CaM-CaMKⅡ信号通路调控小鼠附植前期胚胎HSP72表达的研究

The embryos synthetize heat shock protein (HSP)72 after being heat stressed and the HSP72 plays a crucial role in the embryos for resisting futher elevation of temperature. However, it is not clear, up to now, that how the embryos regulating this HSP72 expression. This project is planed to use preimplantation mice embryos as study subjects, and a number of methods will be employed in treating the experimental embryos, which include inducing HSP72 by heat shock, using agonists and inhibitors and RNA interfere. The techniques of qRT-PCR, Western blot, immunofluorescence histochemistry and electrophoreretic mobility shift assay etc. will be used in the experiments. On the basis of the optimum conditions selected for the expression of CaM, CaMKⅡ and HSP72 the embrionic HSP72 expression under heat shock condition regulating by CaM-CaMKⅡsignaling pathway including the activation of HSF1, the phosphorylation of HSF1 tripolyme and its binding activity to the HSE, which in turn influencing HSP72 expression,will be investigated in the objectives to reveal the mechanisms of signal transduction in the HSP72 expression of mice preimplantation embryos in the molecular level. The results will be useful for futher study the mechanism of embryo lose of the early pregnant mammalian in the hot summer or suffering from fervescence diseases.

胚胎受热应激合成热休克蛋白（HSP）72，HSP72对胚胎耐热起重要的作用，然而胚胎如何调控HSP72表达目前尚不清楚。本项目拟以体外培养的小鼠胚胎为研究对象，通过热休克诱导胚胎HSP72表达、添加激动剂和抑制剂以及RNAi方法，采用荧光定量PCR、Western blot、免疫荧光组化和凝胶阻滞分析等技术，在筛选出CaM、CaMKⅡ和HSP72在胚胎中表达最佳条件的基础上，研究CaM-CaMKⅡ信号通路如何激活HSF1、HSF1三聚体磷酸化及其与DNA结合活性，进而影响HSP72表达，以阐明附植前小鼠胚胎HSP72表达信号转导的分子机制。研究成果将为深入研究热应激和热性疾病导致的哺乳动物妊娠早期胚胎死亡奠定理论基础。

胚胎受热应激合成热休克蛋白（HSP）72，HSP72对胚胎耐热起重要的作用，然而胚胎如何调控HSP72表达目前尚不清楚。项目以体外培养的小鼠胚胎为研究对象，通过热休克诱导胚胎HSP72表达、添加激动剂和抑制剂以及RNAi方法，采用荧光定量PCR、Western blot、免疫荧光组化等技术，筛选出了CaM、CaMKⅡ和HSP72在胚胎中表达的最佳条件，发现小鼠胚胎受热应激时，CaM通过与HSP70竞争性结合，解离出HSF1，并且Ca2+激活CaM-CaMKⅡ信号通路，从而使HSF1磷酸化，进而增加其与DNA结合活性，调控HSP72表达。此外，项目还明确了一定浓度的黄芩苷可促进胚胎发育及囊胚细胞增殖，培养液中添加黄芩苷可促进小鼠胚胎HSP70、Bcl-2mRAN表达，降低Bax、Caspase-3mRAN表达；上调热应激条件下LLC-PK1细胞HSP70的表达，下调热应激条件下LLC-PK1细胞Bax的表达，适量黄芩苷能上调热应激条件下LLC-PK1细胞Bcl-2的表达，提高Bcl-2和Bax的比值。葛根素可增加抗氧化酶SOD、CAT和GPx的活力，增加HSP72的表达，降低MDA的含量，减轻由热应激引起的氧化损伤，起到保护LLC-PK1细胞的作用。研究成果将为深入研究热应激和热性疾病导致的哺乳动物妊娠早期胚胎死亡奠定了理论基础。