本文選題：LC3　切入點(diǎn)：牙齦卟啉單胞菌　出處：《錦州醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的為了探討自噬與牙齦卟啉單胞菌侵入口腔上皮細(xì)胞存活的關(guān)系,揭示牙齦卟啉單胞菌在口腔上皮細(xì)胞中存活的作用機(jī)制。方法1.MOI(感染復(fù)數(shù))值分別為50:1、100:1、150:1、200:1、250:1的P.gingivalis與KB細(xì)胞共培養(yǎng)3h,洗去未侵入細(xì)胞的P.gingivalis,繼續(xù)培養(yǎng)24h后使用CKK-8法測定不同MOI值的P.gingivalis感染KB細(xì)胞的細(xì)胞存活率。2.KB細(xì)胞轉(zhuǎn)染質(zhì)粒GFP-LC3,24h后于熒光顯微鏡下觀察轉(zhuǎn)染效果,轉(zhuǎn)染成功后,加入MOI值100的P.gingivalis共培養(yǎng)3h后,洗去未侵入細(xì)胞的P.gingivalis繼續(xù)培養(yǎng),于12h、18h、24h、36h時間點(diǎn)觀察GFP-LC3的熒光,以及經(jīng)革蘭氏(G)染色的P.gingivalis,Western blot檢測自噬相關(guān)蛋白LC3和p62的表達(dá)。3.KB細(xì)胞轉(zhuǎn)染質(zhì)粒GFP-p62,24h后于熒光顯微鏡下觀察,轉(zhuǎn)染成功后,加入MOI值100的P.gingivalis共培養(yǎng)3h后,洗去未侵入細(xì)胞的P.gingivalis繼續(xù)培養(yǎng),于12h、24h、36h,通過熒光顯微鏡觀察GFP-p62熒光變化情況。4.KB細(xì)胞分別轉(zhuǎn)染人工合成的p62si RNA、LC3si RNA,干擾p62及LC3表達(dá)水平,轉(zhuǎn)染24h后,通過Western blot檢驗(yàn)是否成功干擾p62及LC3表達(dá)。5.MOI值100的P.gingivalis分別加入到干擾p62表達(dá)的KB細(xì)胞、轉(zhuǎn)染GFP-p62的KB細(xì)胞、干擾LC3表達(dá)的KB細(xì)胞和正常KB細(xì)胞,侵染細(xì)胞3h后,洗去未侵入細(xì)胞的P.gingivalis繼續(xù)培養(yǎng),于12h、24h、36h觀察各組細(xì)胞形態(tài),CCK-8法測定細(xì)胞活性。6.MOI值100的P.gingivalis侵入p62高表達(dá)KB細(xì)胞、干擾p62表達(dá)的KB細(xì)胞、干擾LC3表達(dá)的KB細(xì)胞和KB細(xì)胞,于0h、12h、24h、36h時間點(diǎn),細(xì)胞用PBS洗滌3次,細(xì)胞消化并計(jì)數(shù),使每組細(xì)胞數(shù)相等,裂解細(xì)胞,將裂解液涂于BHI血瓊脂板上,5~6天觀察細(xì)胞內(nèi)的P.gingivalis培養(yǎng)后的菌落數(shù)。結(jié)果1.成功轉(zhuǎn)染GFP-LC3的KB細(xì)胞加入MOI值100的P.gingivalis,共培養(yǎng)3h后,顯微鏡下觀察到細(xì)胞內(nèi)有侵入的被G染色的P.gingivalis,12h與0h相比,熒光顯微鏡下觀察到自噬體熒光點(diǎn)數(shù)量明顯增加(P0.01),Western blot檢測LC3蛋白表達(dá)明顯增加(P0.01),p62蛋白表達(dá)也增加(P0.01);18h與12h相比,自噬體數(shù)量減少不明顯(P0.05),LC3和p62表達(dá)水平變化不明顯(P0.05),24h與18h相比自噬體數(shù)量減少(P0.01),LC3、p62表達(dá)減少(P0.05);36h與24h相比,自噬體數(shù)目明顯減少(P0.01),LC3、p62表達(dá)明顯減少(P0.01)。36h與0h相比,自噬體數(shù)目以及LC3減少表達(dá)無統(tǒng)計(jì)學(xué)意義(P0.05),p62表達(dá)減少(P0.01)。2.P.gingivalis感染轉(zhuǎn)染GFP-LC3的KB細(xì)胞,0h細(xì)胞內(nèi)可觀察到G染色的P.gingivalis,30h后細(xì)胞內(nèi)仍觀察到染色的P.gingivalis,30h細(xì)菌的數(shù)量與0h相比無明顯差別(P0.05)。3.成功轉(zhuǎn)染GFP-p62的KB細(xì)胞加入MOI值100的P.gingivalis,12h與0h相比,可觀察到p62蛋白的聚集融合;24h與12h相比,p62熒光點(diǎn)分散并減弱。36h時鏡下幾乎看不到熒光點(diǎn)。4.KB細(xì)胞轉(zhuǎn)染人工合成的p62si RNA干擾p62表達(dá),轉(zhuǎn)染24h后Western blot結(jié)果顯示,p62的表達(dá)水平低于對照組(P0.01)。5.MOI值100的P.gingivalis分別感染KB細(xì)胞、轉(zhuǎn)染GFP-p62的KB細(xì)胞,干擾p62表達(dá)的KB細(xì)胞,12h時,3組細(xì)胞存活率無明顯差別(P0.05),3組中均較少的細(xì)胞變?yōu)閳A形;24h時,p62高表達(dá)的KB細(xì)胞存活率低于KB細(xì)胞(P0.05),p62表達(dá)干擾的KB細(xì)胞存活率高于KB細(xì)胞(P0.05),p62高表達(dá)的KB細(xì)胞和KB細(xì)胞變?yōu)閳A形的細(xì)胞明顯增多,p62表達(dá)干擾的KB細(xì)胞的細(xì)胞形態(tài)相對較好;36h后,與KB細(xì)胞比較p62高表達(dá)的KB細(xì)胞存活率降低了2.55倍(P0.01),p62表達(dá)干擾的KB細(xì)胞存活率增高了1.83倍(P0.01),KB細(xì)胞與p62高表達(dá)的KB細(xì)胞的細(xì)胞形態(tài)幾乎都變?yōu)閳A形,而p62表達(dá)干擾的KB細(xì)胞很大一部分細(xì)胞保持原來細(xì)胞的形態(tài)。細(xì)胞內(nèi)細(xì)菌在BHI血瓊脂板上生長菌落數(shù)的結(jié)果顯示,0h時3組菌落數(shù)無差別(P0.05);12h時p62高表達(dá)組的菌落數(shù)與KB組相比無差別(P0.05),干擾p62組菌落數(shù)小于KB組(P0.05),24h時p62高表達(dá)組與KB組相比菌落數(shù)增多(P0.05),干擾p62組菌落數(shù)較KB組明顯減少(P0.01);36h時p62高表達(dá)組菌落數(shù)與KB組相比明顯增多(P0.01),干擾p62組較KB組菌落數(shù)減少了3.56倍。(P0.01)6.MOI值100的P.gingivalis分別感染KB細(xì)胞、干擾LC3表達(dá)的KB細(xì)胞,12h時,2組均有較少的細(xì)胞形態(tài)變?yōu)閳A形,2組細(xì)胞存活率比較無差別(P0.05);24h時,KB細(xì)胞形態(tài)變?yōu)閳A形細(xì)胞明顯增多,而干擾LC3的KB細(xì)胞形態(tài)相對較好,干擾LC3的KB細(xì)胞與KB細(xì)胞相比存活率較高(P0.05);36h后KB細(xì)胞形態(tài)幾乎都變?yōu)閳A形,而干擾LC3的KB細(xì)胞很大一部分細(xì)胞保持原細(xì)胞形態(tài),干擾LC3的KB細(xì)胞存活率是KB細(xì)胞的2.7倍(P0.01)。c內(nèi)細(xì)菌在BHI血瓊脂板上生長菌落數(shù)的結(jié)果顯示,12h時LC3干擾組菌落數(shù)較KB組少(P0.05),24h時LC3干擾組菌落數(shù)較KB組比較減少了2.29倍(P0.01);36h后與KB組比較LC3干擾組菌落數(shù)減少了5.31倍(P0.01)。結(jié)論P(yáng).gingivalis可在KB細(xì)胞的自噬體中存活;自噬水平降低,P.gingivalis在KB細(xì)胞內(nèi)存活或增殖能力減弱,KB細(xì)胞存活率增加。KB細(xì)胞中p62對P.gingivalis持續(xù)存活起到促進(jìn)作用。
[Abstract]:Objective to investigate autophagy and Porphyromonas gingivalis invasion between oral epithelial cell survival, reveal mechanism of Porphyromonas gingivalis survival in oral epithelial cells. Methods 1.MOI (multiplicity of infection) were 3H P.gingivalis co cultured with KB cells 50:1100:1150:1200:1250:1, washing away the non invasion of cells to P.gingivalis, rate of.2.KB cells transfection of plasmid GFP-LC3,24h after transfection was observed under fluorescence microscope on survival after 24h culture, the use of CKK-8 P.gingivalis method for the determination of different values of MOI infected KB cells transfected cells, after the success of joining the MOI value of 100 P.gingivalis 3h after co culture, washing away the non invasion of cells cultured in P.gingivalis, 12h, 18h, 24h, 36h time point to observe the fluorescence of GFP-LC3 (G), and by Gram staining of P.gingivalis expression in.3.KB cells transfected with Western blot detection of autophagy related protein LC3 and p62 Plasmid GFP-p62,24h was observed after transfection under fluorescent microscopy, after the successful accession to the MOI value of 100 P.gingivalis 3h after co culture, washing away the non invasion of cells cultured in P.gingivalis, 12h, 24h, 36h, respectively, transfection of synthetic p62si RNA by fluorescence microscopy GFP-p62 fluorescence changes of.4.KB LC3si RNA cells, p62 interference and the expression of LC3, 24h after transfection by Western blot test the success of p62 interference and the expression of LC3.5.MOI cell KB 100 P.gingivalis were added to interfere the expression of p62, KB cells transfected with GFP-p62, interference of LC3 expression in KB cells and normal KB cells infected 3H cells after washing away the non invasion of cells to P.gingivalis in culture, 12h, 24h, 36h, cell morphology was observed, the CCK-8 method for the determination of P.gingivalis value of 100 invasive p62 high expression of KB cell activity in.6.MOI cells, KB expression by p62 interference, the interference of LC3 expression The KB and KB cells, 0h, 12h, 24h, 36h time points, cells were washed with PBS 3 times, and counting the number of cells in the digestive cells in each group, the same number of cell lysis, the lysates were coated on BHI blood agar plate, 5~6 day number of observed cells after P.gingivalis culture. Results KB 1. cells successfully transfected GFP-LC3 into MOI 100 P.gingivalis, after 3H co cultured cells were observed under microscope by G staining in the invasion of P.gingivalis, 12h compared with 0h, fluorescence microscopy showed that autophagy significantly increased the number of fluorescent spots (P0.01), to detect the expression of LC3 protein increased significantly (Western blot P0.01), the expression of p62 protein also increased (P0.01); 18h compared with 12h, autophagosomes were not decreased obviously (P0.05), LC3 and p62 expression levels did not change significantly (P0.05), 24h and 18h compared to the decrease in the number of autophagosomes (P0.01), LC3, p62 expression decreased (P0.05) compared with 24h 36h; the number of autophagosomes. Obviously reduced (P0.01), LC3, p62 significantly decreased the expression of.36h (P0.01) compared with 0h, LC3 and the number of autophagosomes reduced expression was not statistically significant (P0.05), p62 (P0.01) KB cells decreased expression of.2.P.gingivalis transfected with GFP-LC3 infection, 0h cells can be observed in G staining of P.gingivalis cells after 30h still, P.gingivalis 30h staining was observed, the number of bacteria had no significant difference compared with 0h (P0.05) KB.3. was transfected into GFP-p62 cells with MOI value of 100 P.gingivalis 12h, compared with 0h, observed p62 protein aggregation and fusion; 24h compared with 12h, p62 and.36h decreased when the dispersed fluorescence microscope almost invisible fluorescent spots in.4.KB cells transfected with synthetic p62si RNA interference p62 expression display Western blot 24h after the transfection of the expression level of p62 is lower than that of the control group (P0.01).5.MOI 100 P.gingivalis were infected with KB cells, KB cells transfected with GFP-p62 The expression of p62 cells, KB interference, 12h, 3 groups of cell survival rate showed no significant difference (P0.05), the 3 groups had fewer cells became round; 24h, KB cell survival rate was lower than the high expression of p62 KB cells (P0.05), the expression of p62 cells by KB interference (higher survival rate than KB cells P0.05), KB cells and KB cells with high p62 expression into circular cells significantly increased the expression of p62 cells, KB interference cell morphology is relatively good; 36h cells, KB KB cells with high expression of p62 decrease the survival rate of 2.55 times (P0.01), the expression of p62 cell survival rate increased by KB interference 1.83 times (P0.01), KB cells with high expression of KB cells and p62 cells form almost into a circle, and the expression of p62 interference in KB cells of a large part of the cell to maintain the original cell morphology. Results of bacterial colonies in BHI blood agar plate cells showed that the 0h of the 3 groups of colonies no difference (P0.05); 12h 鏃秔62楂樿〃杈劇粍鐨勮弻钀芥暟涓嶬B緇勭浉姣旀棤宸埆(P0.05),騫叉壈p62緇勮弻钀芥暟灝忎簬KB緇，

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