幽门螺杆菌组分对胃上皮细胞增殖与凋亡的影响
发布时间：2019-01-27 08:28
【摘要】：【目的】研究幽门螺杆菌(Hp)不同组分:分泌成分、菌膜及胞浆内容和LPS对胃上皮细胞增殖和凋亡的影响,并进一步探寻致细胞增殖的Hp 组分。 【方法】1)利用超声破碎法提取NCTC11637 Hp(cagA~+)菌株不同组分,包括分泌成分、菌膜及胞浆内容,用Bradford蛋白浓度测定试剂盒测定Hp菌细胞不同组分蛋白浓度。用热酚水法提取Hp 的LPS(H-LPS)和大肠杆菌LPS(E-LPS),比浊法测定LPS浓度。调整分泌成分、菌膜及胞浆内容的蛋白浓度并进行梯度稀释,分别加入SGC-7901细胞,使蛋白终浓度为10-0.05ug/ml;调整H-LPS浓度至5-120EU/ml加入SGC-7901细胞,以E-LPS为对照,均共育24h,检测Hp各细胞组分对SGC-7901细胞增殖(用MTT法)和凋亡(用细胞形态观察及凋亡细胞DNA电泳实验)的影响。2)提取NCTC11639 Hp(cagA~-)和大肠杆菌菌膜,与上述提取的cag A~+Hp菌膜分别作用于SGC-7901细胞,同时将H-LPS以Hp菌膜中所含的LPS的量加入SGC-7901细胞作为对照,共育24h后,用MTT技术检测细胞增殖的情况。3)用细菌蛋白提取试剂盒(P-PEK)分离NCTC 11637 Hp菌膜蛋白,获得4个不同溶解度膜蛋白组分,分别或两两组合加入SGC-7901细胞,用MTT法和Ki-67细胞免疫组化实验检测细胞增殖情况。 【结果】(1) cagA~+Hp 细胞不同组分对SGC-7901 细胞增殖的影响,结果显示Hp 分泌成分和胞浆内容对SGC-7901 细胞均具有抑制细胞增殖的作用,且随着浓度增加,细胞的存活率逐渐降低;Hp 菌膜高浓度时(≥2.5ug/ml)对细胞增殖也具有抑制作用,但当菌膜浓度降至1 ug/ml 时则出现促细胞增殖现象; H-LPS(≥15 EU/ml)具有抑制细胞增殖的作用。Hp 菌膜的3 个不同溶解度蛋白组分当分别单独作用于细胞时,均抑制细胞增殖,且随浓度增高抑制作用增强;但菌膜的中溶解度和难溶解度组分组合后(fraction2-4),在低剂量(0.05、0.01ug/ul)时可促进细胞增殖,细胞免疫组化实验显示其Ki-67 抗原阳性表达率显著增高(p㩳0.05)。(2)cagA~+Hp、cagA~-Hp 和大肠杆菌菌膜对SGC-7901 细胞作用的结果显示:菌膜组分浓度≥2.5ug/ml 时,cagA~+Hp 菌膜组分出现抑制细胞增殖现象,而cagA~-Hp 菌膜对细胞增殖无影响,低剂量(0.1、0.2ug/ul)cagA~+和cagA~-Hp 菌膜
[Abstract]:[objective] to study the effects of different components of Helicobacter pylori (Hp): secretory components, membrane and cytoplasm contents and LPS on the proliferation and apoptosis of gastric epithelial cells, and to further explore the components of Hp that induce the proliferation of gastric epithelial cells. [methods] 1) the different components of NCTC11637 Hp (cagA~) were extracted by ultrasonic fragmentation method, including secretory components, membrane and cytoplasm, and the concentrations of different components of Hp cells were measured by Bradford protein concentration assay kit. LPS (H-LPS) and Escherichia coli LPS (E-LPS) of Hp were extracted by thermophenol water method. LPS concentration was determined by turbidimetric method. The protein concentration of secreting components, membrane and cytoplasm was adjusted and diluted by gradient dilution. The final concentration of protein was 10-0.05ugr / ml by adding SGC-7901 cells respectively. The concentration of H-LPS was adjusted until 5-120EU/ml was added to SGC-7901 cells, and E-LPS was used as control, all of them were bred for 24 hours. The effects of various cell components of Hp on the proliferation of SGC-7901 cells (MTT method) and apoptosis (cell morphology observation and DNA electrophoresis of apoptotic cells) were detected. 2) NCTC11639 Hp (cagA~-) and Escherichia coli membrane were extracted. SGC-7901 cells were treated with the cag A ~ Hp membrane extracted above, and the amount of LPS in the Hp membrane was added to SGC-7901 cells as control. After 24 hours of incubation, the cells were incubated with H-LPS. MTT technique was used to detect cell proliferation. 3) the membrane protein of NCTC 11637 Hp was isolated by bacterial protein extraction kit (P-PEK), and four components of membrane protein with different solubility were obtained. MTT method and Ki-67 cell immunohistochemistry were used to detect cell proliferation. [results] (1) the effect of different components of cagA~ Hp cells on the proliferation of SGC-7901 cells. The results showed that Hp secreting components and cytoplasmic contents could inhibit the proliferation of SGC-7901 cells. The survival rate of cells decreased gradually. Hp membrane (鈮，
本文编号：2416095
[Abstract]:[objective] to study the effects of different components of Helicobacter pylori (Hp): secretory components, membrane and cytoplasm contents and LPS on the proliferation and apoptosis of gastric epithelial cells, and to further explore the components of Hp that induce the proliferation of gastric epithelial cells. [methods] 1) the different components of NCTC11637 Hp (cagA~) were extracted by ultrasonic fragmentation method, including secretory components, membrane and cytoplasm, and the concentrations of different components of Hp cells were measured by Bradford protein concentration assay kit. LPS (H-LPS) and Escherichia coli LPS (E-LPS) of Hp were extracted by thermophenol water method. LPS concentration was determined by turbidimetric method. The protein concentration of secreting components, membrane and cytoplasm was adjusted and diluted by gradient dilution. The final concentration of protein was 10-0.05ugr / ml by adding SGC-7901 cells respectively. The concentration of H-LPS was adjusted until 5-120EU/ml was added to SGC-7901 cells, and E-LPS was used as control, all of them were bred for 24 hours. The effects of various cell components of Hp on the proliferation of SGC-7901 cells (MTT method) and apoptosis (cell morphology observation and DNA electrophoresis of apoptotic cells) were detected. 2) NCTC11639 Hp (cagA~-) and Escherichia coli membrane were extracted. SGC-7901 cells were treated with the cag A ~ Hp membrane extracted above, and the amount of LPS in the Hp membrane was added to SGC-7901 cells as control. After 24 hours of incubation, the cells were incubated with H-LPS. MTT technique was used to detect cell proliferation. 3) the membrane protein of NCTC 11637 Hp was isolated by bacterial protein extraction kit (P-PEK), and four components of membrane protein with different solubility were obtained. MTT method and Ki-67 cell immunohistochemistry were used to detect cell proliferation. [results] (1) the effect of different components of cagA~ Hp cells on the proliferation of SGC-7901 cells. The results showed that Hp secreting components and cytoplasmic contents could inhibit the proliferation of SGC-7901 cells. The survival rate of cells decreased gradually. Hp membrane (鈮，
本文编号：2416095
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