自噬与侵入KB细胞内的牙龈卟啉单胞菌存活关系的研究

发布时间：2018-03-14 09:01

  本文选题：LC3　切入点：牙龈卟啉单胞菌　出处：《锦州医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的为了探讨自噬与牙龈卟啉单胞菌侵入口腔上皮细胞存活的关系,揭示牙龈卟啉单胞菌在口腔上皮细胞中存活的作用机制。方法1.MOI(感染复数)值分别为50:1、100:1、150:1、200:1、250:1的P.gingivalis与KB细胞共培养3h,洗去未侵入细胞的P.gingivalis,继续培养24h后使用CKK-8法测定不同MOI值的P.gingivalis感染KB细胞的细胞存活率。2.KB细胞转染质粒GFP-LC3,24h后于荧光显微镜下观察转染效果,转染成功后,加入MOI值100的P.gingivalis共培养3h后,洗去未侵入细胞的P.gingivalis继续培养,于12h、18h、24h、36h时间点观察GFP-LC3的荧光,以及经革兰氏(G)染色的P.gingivalis,Western blot检测自噬相关蛋白LC3和p62的表达。3.KB细胞转染质粒GFP-p62,24h后于荧光显微镜下观察,转染成功后,加入MOI值100的P.gingivalis共培养3h后,洗去未侵入细胞的P.gingivalis继续培养,于12h、24h、36h,通过荧光显微镜观察GFP-p62荧光变化情况。4.KB细胞分别转染人工合成的p62si RNA、LC3si RNA,干扰p62及LC3表达水平,转染24h后,通过Western blot检验是否成功干扰p62及LC3表达。5.MOI值100的P.gingivalis分别加入到干扰p62表达的KB细胞、转染GFP-p62的KB细胞、干扰LC3表达的KB细胞和正常KB细胞,侵染细胞3h后,洗去未侵入细胞的P.gingivalis继续培养,于12h、24h、36h观察各组细胞形态,CCK-8法测定细胞活性。6.MOI值100的P.gingivalis侵入p62高表达KB细胞、干扰p62表达的KB细胞、干扰LC3表达的KB细胞和KB细胞,于0h、12h、24h、36h时间点,细胞用PBS洗涤3次,细胞消化并计数,使每组细胞数相等,裂解细胞,将裂解液涂于BHI血琼脂板上,5~6天观察细胞内的P.gingivalis培养后的菌落数。结果1.成功转染GFP-LC3的KB细胞加入MOI值100的P.gingivalis,共培养3h后,显微镜下观察到细胞内有侵入的被G染色的P.gingivalis,12h与0h相比,荧光显微镜下观察到自噬体荧光点数量明显增加(P0.01),Western blot检测LC3蛋白表达明显增加(P0.01),p62蛋白表达也增加(P0.01);18h与12h相比,自噬体数量减少不明显(P0.05),LC3和p62表达水平变化不明显(P0.05),24h与18h相比自噬体数量减少(P0.01),LC3、p62表达减少(P0.05);36h与24h相比,自噬体数目明显减少(P0.01),LC3、p62表达明显减少(P0.01)。36h与0h相比,自噬体数目以及LC3减少表达无统计学意义(P0.05),p62表达减少(P0.01)。2.P.gingivalis感染转染GFP-LC3的KB细胞,0h细胞内可观察到G染色的P.gingivalis,30h后细胞内仍观察到染色的P.gingivalis,30h细菌的数量与0h相比无明显差别(P0.05)。3.成功转染GFP-p62的KB细胞加入MOI值100的P.gingivalis,12h与0h相比,可观察到p62蛋白的聚集融合;24h与12h相比,p62荧光点分散并减弱。36h时镜下几乎看不到荧光点。4.KB细胞转染人工合成的p62si RNA干扰p62表达,转染24h后Western blot结果显示,p62的表达水平低于对照组(P0.01)。5.MOI值100的P.gingivalis分别感染KB细胞、转染GFP-p62的KB细胞,干扰p62表达的KB细胞,12h时,3组细胞存活率无明显差别(P0.05),3组中均较少的细胞变为圆形;24h时,p62高表达的KB细胞存活率低于KB细胞(P0.05),p62表达干扰的KB细胞存活率高于KB细胞(P0.05),p62高表达的KB细胞和KB细胞变为圆形的细胞明显增多,p62表达干扰的KB细胞的细胞形态相对较好;36h后,与KB细胞比较p62高表达的KB细胞存活率降低了2.55倍(P0.01),p62表达干扰的KB细胞存活率增高了1.83倍(P0.01),KB细胞与p62高表达的KB细胞的细胞形态几乎都变为圆形,而p62表达干扰的KB细胞很大一部分细胞保持原来细胞的形态。细胞内细菌在BHI血琼脂板上生长菌落数的结果显示,0h时3组菌落数无差别(P0.05);12h时p62高表达组的菌落数与KB组相比无差别(P0.05),干扰p62组菌落数小于KB组(P0.05),24h时p62高表达组与KB组相比菌落数增多(P0.05),干扰p62组菌落数较KB组明显减少(P0.01);36h时p62高表达组菌落数与KB组相比明显增多(P0.01),干扰p62组较KB组菌落数减少了3.56倍。(P0.01)6.MOI值100的P.gingivalis分别感染KB细胞、干扰LC3表达的KB细胞,12h时,2组均有较少的细胞形态变为圆形,2组细胞存活率比较无差别(P0.05);24h时,KB细胞形态变为圆形细胞明显增多,而干扰LC3的KB细胞形态相对较好,干扰LC3的KB细胞与KB细胞相比存活率较高(P0.05);36h后KB细胞形态几乎都变为圆形,而干扰LC3的KB细胞很大一部分细胞保持原细胞形态,干扰LC3的KB细胞存活率是KB细胞的2.7倍(P0.01)。c内细菌在BHI血琼脂板上生长菌落数的结果显示,12h时LC3干扰组菌落数较KB组少(P0.05),24h时LC3干扰组菌落数较KB组比较减少了2.29倍(P0.01);36h后与KB组比较LC3干扰组菌落数减少了5.31倍(P0.01)。结论P.gingivalis可在KB细胞的自噬体中存活;自噬水平降低,P.gingivalis在KB细胞内存活或增殖能力减弱,KB细胞存活率增加。KB细胞中p62对P.gingivalis持续存活起到促进作用。
[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〃杈剧粍鐨勮弻钀芥暟涓嶬B缁勭浉姣旀棤宸�鍒�(P0.05),骞叉壈p62缁勮弻钀芥暟灏忎簬KB缁，

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