本文選題：骨骺干細胞 + SV40T　； 參考：《華中科技大學》2010年博士論文

【摘要】： 目的建立永生化大鼠骨骺干細胞株,并從中克隆Sox9基因,構建真核表達載體,進而轉染骨髓基質細胞,探討Sox9誘導骨髓基質細胞向骨骺干細胞分化并停留在該次分化狀態(tài)的可能性。為研究骨骺干細胞的分化機制及臨床應用奠定基礎。 方法將含有SV40T抗原基因的真核表達載體pCMVSV40T／PUR導入經(jīng)免疫磁珠分選出的原代PSCs進行穩(wěn)定表達,用嘌呤霉素篩選出陽性克隆并擴大培養(yǎng),觀察細胞形態(tài)及生長狀況,繪制細胞生長曲線,用免疫細胞化學方法和RT-PCR鑒定SV40T抗原基因在轉染細胞中的表達。提取永生化PSCs總RNA,以RT-PCR方法獲得Sox9基因的全長,插入pGEM-T Easy克隆載體中進行序列測定,測序正確后將其亞克隆至表達載體pEGFP-IRES2構建重組復合質粒。然后復合質粒以脂質體法轉染骨髓基質細胞,觀察轉染效率,Sox9基因、蛋白的表達,轉化后第12天檢測所轉化細胞的FGFR-3的表達情況及II型膠原、X型膠原的表達。流式細胞檢測細胞分群以及細胞周期。MTT法檢測細胞增殖活性。 結果分離獲得轉化細胞陽性克隆,用免疫組化證實FGFR-3表達陽性,提取RNA后用RT-PCR法成功擴增出588 bp的片段。轉染細胞經(jīng)擴大培養(yǎng),命名為永生化骨骺干細胞。貼壁培養(yǎng)的轉染細胞群體倍增時間為(22.98±2.77)h,傳代、凍存和復蘇對細胞形態(tài)及生長無明顯影響。從永生化骨骺干細胞中提取總RNA經(jīng)電泳可得28s、18s和5s共3條帶,測吸光度值為0.2635,A260／A280為1.8741,說明提取的總RNA完整性較好,純度高,符合RT-PCR的要求。PCR產物經(jīng)電泳可得到約2000bp的特異性條帶,與預期大小一致。經(jīng)限制性內切酶酶切圖譜分析DNA序列測定證實的目的基因已經(jīng)插入重組質粒,成功地構建了Sox9質粒。經(jīng)熒光顯微鏡下觀察證實：成功地對骨髓基質質干細胞實現(xiàn)了Sox9基因的轉染。計算轉染效率約為50%,轉染后的細胞穩(wěn)定表達Sox9基因、Sox9蛋白。轉染后的細胞免疫組化及Western blot檢測顯示FGFR-3表達陽性,而Ⅱ型膠、X型膠原的表達為陰性。流式細胞檢查發(fā)現(xiàn)：從轉染后一直到第30天,穩(wěn)定的表現(xiàn)為大部分細胞處于第1象限,提示為FGFR-3陽性,為骨骺干細胞。MTT法檢測其增殖活性與骨骺干細胞無異。 結論在體外培養(yǎng)條件下,可以從新生大鼠干骺端中分離、培養(yǎng)出骨骺干細胞,pCMVSV40T／PUR轉染能使其永生化。從IPSCs克隆出Sox9基因并成功構建了Sox9基因真核表達載體,并利用其成功轉染了骨髓基質干細胞,轉染后的骨髓基質細胞分化為骨骺干細胞并具有骨骺干細胞的特性,這為調控骨骺干細胞的分化及為再生骨骺、完成自體骨骺干細胞移植奠定了堅實的基礎。
[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 鹵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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