本文關(guān)鍵詞：肺炎鏈球菌溶血素經(jīng)MAPK途徑誘導(dǎo)人臍靜脈內(nèi)皮細胞凋亡　出處：《重慶醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肺炎鏈球菌 溶血素 凋亡 人臍靜脈內(nèi)皮細胞 MAPK

【摘要】：目的 肺炎鏈球菌溶血素(Pneumolysin,PLY)是由肺炎鏈球菌(Streptococcus pneumoniae,S.pn)產(chǎn)生的一種重要的溶細胞毒素。近年來研究證實小劑量PLY可誘導(dǎo)宿主細胞凋亡,但其機制尚不清楚。本研究通過體外表達純化PLY重組蛋白并選用人臍靜脈內(nèi)皮細胞(The Human umbilical vein endothelial cell line, HUVEC)作為體外細胞模型,觀察PLY重組蛋白是否能誘導(dǎo)HUVEC凋亡,且該過程是否有caspase途徑和MAPK途徑的參與。 方法 1.在E.coli BL21中表達融合6個組氨酸標(biāo)簽的PLY重組蛋白,采用Ni-NTA樹脂純化重組蛋白,通過SDS-PAGE和溶血試驗鑒定其分子量和溶血活性。 2.觀察PLY對HUVEC的增殖抑制和誘導(dǎo)凋亡作用:采用MTT法檢測HUVEC的增殖情況,采用Annexin V/PI雙標(biāo)記染色法檢測凋亡率,并提取細胞DNA進行瓊脂糖凝膠電泳觀察細胞核染色質(zhì)DNA的斷裂。 3.采用ELISA檢測PLY作用細胞后的caspase-3,8,9的活性變化,采用免疫細胞組化檢測細胞ERK1/2、p38MAPK的表達水平,以Western Blot檢測經(jīng)PLY作用后ERK1/2和p38MAPK的總蛋白水平和磷酸化水平變化。 結(jié)果 1.成功構(gòu)建質(zhì)粒Ply-pW28并實現(xiàn)了PLY蛋白的可溶性表達,所獲重組蛋白純化后純度達到95%。溶血實驗顯示PLY對人紅細胞具有極高的溶血活性,并呈明顯劑量依賴性(p0.05),在100ng/ml時溶血率為100%。 2. PLY重組蛋白與HUVEC共孵育后,HUVEC的增殖受到了明顯的抑制,并呈明顯的時間和劑量依賴性,IC50(24h)為1.525μg/ml。PLY處理后HUVEC黏附能力降低,部分細胞皺縮變圓,失去典型“鵝卵石樣”形態(tài)特點,并呈明顯劑量依賴性。Annexin V/PI雙標(biāo)記染色法檢測到0.5μg/ml、1μg/ml和2.5μg/ml的PLY與HUVEC共孵育24h后細胞的早期凋亡率分別為8.91%、27.43%、31.50%,與對照組細胞凋亡率為2.67%相比較顯著增高(P0.05)。瓊脂糖凝膠電泳可見凋亡細胞DNA的斷裂。 3. 1μg/ml PLY與HUVEC孵育24h后caspase-3,8,9活性無明顯變化。免疫細胞組化結(jié)果顯示:與對照組相比,PLY處理組ERK1/2表達減弱,而p38MAPK則表達增加。Western Blot結(jié)果顯示:與對照組相比,PLY處理組的ERK1/2和p-ERK1/2的表達水平均顯著下降,而p38MAPK和p-p38MAPK表達水平均增加。 結(jié)論 PLY可抑制HUVEC增殖并誘導(dǎo)其凋亡,該效應(yīng)至少部分是通過抑制ERK1/2信號途徑和激活p38MAPK信號途徑而實現(xiàn)的,但該效應(yīng)并不依賴于caspase-3,8,9的活化。
[Abstract]:Purpose Streptococcus pneumoniae (Streptococcus pneumoniae) was isolated from Streptococcus pneumoniae. S. pn) is an important cytotoxin. In recent years, it has been proved that small dose of PLY can induce apoptosis of host cells. However, its mechanism is not clear. In this study, the recombinant PLY protein was expressed and purified in vitro and human umbilical vein endothelial cells (HUVEC) were selected. The Human umbilical vein endothelial cell line. HUVECs were used as cell models in vitro to observe whether PLY recombinant protein could induce apoptosis of HUVEC and whether the caspase pathway and MAPK pathway were involved in the process. Method 1. The recombinant PLY protein was expressed in E. coli BL21 and purified by Ni-NTA resin. Its molecular weight and hemolytic activity were identified by SDS-PAGE and hemolysis test. 2.To observe the effect of PLY on the proliferation and apoptosis of HUVEC: MTT assay was used to detect the proliferation of HUVEC. The apoptosis rate was detected by Annexin V / Pi double labeling method, and cell DNA was extracted for agarose gel electrophoresis to observe the fragmentation of nuclear chromatin DNA. 3. ELISA was used to detect the activity of caspase-3 and caspase-8, and ERK1/2 was detected by immunocytochemistry. The expression level of p38 MAPK was detected by Western Blot. The total protein and phosphorylation level of ERK1/2 and p38 MAPK were detected by PLY. Results 1. The plasmid Ply-pW28 was successfully constructed and the soluble expression of PLY protein was realized. The purity of the purified recombinant protein was 95%. The hemolysis test showed that PLY had extremely high hemolytic activity on human erythrocytes in a dose-dependent manner (p 0.05). At 100 ng / ml, the hemolysis rate was 100 g / ml. 2. The proliferation of PLY was inhibited in a time-and dose-dependent manner after being co-incubated with HUVEC. The adhesion ability of HUVEC was decreased after treatment of 1.525 渭 g / ml. PLY for 24 h, and some cells shrank and rounded, thus losing the typical "cobblestone" morphology. In a dose-dependent manner, 0.5 渭 g / ml was detected by Annexin V / Pi double labeling staining. After 1 渭 g / ml and 2.5 渭 g / ml PLY were incubated with HUVEC for 24 hours, the early apoptosis rate was 8.91% and 27.43%, 31.50%, respectively. Compared with the control group, the apoptotic rate was significantly higher than that of the control group (2.67%). The DNA fragmentation of apoptotic cells was observed by agarose gel electrophoresis. 3. 1 渭 g / ml PLY was incubated with HUVEC for 24 hours, and the activity of caspase-3t8 was not changed significantly. The immunohistochemical results showed that compared with the control group, there was no significant change in the activity of caspase-38. The expression of p38 MAPK was decreased in PLY treatment group, while p38 MAPK expression was increased. Western Blot showed that compared with control group, the expression of p38 MAPK was higher than that of control group. The expression levels of ERK1/2 and p-ERK1 / 2 in PLY treatment group were significantly decreased, while p38 MAPK and p-p38 MAPK expression levels were increased. Conclusion PLY inhibits the proliferation of HUVEC and induces its apoptosis, at least in part by inhibiting the ERK1/2 signaling pathway and activating the p38 MAPK signaling pathway. However, this effect does not depend on the activation of caspase-3 and caspase-8.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378

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