【作者】 王芳；

【导师】 华进联；

【作者基本信息】 西北农林科技大学 ， 动物生物技术， 2011， 硕士

【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(U<500 V)情况下,电压愈高,存活细胞愈少;高压(U>500 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。更多还原

【Abstract】 Bone marrow mesenchymal stem cells (BMSCs) are a well-characterized population of adult stem cells that can be maintained and propagated in culture for a long time, with the capacity to form a variety of tissue-forming cells such as bone, muscle, cartilage, nerve cells, etc. At present, there are reports that BMSCs derived from human or mice could be cultured and transdifferentiated into multilineage cells. However, there were few reports about dairy goat BMSCs, especially about dairy goat BMSCs being able to transdifferentiate into germ cells. Goats are important economic animal, with the production for meat, wool, milk and other economic value. Furthermore, dairy goats farming is one of the advantages of livestock industry. Using stem cells as seed cells in investigation of genetic breeding and transgenic clone is a shortcut in accelerating good quality and high output goat propagation. In order to provide materials for research on development and differentiation of dairy goat cells as well as animal breeding, and animal disease models. we carried out the following experimental work.1. The cells were isolated from dairy goat bone marrow by different adherent culture and identified by morphology, growth curves, flow cytometry, immunocytochemistry and RT-PCR. The positive rates of non-directional and directed differentiation cells were evaluated by immunocytochemical method. The experiment results showed that BMSCs had a fibroblast-like morphology. The markers were identified in MSCs as same as embryonic stem cells (ESCs) such as OCT4, Nanog, C-Myc and TERT. Flow cytometry results were indicated that CD29、CD44、CD166 were positive and CD71、CD45、CD34 were negative. The spontaneously differentiated cells derived from embryoid bodies expressed markers of three embryonic germ layers. BMSCs were induced to differentiate into early neural-like cells by chemical reagents and were positive forβ-ⅢTubulin. Myocardial cells were obtained from BMSCs and appeared myotube-like cells, which were positive for CT3 and cardiacα-actin. Through RA-induction, the differentiated cells expressed the specific markers of pre-meiotic germ cells including VASA, SCP3 and CD49f, the positive rates of which were 35.7 %, 24 % and 14 % respectively. These results demonstrated that typical mesenchymal stem cells that isolated from dairy goat bone marrow possessed the characteristics of multipotent stem cells, and could lay the foundation for further experiments. 2. According to the protocol of transfection reagent of Fugene?HD, Lipofecter and Lipofectamine? 2000, our results show that Lipofectamine? 2000 were the best, with a efficiency of 44.88 %. Electro transfection was proceeded with the voltage model of 400 V/450 V/500 V/550/650 V/750 V, pulse duration of 3 s, pulse frequency of 3 times, pulse nterval of 1 min, which showed that when the voltage was lower than 500V,the higher voltage, the less alive cells. Higher voltage was beneficial to cell transfection, would lead to unexpected differentiation. In conclusion, transfection with reagents of Lipofectamine? 2000 was the best way to accomplish our experiments to transfect pStra8-EGFP into BMSCs.3. Transfection with Lipofectamine? 2000, a series of inducing condition including RA inducing, combination inducing(RA+GE) and GE monolayer inducing were carried out. These results showed that BMSCs expressed Stra8 and EGFP with RA inducing for 7 days. After RA inducing for 14 days, round cells expressed EGFP and sperm-like cells. In these induced cells, Stra8 and Prm1 could be detected in transcriptional level, and GFR-1, Stra8, VASA, SCP3, DZAL and ACR also could be discovered in protein level. Meanwhile, these induced cells retained the same cell cycle as dairy goat spermatogonia, These results showed that BMSCs could be induced to differentiate into spermatogonia in vitro, and had the hope of developing into meiosis anaphase.4. 25 mice with the defect of sperm production were used to transplant BMSCs into testicle by convoluted seminiferous tubule injection. 15 days, 30 days, and 90 days after transplantation, these mouse were executed by removing cervical to obtain testis. Testis from experiment group and control group were checked by weight, developmental stages of spermatogenic cells, and atrophy of convoluted seminiferous tubule. These data suggested that BMSCs could accelerate development and differentiation of sperm cells in sperm production defection mice 30 days after transplant. BMSCs labeled with CM-DIL also confirmed the conclusion in the same experiment, in which BMSCs were located in basal of convoluted seminiferous tubule under immunofluorescence observation. In addition, PCNA, Stra8 and GFRα-1 were expressed except ACR, indicating allograft BMSCs had the potential to differentiate into sperm cells.更多还原