人Sox2和Nanog基因克隆及其对胎肺成纤维细胞分化的影响
发布时间：2018-08-18 08:17
【摘要】：人Sox2和Nanog基因作为维持胚胎干细胞自我更新与全能性的关键基因,一直是科学研究工作的热点。克隆人Sox2和Nanog基因,研究其分子生物学功能是本实验的重点,得到目的序列与载体质粒pSG连接,在大肠杆菌中扩增来获得重组质粒pSG5-Sox2及pSG5-Nanog,为下一步转染实验做好准备。本实验选取胎儿肺间质成纤维细胞作为宿主细胞,将测序成功的重组质粒pSG5-Sox2及pSG5-Nanog转染上述宿主细胞,观察干细胞相关基因Sox2和Nanog对于终末分化的体细胞形态及功能是否产生影响。 目的 1克隆人Sox2、Nanog基因编码序列,构建pSG5-Sox2及pSG5-Nanog真核表达重组质粒。 2原代培养胎肺间质成纤维细胞并传代作为宿主细胞,转染重组质粒pSG5-Sox2、pSG5-Nanog后检测目的基因表达,观察并鉴定宿主细胞分化方向。 方法 1应用RT-PCR方法分别从4-6周龄流产胎儿脑组织和膀胱癌细胞中扩增出Sox2及Nanog基因编码全序列,琼脂糖凝胶电泳鉴定。 2目的基因片段Sox2及Nanog双酶切纯化后将其分别连接到pSG5表达载体,构成重组质粒pSG5-Sox2、pSG5-Nanog,转化、扩增后进行菌落PCR和双酶切鉴定后测序及序列比对。 3原代培养胎肺成纤维细胞并传代,实验组将重组质粒pSG5-Sox2及pSG5-Nanog用FuGENE HD转染试剂转染胎肺成纤维细胞,以空质粒pSG5用上述方法转染细胞作为空白对照,未转染细胞加入等量的低糖DMEM培养基作为阴性对照,倒置显微镜观察转染前后的细胞形态学变化。 4转染48h后,将3组胎肺成纤维细胞在六孔板中进行爬片,采用RT-PCR方法,免疫细胞化学检测Sox2及Nanog基因表达情况。过表达重组质粒,15-30d后进行角蛋白(广谱),巢蛋白,胶质纤维酸性蛋白、神经元特异性烯醇化酶免疫细胞化学染色,利用免疫反应对细胞中相应蛋白的表达进行定位。 结果 1琼脂糖凝胶电泳表明获得人Sox2基因编码序列(约1kb)和Nanog基因编码序列(约1kb)。重组质粒pSG5-Sox2、pSG5-Nanog菌落PCR产物电泳分别约1kb,双酶切获得质粒片段pSG5(约4.1kb)及Sox2基因片段(约1kb)、Nanog基因片段(约lkb),与理论值人Sox2基因编码序列(957bp),人Nanog基因编码序列(934bp)及质粒pSG5(4076bp)相符。重组质粒测序后,在NCBI中BLAST比对相似性均达到99%,表明所插入的目的片段正确。 2获得稳定传代的胎肺成纤维细胞,RT-PCR方法,免疫细胞化学检测实验组Sox2、Nanog阳性表达,而空白对照组和阴性对照组中未检测到相应基因表达。 3倒置显微镜下观察转染前胎肺成纤维细胞呈典型的栅栏、漩涡状生长。过表达重组质粒转染pSG5-Sox2、pSG5-Nanog8d后实验组细胞形态学先呈现干细胞样变化,15d后进而分化为上皮样细胞,排列紧密,呈扁平多角形,免疫细胞化学角蛋白(广谱)染色阳性；转染20-30d后有向神经细胞分化的趋势,轴突明显,部分细胞呈蜘蛛样,胞质突细长,免疫细胞化学巢蛋白、胶质酸性纤维蛋白、神经元特异性烯醇化酶染色阳性。空白对照和阴性对照组未观察有上述形态学改变,相关免疫细胞化学染色阴性。 结论 本研究克隆了人Sox2、Nanog基因,构建重组质粒pSG5-Sox2、pSG5-Nanog,转染胎肺成纤维细胞并检测到基因阳性表达,过表达重组质粒后胎肺间质成纤维细胞呈干细胞样生长并有向上皮及神经样细胞分化的趋势,为进一步研究Sox2和Nanog基因功能及重编程细胞分化奠定了基础。
[Abstract]:Cloning human Sox2 and Nanog genes and studying their molecular biological functions are the focus of this experiment. The target sequence is linked to vector plasmid pSG and amplified in E.coli to obtain recombinant plasmid pSG5-S. Ox2 and pSG5-Nanog were selected as host cells to transfect the recombinant plasmids pSG5-Sox2 and pSG5-Nanog into the host cells. The morphological and functional effects of the stem cell-related genes Sox2 and Nanog on the end-differentiated somatic cells were observed.
objective
1 clone human Sox2, Nanog gene coding sequence, construct pSG5-Sox2 and pSG5-Nanog eukaryotic expression plasmid.
2. Primary cultured fetal lung interstitial fibroblasts were subcultured as host cells. After transfected with recombinant plasmid pSG5-Sox2 and pSG5-Nanog, the target gene expression was detected and the differentiation direction of host cells was observed and identified.
Method
1. Sox2 and Nanog genes were amplified from brain tissues and bladder cancer cells of 4-6 weeks old aborted fetuses by RT-PCR and identified by agarose gel electrophoresis.
The recombinant plasmids pSG5-Sox2 and pSG5-Nanog were constructed by digesting and purifying the target gene fragments Sox2 and Nanog respectively. The recombinant plasmids pSG5-Sox2 and pSG5-Nanog were transformed, amplified, identified by colony PCR and double digestion, sequenced and aligned.
3. Primary cultured fetal lung fibroblasts were subcultured. The recombinant plasmids pSG5-Sox2 and pSG5-Nanog were transfected into fetal lung fibroblasts with FuGENE HD transfection reagent in the experimental group. The cells transfected with blank plasmid pSG5 were used as blank control, and the cells were not transfected with the same amount of DMEM medium as negative control. Morphological changes of cells before and after.
After 48 hours of transfection, three groups of fetal lung fibroblasts were sliced on a six-well plate. The expression of Sox2 and Nanog genes was detected by RT-PCR and immunocytochemistry. After overexpression of the recombinant plasmid, keratin (broad-spectrum), nestin, glial fibrillary acidic protein and neuron-specific enolase were immunocytochemically stained for 15-30 days. The expression of the corresponding proteins in the cells was localized by the epidemic reaction.
Result
The recombinant plasmid pSG5-Sox2 and pSG5-Nanog colony PCR products electrophoresis were about 1 kb, respectively. The plasmid fragments pSG5 (about 4.1 kb) and Sox2 gene fragments (about 1 kb) and Nanog Gene Fragments (about lkb) were obtained by double enzyme digestion. The recombinant plasmid pSG5-Sox2 and pSG5-Nanog colony PCR products were sequenced with the theoretical value of human Sox2 gene. Column 957 bp, human Nanog gene coding sequence 934 BP and plasmid pSG5 (4076 bp) were identical. After sequencing, BLAST alignment in NCBI was 99%, indicating that the inserted target fragment was correct.
2. Stable passage of fetal lung fibroblasts was obtained. The expression of Sox2 and Nanog was detected by RT-PCR and immunocytochemistry. No corresponding gene expression was detected in blank control group and negative control group.
After transfection of pSG5-Sox2 and pSG5-Nanog for 8 days, the morphology of the cells in the experimental group showed stem cell-like changes, and then differentiated into epithelioid cells 15 days later. The cells were arranged in a compact, flat polygonal and immunocytochemical keratin (broad spectrum). Positive staining; 20-30 days after transfection, there was a tendency to differentiate into neural cells, axons were obvious, some cells were spider-like, cytoplasmic processes slender, immunocytochemical nestin, glial acidic fibrin, neuron-specific enolase staining positive. Blank control and negative control group did not observe the above morphological changes, related immunocytochemistry. Learn to stain negative.
conclusion
In this study, human Sox2 and Nanog genes were cloned and recombinant plasmids pSG5-Sox2 and pSG5-Nanog were constructed. Fetal lung fibroblasts were transfected with the recombinant plasmids and positive expression of the genes was detected. After overexpression of the recombinant plasmids, fetal lung interstitial fibroblasts grew like stem cells and tended to differentiate into epithelial and neuron-like cells. And lay the foundation for reprogramming cell differentiation.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R362
[Abstract]:Cloning human Sox2 and Nanog genes and studying their molecular biological functions are the focus of this experiment. The target sequence is linked to vector plasmid pSG and amplified in E.coli to obtain recombinant plasmid pSG5-S. Ox2 and pSG5-Nanog were selected as host cells to transfect the recombinant plasmids pSG5-Sox2 and pSG5-Nanog into the host cells. The morphological and functional effects of the stem cell-related genes Sox2 and Nanog on the end-differentiated somatic cells were observed.
objective
1 clone human Sox2, Nanog gene coding sequence, construct pSG5-Sox2 and pSG5-Nanog eukaryotic expression plasmid.
2. Primary cultured fetal lung interstitial fibroblasts were subcultured as host cells. After transfected with recombinant plasmid pSG5-Sox2 and pSG5-Nanog, the target gene expression was detected and the differentiation direction of host cells was observed and identified.
Method
1. Sox2 and Nanog genes were amplified from brain tissues and bladder cancer cells of 4-6 weeks old aborted fetuses by RT-PCR and identified by agarose gel electrophoresis.
The recombinant plasmids pSG5-Sox2 and pSG5-Nanog were constructed by digesting and purifying the target gene fragments Sox2 and Nanog respectively. The recombinant plasmids pSG5-Sox2 and pSG5-Nanog were transformed, amplified, identified by colony PCR and double digestion, sequenced and aligned.
3. Primary cultured fetal lung fibroblasts were subcultured. The recombinant plasmids pSG5-Sox2 and pSG5-Nanog were transfected into fetal lung fibroblasts with FuGENE HD transfection reagent in the experimental group. The cells transfected with blank plasmid pSG5 were used as blank control, and the cells were not transfected with the same amount of DMEM medium as negative control. Morphological changes of cells before and after.
After 48 hours of transfection, three groups of fetal lung fibroblasts were sliced on a six-well plate. The expression of Sox2 and Nanog genes was detected by RT-PCR and immunocytochemistry. After overexpression of the recombinant plasmid, keratin (broad-spectrum), nestin, glial fibrillary acidic protein and neuron-specific enolase were immunocytochemically stained for 15-30 days. The expression of the corresponding proteins in the cells was localized by the epidemic reaction.
Result
The recombinant plasmid pSG5-Sox2 and pSG5-Nanog colony PCR products electrophoresis were about 1 kb, respectively. The plasmid fragments pSG5 (about 4.1 kb) and Sox2 gene fragments (about 1 kb) and Nanog Gene Fragments (about lkb) were obtained by double enzyme digestion. The recombinant plasmid pSG5-Sox2 and pSG5-Nanog colony PCR products were sequenced with the theoretical value of human Sox2 gene. Column 957 bp, human Nanog gene coding sequence 934 BP and plasmid pSG5 (4076 bp) were identical. After sequencing, BLAST alignment in NCBI was 99%, indicating that the inserted target fragment was correct.
2. Stable passage of fetal lung fibroblasts was obtained. The expression of Sox2 and Nanog was detected by RT-PCR and immunocytochemistry. No corresponding gene expression was detected in blank control group and negative control group.
After transfection of pSG5-Sox2 and pSG5-Nanog for 8 days, the morphology of the cells in the experimental group showed stem cell-like changes, and then differentiated into epithelioid cells 15 days later. The cells were arranged in a compact, flat polygonal and immunocytochemical keratin (broad spectrum). Positive staining; 20-30 days after transfection, there was a tendency to differentiate into neural cells, axons were obvious, some cells were spider-like, cytoplasmic processes slender, immunocytochemical nestin, glial acidic fibrin, neuron-specific enolase staining positive. Blank control and negative control group did not observe the above morphological changes, related immunocytochemistry. Learn to stain negative.
conclusion
In this study, human Sox2 and Nanog genes were cloned and recombinant plasmids pSG5-Sox2 and pSG5-Nanog were constructed. Fetal lung fibroblasts were transfected with the recombinant plasmids and positive expression of the genes was detected. After overexpression of the recombinant plasmids, fetal lung interstitial fibroblasts grew like stem cells and tended to differentiate into epithelial and neuron-like cells. And lay the foundation for reprogramming cell differentiation.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R362
【参考文献】
相关期刊论文 前10条
1 胡敏华;郭欣政;张守全;;同源结构蛋白Nanog基因的研究进展[J];黑龙江畜牧兽医;2011年03期
2 罗虎;李泽桂;;诱导多能干细胞建系过程中关键转录因子的作用[J];基础医学与临床;2011年04期
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