人谷氨酰半胱氨酸合成酶通过抑制JNK防止紫外刺激诱导的细胞死亡的研究

发布时间：2018-11-18 20:09

【摘要】：细胞死亡通路对于胚胎发育和正常组织稳态的维持是必需的。在分子水平,细胞凋亡受到严格的调控。凋亡不足或过量凋亡都会引起各种疾病的发生比如:肿瘤、自体免疫性疾病或神经退行性疾病的发生。因此,细胞凋亡在生物医药科学研究中也已成为重要的研究领域。对凋亡信号通路及其调节机制的研究为各种疾病的治疗提供了新的思路。为研究谷胱甘肽合成酶类谷氨酰半胱氨酸合成酶对紫外诱导的HEK293细胞死亡的影响,本实验分为以下两部分: 一:谷胱甘肽合成酶类基因的克隆与诱导表达通过PCR技术从人cDNA库中扩增出GCLC(谷氨酰半胱氨酸合成酶催化亚基)和GSS(谷胱甘肽合成酶),并分别将他们插到真核和原核质粒的MCS位点中。利用这些重组载体的构建能够为下一步研究GCLC对紫外刺激诱导细胞凋亡影响做好基础。构建的原核重组载体分别用IPTG进行诱导表达能够得到大量表达的目的蛋白,也可为两种蛋白抗体的制备做好准备,从而在将来的工作里能够有效的检测到内源和外源蛋白表达的变化。二:人谷氨酰半胱氨酸合成酶防止紫外刺激诱导HEK293细胞死亡的机制研究人的谷氨酰半胱氨酸合成酶(γ-GCS)是谷胱甘肽合成的限速酶,而GCLC具有γ-GCS所有的催化活性。从形态上观察GCLC能够显著地抑制紫外刺激诱导的HEK293细胞的死亡。MTS实验和FACS检测的结果也表明GCLC和JNK1~(APF)的表达能够提高细胞对紫外刺激的耐受性。通过Western blotting技术检测到UV能够强烈的激活JNK、p38和caspase-3。有趣的是,检测结果表明GCLC能够抑制UV诱导的JNK1的磷酸化,而对p38的磷酸化没有任何影响。因此,大量表达GCLC能够保护HEK293细胞免受紫外刺激诱导的细胞死亡主要是通
[Abstract]:Cell death pathways are essential for embryonic development and the maintenance of normal tissue homeostasis. At the molecular level, apoptosis is strictly regulated. Insufficient or excessive apoptosis can lead to various diseases such as tumor, autoimmune disease or neurodegenerative disease. Therefore, apoptosis has become an important research field in biomedical science. The study of apoptotic signaling pathway and its regulatory mechanism provides a new idea for the treatment of various diseases. To study the effect of glutathione synthase glutamyl cysteine synthase on UV-induced death of HEK293 cells. This experiment is divided into two parts as follows: firstly, the cloning and induced expression of glutathione synthase genes were amplified from human cDNA library by PCR technique. Acylcysteine synthase catalytic subunits and GSS (glutathione synthetase), They were inserted into the MCS site of eukaryotic and prokaryotic plasmids, respectively. The construction of these recombinant vectors can provide a good basis for the further study of the effect of GCLC on UV-stimulated apoptosis. The constructed prokaryotic recombinant vectors were induced by IPTG to express a large number of expressed target proteins, which could also be prepared for the preparation of two kinds of protein antibodies. Therefore, the changes of endogenous and exogenous protein expression can be detected effectively in the future work. Second: the mechanism of human glutamyl cysteine synthase against UV-stimulated HEK293 cell death; Human glutamyl cysteine synthase (纬-GCS) is a rate-limiting enzyme for glutathione synthesis. GCLC has all the catalytic activity of 纬-GCS. Morphologically, GCLC could significantly inhibit the death of HEK293 cells induced by UV stimulation, and the expression of GCLC and JNK1~ (APF) could improve the tolerance of HEK293 cells to UV stimulation by MTS assay and FACS assay. UV can be strongly activated by JNK,p38 and caspase-3. detected by Western blotting technique Interestingly, the results showed that GCLC could inhibit the phosphorylation of JNK1 induced by UV, but had no effect on the phosphorylation of p38. Therefore, the high expression of GCLC can protect HEK293 cells from UV-induced cell death.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329

【相似文献】