CDX2基因修饰的脐带间充质干细胞向杯状细胞分化的可行性研究

Feasibility Study of Umbilical Cord Mesenchymal Stem Cells Modified by CDX2 Differentiating into Goblet Cells

目的:研究尾型同源盒2(CDX2)在脐带间充质干细胞(UC-MSC)中过表达后粘蛋白2(MUC2)的表达情况,探寻利用基因修饰使UC-MSC大量表达MUC2的实验方法,了解诱导UC-MSC向内胚层分化对UC-MSC大量表达MUC2的必要性,为研究诱导UC-MSC向杯状细胞分化奠定基础。方法:实验分为5个组,分别为单转组(CDX2转染UC-MSC)、单诱组(仅诱导UC-MSC向内胚层细胞分化)、诱转组(诱导UC-MSC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)和对照组(UC-MSC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须转染试剂处理,但不加质粒;各组最后检测CDX2、MUC2的mRNA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:单转组、诱转组和空转组细胞在转染48 h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 d后,内胚层标志物GATA4的表达增加27倍,Sox17的表达增加54倍。对5组细胞行RT-PCR检测MUC2与CDX2的mRNA表达情况,结果单转组CDX2升高113倍,MUC2升高30倍;诱转组CDX2升高196倍,MUC2升高98倍;空转组、单诱组中CDX2与MUC2的表达无明显变化。结论:在UC-MSC中过表达CDX2能够增加杯状细胞标志物MUC2的表达,而事先诱导UC-MSC向内胚层细胞分化,能进一步增加MUC2的表达。利用CDX2的过表达有使UC-MSC向杯状细胞分化的可能性,而Activin A、Wnt3a的诱导处理能促进其分化能力。

Objective： To research the expression of mucin-2（MUC2） after the overexpression of caudal type ho-meobox 2（CDX2） in umbilical cord mesenchymal stem cells（UC-MSC）, explore the method of making MUC2 over-express in MSC by genetical modification,study the necessity that inducing UC-MSC differentiate into endodermal cell for overexpressing MUC2, and lay the foundation for studying UC-MSC differentiate into goblet cells in intesti-nal mucosa. Methods： The experiment was divided into 5 groups, they were single-transfection group（CDX2 was transfected into UC-MSC）, single-induction group（induce UC-MSC differentiate into endodermal cells）, induction and transfection group（CDX2 was transfected into UC-MSC after induction）, single-induction group（just transfect-ing vector without CDX2） and control group（UC-MSC without any treatment）. Single-induction group should be treated by transfection reagent in order to exclude the effect of transfection reagen. Finally, the mRNA of MUC 2 and CDX2 were detected in each group. The endodermal cells was induced by recombinant human Activin A, re-combinant human Wnt3a. Results： Green fluorescence can be seen by fluorescence microscope in single-transfec-tion group, induction and transfection group and single-induction group after 48 h. GATA4 and Sox17 increase by 27 fold and 54 fold respectively, which both were marker of endoderm until the 5th day when UC-MSC was in-duced in single-transfection group and induction and transfection group. The mRNA of five groups was detected by RT-PCR, the result showed that CDX2 increase by 113 fold, MUC2 increase by 30 fold in single-transfection group; CDX2 increase by 196 fold, MUC2 increase by 98 fold in induction and transfection group. The expression of CDX2 and MUC2 didn＇t change significantly in the other three groups. Conclusion： Overexpression of CDX2 can increase the expression of MUC2 that is marker of goblet cell in UC-MSC. The expression of MUC2 can be increased if UC-MSC is induced to endodermal cells in advace. So it is possible to induce UC-MSC differentiate into goblet cells by overepression of CDX2 and induction to endodermal cells can make it easier.