米非司酮对早孕绒毛Oct4、Sox2、Nanog 表达影响*

目的:探讨米非司酮对早孕绒毛组织Oct4、Sox2、Nanog mRNA和蛋白水平表达的影响。方法:要求终止妊娠的正常早孕妇女60例,分为:负压吸宫组及服用米非司酮150mg及米索前列醇联合行药物流产组。运用Real-time PCR方法检测两组早孕绒毛组织中Oct4、Sox2、Nanog mRNA的表达;采用免疫组织化学方法检测两组早孕绒毛组织Oct4、Sox2、Nanog蛋白的定位及半定量表达情况,比较两组差异。结果:Oct4、Nanog、Sox2 mRNA在药物流产组早孕绒毛中相对表达量明显低于负压吸宫组,差异有统计学意义(相对表达量分别为:0.15±0.045;0.37±0.053;0.23±0.040,P值均<0.05);Oct4、Nanog、Sox2在药物流产组早孕绒毛蛋白表达量亦明显低于负压吸宫组,差异有统计学意义(药物流产组蛋白表达量分别为13869±541、19251±1503、139492±918明显低于负压吸宫组22017±235、30543±729、37237±710)。结论:米非司酮可以通过抑制早孕绒毛中Oct4、Sox2、Nanog表达,发挥抗早孕作用。     

Inactivation of Klf5 by zinc finger nuclease downregulates expression of pluripotent genes and attenuates colony formation in embryonic stem cells

Recent studies suggest that Klf5 is required to maintain embryonic stem (ES) cells in an undifferentiated state. However, whether Klf5 can be inactivated by novel fusion technology of zinc finger nucleases (ZFN) has never before been examined. Therefore, we used ZFN technology to target the Klf5 gene in mouse ES cells, and examined the effects of the Klf5 gene on the expression of pluripotency-related genes, Oct3/4, Nanog, and Sox2 and on the self-renewal of ES cells. In Klf5–ZFN-transfected cells, expression of the Klf5 mRNA was downregulated by ~80 % compared to the control. Furthermore, expression of the Oct3/4 and Nanog mRNAs was significantly decreased in the Klf5–ZFN-targeted cells. RT-PCR analysis, however, showed no significant change in the level of Sox2 mRNA, but a decreased trend was evident in the Klf5–ZFN-targeted cells. Moreover, we observed the spontaneous differentiation of Klf5–ZFN-transfected cells and quantitative analysis revealed a significant decrease in colony formation in Klf5–ZFN-transfected cells. In conclusion, our data suggest that ZFN methodology is an effective approach to target the Klf5 gene and that Klf5 plays an important role in the maintenance of ES cell self-renewal.     

Expression of Oct4 in HCC and modulation to wnt/β-catenin and TGF-β signal pathways

Hepatocellular carcinoma (HCC) is considered as a disease of dysfunction of the stem cells. Studies on stem cells have demonstrated that Oct4 plays a pivotal role in embryo regulation. In order to understand the role of Oct4 in HCC and the relationship among Oct4 and wnt/β-catenin and TGF-β signal pathways, we have detected the expression of Oct4, Nanog, Sox2, STAT3 as well as the genes in wnt/β-catenin, and TGF-β families in HCC cell lines and in tumor specimens from HCC patients. The authors found that Oct4 was expressed in all of the four HCC cell lines and the tumor specimens from HCC patients. Some other genes were also expressed in them with different level including Nanog, Sox2, STAT3 and TCF3, wnt10b, β-catenin, ELF, Smad3 and Smad4. The ability of the clone formation and migration of the HepG2 decreased after Oct4 was knockdowned. Silencing of Oct4 and TCF3 in HCC cell line HepG2 revealed that there were complicated relationships among Oct4, wnt/β-catenin family and TGF-β family genes. Knockdowning Oct4 reduced the expression of TGF-β family genes ELF, Smad3, Smad4 and wnt/β-catenin family genes, wnt10b, and β-catenin but increased TCF3. In reverse, knockdowning TCF3 led to the increased expression of Oct4 and TGF-β family genes. In conclusion, the expression of Oct4 in HCC may play an important role as in stem cell. Because Oct4 improves not only the function of wnt/β-catenin, but also the TGF-β signal pathways, the significance of its expression in HCC might be more complicated than we evinced before.