肝细胞分泌的HMGB1蛋白的分离、纯化、鉴定及对小鼠巨噬细胞的作用

发布时间：2018-07-06 11:50

本文选题：高迁移率族蛋白B1 + 分离纯化　；参考：《中南大学》2012年博士论文

【摘要】：目的：(1)分离、纯化肝细胞系HepG2细胞分泌的高迁移率族蛋白1(high mobility group box-1protein, HMGB1),并加以鉴定。(2)探讨HMGB1在体外对巨噬细胞RAW264.7增殖、释放乳酸脱氢酶(lactate Dehydrogenase, LDH)、凋亡以及分泌肿瘤坏死因子-α(tumor necrosis factor-a, TNF-α)、白介素-1β(interleukin-1beta, IL-1β)和白介素-6(IL-6)的作用。方法：(1)分别培养人肝细胞系HepG2细胞与免疫细胞系U937细胞,采用400ng/mL的脂多糖(lipopolysaccharide, LPS)刺激20h后收集细胞培养液上清。依次采用超滤浓缩、阳离子交换层析(CM-Sepharose cation exchange chromatography)、阴离子交换层(DEAE-Sepharose cation exchange chromatography)、Sephadex G75凝胶过滤层析(Sephadex G75-gel filtration chromatography),结合免疫沉淀的方法,进行分离纯化。采用聚丙烯酰胺凝胶电泳(SDS-PAGE)及western印迹法进行蛋白分子质量及性质鉴定。(2)体外经不同浓度的HMGB1(10,50,100ng/mL)刺激24h,并以100ng/mL于不同时间点(8h,24h,48h)刺激巨噬细胞RAW264.7,采用MMT法检测巨噬细胞的增殖,收集培养上清,测定上清中的LDH含量。(3)体外经不同浓度的HMGB1(10,50,100ng/mL)刺激24h,或以100ng/mL的HMGB1作用不同时间(8h,24h,48h)后,采用TUNEL法检测巨噬细胞凋亡的情况。(4)体外经不同浓度的HMGB1(10,50,100ng/mL)刺激24h,或以100ng/mL作用不同时间(8h,24h,48h),采用酶联免疫吸附法(ELISA)测定细胞培养上清液中TNF-α IL-1β以及IL-6水平的变化。结果：(1)从2株细胞培养上清分离纯化得到的蛋白经SDS-PAGE鉴定,其纯度达90%以上,分子质量约为26kD, Western印迹法鉴定为HMGB1蛋白。(2)MTT结果显示,HMGB1不同剂量(10,50,100ng/mL)组以及100ng/mLHMGB1作用不同时间点(8h,24h,48h),巨噬细胞的增殖能力均明显低于巨噬细胞空白对照组(P0.05)。(3)LDH检测结果显示,HMGB1不同剂量(10,50,100ng/mL)组以及100ng/mLHMGB1作用不同时间点(8h,24h,48h),巨噬细胞释放LDH的量均明显高于巨噬细胞空白对照组(P0.05)。(4)经不同浓度的HMGB1刺激24h,其中100ng/mL组HMGB1诱导巨噬细胞凋亡效应最强,与对照组比较有显著性差异(P0.05)。100ng/mL的HMGB1作用巨噬细胞不同时间后,以48h组凋亡率发生最高,明显高于对照组(P0.05)。(5)经不同浓度的HMGB1刺激24h,可促进巨噬细胞TNF-α、IL-1β和IL-6表达增强,与对照组比较有显著性差异(P0.01),其中HMGB1剂量为100ng/mL时作用最强,呈剂量-效应关系。采用100ng/mL的HMGB1作用不同时间后,培养上清液中TNF-α,IL-1β和IL-6的表达水平在48h达高峰(P0.01),呈时间-效应关系。结论：(1)该纯化方法简便、可行、效果好,可以获得纯度较高的HMGB1。(2)HMGB1能抑制巨噬细胞的增殖,促进巨噬细胞LDH的释放,诱导巨噬细胞的凋亡和促进TNF-α、IL-1β与IL-6的释放,并呈时间-浓度依赖性。
[Abstract]:Objective: (1) to isolate and purify high mobility group protein 1 (high mobility group box-1 protein (HMGB1) secreted by HepG2 cells, and to identify HMGB1. (2) to investigate the proliferation of macrophage RAW264.7 by HMGB1 in vitro. Release of lactate dehydrogenase (LDH), apoptosis and secretion of tumor necrosis factor- 伪 (tumor necrosis factor-a (TNF- 伪), interleukin-1 beta (IL-1 尾) and interleukin-6 (IL-6). Methods: (1) HepG2 cell line and U937 cell line were cultured respectively. The supernatants were collected after 20 h stimulation with 400 ng / mL lipopolysaccharide (LPS). In turn, ultrafiltration concentration, CM-Sepharose cation exchange chromatography), anion exchange layer (DEAE-Sepharose cation exchange chromatography) Sephadex G75 gel filtration chromatography (Sephadex G75-gel filtration chromatography), combined with immunoprecipitation) were used for separation and purification. Polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were used to identify the molecular weight and properties of macrophages. (2) the macrophages were stimulated with HMGB1 (1050ng / mL) for 24 h in vitro and RAW264.7 at different time points (8 h or 24 h / 48 h) with 100ng / mL HMGB1 (1050ng / mL). The proliferation of macrophages was detected by MMT method. The contents of LDH in supernatants were collected and determined. (3) the supernatants were stimulated with HMGB1 (1050ng-1 / mL) for 24 h in vitro, or HMGB1 (100ng / mL HMGB1) for different time (8h 24 h or 48h). Tunel method was used to detect the apoptosis of macrophages. (4) the levels of TNF- 伪 IL-1 尾 and IL-6 in the supernatant of cell culture were measured by enzyme-linked immunosorbent assay (Elisa) after 24 h stimulation with different concentrations of HMGB1 (10 ~ 50 ng / mL) or 100 ng / mL at different time (8 h or 24 h ~ 48 h). Results: (1) the protein isolated from the supernatant of two cell lines was identified by SDS-PAGE and its purity was over 90%. The molecular weight of HMGB1 protein was about 26kD. (2) the results showed that the proliferation ability of macrophages was significantly lower than that of the control group (P0.05). (3) at different doses of HMGB1 (1050ng / mL) and 100ng / mLHMGB1 at different time points (8hmLHMGB1) (8hmLHMGB1 for 48h). In different doses of HMGB1 (1050ng-1 / mL) and 100ng / mLHMGB1 at different time points (8hmLHMGB1), the amount of LDH released by macrophages was significantly higher than that of macrophages blank control group (P0.05). (4) stimulated with different concentrations of HMGB1 for 24 h, and the effect of HMGB1 on macrophage apoptosis was the strongest in 100ng- mL group. Compared with the control group, there was a significant difference (P0.05). 100 ng / mL HMGB1 had the highest apoptotic rate at 48h, which was significantly higher than that in the control group (P0.05). (5) stimulated by HMGB1 at different concentrations for 24 h, and the expression of IL-1 尾 and IL-6 in macrophages was enhanced. There was significant difference between HMGB1 and the control group (P0.01). HMGB1 had the strongest effect when the dose was 100 ng / mL, showing a dose-effect relationship. After treated with 100ng / mL HMGB1 for different time, the expression levels of TNF- 伪, IL-1 尾 and IL-6 in the supernatant reached a peak at 48 h (P0.01), showing a time-effect relationship. Conclusion: (1) the purification method is simple, feasible and effective. HMGB1 can obtain high purity HMGB1. (2) HMGB1 can inhibit the proliferation of macrophages, promote the release of LDH, induce apoptosis of macrophages and promote the release of TNF- 伪 IL-1 尾 and IL-6. And in a time-concentration dependent manner.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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