人TLR4胞外段的真核表达及其对LPS所致炎症反应的影响

发布时间：2018-11-23 08:16

【摘要】： 严重创伤、大手术后及其他危重病人往往伴有严重的革兰氏阴性菌感染,最终可发展为内毒素血症,甚至内毒素休克,是目前危重症治疗领域非常棘手的问题。革兰氏阴性菌的致病物质基础是被称为内毒素的脂多糖(LPS),近年研究证明,LPS主要通过一种跨膜蛋白Toll-like receptor 4(TLR4)将炎症信号传入胞内,进一步激发一系列炎症反应。若能在TLR4水平阻断该信号转导通路,那么就有可能抑制过度的炎症反应。基于此,本实验拟通过真核系统表达TLR4胞外段,并观察它对LPS所致炎症反应的影响。方法:首先通过PCR技术从携带TLR4全长cDNA的质粒pCMV-TLR4上扩增获得TLR4胞外段cDNA,将后者克隆入真核表达载体pcDNA3.1(+),构建真核表达质粒pcDNA3.1(+)-eTLR4,通过酶切分析及测序给予鉴定。然后将pcDNA3.1(+)-eTLR4通过脂质体转染法转入HEK293细胞,在mRNA水平观察TLR4胞外基因的表达。最后我们用G418筛选稳定表达TLR4胞外段HEK293细胞,用培养液上清作用于诱导分化成熟的U937细胞,通过ELISA观察该细胞TNF-α的分泌变化。结果:1.从携带TLR4全长cDNA的质粒pCMV-TLR4上成功扩增获得TLR4胞外段cDNA,片段大小1.8kb,与理论值一致。2.将TLR4胞外段cDNA克隆入真核表达载体pcDNA3.1(+),通过酶切分析及测序证明成功构建了真核表达质粒cDNA3.1(+)-eTLR4,其插入片段的碱基序列、两端的酶切位点及读码框完全正确。3.通过脂质体转染法,将pcDNA3.1(+)-eTLR4和pcDNA3.1(+)分别转染HEK293细胞后,在mRNA水平证明:前者有TLR4胞外基因表达,而后者无任何表达。4.LPS能刺激诱导分化成熟的U937细胞TNF-α的分泌量增加(p0.01),其分泌量随LPS剂量增加而增加。5.稳定表达TLR4胞外段的HEK293细胞培养液上清能减少诱导分化成熟的U937细胞TNF-α的分泌量(p0.01),并随剂量增加而降低;而稳定表达TLR4胞外段的HEK293细胞裂解液对TNF-α的分泌无明显影响;同时转染空载体的细胞培养液上清对TNF-α的分泌亦无明显影响。6.细胞培养液上清在不同时刻给予,对诱导分化成熟的U937细胞TNF-α的分泌量有所影响,随给药时间延迟TNF-α的分泌量有逐渐降低的趋势。
[Abstract]:Severe trauma, major surgery and other critical patients are often accompanied by severe Gram-negative bacteria infection, which can eventually develop into endotoxemia or even endotoxic shock, which is a very difficult problem in the field of critical and severe treatment at present. The pathogenic substance of Gram-negative bacteria is the lipopolysaccharide (LPS),) which is called endotoxin. It has been proved in recent years that LPS transmembrane protein Toll-like receptor 4 (TLR4) sends the inflammatory signal into the cell and further stimulates a series of inflammatory reactions. If the signal transduction pathway is blocked at TLR4 level, it may inhibit excessive inflammation. Based on this, we intend to express the extracellular segment of TLR4 through eukaryotic system and observe its effect on the inflammatory response induced by LPS. Methods: firstly, the extracellular cDNA, of TLR4 was amplified from the plasmid pCMV-TLR4 carrying the full-length cDNA of TLR4 by PCR technique. The latter was cloned into the eukaryotic expression vector pcDNA3.1 (), to construct the eukaryotic expression plasmid pcDNA3.1 () -eTLR4,. The identification was carried out by restriction enzyme digestion and sequencing. Then pcDNA3.1 ()-eTLR4 was transfected into HEK293 cells by liposome transfection and the expression of TLR4 extracellular genes was observed at the mRNA level. Finally, we used G418 to screen the HEK293 cells stably expressing the extracellular segment of TLR4. The supernatant was used to induce the differentiation of U937 cells. The secretion of TNF- 伪 was observed by ELISA. Results: 1. The extracellular cDNA, fragment of TLR4 was amplified successfully from the plasmid pCMV-TLR4 carrying the full-length cDNA of TLR4, which was 1.8kb in size, which was in agreement with the theoretical value of 2.2. The extracellular cDNA of TLR4 was cloned into the eukaryotic expression vector pcDNA3.1 (),. It was proved by restriction endonuclease analysis and sequencing that the base sequence of the eukaryotic expression plasmid cDNA3.1 ()-eTLR4, was successfully constructed. The endonuclease sites and reading frames are completely correct. 3. After transfection of pcDNA3.1 ()-eTLR4 and pcDNA3.1 () into HEK293 cells by liposome transfection, it was proved at mRNA level that the former had extracellular TLR4 gene expression. 4.LPS stimulated the secretion of TNF- 伪 in differentiated U937 cells (p0.01), and the secretion of TNF- 伪 increased with the increase of LPS dose. The supernatant of HEK293 cells stably expressing extracellular segments of TLR4 decreased the secretion of TNF- 伪 in U937 cells (p0.01), and decreased with the increase of dosage. However, the HEK293 cell lysate, which stably expressed the extracellular segment of TLR4, had no significant effect on the secretion of TNF- 伪, and the supernatant of the cell culture medium transfected with empty vector had no significant effect on the secretion of TNF- 伪. The supernatant of cell culture medium at different time affected the secretion of TNF- 伪 in U937 cells, and the secretion of TNF- 伪 decreased gradually with the delay of drug administration.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R362

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