猪脐带间充质干细胞的分离培养与鉴定

发布时间：2018-03-27 21:30

本文选题：脐带间充质干细胞　切入点：猪　出处：《东北农业大学》2012年硕士论文

【摘要】：脐带间充质干细胞(Umbilical cord mesnchymal stem cells, UCMSCs)是一种位于脐带沃顿氏胶(Wharton's jelly)内和血管周围,来源于发育早期的中胚层和外胚层,具有自我更新能力和多分化潜能的一类成体干细胞。在体外培养中发现,脐带间充质干细胞具有分化为三个胚层细胞的能力,相对高量表达多能性基因Oct-4,sox-2和Nanog;同时,较低的免疫排斥反应使得脐带间充质干细胞在细胞替代治疗和组织工程方面,成为替代有年龄和疾病限制的骨髓间充质干细胞的种子细胞。由于其在临床应用上的巨大潜力,关于人脐带间充质干细胞的研究很多,但多局限于体外实验,而人的临床实验受到限制,因此,在临床应用前寻找一种大动物模型成为迫切的需要；但在人以外的其他物种相关研究报道匮乏。猪是除灵长类动物外,在生理和解剖结构上与人类最相似的动物,是一种理想的大动物模型,因此其用于基础理论研究和临床应用研究的潜力巨大。本实验以新生大白猪的脐带为实验材料,比较不同的酶消化法和组织培养法对猪脐带间充质干细胞的分离效果,同时,添加生长因子EGF和bFGF,观察生长因子对脐带间充质干增殖速度和生长状态的影响,在此基础上,确定猪脐带间充质干细胞的培养体系,观察细胞的生长特性。实验还通过免疫荧光和PCR方法,在蛋白水平和分子检测了细胞多能性基因Oct-4,sox-2和Nanog的表达情况,同时跟踪了其表达量的变化及趋势。最后,为确定猪脐带间充质干细胞的分化能力,对体外培养的细胞进行了成脂和成软骨的诱导分化。实验结果表明：在原代细胞的分离培养中,胶原酶Ⅰ和透明质酸酶相结合的酶处理方法更适宜于猪脐带间充质干细胞的分离,而通过沃顿氏胶消化法得到的原代细胞纯度更高,且生长能力更强；在观察生长因子EGF和bFGF对猪脐带间充质干细胞增殖速度和生长状态影响的实验中发现,单独添加EGF和bFGF的细胞与对照组相比,无明显变化,而两种因子同时添加的细胞在做生长曲线的检测时,明显高于对照组和单独添加组别,说明EGF和bFGF协同作用可以促进维持细胞本身的自我更新能力；而后对细胞进行传代培养,发现传代培养细胞在接种6-12h后贴壁,细胞单层漩涡状生长,呈现出典型的成纤维样,但少量细胞,呈现出扁平,扩张的形态；细胞具有一定的增殖能力,目前已有培养至13代的细胞,细胞形态维持成纤维样,但增殖速度逐渐下降。在多能性基因的检测中发现,通过免疫荧光的方法,可以看到多能性基因sox-2, nanog和nestin的表达,通过分子水平的检测,可以看到Oct-4,sox-2和nanog的表达,且其表达量随传代次数的增加而减少；最后对猪脐带间充质干细胞进行了成脂和成软骨的诱导分化,通过油红0和阿辛蓝的检测,证明其具有分化为成脂细胞和成软骨细胞的能力。
[Abstract]:Umbilical cord mesenchymal cord mesnchymal stem cells (UCMSCs) are a kind of adult stem cells, which are located in and around the blood vessels of Whartonia jelly. they are derived from mesoderm and ectoderm in the early stage of development, and have the ability of self-renewal and multi-differentiation. It was found in vitro that umbilical cord mesenchymal stem cells had the ability to differentiate into three embryonic layer cells, and expressed the multipotent genes Oct-4, sox-2 and Nanog. at the same time, Due to low immune rejection, umbilical cord mesenchymal stem cells are used as seed cells to replace bone marrow mesenchymal stem cells with age and disease limitation in cell substitution therapy and tissue engineering. Because of its great potential in clinical application, there are many researches on human umbilical cord mesenchymal stem cells, but most of them are confined to in vitro experiments, but human clinical trials are limited. It is an urgent need to find a large animal model before clinical application, but there are few reports about other species other than human. Pigs are the most physiologically and anatomically similar animals except primates. It is an ideal large animal model, so it has great potential for basic theory research and clinical application research. In this experiment, the umbilical cord of newborn white pig was used as experimental material to compare the isolation effects of different enzyme digestion and tissue culture methods on porcine umbilical cord mesenchymal stem cells. The effects of EGF and bFGF on the growth rate and growth state of umbilical cord mesenchymal stem cells were observed, and the culture system of porcine umbilical cord mesenchymal stem cells was established. The cell growth characteristics were observed. The expression of the multipotent gene Oct-4Osox-2 and Nanog was detected at the protein level and molecular level by immunofluorescence and PCR methods, and the changes and trends of the expression of Oct-4Osox-2 and Nanog were followed. In order to determine the differentiation ability of porcine umbilical cord mesenchymal stem cells, adipogenic and chondrogenic differentiation of cultured cells was carried out. The results showed that collagenase I combined with hyaluronidase was more suitable for isolation of porcine umbilical cord mesenchymal stem cells. The primary cells obtained by Wharton's gum digestion method were of higher purity and stronger growth ability, and the effects of growth factors EGF and bFGF on the proliferation rate and growth state of porcine umbilical cord mesenchymal stem cells were observed. Compared with the control group, the cells supplemented with EGF and bFGF alone had no significant change, but the growth curves of the cells added with the two factors at the same time were significantly higher than those in the control group and the added group alone. The results showed that the synergistic effect of EGF and bFGF could promote the self-renewal ability of the cells, and then the cells were subcultured, and the cells adhered to the wall 6-12 hours after inoculation, and the monolayer whirlpool grew, showing a typical fibroblast. However, a small number of cells showed flat and dilated morphology, and the cells had the ability to proliferate to a certain extent. At present, the cells were cultured to the 13th passage, the morphology of the cells remained fibroid, but the proliferation rate decreased gradually. In the detection of multipotent genes, the expression of Sox-2, nanog and nestin could be seen by immunofluorescence, and Oct-4sox-2 and nanog could be detected by molecular level. And its expression decreased with the increase of passage times. Finally, adipogenic and chondrogenic differentiation of porcine umbilical cord mesenchymal stem cells was carried out, which was detected by oil red 0 and acinyl blue. It is proved that it has the ability to differentiate into adipocytes and chondroblasts.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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