【作者】 胡伟华；

【导师】 陈安民；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 目的建立永生化大鼠骨骺干细胞株,并从中克隆Sox9基因,构建真核表达载体,进而转染骨髓基质细胞,探讨Sox9诱导骨髓基质细胞向骨骺干细胞分化并停留在该次分化状态的可能性。为研究骨骺干细胞的分化机制及临床应用奠定基础。方法将含有SV40T抗原基因的真核表达载体pCMVSV40T／PUR导入经免疫磁珠分选出的原代PSCs进行稳定表达,用嘌呤霉素筛选出阳性克隆并扩大培养,观察细胞形态及生长状况,绘制细胞生长曲线,用免疫细胞化学方法和RT-PCR鉴定SV40T抗原基因在转染细胞中的表达。提取永生化PSCs总RNA,以RT-PCR方法获得Sox9基因的全长,插入pGEM-T Easy克隆载体中进行序列测定,测序正确后将其亚克隆至表达载体pEGFP-IRES2构建重组复合质粒。然后复合质粒以脂质体法转染骨髓基质细胞,观察转染效率,Sox9基因、蛋白的表达,转化后第12天检测所转化细胞的FGFR-3的表达情况及II型胶原、X型胶原的表达。流式细胞检测细胞分群以及细胞周期。MTT法检测细胞增殖活性。结果分离获得转化细胞阳性克隆,用免疫组化证实FGFR-3表达阳性,提取RNA后用RT-PCR法成功扩增出588 bp的片段。转染细胞经扩大培养,命名为永生化骨骺干细胞。贴壁培养的转染细胞群体倍增时间为(22.98±2.77)h,传代、冻存和复苏对细胞形态及生长无明显影响。从永生化骨骺干细胞中提取总RNA经电泳可得28s、18s和5s共3条带,测吸光度值为0.2635,A260／A280为1.8741,说明提取的总RNA完整性较好,纯度高,符合RT-PCR的要求。PCR产物经电泳可得到约2000bp的特异性条带,与预期大小一致。经限制性内切酶酶切图谱分析DNA序列测定证实的目的基因已经插入重组质粒,成功地构建了Sox9质粒。经荧光显微镜下观察证实：成功地对骨髓基质质干细胞实现了Sox9基因的转染。计算转染效率约为50%,转染后的细胞稳定表达Sox9基因、Sox9蛋白。转染后的细胞免疫组化及Western blot检测显示FGFR-3表达阳性,而Ⅱ型胶、X型胶原的表达为阴性。流式细胞检查发现：从转染后一直到第30天,稳定的表现为大部分细胞处于第1象限,提示为FGFR-3阳性,为骨骺干细胞。MTT法检测其增殖活性与骨骺干细胞无异。结论在体外培养条件下,可以从新生大鼠干骺端中分离、培养出骨骺干细胞,pCMVSV40T／PUR转染能使其永生化。从IPSCs克隆出Sox9基因并成功构建了Sox9基因真核表达载体,并利用其成功转染了骨髓基质干细胞,转染后的骨髓基质细胞分化为骨骺干细胞并具有骨骺干细胞的特性,这为调控骨骺干细胞的分化及为再生骨骺、完成自体骨骺干细胞移植奠定了坚实的基础。更多还原

【Abstract】 Objective To establish immortalized precartilaginous stem cells (IPSCs) from which we clone the Sox9 gene and construct its eukaryotic expression vector. Then we transfect the plasmid into the bone marrow stromal cells (MSCs). We study the possibility of MSCs differentiating to PSCs and remaining in the PSCs state for the further related research on the differentiation mechanism and clinic application of precartilaginous stem cells.Methods pCMVSV40T/PUR was transfected into the primary cultured PSCs isolated by immuniomagnetic beads select system. Colonies were isolated by puromycin selection and expanded by many passages. Investigate the capability of differentiation of the transfected cells. The expression of SV40Tag in expanded cell lines was identified by immunocytochemistry method and RT-PCR. The total RNA was extracted from immortalized PSCs (IPSCs) after identification and the Sox9 gene was obtained by RT-PCR and be inserted into pGEM-T Easy cloning vector. After the sequencing was confirmed, the gene was subcloned to pEGFP-IRES2 to construct recombinant eukaryotic expression vector pEGFP-IRES2. After that, we transfected the plasmid into MSCs by liposome. We observed the transfection efficiency and expression of Sox9 gene and Sox9 protein, then we detected the transfected cell’s expression of FGFR-3 and expression of collagen type II and collagen type X after 12 days after transfection. Detecting the grouping of cells by flow cytometry was applied. Proliferation was determined by MTT.Results A particular anti-puromycin cell clone was acquired, which was confirmed as fibroblast growth factor receptor-3(FGFR-3) positive PSCs. The total RNA were isolated from the positive cell clones, and a 588 bp fragment, which was specific for the SV40T antigene gene, was amplified. The transfected cells were expanded to immortalized cell strain, named as immortalized precartilaginous stem cells(IPSCs). The population doubling time of IPSCs was 22.98±2.77 h, no significant effect of subculture, freezing and recovering had been found. The total RNA extracted from IPSCs through electrophoresis can get 28s、18s、3s three bands. The OD value is 0.2635 and the A260/A280 is 1.8741 which shows that the total RNA extracted has a good integrity and purity, and accord with the request of RT-PCR.The PCR product through electrophoresis can get a 2000bp specificity electrophoretic band which is accord with anticipation. Enzyme digestion analysis and DNA sequencing showed that the target gene had been cloned into recombinant plasmids。The Sox9 recombinant plasmids were constructed successfully. Observing under fluorescence microscope confirmed that the Sox9 gene has transfected the bone marrow stromal stem cells successfully. The transfection efficiency is about 50%. The transfected cells expressing Sox9 gene and protein Sox9 stably. Immunohistochemistry and Western blot tests showed that the transfected cells express of FGFR-3 but notⅡtype of collagen and X type of collagen. Flow cytometry revealed:The majority of cells is in the first quadrant 1 after transfection from 1 day to 40 days, which hint FGFR-3 positive and the cells are PSCs, whose proliferation activity was similar to precartiliaginous stem cells (PSCs) in cells proliferation curve derived from MTT.Conclusion Precartilaginous stem cells could be isolated from neonatal SD rats, cultured in vitro, and immortalized through the transfection of pCMVSV40T/PUR.The Sox9 gene was cloned successfully from IPSCs. The eukaryotic expression plasmid containing Sox9 gene was successfully constructed. The bone marrow stromal stem cells were transfected by Sox9 gene eukaryotic expression vector successfully. The MSCs transfected by Sox9 gene eukaryotic expression vector differentiate to PSCs and have the characteristics of PSCs.Which may be a promising solid foundation for artificially controlling the differentiation of PSCs and regeneration of epiphyseal、autologous stem cell transplantation.更多还原