【摘要】：【目的】研究幽門螺桿菌(Hp)不同組分:分泌成分、菌膜及胞漿內(nèi)容和LPS對(duì)胃上皮細(xì)胞增殖和凋亡的影響,并進(jìn)一步探尋致細(xì)胞增殖的Hp 組分。 【方法】1)利用超聲破碎法提取NCTC11637 Hp(cagA~+)菌株不同組分,包括分泌成分、菌膜及胞漿內(nèi)容,用Bradford蛋白濃度測(cè)定試劑盒測(cè)定Hp菌細(xì)胞不同組分蛋白濃度。用熱酚水法提取Hp 的LPS(H-LPS)和大腸桿菌LPS(E-LPS),比濁法測(cè)定LPS濃度。調(diào)整分泌成分、菌膜及胞漿內(nèi)容的蛋白濃度并進(jìn)行梯度稀釋,分別加入SGC-7901細(xì)胞,使蛋白終濃度為10-0.05ug/ml;調(diào)整H-LPS濃度至5-120EU/ml加入SGC-7901細(xì)胞,以E-LPS為對(duì)照,均共育24h,檢測(cè)Hp各細(xì)胞組分對(duì)SGC-7901細(xì)胞增殖(用MTT法)和凋亡(用細(xì)胞形態(tài)觀察及凋亡細(xì)胞DNA電泳實(shí)驗(yàn))的影響。2)提取NCTC11639 Hp(cagA~-)和大腸桿菌菌膜,與上述提取的cag A~+Hp菌膜分別作用于SGC-7901細(xì)胞,同時(shí)將H-LPS以Hp菌膜中所含的LPS的量加入SGC-7901細(xì)胞作為對(duì)照,共育24h后,用MTT技術(shù)檢測(cè)細(xì)胞增殖的情況。3)用細(xì)菌蛋白提取試劑盒(P-PEK)分離NCTC 11637 Hp菌膜蛋白,獲得4個(gè)不同溶解度膜蛋白組分,分別或兩兩組合加入SGC-7901細(xì)胞,用MTT法和Ki-67細(xì)胞免疫組化實(shí)驗(yàn)檢測(cè)細(xì)胞增殖情況。 【結(jié)果】(1) cagA~+Hp 細(xì)胞不同組分對(duì)SGC-7901 細(xì)胞增殖的影響,結(jié)果顯示Hp 分泌成分和胞漿內(nèi)容對(duì)SGC-7901 細(xì)胞均具有抑制細(xì)胞增殖的作用,且隨著濃度增加,細(xì)胞的存活率逐漸降低;Hp 菌膜高濃度時(shí)(≥2.5ug/ml)對(duì)細(xì)胞增殖也具有抑制作用,但當(dāng)菌膜濃度降至1 ug/ml 時(shí)則出現(xiàn)促細(xì)胞增殖現(xiàn)象; H-LPS(≥15 EU/ml)具有抑制細(xì)胞增殖的作用。Hp 菌膜的3 個(gè)不同溶解度蛋白組分當(dāng)分別單獨(dú)作用于細(xì)胞時(shí),均抑制細(xì)胞增殖,且隨濃度增高抑制作用增強(qiáng);但菌膜的中溶解度和難溶解度組分組合后(fraction2-4),在低劑量(0.05、0.01ug/ul)時(shí)可促進(jìn)細(xì)胞增殖,細(xì)胞免疫組化實(shí)驗(yàn)顯示其Ki-67 抗原陽(yáng)性表達(dá)率顯著增高(p㩳0.05)。(2)cagA~+Hp、cagA~-Hp 和大腸桿菌菌膜對(duì)SGC-7901 細(xì)胞作用的結(jié)果顯示:菌膜組分濃度≥2.5ug/ml 時(shí),cagA~+Hp 菌膜組分出現(xiàn)抑制細(xì)胞增殖現(xiàn)象,而cagA~-Hp 菌膜對(duì)細(xì)胞增殖無(wú)影響,低劑量(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，

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