Sox9基因修饰人脐带间充质干细胞向软骨细胞分化的实验研究
发布时间：2018-11-27 21:23
【摘要】：目的:构建人Sox9基因慢病毒载体,体外Sox9慢病毒载体转染人脐带间充质干细胞(hUC-MSCs),观察Sox9基因转染对hUC-MSCs生物学特性的影响,以及经Sox9基因修饰的hUC-MSCs在单层培养条件下向软骨细胞分化的能力,为软骨组织损伤及退变性疾病的细胞学及基因治疗提供实验依据。 方法:通过PCR的方法获得人Sox9基因编码区片段,将该片段克隆入慢病毒载体Pwpxl-MOD中产生Pwpxl-MOD/Sox9 ,通过酶切测序鉴定慢病毒载体,将Pwpxl-MOD/Sox9、pRsv-REV、pMDlg-pRRE、pMD2G共转染293T细胞,包装成含有Sox9基因的重组慢病毒。采用酶消化法从人脐带基质中获取间充质干细胞,流式细胞仪进行细胞表型鉴定,诱导向成骨、脂肪分化以观察其多向分化潜能。包装好的慢病毒体外转染hUC-MSCs,利用荧光倒置相差显微镜观察转基因细胞的形态变化、绿色荧光表达(GFP)情况,转染后48h确定基因转染效率。逆转录-聚合酶链反应(RT-PCR)法及免疫印迹(Western-blot)检测Sox9在hUC-MSCs中的表达;四甲基偶氮唑盐比色法(MTT)法测定慢病毒对细胞增殖能力的影响。在高密度单层培养条件下,不增加生长因子、地塞米松等软骨诱导液,将Sox9基因修饰的hUC-MSCs培养分化21d, RT-PCR、Western-blot、免疫组化及免疫荧光染色、阿利新兰染色检测特异性软骨细胞分子标志Ⅱ型胶原、Agrrecan的表达以及蛋白多糖的分泌情况。 结果:酶切、测序鉴定证实慢病毒载体Pwpxl-MOD中插人片段为基因Sox9。经检测包装好的慢病毒滴度为6.0×10~7TU/ml;从人脐带基质分离出的hUC-MSCs形态类似于成纤维样细胞,CD29(95.9%)、CD44(96.5%)表达阳性,CD34(3.0%)、CD45(2.6%)造血干细胞表型标志表达为阴性。向脂肪及成骨方向诱导培养21d,油红O染色、碱性磷酸酶染色、Von kossa染色均为强阳性,表明hUC-MSCs具有较强的多向分化潜能。基因转染48h后观察到较强的绿色荧光表达,Lenti-GFP-Sox9及Lenti-GFP的转染率均达到90%以上。RT-PCR、Western-blot检测到目的基因Sox9在hUC-MSCs中出现过表达(P0.001),细胞生长曲线显示转染对hUC-MSCs的增殖活力无明显影响(P0.05)。Sox9基因修饰的hUC-MSCs在单层培养下7d开始出现自发性的细胞聚集现象,到14d细胞集聚明显增大,21d左右,有较大的软骨结节形成。而Lenti-GFP及未转染的细胞无明显的细胞集聚现象产生。RT-PCR、Western-blot、免疫组化及免疫荧光染色检测到Ⅱ型胶原、Agrrecan有较高水平的表达,阿利新兰染色示大量的蛋白多糖的在软骨结节中结聚。 结论:1、成功构建了含有Sox9基因慢病毒载体。2、人脐带间充质干细胞体外培养扩增容易,具有多向分化潜能,易于被外源性基因修饰。2、在单层培养条件下,无生长因子及其他软骨诱导因子的刺激的情况下,经Sox9基因修饰的HU-MSCs具有较强的向软骨细胞分化的能力,表明Sox9基因在促进间充质干细胞向软骨细胞分化过程中发挥了重要的作用。
[Abstract]:Aim: to construct lentivirus vector of human Sox9 gene and transfect Sox9 lentivirus vector into human umbilical cord mesenchymal stem cells (hUC-MSCs) in vitro to observe the effect of Sox9 gene transfection on the biological characteristics of hUC-MSCs. The ability of hUC-MSCs modified by Sox9 gene to differentiate into chondrocytes in monolayer culture provides experimental basis for cytology and gene therapy of cartilage tissue damage and degeneration. Methods: the encoding region of human Sox9 gene was obtained by PCR. The fragment was cloned into lentivirus vector Pwpxl-MOD to produce Pwpxl-MOD/Sox9. The lentivirus vector was identified by restriction endonuclease sequencing, and Pwpxl-MOD/Sox9,pRsv-REV,pMDlg-pRRE, was identified. PMD2G co-transfected 293T cells and packaged into recombinant lentivirus containing Sox9 gene. Mesenchymal stem cells were obtained from human umbilical cord stroma by enzyme digestion. The phenotype of mesenchymal stem cells was identified by flow cytometry. After transfection of lentivirus into hUC-MSCs, in vitro, the morphological changes of transgenic cells were observed by fluorescence phase contrast microscope, the expression of (GFP) was detected by green fluorescence, and the efficiency of gene transfection was determined 48 hours after transfection. The expression of Sox9 in hUC-MSCs was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot (Western-blot), and the effect of lentivirus on cell proliferation was determined by (MTT) assay. Under the condition of high density monolayer culture, the Sox9 gene modified hUC-MSCs was cultured for 21 days, RT-PCR,Western-blot, immunohistochemistry and immunofluorescence staining, without increasing the growth factor, dexamethasone and other cartilage inducers. The expression of type 鈪，
本文编号：2362072
[Abstract]:Aim: to construct lentivirus vector of human Sox9 gene and transfect Sox9 lentivirus vector into human umbilical cord mesenchymal stem cells (hUC-MSCs) in vitro to observe the effect of Sox9 gene transfection on the biological characteristics of hUC-MSCs. The ability of hUC-MSCs modified by Sox9 gene to differentiate into chondrocytes in monolayer culture provides experimental basis for cytology and gene therapy of cartilage tissue damage and degeneration. Methods: the encoding region of human Sox9 gene was obtained by PCR. The fragment was cloned into lentivirus vector Pwpxl-MOD to produce Pwpxl-MOD/Sox9. The lentivirus vector was identified by restriction endonuclease sequencing, and Pwpxl-MOD/Sox9,pRsv-REV,pMDlg-pRRE, was identified. PMD2G co-transfected 293T cells and packaged into recombinant lentivirus containing Sox9 gene. Mesenchymal stem cells were obtained from human umbilical cord stroma by enzyme digestion. The phenotype of mesenchymal stem cells was identified by flow cytometry. After transfection of lentivirus into hUC-MSCs, in vitro, the morphological changes of transgenic cells were observed by fluorescence phase contrast microscope, the expression of (GFP) was detected by green fluorescence, and the efficiency of gene transfection was determined 48 hours after transfection. The expression of Sox9 in hUC-MSCs was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot (Western-blot), and the effect of lentivirus on cell proliferation was determined by (MTT) assay. Under the condition of high density monolayer culture, the Sox9 gene modified hUC-MSCs was cultured for 21 days, RT-PCR,Western-blot, immunohistochemistry and immunofluorescence staining, without increasing the growth factor, dexamethasone and other cartilage inducers. The expression of type 鈪，
本文编号：2362072
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