Oct4、Sox2、c-Myc、Klf4-慢病毒载体构建与包装及感染人皮肤成纤维细胞的实验研究
发布时间：2018-09-13 06:28
【摘要】： 目的本研究构建并包装含有Oct4、Sox2、c-Myc、Klf4四基因的慢病毒载体(lentivirus vector),并进行目的基因过表达慢病毒颗粒感染原代培养的人皮肤成纤维细胞(human foreskin fibroblast HFF),旨在初步探讨诱导性多能干细胞((induced pluripotent stem cell ,iPSC)成功诱导的关键技术。 方法(1)从含有Oct4、Sox2、c-Myc、Klf4的质粒中获取目的基因。将目的基因与酶切线性化的慢病毒载体进行定向连接,其产物转化细菌感受态细胞,对长出的阳性克隆进行测序和比对分析,比对正确的即为构建成功的目的质粒.。然后将构建好的目的质粒进行超纯去内毒素抽提。采用Western Blot法检测目的基因质粒表达。通过三质粒系统共转染293T细胞对慢病毒进行包装并进行滴度测定;(2)采用Ⅰ型胶原酶消化结合组织块培养法分离培养HFF,以胰蛋白酶消化法分离培养小鼠胚胎成纤维细胞(Mouse embryonic fibroblast MEF),分别观察两种细胞的生长情况,并以免疫细胞化学染色SABC法对两种细胞进行鉴定;丝裂霉素C处理MEF用于建立干细胞培养所需要的饲养层;(3)用包装有绿色荧光蛋白的空病毒感染HFF,摸索出慢病毒感染HFF的最佳MOI值;(4)将生长状态良好的第3代HFF随机分为3组:目的基因感染组、空病毒感染组、正常对照组,采用含四种目的基因的慢病毒混合感染HFF并连续感染两次的方法,在二次感染后4天给予HFF细胞干细胞培养条件,在倒置显微镜下观察其形态变化,筛选最佳实验室条件。 结果(1)成功构建并包装出Oct4、Sox2、c-Myc、Klf4 慢病毒载体,基因测序结果与目标序列完全一致,滴度为1×109 TU/ml;(2) 0.02%Ⅰ型胶原酶消化结合组织块贴壁培养法可以在体外建立稳定的HFF系,培养4d时即有细胞游走出,细胞呈长梭形、有分支,6d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。采用0.0625%胰蛋白酶分次消化法可以获得高活力高产量的MEF,细胞呈长梭形、多角形,24h内细胞完全贴壁。免疫细胞化学鉴定两种细胞均呈阳性结果;(3)用含有15ug/ml的丝裂霉素C的培养液1ml处理长满25CM2培养瓶的MEF1.5h,可以获得良好功能的干细胞培养所需的饲养层;(4)摸索出慢病毒感染HFF的最佳MOI值为30;与正常组相比目的基因慢病毒感染HFF组虽未见到明显的多能干细胞,但出现了明显的细胞形态变化,由长梭型变成椭圆形或半椭圆形,相同条件下的空病毒感染组HFF形态未发生明显的变化,仍为典型的长梭型。 结论本研究成功地进行了HFF和MEF的原代培养,构建并包装出高滴度的Oct4、Sox2、c-Myc、Klf4 慢病毒载体,并感染人皮肤成纤维细胞,通过目的基因转导可诱导HFF发生形态变化,初步探讨了iPSC研究的关键技术。
[Abstract]:Objective to construct and package lentivirus vector (lentivirus vector), containing four genes of Oct4,Sox2,c-Myc,Klf4 and overexpression lentivirus particles in primary cultured human skin fibroblasts (human foreskin fibroblast HFF), in order to explore the inducible pluripotency. The key technology of the successful induction of cell (induced pluripotent stem cell / iPSC. Methods (1) the target gene was obtained from the plasmid containing Oct4,Sox2,c-Myc,Klf4. The target gene was directly ligated with the lentivirus vector which was digested by enzyme digestion. The product was transformed into bacterial receptive cells and the positive clones were sequenced and compared. Then the constructed target plasmid was extracted by super pure endotoxin removal. The expression of target gene plasmid was detected by Western Blot method. Packaging and titer determination of lentivirus by three plasmid system co-transfection 293T cells. (2) isolation and culture of HFF, by type I collagenase digestion combined with tissue mass culture and isolation and culture of mouse embryonic fibroblasts by trypsin digestion Cell (Mouse embryonic fibroblast MEF), was used to observe the growth of two kinds of cells. Two kinds of cells were identified by immunocytochemical staining (SABC). Mitomycin C treatment of MEF was used to establish the feeder layer needed for stem cell culture; (3) the best MOI value of lentivirus infected HFF was found by HFF, infected with empty virus wrapped with green fluorescent protein; (4) the third generation HFF with good growth was followed. The machine was divided into three groups: target gene infection group, In the empty virus infection group and the normal control group, HFF cell stem cells were cultured 4 days after the second infection with lentivirus containing four target genes. The morphologic changes were observed under inverted microscope and the optimum laboratory conditions were screened. Results (1) the Oct4,Sox2,c-Myc,Klf4 lentivirus vector was successfully constructed and packaged. The result of gene sequencing was identical with the target sequence. The titer of 1 脳 109 TU/ml; (2) 0.02% collagenase digestion combined with tissue mass adherence culture could establish stable HFF lines in vitro. After 4 days of culture, some cells swam out, and the cells were fusiform. After 6 days of branching, more cells were free out, and the cells were collected into bundles about 10 days after digestion and passage of 0.25% trypsin. MEF, cells with high activity and high yield could be obtained by 0.0625% trypsin fractionation method, and the cells with polygonal shape could be completely adhered to within 24 hours. (3) the culture medium 1ml containing mitomycin C of 15ug/ml can obtain the feeder layer of stem cell culture with good function by treating MEF1.5h, filled with 25CM2 culture flask; (4) to find out the necessary feeder layer for stem cell culture with good function. (4) to find out the necessary feeder layer for the culture of stem cells with good function. (3) to use 1ml as a culture medium containing mitomycin C of 15ug/ml. The optimal MOI value of infected HFF was 30. Compared with the control group, there were no significant pluripotent stem cells in the HFF group infected with the target gene lentivirus. However, there were obvious changes in cell morphology, from long spindle type to elliptic or semi elliptic shape. The HFF morphology of empty virus infected group did not change obviously under the same condition, and it was still a typical long spindle type. Conclusion the primary culture of HFF and MEF was successfully carried out in this study, and the high titer Oct4,Sox2,c-Myc,Klf4 lentivirus vector was constructed and packaged, and infected with human skin fibroblasts. The morphological changes of HFF could be induced by target gene transduction. The key technologies of iPSC research are discussed.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R751
本文编号：2240351
[Abstract]:Objective to construct and package lentivirus vector (lentivirus vector), containing four genes of Oct4,Sox2,c-Myc,Klf4 and overexpression lentivirus particles in primary cultured human skin fibroblasts (human foreskin fibroblast HFF), in order to explore the inducible pluripotency. The key technology of the successful induction of cell (induced pluripotent stem cell / iPSC. Methods (1) the target gene was obtained from the plasmid containing Oct4,Sox2,c-Myc,Klf4. The target gene was directly ligated with the lentivirus vector which was digested by enzyme digestion. The product was transformed into bacterial receptive cells and the positive clones were sequenced and compared. Then the constructed target plasmid was extracted by super pure endotoxin removal. The expression of target gene plasmid was detected by Western Blot method. Packaging and titer determination of lentivirus by three plasmid system co-transfection 293T cells. (2) isolation and culture of HFF, by type I collagenase digestion combined with tissue mass culture and isolation and culture of mouse embryonic fibroblasts by trypsin digestion Cell (Mouse embryonic fibroblast MEF), was used to observe the growth of two kinds of cells. Two kinds of cells were identified by immunocytochemical staining (SABC). Mitomycin C treatment of MEF was used to establish the feeder layer needed for stem cell culture; (3) the best MOI value of lentivirus infected HFF was found by HFF, infected with empty virus wrapped with green fluorescent protein; (4) the third generation HFF with good growth was followed. The machine was divided into three groups: target gene infection group, In the empty virus infection group and the normal control group, HFF cell stem cells were cultured 4 days after the second infection with lentivirus containing four target genes. The morphologic changes were observed under inverted microscope and the optimum laboratory conditions were screened. Results (1) the Oct4,Sox2,c-Myc,Klf4 lentivirus vector was successfully constructed and packaged. The result of gene sequencing was identical with the target sequence. The titer of 1 脳 109 TU/ml; (2) 0.02% collagenase digestion combined with tissue mass adherence culture could establish stable HFF lines in vitro. After 4 days of culture, some cells swam out, and the cells were fusiform. After 6 days of branching, more cells were free out, and the cells were collected into bundles about 10 days after digestion and passage of 0.25% trypsin. MEF, cells with high activity and high yield could be obtained by 0.0625% trypsin fractionation method, and the cells with polygonal shape could be completely adhered to within 24 hours. (3) the culture medium 1ml containing mitomycin C of 15ug/ml can obtain the feeder layer of stem cell culture with good function by treating MEF1.5h, filled with 25CM2 culture flask; (4) to find out the necessary feeder layer for stem cell culture with good function. (4) to find out the necessary feeder layer for the culture of stem cells with good function. (3) to use 1ml as a culture medium containing mitomycin C of 15ug/ml. The optimal MOI value of infected HFF was 30. Compared with the control group, there were no significant pluripotent stem cells in the HFF group infected with the target gene lentivirus. However, there were obvious changes in cell morphology, from long spindle type to elliptic or semi elliptic shape. The HFF morphology of empty virus infected group did not change obviously under the same condition, and it was still a typical long spindle type. Conclusion the primary culture of HFF and MEF was successfully carried out in this study, and the high titer Oct4,Sox2,c-Myc,Klf4 lentivirus vector was constructed and packaged, and infected with human skin fibroblasts. The morphological changes of HFF could be induced by target gene transduction. The key technologies of iPSC research are discussed.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R751
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