丸組織稱重并計算臟器系數(shù);HE染色進(jìn)行病理組織學(xué)觀察;睪丸組織冰凍切片后,采用激光共聚焦顯微鏡觀察BMSCs在細(xì)胞治療組大鼠睪丸組織內(nèi)的定植情況;原子吸收法測定睪丸組織內(nèi)鎘含量;TUNEL法檢測睪丸組織細(xì)胞凋亡;免疫組織化學(xué)法及Western blot法檢測線粒體凋亡相關(guān)蛋白Bim、Bax、Bcl-2、Cytochrome C、Caspase-3、active-Caspase-3和AIF,線粒體自噬相關(guān)蛋白Beclin1、PINK1、Parkin、p-Parkin、LC3B,線粒體生物發(fā)生相關(guān)蛋白SIRT1、PGC-1α的表達(dá);實時定量PCR檢測線粒體基因組(mt DNA)拷貝數(shù)。結(jié)果:流式細(xì)胞儀檢測結(jié)果顯示,第三代BMSCs大部分處于相對不活躍的分裂間期,細(xì)胞基本不表達(dá)CD45,高表達(dá)CD90。細(xì)胞移植2周后,模型組大鼠明顯精神萎靡且少動,細(xì)胞治療組大鼠精神行為狀態(tài)明顯好于模型組,且細(xì)胞治療組大鼠體質(zhì)量顯著高于模型組(P0.05)。模型組大鼠睪丸組織質(zhì)量與臟器系數(shù)與對照組相比無顯著性差異。HE染色結(jié)果顯示,模型組大鼠睪丸組織曲細(xì)精管萎縮,小管內(nèi)細(xì)胞排列紊亂,細(xì)胞層次與成熟生精細(xì)胞減少;細(xì)胞治療組病變明顯改善。激光共聚焦顯微鏡可在細(xì)胞治療組大鼠睪丸組織內(nèi)觀察到CM-Dil紅色熒光標(biāo)記的BMSCs。原子吸收法檢測結(jié)果顯示,模型組與細(xì)胞治療組大鼠睪丸組織內(nèi)鎘蓄積量顯著高于對照組,二者間比較無明顯差別。TUNEL法檢測結(jié)果顯示,細(xì)胞治療組大鼠睪丸組織細(xì)胞凋亡率顯著低于模型組(P0.05)。免疫組織化學(xué)法和Western blot檢測結(jié)果顯示,模型組大鼠睪丸組織線粒體途徑凋亡相關(guān)蛋白Bim、Bax、Cytochrome C、Caspase-3、active-Caspase-3和AIF表達(dá)比其他兩組顯著增多,Bcl-2表達(dá)最少(P0.05);模型組線粒體自噬相關(guān)蛋白Beclin1、PINK1、Parkin、p-Parkin、LC3B蛋白表達(dá)水平顯著高于另外兩組(P0.05);線粒體生物發(fā)生相關(guān)蛋白SIRT1在模型組大鼠睪丸組織表達(dá)減少、PGC-1α去乙酰化水平減少(P0.05)。mt DNA拷貝數(shù)結(jié)果顯示,模型組顯著低于對照和細(xì)胞治療組(P0.05)。結(jié)論:鎘可以進(jìn)入大鼠體內(nèi),在睪丸組織蓄積并導(dǎo)致睪丸組織損傷;靜脈移植的BMSCs可以定位在鎘致?lián)p傷的睪丸組織,并且修復(fù)局部組織損傷;其修復(fù)機(jī)制可能是通過抑制睪丸組織線粒體途徑細(xì)胞凋亡,線粒體途徑細(xì)胞自噬和促進(jìn)線粒體生物發(fā)生實現(xiàn)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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R114

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相關(guān)期刊論文 前10條

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本文編號：1629587