【摘要】：研究背景和目的：尾型同源盒轉(zhuǎn)錄因子2（caudal type homeobox2，CDX2）是一種在腸道黏膜特異性表達(dá)的基因，它也是胚胎發(fā)育過(guò)程中促進(jìn)限制內(nèi)胚層向后腸發(fā)育的關(guān)鍵基因。過(guò)表達(dá)CDX2的食管上皮細(xì)胞可以表達(dá)腸道粘蛋白MUC2，促進(jìn)食管腸上皮化生。將CDX2通過(guò)內(nèi)部核糖體進(jìn)入位點(diǎn)序列（Internal ribosome entry site, IRES）與增強(qiáng)型綠色熒光蛋白（Enhanced Green Fluorescent Portein,EGFP）連接，可以使CDX2與EGFP共表達(dá)。因此可以通過(guò)對(duì)EGFP的直接觀察了解CDX2的表達(dá)情況。本實(shí)驗(yàn)第一部分?jǐn)M通過(guò)分子生物學(xué)的相關(guān)方法構(gòu)建、擴(kuò)增、提取CDX2-IRES-EGFP真核表達(dá)載體，為后續(xù)臍帶間充質(zhì)干細(xì)胞的基因過(guò)表達(dá)奠定基礎(chǔ)。臍帶間充質(zhì)干細(xì)胞（umbilicalcord mesenchymal stem cells，UC-MSCs）主要存在于臍帶血管與外膜之間的間質(zhì)組織，屬于成體干細(xì)胞的一種。UC-MSCs具有自我更新及向不同組織分化的能力。本研究第二部分從人臍帶間質(zhì)中分離并提取UC-MSCs，對(duì)細(xì)胞形態(tài)進(jìn)行觀察，并檢測(cè)UC-MSCs的免疫表型、增殖能力，以確定分離提取的細(xì)胞是UC-MSCs，為下一步探索過(guò)表達(dá)CDX2的臍帶間充質(zhì)干細(xì)胞的MUC2表達(dá)研究奠定基礎(chǔ)。潰瘍性結(jié)腸炎患者腸粘膜杯狀細(xì)胞減少、粘液層變薄、黏蛋白分泌減少，本實(shí)驗(yàn)第三部分?jǐn)M研究CDX2在UC-MSCs中過(guò)表達(dá)后粘蛋白MUC2的表達(dá)情況，探尋利用過(guò)表達(dá)CDX2的UC-MSCs大量表達(dá)MUC2的實(shí)驗(yàn)方法，了解誘導(dǎo)UC-MSCs向內(nèi)胚層分化對(duì)MSCs大量表達(dá)MUC2的影響，為研究UC-MSCs在腸道黏膜定向分化為杯狀細(xì)胞或在腸道大量表達(dá)黏蛋白治療潰瘍性結(jié)腸炎奠定基礎(chǔ)。 研究方法：通過(guò)RT-PCR從cDNA文庫(kù)中擴(kuò)增出CDX2基因的CDS序列，并通過(guò)in-fusion交換法將目的片段克隆入IRES-EGFP骨架質(zhì)粒中。然后將構(gòu)建好的重組質(zhì)粒CDX2-IRES-EGFP通過(guò)熱休克法轉(zhuǎn)化入感受態(tài)細(xì)菌后進(jìn)行抗生素選擇培養(yǎng)。將獲得的陽(yáng)性克隆進(jìn)行菌液PCR初步鑒定，利用DNA測(cè)序技術(shù)進(jìn)一步確定所得載體及基因是否與數(shù)據(jù)庫(kù)公布一致。大量擴(kuò)增陽(yáng)性克隆后，，再通過(guò)堿裂解法提取質(zhì)粒，通過(guò)分光光度法檢測(cè)其濃度及純度，用于后續(xù)實(shí)驗(yàn)。細(xì)胞來(lái)源則是取足月產(chǎn)健康新生兒臍帶，分離間質(zhì)組織，剪碎后置于培養(yǎng)瓶，加DMEM/F12培養(yǎng)基和胎牛血清行組織塊貼壁培養(yǎng)，待有大量細(xì)胞爬出時(shí)進(jìn)行傳代培養(yǎng)。觀察細(xì)胞的形態(tài)，測(cè)定1、3、7、12、16代細(xì)胞的增殖能力。然后對(duì)P3代細(xì)胞行流式細(xì)胞術(shù)檢測(cè)CD45、CD34、CD105、CD90、CD73、HLA-DR等細(xì)胞免疫表型。第三部分MUC2表達(dá)研究實(shí)驗(yàn)中共分5個(gè)組，分別為單轉(zhuǎn)組：CDX2轉(zhuǎn)染UC-MSCs；單誘組：僅誘導(dǎo)UC-MSCs向內(nèi)胚層細(xì)胞分化；誘轉(zhuǎn)組：誘導(dǎo)MSCs向內(nèi)胚層細(xì)胞分化后，再將CDX2轉(zhuǎn)染誘導(dǎo)后細(xì)胞；空轉(zhuǎn)組：不進(jìn)行任何誘導(dǎo)但轉(zhuǎn)染空質(zhì)粒；對(duì)照組：UC-MSCs不做任何處理。為排除轉(zhuǎn)染試劑本身的影響，單誘組在誘導(dǎo)分化后仍需轉(zhuǎn)染試劑處理，但不加質(zhì)粒。各組最后檢測(cè)CDX2、MUC2mRNA的表達(dá)情況。內(nèi)胚層細(xì)胞誘導(dǎo)利用重組人Activin A、重組人Wnt3a實(shí)現(xiàn)。 結(jié)果：目的基因PCR產(chǎn)物長(zhǎng)度962bp，凝膠電泳中條帶位置與預(yù)期一致；測(cè)序結(jié)果與Genebank公布CDX2的CDS序列一致；轉(zhuǎn)化子陽(yáng)性克隆菌液PCR產(chǎn)物預(yù)計(jì)長(zhǎng)度823bp，凝膠電泳中條帶位置與預(yù)期一致。所提取真核表達(dá)載體CDX2-IRES-EGFP濃度為843.6ng/ul，OD260/280=1.74，OD260/230=2.23。在UC-MSCs培養(yǎng)中，貼壁組織周圍在原代培養(yǎng)第7-8天有少量細(xì)胞爬出并貼壁生長(zhǎng)，形態(tài)為梭形和多角形，10-14天組織塊周圍細(xì)胞密集。傳代后的細(xì)胞呈漩渦狀生長(zhǎng)，形態(tài)為長(zhǎng)梭形，且較為一致。細(xì)胞增殖能力強(qiáng)，每4-6天即可進(jìn)行傳代，傳至第16代其增殖能力未見(jiàn)明顯減弱。流式細(xì)胞術(shù)分析所得細(xì)胞免疫表型結(jié)果表明，CD90/CD105/CD73陽(yáng)性，CD45/CD34/HLA-DR陰性。MUC2表達(dá)研究實(shí)驗(yàn)中，單轉(zhuǎn)組、誘轉(zhuǎn)組和空轉(zhuǎn)組細(xì)胞在轉(zhuǎn)染48小時(shí)后在熒光顯微鏡下能看到綠色熒光。單誘組和誘轉(zhuǎn)組經(jīng)誘導(dǎo)培養(yǎng)5天后，內(nèi)胚層標(biāo)志物GATA4表達(dá)增加27.0±7.0倍，Sox17表達(dá)增加54±6.4倍。對(duì)5組細(xì)胞行RT-PCR檢測(cè)MUC2與CDX2表達(dá)情況，結(jié)果單轉(zhuǎn)組CDX2表達(dá)升高113.2±13.5倍，MUC2表達(dá)升高30.2±3.7倍；誘轉(zhuǎn)組CDX2升高196.7±15.4倍，MUC2升高98.8±11.1倍。CDX2與MUC2的表達(dá)在其他三組中無(wú)明顯變化。 結(jié)論：真核表達(dá)載體CDX2-IRES-EGFP構(gòu)建正確，其濃度、純度符合轉(zhuǎn)染要求。人臍帶含有豐富的MSCs，通過(guò)組織塊貼壁法能提取出足量MSCs，該細(xì)胞呈長(zhǎng)梭形，漩渦樣生長(zhǎng)，增殖能力強(qiáng)且純度較高。在UC-MSCs中過(guò)表達(dá)CDX2能夠增加杯狀細(xì)胞標(biāo)志物粘蛋白MUC2的表達(dá)，而利用Activin A與Wnt3a誘導(dǎo)處理能進(jìn)一步增加MUC2的表達(dá)。但是更大量的MUC2表達(dá)還需進(jìn)一步優(yōu)化實(shí)驗(yàn)條件。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R329.2

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本文編號(hào)：2201965