脐带间充质干细胞过表达CDX2后MUC2的表达研究
发布时间：2018-08-24 20:35
【摘要】：研究背景和目的：尾型同源盒转录因子2（caudal type homeobox2，CDX2）是一种在肠道黏膜特异性表达的基因，它也是胚胎发育过程中促进限制内胚层向后肠发育的关键基因。过表达CDX2的食管上皮细胞可以表达肠道粘蛋白MUC2，促进食管肠上皮化生。将CDX2通过内部核糖体进入位点序列（Internal ribosome entry site, IRES）与增强型绿色荧光蛋白（Enhanced Green Fluorescent Portein,EGFP）连接，可以使CDX2与EGFP共表达。因此可以通过对EGFP的直接观察了解CDX2的表达情况。本实验第一部分拟通过分子生物学的相关方法构建、扩增、提取CDX2-IRES-EGFP真核表达载体，为后续脐带间充质干细胞的基因过表达奠定基础。脐带间充质干细胞（umbilicalcord mesenchymal stem cells，UC-MSCs）主要存在于脐带血管与外膜之间的间质组织，属于成体干细胞的一种。UC-MSCs具有自我更新及向不同组织分化的能力。本研究第二部分从人脐带间质中分离并提取UC-MSCs，对细胞形态进行观察，并检测UC-MSCs的免疫表型、增殖能力，以确定分离提取的细胞是UC-MSCs，为下一步探索过表达CDX2的脐带间充质干细胞的MUC2表达研究奠定基础。溃疡性结肠炎患者肠粘膜杯状细胞减少、粘液层变薄、黏蛋白分泌减少，本实验第三部分拟研究CDX2在UC-MSCs中过表达后粘蛋白MUC2的表达情况，探寻利用过表达CDX2的UC-MSCs大量表达MUC2的实验方法，了解诱导UC-MSCs向内胚层分化对MSCs大量表达MUC2的影响，为研究UC-MSCs在肠道黏膜定向分化为杯状细胞或在肠道大量表达黏蛋白治疗溃疡性结肠炎奠定基础。 研究方法：通过RT-PCR从cDNA文库中扩增出CDX2基因的CDS序列，并通过in-fusion交换法将目的片段克隆入IRES-EGFP骨架质粒中。然后将构建好的重组质粒CDX2-IRES-EGFP通过热休克法转化入感受态细菌后进行抗生素选择培养。将获得的阳性克隆进行菌液PCR初步鉴定，利用DNA测序技术进一步确定所得载体及基因是否与数据库公布一致。大量扩增阳性克隆后，，再通过碱裂解法提取质粒，通过分光光度法检测其浓度及纯度，用于后续实验。细胞来源则是取足月产健康新生儿脐带，分离间质组织，剪碎后置于培养瓶，加DMEM/F12培养基和胎牛血清行组织块贴壁培养，待有大量细胞爬出时进行传代培养。观察细胞的形态，测定1、3、7、12、16代细胞的增殖能力。然后对P3代细胞行流式细胞术检测CD45、CD34、CD105、CD90、CD73、HLA-DR等细胞免疫表型。第三部分MUC2表达研究实验中共分5个组，分别为单转组：CDX2转染UC-MSCs；单诱组：仅诱导UC-MSCs向内胚层细胞分化；诱转组：诱导MSCs向内胚层细胞分化后，再将CDX2转染诱导后细胞；空转组：不进行任何诱导但转染空质粒；对照组：UC-MSCs不做任何处理。为排除转染试剂本身的影响，单诱组在诱导分化后仍需转染试剂处理，但不加质粒。各组最后检测CDX2、MUC2mRNA的表达情况。内胚层细胞诱导利用重组人Activin A、重组人Wnt3a实现。 结果：目的基因PCR产物长度962bp，凝胶电泳中条带位置与预期一致；测序结果与Genebank公布CDX2的CDS序列一致；转化子阳性克隆菌液PCR产物预计长度823bp，凝胶电泳中条带位置与预期一致。所提取真核表达载体CDX2-IRES-EGFP浓度为843.6ng/ul，OD260/280=1.74，OD260/230=2.23。在UC-MSCs培养中，贴壁组织周围在原代培养第7-8天有少量细胞爬出并贴壁生长，形态为梭形和多角形，10-14天组织块周围细胞密集。传代后的细胞呈漩涡状生长，形态为长梭形，且较为一致。细胞增殖能力强，每4-6天即可进行传代，传至第16代其增殖能力未见明显减弱。流式细胞术分析所得细胞免疫表型结果表明，CD90/CD105/CD73阳性，CD45/CD34/HLA-DR阴性。MUC2表达研究实验中，单转组、诱转组和空转组细胞在转染48小时后在荧光显微镜下能看到绿色荧光。单诱组和诱转组经诱导培养5天后，内胚层标志物GATA4表达增加27.0±7.0倍，Sox17表达增加54±6.4倍。对5组细胞行RT-PCR检测MUC2与CDX2表达情况，结果单转组CDX2表达升高113.2±13.5倍，MUC2表达升高30.2±3.7倍；诱转组CDX2升高196.7±15.4倍，MUC2升高98.8±11.1倍。CDX2与MUC2的表达在其他三组中无明显变化。 结论：真核表达载体CDX2-IRES-EGFP构建正确，其浓度、纯度符合转染要求。人脐带含有丰富的MSCs，通过组织块贴壁法能提取出足量MSCs，该细胞呈长梭形，漩涡样生长，增殖能力强且纯度较高。在UC-MSCs中过表达CDX2能够增加杯状细胞标志物粘蛋白MUC2的表达，而利用Activin A与Wnt3a诱导处理能进一步增加MUC2的表达。但是更大量的MUC2表达还需进一步优化实验条件。
[Abstract]:BACKGROUND AND OBJECTIVE: caudal type homeobox 2 (CDX2) is a gene specifically expressed in the intestinal mucosa, and it is also a key gene that promotes the development of the endoderm into the hindgut during embryonic development. Overexpression of CDX2 in esophageal epithelial cells can express intestinal mucin MUC2 and promote the development of the esophageal and intestinal epithelium. Metaplasia. The binding of CDX2 to Enhanced Green Fluorescent Portein (EGFP) via the internal ribosome entry site (IRES) enables the co-expression of CDX2 and EGFP. Therefore, the expression of CDX2 can be understood by direct observation of EGFP. The construction, amplification and extraction of CDX2-IRES-EGFP eukaryotic expression vectors lay the foundation for subsequent gene overexpression of umbilical cord mesenchymal stem cells. UC-MSCs have the ability to self-renew and differentiate into different tissues. In the second part of this study, UC-MSCs were isolated from human umbilical cord stroma, and the morphology of UC-MSCs was observed. The immunophenotype and proliferation ability of UC-MSCs were detected to confirm that the isolated cells were UC-MSCs. The third part of this experiment is to study the expression of mucin MUC2 after overexpression of CDX2 in UC-MSCs, to explore the experimental method of overexpression of MUC2 by UC-MSCs overexpression of CDX2, and to understand the direction of inducing UC-MSCs. The effect of endodermal differentiation on the expression of MUC2 in MSCs lays a foundation for the study of UC-MSCs differentiating into goblet cells in intestinal mucosa or expressing mucin in intestinal mucosa to treat ulcerative colitis.
Methods: The CDS sequence of CDX2 gene was amplified from the cDNA library by RT-PCR, and the target fragment was cloned into the IRES-EGFP matrix plasmid by in-fusion exchange. Then the recombinant plasmid CDX2-IRES-EGFP was transformed into the susceptible bacteria by heat shock and the positive clones were selected for antibiotic culture. After a large number of positive clones were amplified, the plasmids were extracted by alkaline lysis method. The concentration and purity of the plasmids were detected by spectrophotometry. The cells were isolated from the umbilical cord of full-term healthy newborns. Mesenchymal tissue was cut and placed in a culture flask, then cultured with DMEM/F12 medium and fetal bovine serum. Cells were subcultured when a large number of cells crawled out. Cell morphology was observed and the proliferative capacity of passages 1, 3, 7, 12 and 16 was measured. Then the cell immunity of passages 3 was detected by flow cytometry. The third part of MUC2 expression experiment was divided into five groups: single-transfection group: CDX2 transfection UC-MSCs; single-inducement group: only UC-MSCs were induced to differentiate into endodermal cells; induced group: MSCs were induced to differentiate into endodermal cells, then CDX2 was transfected into induced cells; blank-transfection group: no induction but transfection of empty plasmids; control group Group A: UC-MSCs did not undergo any treatment. In order to exclude the effect of transfection reagent itself, the single inducer group still needed transfection reagent treatment after induction of differentiation, but did not contain plasmid. The expression of CDX2 and MUC2 mRNA was detected in each group. The induction of endodermal cells was achieved by recombinant human Activin A and recombinant human Wnt3a.
Results: The length of the PCR product was 962 BP and the position of the band in gel electrophoresis was the same as expected. The sequencing results were consistent with the CDS sequence of CDX2 published by Genebank. OD260/280 = 1.74, OD260/230 = 2.23. In UC-MSCs culture, a small number of cells climbed out and grew adhering to the wall around the adherent tissue on the 7th-8th day of primary culture. The cells were spindle-shaped and polygonal in shape, and dense around the tissue block on the 10th-14th day. The results of flow cytometry showed that CD90/CD105/CD73 was positive and CD45/CD34/HLA-DR was negative. In the MUC2 expression study, green fluorescence was observed 48 hours after transfection in the single transfection group, the induced transfection group and the empty transfection group. The expression of GATA4 and Sox17 increased by 27.0 (+ 7.0) folds and 54 (+ 6.4) folds in both groups after induction and culture for 5 days. The expression of MUC2 and CDX2 was detected by RT-PCR in 5 groups. The results showed that the expression of CDX2 and MUC2 increased by 113.2 (+ 13.5) folds, 30.2 (+ 3.7) folds and 196.7 (+ 15.4) folds and 98.8 (+ 11.1) folds in the single-transfection group, respectively. There was no significant change in the expression of.CDX2 and MUC2 in the other three groups.
Conclusion: Eukaryotic expression vector CDX2-IRES-EGFP is constructed correctly, its concentration and purity meet the requirements of transfection. Human umbilical cord contains abundant MSCs, which can be extracted by tissue adherence method. The cells are spindle-shaped, vortex-like growth, strong proliferation ability and high purity. Overexpression of CDX2 in UC-MSCs can increase goblet cell marker sticky eggs. The expression of white MUC2 was further enhanced by Activin A and Wnt3a induction, but more MUC2 expression still needed to be optimized.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R329.2
本文编号：2201965
[Abstract]:BACKGROUND AND OBJECTIVE: caudal type homeobox 2 (CDX2) is a gene specifically expressed in the intestinal mucosa, and it is also a key gene that promotes the development of the endoderm into the hindgut during embryonic development. Overexpression of CDX2 in esophageal epithelial cells can express intestinal mucin MUC2 and promote the development of the esophageal and intestinal epithelium. Metaplasia. The binding of CDX2 to Enhanced Green Fluorescent Portein (EGFP) via the internal ribosome entry site (IRES) enables the co-expression of CDX2 and EGFP. Therefore, the expression of CDX2 can be understood by direct observation of EGFP. The construction, amplification and extraction of CDX2-IRES-EGFP eukaryotic expression vectors lay the foundation for subsequent gene overexpression of umbilical cord mesenchymal stem cells. UC-MSCs have the ability to self-renew and differentiate into different tissues. In the second part of this study, UC-MSCs were isolated from human umbilical cord stroma, and the morphology of UC-MSCs was observed. The immunophenotype and proliferation ability of UC-MSCs were detected to confirm that the isolated cells were UC-MSCs. The third part of this experiment is to study the expression of mucin MUC2 after overexpression of CDX2 in UC-MSCs, to explore the experimental method of overexpression of MUC2 by UC-MSCs overexpression of CDX2, and to understand the direction of inducing UC-MSCs. The effect of endodermal differentiation on the expression of MUC2 in MSCs lays a foundation for the study of UC-MSCs differentiating into goblet cells in intestinal mucosa or expressing mucin in intestinal mucosa to treat ulcerative colitis.
Methods: The CDS sequence of CDX2 gene was amplified from the cDNA library by RT-PCR, and the target fragment was cloned into the IRES-EGFP matrix plasmid by in-fusion exchange. Then the recombinant plasmid CDX2-IRES-EGFP was transformed into the susceptible bacteria by heat shock and the positive clones were selected for antibiotic culture. After a large number of positive clones were amplified, the plasmids were extracted by alkaline lysis method. The concentration and purity of the plasmids were detected by spectrophotometry. The cells were isolated from the umbilical cord of full-term healthy newborns. Mesenchymal tissue was cut and placed in a culture flask, then cultured with DMEM/F12 medium and fetal bovine serum. Cells were subcultured when a large number of cells crawled out. Cell morphology was observed and the proliferative capacity of passages 1, 3, 7, 12 and 16 was measured. Then the cell immunity of passages 3 was detected by flow cytometry. The third part of MUC2 expression experiment was divided into five groups: single-transfection group: CDX2 transfection UC-MSCs; single-inducement group: only UC-MSCs were induced to differentiate into endodermal cells; induced group: MSCs were induced to differentiate into endodermal cells, then CDX2 was transfected into induced cells; blank-transfection group: no induction but transfection of empty plasmids; control group Group A: UC-MSCs did not undergo any treatment. In order to exclude the effect of transfection reagent itself, the single inducer group still needed transfection reagent treatment after induction of differentiation, but did not contain plasmid. The expression of CDX2 and MUC2 mRNA was detected in each group. The induction of endodermal cells was achieved by recombinant human Activin A and recombinant human Wnt3a.
Results: The length of the PCR product was 962 BP and the position of the band in gel electrophoresis was the same as expected. The sequencing results were consistent with the CDS sequence of CDX2 published by Genebank. OD260/280 = 1.74, OD260/230 = 2.23. In UC-MSCs culture, a small number of cells climbed out and grew adhering to the wall around the adherent tissue on the 7th-8th day of primary culture. The cells were spindle-shaped and polygonal in shape, and dense around the tissue block on the 10th-14th day. The results of flow cytometry showed that CD90/CD105/CD73 was positive and CD45/CD34/HLA-DR was negative. In the MUC2 expression study, green fluorescence was observed 48 hours after transfection in the single transfection group, the induced transfection group and the empty transfection group. The expression of GATA4 and Sox17 increased by 27.0 (+ 7.0) folds and 54 (+ 6.4) folds in both groups after induction and culture for 5 days. The expression of MUC2 and CDX2 was detected by RT-PCR in 5 groups. The results showed that the expression of CDX2 and MUC2 increased by 113.2 (+ 13.5) folds, 30.2 (+ 3.7) folds and 196.7 (+ 15.4) folds and 98.8 (+ 11.1) folds in the single-transfection group, respectively. There was no significant change in the expression of.CDX2 and MUC2 in the other three groups.
Conclusion: Eukaryotic expression vector CDX2-IRES-EGFP is constructed correctly, its concentration and purity meet the requirements of transfection. Human umbilical cord contains abundant MSCs, which can be extracted by tissue adherence method. The cells are spindle-shaped, vortex-like growth, strong proliferation ability and high purity. Overexpression of CDX2 in UC-MSCs can increase goblet cell marker sticky eggs. The expression of white MUC2 was further enhanced by Activin A and Wnt3a induction, but more MUC2 expression still needed to be optimized.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R329.2
【参考文献】
相关期刊论文 前2条
1 ;Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells differentiation into nerve-like cells[J];Chinese Medical Journal;2005年23期
2 Michael Gersemann;Eduard Friedrich Stange;Jan Wehkamp;;From intestinal stem cells to inflammatory bowel diseases[J];World Journal of Gastroenterology;2011年27期
本文编号：2201965
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