奶山羊骨髓间充质干细胞的分离培养及向雄性生殖细胞诱导分化

发布时间：2018-06-09 01:55

本文选题：骨髓间充质干细胞 + 生殖细胞　；参考：《西北农林科技大学》2011年硕士论文

【摘要】：骨髓间充质干细胞(BMSCs)是一种具有多向分化潜能的成体干细胞,体内外可以分化为成骨细胞、肌肉细胞、软骨细胞、神经细胞等。目前关于人和小鼠BMSCs的分离培养及诱导已经有大量报道,但关于奶山羊BMSCs报道很少,尤其是奶山羊BMSCs分化为生殖细胞的研究相对较少。山羊作为重要的经济动物,具有生产肉、绒、奶等经济价值,而且奶山羊养殖业是我国的优势畜牧产业之一,所以,利用干细胞作为种子细胞开展奶山羊的遗传育种以及转基因克隆的研究可能是加速优质高产奶山羊扩繁的捷径之一。为了进一步为奶山羊细胞发育分化、育种及动物疾病模型等研究提供材料,我们开展了以下的试验工作。 1.采用贴壁培养法进行BMSCs的分离和培养,通过形态学观察、生长曲线测定、免疫细胞化学、流式分析、RT-PCR技术等进行检测;免疫细胞化学法对自发分化的细胞及定向诱导分化的神经样细胞、心肌样细胞、生殖样细胞进行检测,并计算生殖样细胞的阳性率。实验结果表明分离的奶山羊BMSCs为成纤维样细胞,增殖能力强;其表达MSCs和胚胎干细胞(ESCs)的表面标记如OCT4、Nanog、C-Myc和TERT,细胞表面标记CD29、CD44、CD166呈阳性,CD71、CD45、CD34呈阴性;将该细胞制作成类胚体,经自发分化后表达三胚层标志基因:AFP、NSE、β-ⅢTubulin和心肌α-actin;经化学试剂诱导,定向分化为表达β-ⅢTubulin的早期神经样细胞;表达CT3及心肌α-actin的心肌样细胞,出现肌管样细胞;表达生殖细胞特异性蛋白VASA、SCP3和CD49f(α6整合素),其阳性率分别达到35.7 %,24 %和14 %。结果表明所分离培养的BMSCs具备间充质干细胞的生物学特性,可以为进一步的实验奠定基础。 2.用不同转染体系筛选针对BMSCs的最佳转染条件。根据Fugene?HD转染试剂,Lipofecter脂质体转染试剂, Lipofectamine? 2000转染试剂转染结果证明,Lipofectamine? 2000转染效率较好,达44.88 %;不同电压模式:400 V/450 V/500 V/550/650 V/750 V,脉冲时间:3 S;脉冲次数:3次,脉冲间隔:1 min进行细胞电转,实验结果表明低压(U500 V)情况下,电压愈高,存活细胞愈少;高压(U500 V)情况下不稳定。高压情况下利于细胞转染,最高达26.94 %,但细胞容易分化。综合电转和脂质体转染的结果,后期转染采用Lipofectamine? 2000进行BMSCs的pStra8-EGFP转染(BMSCs-Stra8)。 3.采用Lipofectamine? 2000转染细胞后利用不同的诱导体系,包括RA、睾丸提取液(GE)及组合诱导液(RA+GE)诱导,实验结果表明,与组合诱导、GE诱导相比,诱导7 d后,RA诱导组表达GFP的细胞数量和免疫组化染色表达Stra8的阳性细胞数量呈极显著差异。利用RA单层诱导,诱导第5 d时观察到圆形GFP细胞,后期诱导细胞消失,此后不断有圆形细胞出现,在诱导14 d时出现了精子样细胞。转录水平上表达Stra8和Prm1,蛋白水平上表达GFRα-1, Stra8, VASA, SCP3, DAZL, ACR,细胞周期分析与山羊的精原细胞相似,说明体外诱导BMSCs能够分化为精原细胞,并可能向减数分裂后期发育。 4.制作生精缺陷模型鼠25只,采用曲细精管注射方法将BMSCs注射于生精缺陷模型鼠睾丸,在移植15 d,30 d,90 d时,脱颈致死小鼠,取出双侧睾丸,分别对治疗侧和未治疗侧的睾丸、附睾称重,比较两侧睾丸各级生精细胞发育和曲细精管萎缩程度等,实验结果证明BMSCs移植30 d后能够明显促进生精缺陷模型小鼠睾丸生殖细胞的发育和分化。将BMSCs用CM-DIL(氯甲基苯甲酰胺)标记后采用曲细精管注射生精缺陷模型小鼠睾丸,移植30 d时免疫荧光染色观察,移植细胞定位于曲细精管的基底膜部,且表达PCNA, Stra8, GFRα-1(不表达ACR),说明异体移植BMSCs具有分化为生殖细胞的潜能。
[Abstract]:Bone marrow mesenchymal stem cells ( MSCs ) are a kind of adult stem cells with multi - directional differentiation potential , which can differentiate into osteoblasts , muscle cells , chondrocytes , nerve cells , etc .

1 . Separation and culture of bone marrow cells were carried out by adherent culture . The results showed that the isolated milk goats were detected by means of morphological observation , growth curve determination , immune cell chemistry , flow analysis , RT - PCR technique and so on . The results showed that the isolated milk goats were made into fibroid cells , and the positive rates of CD29 , CD44 and CD166 were detected . The results showed that the isolated cultured bone marrow cells had the biological characteristics of mesenchymal stem cells , which could lay the foundation for further experiments .

2 . The transfection efficiency of Lipofectamine 2000 transfection reagent , Lipofectamine 2000 transfection reagent and Lipofectamine 2000 transfection reagent showed that the transfection efficiency of Lipofectamine 2000 was better than that under high voltage ( U500 V ) .

3 . After transfection of cells with Lipofectamine 2000 , different induction systems were used , including RA , testis extract ( GE ) and combination - induced liquid ( RA + GE ) . The results showed that the number of cells expressing GFP in RA - induced group was significantly different from that in combination - induced and GE - induced .

4 . Twenty - five mice were injected into the rat testis of spermatogenic defect model mice by the injection method of fine tube . The results showed that after 30 days of transplantation , the development and differentiation of spermatogenic cells and the atrophy of spermatogenic cells were observed . The experimental results showed that the transplanted cells were located in the basement membrane of the fine tube and the PCNA , Stra8 and GFR 伪 - 1 ( not expressed ACR ) were injected into the basal membrane of the tubules .
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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