本文選題：骨骺干細(xì)胞 + SV40T��； 參考：《華中科技大學(xué)》2010年博士論文

【摘要】： 目的建立永生化大鼠骨骺干細(xì)胞株,并從中克隆Sox9基因,構(gòu)建真核表達(dá)載體,進(jìn)而轉(zhuǎn)染骨髓基質(zhì)細(xì)胞,探討Sox9誘導(dǎo)骨髓基質(zhì)細(xì)胞向骨骺干細(xì)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(xì)胞的分化機(jī)制及臨床應(yīng)用奠定基礎(chǔ)。 方法將含有SV40T抗原基因的真核表達(dá)載體pCMVSV40T／PUR導(dǎo)入經(jīng)免疫磁珠分選出的原代PSCs進(jìn)行穩(wěn)定表達(dá),用嘌呤霉素篩選出陽(yáng)性克隆并擴(kuò)大培養(yǎng),觀察細(xì)胞形態(tài)及生長(zhǎng)狀況,繪制細(xì)胞生長(zhǎng)曲線,用免疫細(xì)胞化學(xué)方法和RT-PCR鑒定SV40T抗原基因在轉(zhuǎn)染細(xì)胞中的表達(dá)。提取永生化PSCs總RNA,以RT-PCR方法獲得Sox9基因的全長(zhǎng),插入pGEM-T Easy克隆載體中進(jìn)行序列測(cè)定,測(cè)序正確后將其亞克隆至表達(dá)載體pEGFP-IRES2構(gòu)建重組復(fù)合質(zhì)粒。然后復(fù)合質(zhì)粒以脂質(zhì)體法轉(zhuǎn)染骨髓基質(zhì)細(xì)胞,觀察轉(zhuǎn)染效率,Sox9基因、蛋白的表達(dá),轉(zhuǎn)化后第12天檢測(cè)所轉(zhuǎn)化細(xì)胞的FGFR-3的表達(dá)情況及II型膠原、X型膠原的表達(dá)。流式細(xì)胞檢測(cè)細(xì)胞分群以及細(xì)胞周期。MTT法檢測(cè)細(xì)胞增殖活性。 結(jié)果分離獲得轉(zhuǎn)化細(xì)胞陽(yáng)性克隆,用免疫組化證實(shí)FGFR-3表達(dá)陽(yáng)性,提取RNA后用RT-PCR法成功擴(kuò)增出588 bp的片段。轉(zhuǎn)染細(xì)胞經(jīng)擴(kuò)大培養(yǎng),命名為永生化骨骺干細(xì)胞。貼壁培養(yǎng)的轉(zhuǎn)染細(xì)胞群體倍增時(shí)間為(22.98±2.77)h,傳代、凍存和復(fù)蘇對(duì)細(xì)胞形態(tài)及生長(zhǎng)無(wú)明顯影響。從永生化骨骺干細(xì)胞中提取總RNA經(jīng)電泳可得28s、18s和5s共3條帶,測(cè)吸光度值為0.2635,A260／A280為1.8741,說(shuō)明提取的總RNA完整性較好,純度高,符合RT-PCR的要求。PCR產(chǎn)物經(jīng)電泳可得到約2000bp的特異性條帶,與預(yù)期大小一致。經(jīng)限制性內(nèi)切酶酶切圖譜分析DNA序列測(cè)定證實(shí)的目的基因已經(jīng)插入重組質(zhì)粒,成功地構(gòu)建了Sox9質(zhì)粒。經(jīng)熒光顯微鏡下觀察證實(shí)：成功地對(duì)骨髓基質(zhì)質(zhì)干細(xì)胞實(shí)現(xiàn)了Sox9基因的轉(zhuǎn)染。計(jì)算轉(zhuǎn)染效率約為50%,轉(zhuǎn)染后的細(xì)胞穩(wěn)定表達(dá)Sox9基因、Sox9蛋白。轉(zhuǎn)染后的細(xì)胞免疫組化及Western blot檢測(cè)顯示FGFR-3表達(dá)陽(yáng)性,而Ⅱ型膠、X型膠原的表達(dá)為陰性。流式細(xì)胞檢查發(fā)現(xiàn)：從轉(zhuǎn)染后一直到第30天,穩(wěn)定的表現(xiàn)為大部分細(xì)胞處于第1象限,提示為FGFR-3陽(yáng)性,為骨骺干細(xì)胞。MTT法檢測(cè)其增殖活性與骨骺干細(xì)胞無(wú)異。 結(jié)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(xì)胞,pCMVSV40T／PUR轉(zhuǎn)染能使其永生化。從IPSCs克隆出Sox9基因并成功構(gòu)建了Sox9基因真核表達(dá)載體,并利用其成功轉(zhuǎn)染了骨髓基質(zhì)干細(xì)胞,轉(zhuǎn)染后的骨髓基質(zhì)細(xì)胞分化為骨骺干細(xì)胞并具有骨骺干細(xì)胞的特性,這為調(diào)控骨骺干細(xì)胞的分化及為再生骨骺、完成自體骨骺干細(xì)胞移植奠定了堅(jiān)實(shí)的基礎(chǔ)。
[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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