重组瘦素基因对骨髓基质细胞增殖分化的影响

发布时间：2019-01-10 08:25

【摘要】： 目的: 构建重组瘦素基因(Leptin)真核表达质粒pTracerTM-Leptin;转染SD大鼠骨髓基质细胞(marrow stromal cell, MSCs),研究Leptin基因对MSCs增殖分化的影响。方法: 提取人脂肪组织总RNA,根据GeneBank公布的Leptin基因序列设计合成一对引物,采用RT-PCR技术克隆Leptin基因,酶切后重组到带有GFP标记的pTracerTM-CMV/Bsd载体上,构建重组基因真核质粒pTracerTM-Leptin。采用密度梯度离心法体外获得SD大鼠的骨髓基质细胞,将重组真核质粒pTracerTM-Leptin通过脂质体转染法转染骨髓基质细胞,以抗生素Blasticidin加压筛选获得Leptin基因修饰的转基因细胞株,普通光镜及荧光显微镜下观察转基因细胞形态,采用分子生物学法(RT-PCR法)和免疫学法(Western blot法)分别从mRNA水平和蛋白质水平检测目的基因在转基因细胞中的转录和翻译,MTT法检测Leptin基因对MSCs的生长增殖情况,ELISA法检测转基因细胞上清骨钙素(OC)分泌水平,并采用对硝基苯磷酸盐(PNP)试剂盒测定转基因细胞碱性磷酸酶(ALP)活力,半定量RT-PCR法检测转基因细胞中成骨细胞分化相关基因Cbfα1和Cbfβ的表达。结果: 成功获得约450bp的人Leptin基因,PCR、双酶切及序列测定结果显示成功构建了重组基因真核质粒pTracerTM-Leptin,并转染密度梯度分离法获得的大鼠骨髓基质细胞,加压筛选后获得转基因细胞,荧光显微镜下可观察到绿色荧光,并伴有细胞形态变化,实验组的RT-PCR和Western blot结果均出现目的条带,表明脂质体介导的Leptin基因可以在骨髓基质细胞中表达,且促进MSCs增殖,并有一定的时间依赖性,转基因细胞上清中OC含量增加,ALP活力上调,成骨细胞特异性转录因子Cbfα1和CbfβmRNA表达量也增高。结论: 本实验成功构建了重组真核质粒pTracerTM-Leptin,转染骨髓基质细胞后,Leptin基因可以使其增殖,且促进成骨样改变。
[Abstract]:Aim: to construct recombinant (Leptin) eukaryotic expression plasmid of leptin gene (Leptin) and transfect (marrow stromal cell, MSCs), into SD rat bone marrow stromal cells to study the effect of Leptin gene on MSCs proliferation and differentiation. Methods: a pair of primers were designed and synthesized from human adipose tissue total RNA, according to the Leptin gene sequence published by GeneBank. The Leptin gene was cloned by RT-PCR technique. The Leptin gene was digested and recombined into pTracerTM-CMV/Bsd vector marked with GFP. Construction of Recombinant Eukaryotic plasmid pTracerTM-Leptin. Bone marrow stromal cells of SD rats were obtained by density gradient centrifugation in vitro. The recombinant eukaryotic plasmid pTracerTM-Leptin was transfected into bone marrow stromal cells by liposome transfection. Leptin gene modified transgenic cells were obtained by Blasticidin pressurized screening. The morphology of transgenic cells was observed under light microscope and fluorescence microscope. The transcription and translation of target genes in transgenic cells were detected by molecular biology (RT-PCR) and immunological (Western blot (Western blot) from the level of mRNA and protein, respectively. MTT assay was used to detect the growth and proliferation of Leptin gene to MSCs, ELISA method was used to detect the level of osteocalcin (OC) secretion in the supernatant of transgenic cells, and the activity of alkaline phosphatase (ALP) (ALP) in transgenic cells was determined by using p-nitrophenyl phosphate (PNP) kit. The expression of osteoblast differentiation related genes Cbf 伪 1 and Cbf 尾 in transgenic cells was detected by semi quantitative RT-PCR. Results: the human Leptin gene of about 450bp was successfully obtained. The results of PCR, double enzyme digestion and sequencing showed that the recombinant gene eukaryotic plasmid pTracerTM-Leptin, was successfully constructed and transfected into rat bone marrow stromal cells obtained by density gradient separation. After pressurized screening, the transgenic cells were obtained. Green fluorescence was observed under fluorescence microscope, accompanied by cell morphological changes. The results of RT-PCR and Western blot in the experimental group showed the target bands. The results indicated that Leptin gene mediated by liposome could be expressed in bone marrow stromal cells and promoted the proliferation of MSCs in a time-dependent manner. The content of OC in the supernatant of transgenic cells was increased and the activity of ALP was upregulated. The expression of Cbf 伪 1 and Cbf 尾 mRNA in osteoblasts was also increased. Conclusion: after transfection of recombinant eukaryotic plasmid pTracerTM-Leptin, into bone marrow stromal cells, Leptin gene can proliferate and promote osteoblast-like changes.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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