PTEN在胚胎早期心脏发育过程中作用的研究

目的 胚胎早期原肠胚形成期PTEN表达在原条及其而后发生的中胚层组织，本实验通过过表达PTEN探讨其在早期心脏发生发育过程的作用和意义。方法 《改良滤纸鸡胚胎整体培养法》取胚，37℃孵箱孵育待其发育到HH4期，对鸡胚心肌干细胞（Cardiac Stem Cell）进行PTEN电转染，转染成功后一部分鸡胚37℃孵箱继续培养30 h，待其发育形成心管，观察过表达PTEN对心管发生的影响；另一部分鸡胚在超净台内完成心肌干细胞的离体，体外培养使其分化发育成心肌组织1]，主要研究PTEN在细胞增殖、分化和凋亡过程中的作用。结果 30 h后鸡胚整体的免疫组织化学（N-cadherin）结果显示，PTEN-WT组和PTEN-G129E组鸡胚心管出现了反向环化、多囊化等畸形，GFP组和PTEN-C124S组心管发育受到的影响较小；转染后离体培养得到的心肌组织的跳动频率与GFP组下降的速度加快，且维持时间缩短，GFP组与PTEN-WT组有显著差异（P<0.05），PTEN-WT组与PTEN-C124S组、PTEN-G129E组无显著差异（P＞0.05）。结论 PTEN蛋白酯酶可能参与早期胚胎心肌干细胞之间的粘附分子的表达从而调节细胞迁移、分化发育，并加速了心肌组织的衰老。

PTEN function on embryonic early heart development

Abstract Objective：Endogenous PTEN express in primitive streak during chick embryo gastrulation and mesoderm derived structure later on including heart tubes. We investigated the possibility that PTEN is involved in heart formation on the period of early embryo development. Method：Whole un-incubated chicken embryos are cultured using the modified filter paper method at 37℃ incubator until the chick embryos to reach HH4 stage, and then transfected PTENs to cardiogenesis area around anterior primitive streak including cardiac stem cells, in which half of transfected embryos were fostered at 37℃ for about 30hs continuously until the heart tube formation to study the role of PTEN in the process of cell adhesion and migration; CSC from the other part PTENs-transfected emrbyos were isolated and cultured at 37℃ CO2 incubator in vitro until differentiating into myocardial tissue with beating pulse in order to study the role of PTEN in the process of cardiac cell proliferation, differentiation and apoptosis. Result：The result of whole chick embryos immunohistochemistry (N-cadherin) showed that chick heart tubes transfected by PTEN-WT and PTEN-G129E appeared in reverse cyclization, polycystic malformation and so on while the heart tubes from GFP group and PTEN-C124S group developed roughly normally; After transfection of PTEN, the beating frequency of myocardial tissue cultured in vitro decline faster than GFP group and the beating time was not capable of maintaining as log as control group. There is significantly difference (P＜0.05) between PTEN-WT group and the GFP group, while there is no significant difference (P＞0.05) among PTEN-WT group, PTEN-C124S group and PTEN-G129E. Conclusion：Our data suggest that PTEN might increase the cell adhesion between the early embryo CSC and cell matrix and interfere with cell migration while promote the differentiation CSC and accelerate the aging of myocardium.