Ⅱ fimA型P.gingivalis重组菌毛蛋白对平滑细胞源外泌体作用下内皮细胞相关功能的影响

发布时间：2018-05-07 18:00

本文选题：牙龈卟啉单胞菌/菌毛蛋白 + 人脐静脉内皮细胞　；参考：《遵义医学院》2017年硕士论文

【摘要】：研究背景及目的:P.gingivalis作为慢性牙周炎最主要的致病菌,除了导致口腔局部炎症外,还可能影响心血管系统健康。大量研究表明P.gingivalis及其毒力因子与内皮功能障碍有直接的关系。有研究表明内皮细胞与平滑肌细胞间的信息交流在As发生发展过程中起重要作用,而外泌体作为细胞旁分泌的重要介质,广泛参与了各类信息物质的分泌和摄取过程,介导着细胞间信号转导过程。本研究通过分析IIfim A型P.gingivalis的主要毒力因子菌毛蛋白对平滑肌细胞源外泌体作用下内皮细胞相关功能的影响,进一步探讨P.gingivalis在As发生和发展过程中的可能作用。方法:(1)采用酶消化法体外培养HUVECs,组织块贴壁法体外培养HUASMCs,免疫荧光鉴定所培养的细胞;用PEG法提取平滑肌细胞源外泌体(HUASMCs-Exo),并行Western blot鉴定其表面标记蛋白CD63、CD9的表达情况,以电镜鉴定其大小和形态,以Di I染色外泌体,并将之与HUVECs共培养24h,观察HUVECs对HUASMCs-Exo的内化摄取情况。(2)用不同浓度(0.1、1、5、10μg/ml)的IIfim A型P.gingivalis-r Fim A处理HUVECs不同时间(2、8、24、48h),采用CCK-8检测细胞的增殖活性,并确定P.gingivalis-r Fim A处理HUVECs的最适浓度和时间;后续实验分为4组:(1)正常组(Normal组):HUVECs不予任何处理;(2)HUASMCs-Exo组:HUASMCs-Exo与HUVECs共培养;(3)r Fim A组:r Fim A处理HUVECs;(4)HUASMCs-Exo+r Fim A组:HUASMCs-Exo与HUVECs共培养后,加入r Fim A处理;FACS检测各组细胞总体凋亡率、存活率及坏死率,RT-PCR检测各组细胞内IL-18及Caspase-3 m RNA的表达情况,Western blot检测各组细胞内Cleaved caspase-3蛋白表达情况,ELISA检测各组细胞上清液中IL-18的表达情况。结果:(1)原代培养的HUVECs和HUASMCs两种细胞生长状态良好,并均经形态学和免疫荧光鉴定确认;采用PEG法成功提取、鉴定了HUASMCs-Exo,并可被HUVECs内化摄取,可用于后续实验。(2)CCK-8结果示10μg/ml浓度的r Fim A处理HUVECs48h时,细胞增殖活性最低,所以用10μg/ml浓度的r Fim A处理HUVECs48h行后续实验。FACS结果示:与Normal组相比,HUASMCs-Exo组细胞总体凋亡率无明显差异(P0.05),而r Fim A组和HUASMCs-Exo+r Fim A组均可导致HUVECs总体凋亡率升高(P0.05);与r Fim A组相比,HUASMCs-Exo+r Fim A组可下调r Fim A作用下HUVECs的总体凋亡率。RT-PCR、Western blot和ELISA结果示:Normal组与HUASMCs-Exo组细胞IL-18m RNA和Caspase-3 m RNA、凋亡主要效应蛋白Cleaved caspase-3及细胞上清液中IL-18水平均无明显差异(P0.05);与Normal组相比,r Fim A组和HUASMCs-Exo+r Fim A组细胞IL-18m RNA和Caspase-3 m RNA、Cleaved caspase-3及细胞上清液中IL-18水平均增加(P0.05),而HUASMCs-Exo+r Fim A组可下调r Fim A作用下各检测指标的表达量(P0.05)。结论:IIfim A型P.gingivalis-r Fim A可诱导HUVECs凋亡和炎症因子IL-18的表达,而HUASMCs-Exo虽然可在一定程度上调控P.gingivalis-r Fim A诱导的HUVECs凋亡和炎症反应,仍无法改变HUVECs功能异常的结果。因此,P.gingivalis的主要毒力因子菌毛蛋白可能在促进As发生、发展中发挥一定的作用。
[Abstract]:Background and purpose: P.gingivalis, as the most important pathogenic bacteria in chronic periodontitis, may also affect the health of the cardiovascular system in addition to local inflammation in the oral cavity. A large number of studies have shown that P.gingivalis and its virulence factors have a direct relationship with endothelial dysfunction. It plays an important role in the development of As. As an important mediator of cell paracrine, exocrine participates in the process of secretion and uptake of various information materials and mediates the process of intercellular signal transduction. The main toxicity of the IIfim A type P.gingivalis is analyzed by the analysis of the exocrine effect of the subhair protein on the smooth muscle cells. The possible role of P.gingivalis in the development and development of As was further explored. Methods: (1) HUVECs was cultured in vitro by enzyme digestion, HUASMCs was cultured in vitro by tissue block adherence, immunofluorescence was used to identify the cultured cells; PEG method was used to extract the extracellular secretory (HUASMCs-Exo) of smooth muscle cells (HUASMCs-Exo), and Wester was used in Wester. N blot identified the expression of its surface labeled protein CD63, CD9, identified its size and morphology by electron microscopy, stained the Exocyst with Di I, and co cultured 24h with HUVECs, and observed the internalization of HUVECs on HUASMCs-Exo. (2) different concentrations were treated with different concentrations (0.1,1,5,10 micron g/ml). CCK-8 was used to detect the proliferation activity of the cells and determine the optimum concentration and time for the treatment of HUVECs by P.gingivalis-r Fim A; the follow-up experiments were divided into 4 groups: (1) the normal group (group Normal): HUVECs was not treated with any treatment; (2) HUASMCs-Exo group: HUASMCs-Exo and HUVECs co culture; (3) r Fim. After HUVECs co culture, R Fim A was added, and FACS was used to detect the total apoptosis rate, survival rate and necrosis rate of each group. RT-PCR was used to detect the expression of IL-18 and Caspase-3 m RNA in each group, and Western blot to detect the expression of the protein in each cell. Results: (1 The two cells of primary culture HUVECs and HUASMCs have good growth state and are identified by morphological and immunofluorescence identification; HUASMCs-Exo is successfully extracted by PEG method, and can be absorbed by HUVECs and can be used in the follow-up experiment. (2) CCK-8 results show that the proliferation activity of R Fim A is the lowest when the concentration of R Fim A is 10 mu g/ml, so the use of CCK-8 is the lowest. Compared with the Normal group, there was no significant difference in the total apoptosis rate between the HUASMCs-Exo group and the Normal group, and the overall apoptosis rate of the HUASMCs-Exo group was no significant difference compared with the Normal group. Compared with the Normal group, the overall apoptosis rate of the HUASMCs-Exo group could lead to a higher apoptosis rate in the HUASMCs-Exo group than the Normal group. Compared with the Normal group, the overall apoptosis rate of the HUASMCs-Exo group was higher than that in the Normal group. The overall apoptosis rate of UVECs,.RT-PCR, Western blot and ELISA showed that there was no significant difference between the IL-18m RNA and Caspase-3 m RNA in the Normal group and the HUASMCs-Exo group, and there was no significant difference between the apoptotic major effector proteins and the cell supernatants. The level of IL-18 in se-3 m RNA, Cleaved caspase-3 and cell supernatant increased (P0.05), while HUASMCs-Exo+r Fim A group could down regulate the expression of every detection index under R Fim. Gingivalis-r Fim A induced HUVECs apoptosis and inflammatory response can not change the result of abnormal HUVECs function. Therefore, the main virulence factor of P.gingivalis may play a role in promoting the development of As.

【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.4

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