人包皮成纤维细胞体外诱导重编程为多能干细胞的基础研究

发布时间：2018-02-10 00:39

本文关键词： 人包皮成纤维细胞诱导性多能干细胞慢病毒饲养层　出处：《福建医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的本研究建立原代培养人包皮成纤维细胞(human postnatal foreskin fibroblast hPFF)体系,丝裂霉素-C(Mitomycin C ,MMC)处理hPFF制备人体组织来源细胞饲养层;将分别表达Oct4、Sox2、c-Myc、Klf4基因慢病毒颗粒感染hPFF,探讨目的基因过表达慢病毒颗粒感染hPFF体外重编程为诱导性多能干细胞(induced pluripotent stem cell iPSC)的关键技术。方法(1)采用Ⅰ型胶原酶消化法结合组织块培养法分离培养hPFF,在倒置显微镜观察细胞生长情况,以台盼蓝染色检测细胞活力、免疫细胞化学方法染色链酶亲和素-生物素-过氧化物酶复合法(Strept actividin-biotin complex SABC法)细胞鉴定。四甲基偶氮唑盐微量酶反应比色法(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl- trazolium bromide MTT法)测定细胞生长曲线。采用MMC处理hPFF制备细胞饲养层,MTT法筛选MMC作用的最佳浓度和时间(;2)将过表达绿色荧光蛋白(Green fluorescent protein,GFP)的空病毒颗粒感染hPFF,在荧光倒置显微镜下观察感染效率及荧光表达情况,探索最佳感染条件及感染复数(Multiplicity of infection,MOI值);(3)将生长状态良好的hPFF随机分为3组:阴性对照组、含绿色荧光蛋白空病毒感染组、目的基因感染组,采用含连续感染方式,在二次感染后4天给予hPFF干细胞培养条件,在倒置显微镜下动态观察其形态变化,拉曼光谱技术检测三组细胞光谱,探索最佳培养条件。结果(1)0.025%Ⅰ型胶原酶消化法结合组织块培养法可以在体外建立稳定的hPFF系,培养5d时即有细胞游走出,细胞呈长梭形、核仁明显,7d时有较多的细胞游离出,汇集成束,10d左右细胞铺满瓶底,0.25%胰蛋白酶消化传代。免疫细胞化学法SABC法鉴定细胞呈阳性结果。丝裂霉素C 10μg/ml作用3.5h或15μg/ml作用2.5h能抑制hPFF增殖,细胞在1周内维持稳定的水平;(2)本实验室培养条件下,慢病毒感染hPFF的最佳MOI值为40;(3)同等感染条件下,含绿色荧光蛋白空病毒感染组、阴性对照组的hPFF倒置显微镜下观察形态未发生明显改变,四个目的基因慢病毒混合感染组细胞伪足明显收缩,形态由长梭形变为圆形。拉曼光谱技术检测也发现阴性对照组与含绿色荧光蛋白空病毒感染组光谱图没有明显差异,目的基因组与阴性对照组有差异。结论本研究成功地进行了hPFF的原代培养,体外建立稳定的hPFF系,制备的人体组织来源的细胞饲养层;实验表明慢病毒颗粒可成功感染hPFF,四个目的基因转导可诱导hPFF形态变化。
[Abstract]:Objective to establish a primary culture system of human postnatal foreskin fibroblast hPFF, and to prepare human tissue derived cell feeder layer by mitomycin C mitomycin C (MMC) treatment. The lentivirus particles expressing Oct4 / Sox2nc-Mycf4 gene were infected with lentivirus particles (hPFFs), and the reprogramming of lentivirus particles infected with lentivirus particles was studied as a key technique for inducing pluripotent stem cell (IP SCC) of pluripotent stem cells in vitro. Methods 1) hPFF was isolated by collagenase digestion and tissue mass culture. Cell growth was observed under inverted microscope and cell viability was detected by trypan blue staining. The cell growth curve was determined by strept actividin-biotin complex SABC method using strept actividin-biotin complex SABC method. The cell growth curve was determined by trimethylazolium microzyme reaction colorimetric method. The cell growth curve was determined by the method of 4-dimethylazolazolyl thiazol-2-ylthiazol-2-ylthiazol-2-diphenyl5-diphenyl- trazolium bromide MTT. The best concentration and time for screening the action of MMC by MMC treatment with hPFF was determined. The empty viral particles expressing green fluorescent protein (GFP) were infected with MMC, and the infection efficiency and fluorescence expression were observed under fluorescence inverted microscope. To explore the optimal infection conditions and the infection plural infection of moi, the hPFF with good growth state was randomly divided into three groups: negative control group, empty virus infection group containing green fluorescent protein, and target gene infection group, with continuous infection. HPFF stem cells were cultured 4 days after secondary infection. Morphological changes were observed dynamically under inverted microscope. Raman spectroscopy was used to detect the spectrum of three groups of cells and to explore the best culture conditions. Results A stable hPFF cell line could be established in vitro by collagenase digestion with 0.025% collagenase digestion and tissue mass culture. After 5 days of culture, the cells swam out, the cells were fusiform, and more cells were free from the nucleoli at 7 days. The cells were collected into bundles for about 10 days, the cells were filled with 0.25% trypsin digestion and passaged. The positive results were identified by immunocytochemistry SABC method. Mitomycin C 10 渭 g / ml could inhibit the proliferation of hPFF in 3.5 h or 15 渭 g / ml for 2.5 h, and the cell proliferation was inhibited by mitomycin C (10 渭 g / ml) or 15 渭 g / ml (2.5 h). The optimal MOI value of lentivirus infected with hPFF was 40 ~ 3) under the same infection condition, the group containing green fluorescent protein empty virus (GFP) was infected with green fluorescent protein (GFP). The morphology of the negative control group was not changed under inverted hPFF microscope, but the pseudopodia contracted significantly in the four target gene lentivirus mixed infection groups. Raman spectroscopy also showed that there was no significant difference between the negative control group and the green fluorescent protein empty virus infection group, but the target genome was different from the negative control group. Conclusion the primary culture of hPFF was successfully carried out in this study, and a stable hPFF line was established in vitro, and the human tissue derived cell feeder layer was prepared, and the results showed that lentivirus particles could be successfully infected with hPFF, and four target gene transduction could induce the morphological changes of hPFF.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【相似文献】