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1、天津医科大学硕士学位论文中文摘要研究目的利用肺炎衣原体(Chlamydiapneumoniae，Cpn)体外感染大鼠原代血管平 滑肌细胞(Vascular smooth muscle cell，VSMC)模型；观察Cpn感染对VSMC 迁移能力的影响，研究磷脂酰肌醇3激酶(Phosphoinositide 3-kinase，P13K) 丝氨酸苏氨酸蛋白激酶(Serinethreonine hnaSe，Akt)和Toll受体2(Toll likereceptor 2，TLR2)P13KAkt信号转导通路在Cpn感染诱导VSMC迁移中的作 用，探讨其相关分子机制。内容和方法1Cp，2增殖培养后体外
2、感染大鼠原代VSMC；2C矽胛感染VSMC后，利用透射电镜(Ttransmission electron microscope，TEM)确定感染模型成功建立；3Woundhealing实验和Transwell实验从二维和三维水平分别观察C矽九感染 VSMC后其迁移能力的变化：4逆转录聚合酶链反响(Reverse transcription polymerase chain reaction，RT-PCR)实验检测Cpn感染后各组VSMC P13K mRNA表达水平；5Western blot实验检测Cpn感染VSMC后不同时间点Akt的磷酸化水平；6用P13K特异性抑制剂LY294002抑制P
3、13K的活性，Western blot实验检测 各组VSMC磷酸化Akt的表达变化，Woundhealing实验和Transwell实验观察各组VSMC迁移能力的改变；7用TLR2中和抗体抑制TLR2的活性，Westem blot实验检测Cpn感染后各 组VSMC Akt的磷酸化水平，确定TLR2在介导Cpn感染诱导P13KAkt磷 酸化中的作用。天津医科大学硕士学位论文p士田：日7K1Cpn感染VSMC后，透射电镜下可见胞浆内出现典型Cpn原体(Elementary body，EB)，提示Cpn感染VSMC模型成功建立；2Woundhealing实验结果显示，Cpn感染VSMC后，细胞向划痕
4、中央迁移的 面积明显大于正常对照组(尸005)。Transwell实验结果显示，Cpn感 染组的细胞穿膜数明显多于正常对照组(PO05)；3P13K特异性抑制剂LY294002预处理VSMC后，RT-PCR实验结果显示，Cpn 感染组P13K mRNA的条带亮度与正常对照组相比拟无明显区别；使用 LY294002预处理的Cp，7感染组VSMC P13K mRNA的条带与单纯Cpn感染 组比照，亮度也无明显改变，差异均无统计学意义；4分别于Cpn感染VSMC 0 h、15 min、30 min、1 h、2 h、3 h、6 h、12 h和 24 h后提取总蛋白，Western blot实验结果显示
5、，Cpn感染VSMC 30 min后 PAkt表达水平开始升高；随着感染时间延长，p-Akt表达水平逐渐升高，1 h 到达顶峰，6 h开始下降，并持续至24 h；各组VSMC中总Akt表达水平无显著差异；5Western blot实验结果显示，用LY294002预处理的Cpn感染组VSMC p-Akt 的条带灰度较单纯Cpn感染组明显减弱(尸005)，差异有统计学意义， 各组VSMC中总Akt条带灰度无显著差异；6Woundhealing实验结果显示，LY294002预处理的Cpn感染组VSMC向划 痕中央迁移的面积明显小于单纯C矽甩感染组(尸O05)。Transwell实验 结果显示，LY2
6、94002预处理的Cpn感染组VSMC穿膜细胞数明显低于单 纯C矽n感染组(尸005)，差异有统计学意义；7使用TLR2中和抗体预处理VSMC，Western blot实验结果显示，Cpn感染 VSMC后，用TLR2中和抗体预处理的Cpn感染组VSMC的PAkt的条带 灰度较单纯Cpn感染组减弱(P005)，差异有统计学意义，各组VSMC 中总Akt的条带灰度无显著差异。II天津医科大学硕士学位论文结论C玎感染经由TLR2通过P13KAkt信号通路促进VSMC迁移。关键词：P13KAkt：肺炎衣原体；血管平滑肌细胞；细胞迁移；TLR2III天津医科大学硕士学位论文Abstract Object
7、 i veUsing the model of Rat primary vascular smooth muscle cell(VSMC)infected with Chlamydia pneumoniae(Cpn)in vitro，to observe the effects of Cpn infection on VSMC migration，to investigate the roles of phosphoinositide 3-kinase(PISK) Serinethreonine kinase(Akt)signaling pathway and Toll like recept
8、or 2(TLR2)in VSMC migration induced by Cpn infection，to explore the related molecular mechanismsMethods1VSMCs were infected wi也Cpn in vitro after the culture and propagation of Cpn 2Successful infection of VSMCs with Cpn was identified by the observation ofCpn inclusions under Ttransmission electron
9、 microscope(TEM)3Wound-healing assay and Transwell assay were performed to investigate the effectof Cpn infection on VSMC mi铲ation4Reverse transcriptionpolymerase chain reaction(RT-PCR)Was used to determinethe mRNA expression level of P13K after Cpn infected VSMCs5Western blot was performed to detec
10、t the PAkt expression level in the VSMCs atdifferent time potins after Cpn infection6After the P13K activity was inhibited by P13K specific inhibitors，LY294002， Western blot was performed to detect the P-Akt expression level in the infected VSMCs；Woundhealing assay and Transwell assay were performed
11、 to explore the changes in VSMC migration induced by Cpn infection7The activity of TLR2 Was blocked by a TLR2 neutral antibody，and then Western blot was performed to detect the p-Akt expression level in order to explore the roles of P13KAkt signaling pathway and TLR2 in VSMC migration induced by Cpn
12、 infection天津医科大学硕士学位论文Resu l tS1The typical Cpn elementary body，were observed in C,pn inclusions in the cytoplasm of the infected VSMCs，indicating the successful infection of VSMCs with Cpn2The migration area of Cpn-infected VSMCs was significantty larger than that of control group in a wound-healin
13、g assay fP005)In a Transwell assay， VSMCs infected with Cpn were found to migrme more than the control group(PO05)3RT-PCR results showed that there was no significant difference in the mRNA expression of P13K in the VSMCs between Cpn infection group pretreated with LY294002 and the control groupAlso
14、 no significant difference of the P13KmRNA expression in the VSMCs between Cpn infection group pretreated withLY294002 and Cpn infection group4Western blot results showed that the significant activation of Akt was observed in the Cpn-infected VSMCsCpn stimulated phosphorylation of Akt occurred as early as 3 0 min and increased gradually to reach a peak at 1 h postinfection and lasted up to 6 h，and then decreased until 24 h after infectionNo significant changes in total Akt protein expression were detected during the infection5Western blot results showed that
