本文選題：牙齦卟啉單胞菌/菌毛蛋白 + 人臍靜脈內(nèi)皮細胞�。� 參考：《遵義醫(yī)學(xué)院》2017年碩士論文

【摘要】：研究背景及目的:P.gingivalis作為慢性牙周炎最主要的致病菌,除了導(dǎo)致口腔局部炎癥外,還可能影響心血管系統(tǒng)健康。大量研究表明P.gingivalis及其毒力因子與內(nèi)皮功能障礙有直接的關(guān)系。有研究表明內(nèi)皮細胞與平滑肌細胞間的信息交流在As發(fā)生發(fā)展過程中起重要作用,而外泌體作為細胞旁分泌的重要介質(zhì),廣泛參與了各類信息物質(zhì)的分泌和攝取過程,介導(dǎo)著細胞間信號轉(zhuǎn)導(dǎo)過程。本研究通過分析IIfim A型P.gingivalis的主要毒力因子菌毛蛋白對平滑肌細胞源外泌體作用下內(nèi)皮細胞相關(guān)功能的影響,進一步探討P.gingivalis在As發(fā)生和發(fā)展過程中的可能作用。方法:(1)采用酶消化法體外培養(yǎng)HUVECs,組織塊貼壁法體外培養(yǎng)HUASMCs,免疫熒光鑒定所培養(yǎng)的細胞;用PEG法提取平滑肌細胞源外泌體(HUASMCs-Exo),并行Western blot鑒定其表面標記蛋白CD63、CD9的表達情況,以電鏡鑒定其大小和形態(tài),以Di I染色外泌體,并將之與HUVECs共培養(yǎng)24h,觀察HUVECs對HUASMCs-Exo的內(nèi)化攝取情況。(2)用不同濃度(0.1、1、5、10μg/ml)的IIfim A型P.gingivalis-r Fim A處理HUVECs不同時間(2、8、24、48h),采用CCK-8檢測細胞的增殖活性,并確定P.gingivalis-r Fim A處理HUVECs的最適濃度和時間;后續(xù)實驗分為4組:(1)正常組(Normal組):HUVECs不予任何處理;(2)HUASMCs-Exo組:HUASMCs-Exo與HUVECs共培養(yǎng);(3)r Fim A組:r Fim A處理HUVECs;(4)HUASMCs-Exo+r Fim A組:HUASMCs-Exo與HUVECs共培養(yǎng)后,加入r Fim A處理;FACS檢測各組細胞總體凋亡率、存活率及壞死率,RT-PCR檢測各組細胞內(nèi)IL-18及Caspase-3 m RNA的表達情況,Western blot檢測各組細胞內(nèi)Cleaved caspase-3蛋白表達情況,ELISA檢測各組細胞上清液中IL-18的表達情況。結(jié)果:(1)原代培養(yǎng)的HUVECs和HUASMCs兩種細胞生長狀態(tài)良好,并均經(jīng)形態(tài)學(xué)和免疫熒光鑒定確認;采用PEG法成功提取、鑒定了HUASMCs-Exo,并可被HUVECs內(nèi)化攝取,可用于后續(xù)實驗。(2)CCK-8結(jié)果示10μg/ml濃度的r Fim A處理HUVECs48h時,細胞增殖活性最低,所以用10μg/ml濃度的r Fim A處理HUVECs48h行后續(xù)實驗。FACS結(jié)果示:與Normal組相比,HUASMCs-Exo組細胞總體凋亡率無明顯差異(P0.05),而r Fim A組和HUASMCs-Exo+r Fim A組均可導(dǎo)致HUVECs總體凋亡率升高(P0.05);與r Fim A組相比,HUASMCs-Exo+r Fim A組可下調(diào)r Fim A作用下HUVECs的總體凋亡率。RT-PCR、Western blot和ELISA結(jié)果示:Normal組與HUASMCs-Exo組細胞IL-18m RNA和Caspase-3 m RNA、凋亡主要效應(yīng)蛋白Cleaved caspase-3及細胞上清液中IL-18水平均無明顯差異(P0.05);與Normal組相比,r Fim A組和HUASMCs-Exo+r Fim A組細胞IL-18m RNA和Caspase-3 m RNA、Cleaved caspase-3及細胞上清液中IL-18水平均增加(P0.05),而HUASMCs-Exo+r Fim A組可下調(diào)r Fim A作用下各檢測指標的表達量(P0.05)。結(jié)論:IIfim A型P.gingivalis-r Fim A可誘導(dǎo)HUVECs凋亡和炎癥因子IL-18的表達,而HUASMCs-Exo雖然可在一定程度上調(diào)控P.gingivalis-r Fim A誘導(dǎo)的HUVECs凋亡和炎癥反應(yīng),仍無法改變HUVECs功能異常的結(jié)果。因此,P.gingivalis的主要毒力因子菌毛蛋白可能在促進As發(fā)生、發(fā)展中發(fā)揮一定的作用。
[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.

【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R781.4

【相似文獻】

相關(guān)期刊論文 前10條

1 ;Effect of Porphyromonas gingivalis PrtC on cytokine expression in ECV304 endothelial cells and its level in subgingival plaques from patients with chronic periodontitis[J];Acta Pharmacologica Sinica;2007年07期

2 王曉燕;王俊蓮;丁鰲;孫燕;王勤濤;;P．gingivalis對人臍靜脈血管內(nèi)皮細胞分泌炎性因子的影響[J];上�？谇会t(yī)學(xué);2008年04期

3 Joseph Katz;Mairelys D. Onate;Kaleb M. Pauley;Indraneel Bhattacharyya;Seunghee Cha;;Presence of Porphyromonas gingivalis in gingival squamous cell carcinoma[J];International Journal of Oral Science;2011年04期

4 ;Association between infection of different strains of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in subgingival plaque and clinical parameters in chronic periodontitis[J];Journal of Zhejiang University(Science B:An International Biomedicine & Biotechnology Journal);2007年02期

5 David Ojcius;Association between coinfection of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Treponema denticola and periodontal tissue destruction in chronic periodontitis[J];Chinese Medical Journal;2005年11期

6 張冬梅;趙戩;潘亞萍;林莉;李琛;;P. gingivalis感染對血管內(nèi)皮細胞VCAM-1表達的影響[J];實用口腔醫(yī)學(xué)雜志;2009年03期

7 王琳源;靳贏;林曉萍;;CD4~+T細胞在不同菌株誘導(dǎo)小鼠牙周炎中的表達[J];上海口腔醫(yī)學(xué);2014年01期

8 郭淑娟;苗棣;徐屹;;膜型基質(zhì)金屬蛋白酶-1與牙周炎的關(guān)系研究進展[J];口腔醫(yī)學(xué)研究;2008年03期

9 林曉萍;韓曉哲;魏巍;周曉佳;Martin A.Taubman;;抗RANKL多克隆抗體對P.gingivalis感染的大鼠牙周骨吸收的抑制作用[J];解剖科學(xué)進展;2009年03期

10 Carol L Fischer;Katherine S Walters;David R Drake;Deborah V Dawson;Derek R Blanchette;Kim A Brogden;Philip W Wertz;;Oral mucosal lipids are antibacterial against Porphyromonas gingivalis, induce ultrastructural damage, and alter bacterial lipid and protein compositions[J];International Journal of Oral Science;2013年03期

相關(guān)會議論文 前4條

1 Chong Cao;LiangJun Zhong;;Effects of Gingipains on the Vascular Smooth Muscle Cells Proliferation[A];第十次全國牙周病學(xué)學(xué)術(shù)會議論文摘要匯編[C];2014年

2 ;Research on Lys-gingipain Induced Apoptosis in HPDLCs[A];中華口腔醫(yī)學(xué)會第14次全國口腔醫(yī)學(xué)學(xué)術(shù)會議（2012年會）論文匯編[C];2012年

3 胡琳琳;顧飛;葛頌;;牙齦卟啉單胞菌菌毛蛋白誘導(dǎo)內(nèi)皮細胞炎癥反應(yīng)的信號通路研究[A];第十次全國牙周病學(xué)學(xué)術(shù)會議論文摘要匯編[C];2014年

4 李霞;雷鄧;葛頌;;牙齦卟啉單胞菌脂多糖誘導(dǎo)內(nèi)皮細胞炎癥反應(yīng)的信號通路及產(chǎn)生內(nèi)皮素1和一氧化氮的研究[A];第十次全國牙周病學(xué)學(xué)術(shù)會議論文摘要匯編[C];2014年

相關(guān)博士學(xué)位論文 前7條

1 孔凡芝;牙齦卟啉單胞菌Rag基因溯源研究及其蛋白免疫原性分析[D];江蘇大學(xué);2012年

2 張冬梅;牙齦卟啉單胞菌侵入血管內(nèi)皮細胞后粘附分子表達及信號調(diào)控的研究[D];中國醫(yī)科大學(xué);2008年

3 郭永華;牙齦卟啉單胞菌菌毛fimA基因遺傳多態(tài)性和致病性的研究[D];四川大學(xué);2005年

4 寇育榮;牙齦卟啉單胞菌對牙齦上皮細胞粘附和炎癥介質(zhì)的影響以及多酚對細胞炎癥反應(yīng)調(diào)節(jié)的體外研究[D];中國醫(yī)科大學(xué);2008年

5 趙蕾;不同fimA基因型牙齦卟啉單胞菌致病機理的研究[D];四川大學(xué);2007年

6 雷浪;高脂血癥影響載脂蛋白E基因敲除小鼠對牙齦卟啉單胞菌的先天免疫并促進牙周病進展的研究[D];福建醫(yī)科大學(xué);2012年

7 張鳳秋;牙齦卟啉菌牙齦蛋白酶K催化結(jié)構(gòu)域基因克隆、表達、純化及預(yù)防牙周炎的初步研究[D];第四軍醫(yī)大學(xué);2004年

相關(guān)碩士學(xué)位論文 前10條

1 喻麗;不同fimA型牙齦卟啉單胞菌脂多糖對人臍動脈平滑肌細胞功能的影響[D];遵義醫(yī)學(xué)院;2016年

2 陳江曼;牙齦卟啉單胞菌重組菌毛蛋白對人外周血單個核細胞中TNF-α和IL-6的影響[D];錦州醫(yī)科大學(xué);2017年

3 艾麗瓊;Ⅱ fimA型P.gingivalis重組菌毛蛋白對平滑細胞源外泌體作用下內(nèi)皮細胞相關(guān)功能的影響[D];遵義醫(yī)學(xué)院;2017年

4 楊靜;P.gingivalis感染對小鼠動脈粥樣硬化形成的研究[D];遵義醫(yī)學(xué)院;2017年

5 李婧茜;不同fimA型牙齦卟啉單胞菌菌毛蛋白對人臍動脈平滑肌細胞生物學(xué)行為的影響[D];遵義醫(yī)學(xué)院;2017年

6 林玉祥;牙齦卟啉單胞菌脂多糖對血管內(nèi)皮細胞功能影響的實驗研究[D];遵義醫(yī)學(xué)院;2010年

7 鄭重陽;血鏈球菌和/或牙齦卟啉單胞菌對血小板聚集作用的研究[D];浙江大學(xué);2011年

8 蔡樹玉;不同fimA基因型牙齦卟啉單胞菌對血管內(nèi)皮細胞功能影響的實驗研究[D];遵義醫(yī)學(xué)院;2009年

9 費曉露;具核梭桿菌黏附和侵入上皮細胞的初步研究[D];四川大學(xué);2005年

10 楊潔;牙齦卟啉單胞菌感染的動脈粥樣硬化患者調(diào)節(jié)性T細胞的分布特征[D];南京大學(xué);2013年

，

本文編號：1857913