肺炎链球菌溶血素经MAPK途径诱导人脐静脉内皮细胞凋亡

发布时间：2018-01-07 23:30

本文关键词：肺炎链球菌溶血素经MAPK途径诱导人脐静脉内皮细胞凋亡　出处：《重庆医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的肺炎链球菌溶血素(Pneumolysin,PLY)是由肺炎链球菌(Streptococcus pneumoniae,S.pn)产生的一种重要的溶细胞毒素。近年来研究证实小剂量PLY可诱导宿主细胞凋亡,但其机制尚不清楚。本研究通过体外表达纯化PLY重组蛋白并选用人脐静脉内皮细胞(The Human umbilical vein endothelial cell line, HUVEC)作为体外细胞模型,观察PLY重组蛋白是否能诱导HUVEC凋亡,且该过程是否有caspase途径和MAPK途径的参与。方法 1.在E.coli BL21中表达融合6个组氨酸标签的PLY重组蛋白,采用Ni-NTA树脂纯化重组蛋白,通过SDS-PAGE和溶血试验鉴定其分子量和溶血活性。 2.观察PLY对HUVEC的增殖抑制和诱导凋亡作用:采用MTT法检测HUVEC的增殖情况,采用Annexin V/PI双标记染色法检测凋亡率,并提取细胞DNA进行琼脂糖凝胶电泳观察细胞核染色质DNA的断裂。 3.采用ELISA检测PLY作用细胞后的caspase-3,8,9的活性变化,采用免疫细胞组化检测细胞ERK1/2、p38MAPK的表达水平,以Western Blot检测经PLY作用后ERK1/2和p38MAPK的总蛋白水平和磷酸化水平变化。结果 1.成功构建质粒Ply-pW28并实现了PLY蛋白的可溶性表达,所获重组蛋白纯化后纯度达到95%。溶血实验显示PLY对人红细胞具有极高的溶血活性,并呈明显剂量依赖性(p0.05),在100ng/ml时溶血率为100%。 2. PLY重组蛋白与HUVEC共孵育后,HUVEC的增殖受到了明显的抑制,并呈明显的时间和剂量依赖性,IC50(24h)为1.525μg/ml。PLY处理后HUVEC黏附能力降低,部分细胞皱缩变圆,失去典型“鹅卵石样”形态特点,并呈明显剂量依赖性。Annexin V/PI双标记染色法检测到0.5μg/ml、1μg/ml和2.5μg/ml的PLY与HUVEC共孵育24h后细胞的早期凋亡率分别为8.91%、27.43%、31.50%,与对照组细胞凋亡率为2.67%相比较显著增高(P0.05)。琼脂糖凝胶电泳可见凋亡细胞DNA的断裂。 3. 1μg/ml PLY与HUVEC孵育24h后caspase-3,8,9活性无明显变化。免疫细胞组化结果显示:与对照组相比,PLY处理组ERK1/2表达减弱,而p38MAPK则表达增加。Western Blot结果显示:与对照组相比,PLY处理组的ERK1/2和p-ERK1/2的表达水平均显著下降,而p38MAPK和p-p38MAPK表达水平均增加。结论 PLY可抑制HUVEC增殖并诱导其凋亡,该效应至少部分是通过抑制ERK1/2信号途径和激活p38MAPK信号途径而实现的,但该效应并不依赖于caspase-3,8,9的活化。
[Abstract]:Purpose Streptococcus pneumoniae (Streptococcus pneumoniae) was isolated from Streptococcus pneumoniae. S. pn) is an important cytotoxin. In recent years, it has been proved that small dose of PLY can induce apoptosis of host cells. However, its mechanism is not clear. In this study, the recombinant PLY protein was expressed and purified in vitro and human umbilical vein endothelial cells (HUVEC) were selected. The Human umbilical vein endothelial cell line. HUVECs were used as cell models in vitro to observe whether PLY recombinant protein could induce apoptosis of HUVEC and whether the caspase pathway and MAPK pathway were involved in the process. Method 1. The recombinant PLY protein was expressed in E. coli BL21 and purified by Ni-NTA resin. Its molecular weight and hemolytic activity were identified by SDS-PAGE and hemolysis test. 2.To observe the effect of PLY on the proliferation and apoptosis of HUVEC: MTT assay was used to detect the proliferation of HUVEC. The apoptosis rate was detected by Annexin V / Pi double labeling method, and cell DNA was extracted for agarose gel electrophoresis to observe the fragmentation of nuclear chromatin DNA. 3. ELISA was used to detect the activity of caspase-3 and caspase-8, and ERK1/2 was detected by immunocytochemistry. The expression level of p38 MAPK was detected by Western Blot. The total protein and phosphorylation level of ERK1/2 and p38 MAPK were detected by PLY. Results 1. The plasmid Ply-pW28 was successfully constructed and the soluble expression of PLY protein was realized. The purity of the purified recombinant protein was 95%. The hemolysis test showed that PLY had extremely high hemolytic activity on human erythrocytes in a dose-dependent manner (p 0.05). At 100 ng / ml, the hemolysis rate was 100 g / ml. 2. The proliferation of PLY was inhibited in a time-and dose-dependent manner after being co-incubated with HUVEC. The adhesion ability of HUVEC was decreased after treatment of 1.525 渭 g / ml. PLY for 24 h, and some cells shrank and rounded, thus losing the typical "cobblestone" morphology. In a dose-dependent manner, 0.5 渭 g / ml was detected by Annexin V / Pi double labeling staining. After 1 渭 g / ml and 2.5 渭 g / ml PLY were incubated with HUVEC for 24 hours, the early apoptosis rate was 8.91% and 27.43%, 31.50%, respectively. Compared with the control group, the apoptotic rate was significantly higher than that of the control group (2.67%). The DNA fragmentation of apoptotic cells was observed by agarose gel electrophoresis. 3. 1 渭 g / ml PLY was incubated with HUVEC for 24 hours, and the activity of caspase-3t8 was not changed significantly. The immunohistochemical results showed that compared with the control group, there was no significant change in the activity of caspase-38. The expression of p38 MAPK was decreased in PLY treatment group, while p38 MAPK expression was increased. Western Blot showed that compared with control group, the expression of p38 MAPK was higher than that of control group. The expression levels of ERK1/2 and p-ERK1 / 2 in PLY treatment group were significantly decreased, while p38 MAPK and p-p38 MAPK expression levels were increased. Conclusion PLY inhibits the proliferation of HUVEC and induces its apoptosis, at least in part by inhibiting the ERK1/2 signaling pathway and activating the p38 MAPK signaling pathway. However, this effect does not depend on the activation of caspase-3 and caspase-8.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

【参考文献】