永生化骨骺干细胞株的建立及Sox9基因诱导骨髓基质细胞定向分化的研究

发布时间：2018-04-23 14:40

  本文选题：骨骺干细胞 + SV40T　； 参考：《华中科技大学》2010年博士论文

【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。
[Abstract]:Objective to establish immortalized rat epiphyseal stem cell line, clone Sox9 gene from it, construct eukaryotic expression vector, and then transfect bone marrow stromal cells. To investigate the possibility of Sox9 inducing bone marrow stromal cells to differentiate into epiphyseal stem cells and remain in the secondary differentiation state. It lays a foundation for studying the differentiation mechanism and clinical application of epiphyseal stem cells. Methods the eukaryotic expression vector pCMVSV40T/PUR containing SV40T antigen gene was introduced into the primary PSCs isolated by immunomagnetic beads for stable expression. The positive clones were screened by purine mycin and the culture was expanded. The morphology and growth of the cells were observed. The cell growth curve was drawn and the expression of SV40T antigen gene in transfected cells was identified by immunocytochemistry and RT-PCR. The total PSCs of immortalized PSCs was extracted, the full length of Sox9 gene was obtained by RT-PCR method, and inserted into the pGEM-T Easy clone vector for sequencing. After sequencing correctly, the Sox9 gene was subcloned into the expression vector pEGFP-IRES2 to construct the recombinant plasmid. The expression of Sox9 gene and protein in bone marrow stromal cells was observed by liposome method. The expression of FGFR-3 and type X collagen type II collagen were detected on the 12th day after transformation. Flow cytometry was used to detect cell differentiation and cell cycle. MTT assay was used to detect cell proliferation activity. Results the positive clones of transformed cells were isolated and the positive expression of FGFR-3 was confirmed by immunohistochemistry. The fragment of 588bp was successfully amplified by RT-PCR method after RNA was extracted. The transfected cells were expanded and named as immortalized epiphyseal stem cells. The population doubling time of the transfected cells in adherent culture was 22.98 卤2.77 h.The passage, cryopreservation and resuscitation had no significant effect on cell morphology and growth. The total RNA extracted from immortalized epiphyseal stem cells could be obtained by electrophoresis for 28 s / 18 s and 5 s, and the absorbance value was 0.2635% A260 / A280 = 1.8741, which indicated that the total RNA extracted was of good integrity and high purity, and the specific bands of reduced 2000bp could be obtained by electrophoresis. The size is in line with the expected size. The target gene confirmed by DNA sequence analysis by restriction endonuclease digestion was inserted into the recombinant plasmid and the Sox9 plasmid was successfully constructed. Fluorescence microscopy showed that Sox9 gene was successfully transfected into bone marrow stromal stem cells. The transfection efficiency was about 50. The transfected cells stably expressed Sox9 gene and Sox9 protein. After transfection, the expression of FGFR-3 was positive by immunohistochemistry and Western blot, but the expression of type 鈪，

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