人Sox2和Nanog基因克隆及其對胎肺成纖維細胞分化的影響
發(fā)布時間：2018-08-18 08:17
【摘要】：人Sox2和Nanog基因作為維持胚胎干細胞自我更新與全能性的關鍵基因,一直是科學研究工作的熱點�？寺∪薙ox2和Nanog基因,研究其分子生物學功能是本實驗的重點,得到目的序列與載體質粒pSG連接,在大腸桿菌中擴增來獲得重組質粒pSG5-Sox2及pSG5-Nanog,為下一步轉染實驗做好準備。本實驗選取胎兒肺間質成纖維細胞作為宿主細胞,將測序成功的重組質粒pSG5-Sox2及pSG5-Nanog轉染上述宿主細胞,觀察干細胞相關基因Sox2和Nanog對于終末分化的體細胞形態(tài)及功能是否產生影響。 目的 1克隆人Sox2、Nanog基因編碼序列,構建pSG5-Sox2及pSG5-Nanog真核表達重組質粒。 2原代培養(yǎng)胎肺間質成纖維細胞并傳代作為宿主細胞,轉染重組質粒pSG5-Sox2、pSG5-Nanog后檢測目的基因表達,觀察并鑒定宿主細胞分化方向。 方法 1應用RT-PCR方法分別從4-6周齡流產胎兒腦組織和膀胱癌細胞中擴增出Sox2及Nanog基因編碼全序列,瓊脂糖凝膠電泳鑒定。 2目的基因片段Sox2及Nanog雙酶切純化后將其分別連接到pSG5表達載體,構成重組質粒pSG5-Sox2、pSG5-Nanog,轉化、擴增后進行菌落PCR和雙酶切鑒定后測序及序列比對。 3原代培養(yǎng)胎肺成纖維細胞并傳代,實驗組將重組質粒pSG5-Sox2及pSG5-Nanog用FuGENE HD轉染試劑轉染胎肺成纖維細胞,以空質粒pSG5用上述方法轉染細胞作為空白對照,未轉染細胞加入等量的低糖DMEM培養(yǎng)基作為陰性對照,倒置顯微鏡觀察轉染前后的細胞形態(tài)學變化。 4轉染48h后,將3組胎肺成纖維細胞在六孔板中進行爬片,采用RT-PCR方法,免疫細胞化學檢測Sox2及Nanog基因表達情況。過表達重組質粒,15-30d后進行角蛋白(廣譜),巢蛋白,膠質纖維酸性蛋白、神經元特異性烯醇化酶免疫細胞化學染色,利用免疫反應對細胞中相應蛋白的表達進行定位。 結果 1瓊脂糖凝膠電泳表明獲得人Sox2基因編碼序列(約1kb)和Nanog基因編碼序列(約1kb)。重組質粒pSG5-Sox2、pSG5-Nanog菌落PCR產物電泳分別約1kb,雙酶切獲得質粒片段pSG5(約4.1kb)及Sox2基因片段(約1kb)、Nanog基因片段(約lkb),與理論值人Sox2基因編碼序列(957bp),人Nanog基因編碼序列(934bp)及質粒pSG5(4076bp)相符。重組質粒測序后,在NCBI中BLAST比對相似性均達到99%,表明所插入的目的片段正確。 2獲得穩(wěn)定傳代的胎肺成纖維細胞,RT-PCR方法,免疫細胞化學檢測實驗組Sox2、Nanog陽性表達,而空白對照組和陰性對照組中未檢測到相應基因表達。 3倒置顯微鏡下觀察轉染前胎肺成纖維細胞呈典型的柵欄、漩渦狀生長。過表達重組質粒轉染pSG5-Sox2、pSG5-Nanog8d后實驗組細胞形態(tài)學先呈現干細胞樣變化,15d后進而分化為上皮樣細胞,排列緊密,呈扁平多角形,免疫細胞化學角蛋白(廣譜)染色陽性；轉染20-30d后有向神經細胞分化的趨勢,軸突明顯,部分細胞呈蜘蛛樣,胞質突細長,免疫細胞化學巢蛋白、膠質酸性纖維蛋白、神經元特異性烯醇化酶染色陽性�？瞻讓φ蘸完幮詫φ战M未觀察有上述形態(tài)學改變,相關免疫細胞化學染色陰性。 結論 本研究克隆了人Sox2、Nanog基因,構建重組質粒pSG5-Sox2、pSG5-Nanog,轉染胎肺成纖維細胞并檢測到基因陽性表達,過表達重組質粒后胎肺間質成纖維細胞呈干細胞樣生長并有向上皮及神經樣細胞分化的趨勢,為進一步研究Sox2和Nanog基因功能及重編程細胞分化奠定了基礎。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R362
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相關期刊論文 前10條
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2 羅虎;李澤桂;;誘導多能干細胞建系過程中關鍵轉錄因子的作用[J];基礎醫(yī)學與臨床;2011年04期
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