本文選題：高遷移率族蛋白B1 + 分離純化　； 參考：《中南大學(xué)》2012年博士論文

【摘要】：目的：(1)分離、純化肝細(xì)胞系HepG2細(xì)胞分泌的高遷移率族蛋白1(high mobility group box-1protein, HMGB1),并加以鑒定。(2)探討HMGB1在體外對(duì)巨噬細(xì)胞RAW264.7增殖、釋放乳酸脫氫酶(lactate Dehydrogenase, LDH)、凋亡以及分泌腫瘤壞死因子-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)分別培養(yǎng)人肝細(xì)胞系HepG2細(xì)胞與免疫細(xì)胞系U937細(xì)胞,采用400ng/mL的脂多糖(lipopolysaccharide, LPS)刺激20h后收集細(xì)胞培養(yǎng)液上清。依次采用超濾濃縮、陽離子交換層析(CM-Sepharose cation exchange chromatography)、陰離子交換層(DEAE-Sepharose cation exchange chromatography)、Sephadex G75凝膠過濾層析(Sephadex G75-gel filtration chromatography),結(jié)合免疫沉淀的方法,進(jìn)行分離純化。采用聚丙烯酰胺凝膠電泳(SDS-PAGE)及western印跡法進(jìn)行蛋白分子質(zhì)量及性質(zhì)鑒定。(2)體外經(jīng)不同濃度的HMGB1(10,50,100ng/mL)刺激24h,并以100ng/mL于不同時(shí)間點(diǎn)(8h,24h,48h)刺激巨噬細(xì)胞RAW264.7,采用MMT法檢測巨噬細(xì)胞的增殖,收集培養(yǎng)上清,測定上清中的LDH含量。(3)體外經(jīng)不同濃度的HMGB1(10,50,100ng/mL)刺激24h,或以100ng/mL的HMGB1作用不同時(shí)間(8h,24h,48h)后,采用TUNEL法檢測巨噬細(xì)胞凋亡的情況。(4)體外經(jīng)不同濃度的HMGB1(10,50,100ng/mL)刺激24h,或以100ng/mL作用不同時(shí)間(8h,24h,48h),采用酶聯(lián)免疫吸附法(ELISA)測定細(xì)胞培養(yǎng)上清液中TNF-α IL-1β以及IL-6水平的變化。 結(jié)果：(1)從2株細(xì)胞培養(yǎng)上清分離純化得到的蛋白經(jīng)SDS-PAGE鑒定,其純度達(dá)90%以上,分子質(zhì)量約為26kD, Western印跡法鑒定為HMGB1蛋白。(2)MTT結(jié)果顯示,HMGB1不同劑量(10,50,100ng/mL)組以及100ng/mLHMGB1作用不同時(shí)間點(diǎn)(8h,24h,48h),巨噬細(xì)胞的增殖能力均明顯低于巨噬細(xì)胞空白對(duì)照組(P0.05)。(3)LDH檢測結(jié)果顯示,HMGB1不同劑量(10,50,100ng/mL)組以及100ng/mLHMGB1作用不同時(shí)間點(diǎn)(8h,24h,48h),巨噬細(xì)胞釋放LDH的量均明顯高于巨噬細(xì)胞空白對(duì)照組(P0.05)。(4)經(jīng)不同濃度的HMGB1刺激24h,其中100ng/mL組HMGB1誘導(dǎo)巨噬細(xì)胞凋亡效應(yīng)最強(qiáng),與對(duì)照組比較有顯著性差異(P0.05)。100ng/mL的HMGB1作用巨噬細(xì)胞不同時(shí)間后,以48h組凋亡率發(fā)生最高,明顯高于對(duì)照組(P0.05)。(5)經(jīng)不同濃度的HMGB1刺激24h,可促進(jìn)巨噬細(xì)胞TNF-α、IL-1β和IL-6表達(dá)增強(qiáng),與對(duì)照組比較有顯著性差異(P0.01),其中HMGB1劑量為100ng/mL時(shí)作用最強(qiáng),呈劑量-效應(yīng)關(guān)系。采用100ng/mL的HMGB1作用不同時(shí)間后,培養(yǎng)上清液中TNF-α,IL-1β和IL-6的表達(dá)水平在48h達(dá)高峰(P0.01),呈時(shí)間-效應(yīng)關(guān)系。 結(jié)論：(1)該純化方法簡便、可行、效果好,可以獲得純度較高的HMGB1。(2)HMGB1能抑制巨噬細(xì)胞的增殖,促進(jìn)巨噬細(xì)胞LDH的釋放,誘導(dǎo)巨噬細(xì)胞的凋亡和促進(jìn)TNF-α、IL-1β與IL-6的釋放,并呈時(shí)間-濃度依賴性。
[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- 偽 (tumor necrosis factor-a (TNF- 偽), 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- 偽 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- 偽, 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- 偽 IL-1 尾 and IL-6. And in a time-concentration dependent manner.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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