HMGB1通过TREM-1激活Kupffer细胞向M2型极化的机制研究

发布时间：2018-07-28 15:03

【摘要】：第一部分:HMGB1刺激Kupper细胞后功能变化目的:观察HMGB1刺激后Kupffer细胞(KCs)吞噬功能、膜蛋白表达及分泌功能变化。方法:分离大鼠KCs,鉴定KCs的纯度,判断KCs的活性及吞噬功能。将分离、纯化的KCs以每孔3-4×106接种于培养板中,随机分为2组:(1)HMGB1刺激组:给予500ng/ml HMGB1刺激;(2)对照组:给予PBS刺激。分别在处理24小时后收获细胞。吞墨实验检测KCs吞噬功能。流式细胞术检测KCs膜蛋白表面分子F4/80,CD206表达情况。Western blot法检测Arg-1,VEGF,TGF-β,MMP-9等蛋白表达水平。ELISA法检测细胞培养上清液中IL-6,IL-8,IL-10分泌情况。Transwell小室法检测共培养Walker256侵袭及迁移能力变化。重复上述检测。结果:(1)所提取KCs纯度大于91.0%,活力均在97.8%以上;(2)HMGB1组KCs M2表型表面分子CD206明显高于对照组,Arg-1酶活性表达明显高于对照组(P0.05)。HMGB1组细胞培养上清液IL-6水平明显低于对照组;IL-10水平明显高于对照组(P0.05)。HMGB1组Arg-1,TGF-β,MMP-9蛋白水平显著高于对照组(P0.05);VEGF蛋白水平及IL-8分泌量差异不明显(P0.05)。(3)HMGB1组共培养Walker256侵袭及迁移能力均显著高于对照组(P0.05)。结论:HMGB1可激活KCs向M2表型极化,表现为吞噬功能下降,膜表达CD206增加,TGF-β、MMP-9蛋白表达增加,IL-10分泌升高,IL-6分泌减少,增强共培养大鼠肝癌细胞株迁移及侵袭能力。第二部分:抑制TREM-1对HMGB1诱导Kupffer细胞向M2型极化的影响目的:观察特异抑制TREM-1后HMGB1刺激KCs细胞膜蛋白表达、吞噬功能及分泌功能变化。方法:将获取的KCs以每孔3-4×106接种于培养板中,并随机分为4组:(1)HMGB1组(500ng/ml,HMGB1);(2)LP17+HMGB1组(100μmol/L,LP17+500ng/ml,HMGB1);(3)sh RNA-TREM-1+HMGB1组(sh RNA-TREM-1+HMGB1);(4)sh RNA-NC+HMGB1组(sh RNA-NC+HMGB1)。荧光显微镜鉴定转染效果。分别在处理后24小时收获细胞。流式细胞术检测KCs膜蛋白表面分子F4/80,CD206表达情况。Western blot法检测VEGF,TGF-β,MMP-9等蛋白表达水平。ELISA法检测细胞培养上清液中IL-6,IL-8,IL-10分泌情况。Transwell小室法检测共培养Walker256侵袭及迁移能力变化。重复上述检测。结果:(1)转染sh RNA-TREM-1 24h后仅有少量绿色荧光蛋白表达,48h后绿色荧光蛋白表达明显增加,72h后达高峰,转染率达83%。(2)HMGB1组、sh RNA-NC+HMGB1组KCs膜蛋白CD206比例显著高于LP17+HMGB1组、sh RNA-TREM-1+HMGB1组(P0.05)。HMGB1组及sh RNA-NC+HMGB1组TGF-β、MMP-9蛋白表达水平显著高于LP17+HMGB1组、sh RNA-TREM-1+HMGB1组(P0.05);LP17+HMGB1组与sh RNA-TREM-1+HMGB1组间无显著性差异(P0.05),HMGB1组与sh RNA-TREM-1+HMGB1组间无显著性差异(P0.05)。HMGB1组IL-6水平明显低于LP17+HMGB1组及sh RNA-TREM1组,差异具有统计学意义(P0.05);而与sh RNA-NC组无显著性差异(P0.05)。HMGB1组IL-10蛋白水平(528.6±14.3pg/ml)明显高于LP17+HMGB1组及sh RNA-TREM1组,(P0.05);而与sh RNA-NC组,但差异不明显(P0.05)。(3)HMGB1组共培养Walker256迁移力和侵袭显著高于LP17+HMGB1组和sh RNA-TREM1组(P0.05);而与sh RNA-NC组无显著性差异(P0.05)。结论:HMGB1可引起KCs向M2型极化;沉默或特异性拮抗KCs TREM-1可阻断该作用。提示HMGB1是通过结合KCs上TREM-1发挥胞内信号传导作用的。第三部分:JAK-STAT信号通路在HMGB1/TREM-1激活Kupffer细胞向M2型极化中的作用目的:研究JAK-STAT信号通路在HMGB1/TREM-1诱导KCs向M2型极化中的作用。方法:选取2014年6月至2015年2月到重庆医科大学附属第二医院就诊的HCC行HIFU治疗后患者145例。根据纳入及排除标准,入组HCC行HIFU治疗后患者15例,设立15例健康对照人群,测定外周血HMGB1及s TREM1水平。免疫组化法检测3例HCC行HIFU治疗后再手术切除病灶的肿瘤组织及癌旁组织中HMGB1、TREM-1、DAP12、STAT6、PPARg表达情况。将获取的大鼠KCs以每孔3-4×106接种于培养板中,随机分为以下四组:(1)HMGB1组:(500ng/ml,HMGB1);(2)LP17+HMGB1组:(100μmol/L,LP17+500ng/ml,HMGB1);(3)HMGB1+GW9662组:(25μmol/L,GW9662+HMGB1);(4)对照组(Control组)。各组于处理24h后收获细胞。流式细胞术检测膜蛋白表面分子F4/80,CD206表达情况;Western blot检测DAP12、STAT6、VEGF、TGF-β、MMP-9蛋白表达水平变化;ELISA检测细胞上清液中IL-6、IL-8及IL-10的含量变化。重复上述检测。结果:(1)HCC组外周血HMGB1及s TREM1水平显著高于健康人群组(P0.05)。(2)与癌旁组织相比较,肿瘤组织内HMGB1、TREM1、DAP12、STAT6及PPARγ表达均明显增高(P0.05)。(3)体外细胞实验HMGB1组细胞培养上清液IL-10水平显著高于LP17+HMGB1组、Control组及GW9662+HMGB1组(P0.05);IL-6水平显著低于LP17+HMGB1组、Control组及GW9662+HMGB1组(P0.05)。流式细胞检测结果显示:HMGB1组KCs膜蛋白CD206比例显著高于LP17+HMGB1组、GW9662+HMGB1组及Control组(P0.05)。HMGB1组、GW9662+HMGB1组DAP12蛋白表达量均显著高于Control组、LP17+HMGB1组(P0.05);HMGB1组DAP12蛋白表达量稍高于GW9662+HMGB1组,但差异不明显(P0.05);Control组、LP17+HMGB1组DAP12蛋白表达量,但差异不明显(P0.05)。HMGB1组、GW9662+HMGB1组STAT6蛋白表达量显著均高于Control组、LP17+HMGB1组(P0.05);HMGB1组STAT6蛋白表达量高于HMGB1组STAT6蛋白表达量高于GW9662+HMGB1组,但差异不明显(P0.05);Control组、LP17+HMGB1组STAT6蛋白表达量,但差异不明显(P0.05)。HMGB1组MMP-9、TGF-β蛋白表达量显著均高于Control组、LP17+HMGB1组及GW9662+HMGB1组(P0.05);Control组、LP17+HMGB1组及GW9662+HMGB1组三组间MMP-9、TGF-β蛋白表达量,但差异不明显(P0.05)。结论:(1)肝癌HIFU治疗后外周血存在HMGB1及s TREM1蛋白水平显著升高;癌组织中HMGB1、TREM1、DAP12、STAT6及PPARγ较癌旁组织表达明显增高;(2)刺激KCs 24小时后,KCS向M2型极化;LP17,GW9662可明显抑制HMGB1的刺激作用。(3)HMGB1与TREM-1结合,是通过JAK-STAT6途径激活PPARg,引起KCs向M2型极化。
[Abstract]:The first part: HMGB1 stimulation of Kupper cell post function change objective: To observe the phagocytic function, membrane protein expression and secretory function of Kupffer cell (KCs) after HMGB1 stimulation. Methods: isolate rat KCs, identify the purity of KCs, judge the activity and phagocytosis of KCs, and inoculate the purified KCs with 3-4 x per pore in the culture plate and divide them into 2 groups randomly. (1) HMGB1 stimulation group: 500ng/ml HMGB1 stimulation was given; (2) control group was given PBS stimulation. The cells were harvested after 24 hours of treatment. The phagocytosis of KCs was detected by ink swallowing experiment. Flow cytometry was used to detect the F4/80 of the surface molecule of KCs membrane protein, and the.Western blot method of CD206 expression was tested for Arg-1, VEGF, beta, and other protein expression levels were detected. The secretion of IL-6, IL-8, IL-10 in the supernatant of cell culture was detected by.Transwell cell method, and the changes of the invasion and migration of Walker256 were detected. The results were as follows: (1) the purity of the extracted KCs was more than 91% and the activity was above 97.8%; (2) the M2 phenotypic surface subdivision of KCs in HMGB1 group was obviously higher than that of the control group, and the activity expression of Arg-1 enzyme was obviously higher than that of the control group. The level of IL-6 in cell culture supernatant of control group (P0.05).HMGB1 was significantly lower than that of control group, and the level of IL-10 was significantly higher than that of control group (P0.05).HMGB1 group Arg-1, TGF- beta, MMP-9 protein level was significantly higher than that of control group (P0.05), VEGF protein level and IL-8 secretion difference was not obvious. (3) the invasion and migration ability of the group was significantly higher than that of the control group. Higher than the control group (P0.05). Conclusion: HMGB1 can activate the phenotypic polarization of KCs to M2, showing the decrease of phagocytosis, the increase of membrane expression CD206, the increase of TGF- beta, the increase of MMP-9 protein, the increase of IL-10 secretion, and the decrease of IL-6 secretion. The second part: inhibiting TREM-1 to HMGB1 inducible Kupffer cells Polarization effect Objective: To observe the HMGB1 stimulation of KCs cell membrane protein expression, phagocytic function and secretory function after specific inhibition of TREM-1. Methods: the obtained KCs was inoculated in the culture plate with 3-4 x 106 per pore, and randomly divided into 4 groups: (1) HMGB1 group (500ng/ml, HMGB1); (2) LP17+HMGB1 group (100 mu mol/L, LP17+500ng/ml, HMGB1); (3) sh Group (SH RNA-TREM-1+HMGB1); (4) sh RNA-NC+HMGB1 group (SH RNA-NC+HMGB1). The transfection effect was identified by fluorescence microscope. The cells were harvested at 24 hours after treatment. Flow cytometry was used to detect the surface molecule F4/80 of KCs membrane protein. CD206 expression was detected by.Western blot method. The secretion of IL-6, IL-8 and IL-10 in the liquid was detected by.Transwell chamber method. The changes of the invasion and migration of Walker256 were detected. The results were as follows: (1) only a small amount of green fluorescent protein was expressed after transfection of SH RNA-TREM-1 24h, and the expression of green fluorescent protein was obviously increased after 48h, and the peak was reached after 72h, and the transfection rate reached 83%. (2) HMGB1 group. The ratio of KCs membrane protein CD206 was significantly higher than that of group LP17+HMGB1, and sh RNA-TREM-1+HMGB1 group (P0.05).HMGB1 group and sh RNA-NC+HMGB1 group TGF- beta, MMP-9 protein expression level was significantly higher than that of LP17+HMGB1 group. No significant difference (P0.05) in group.HMGB1 was significantly lower than that in group LP17+HMGB1 and sh RNA-TREM1 (P0.05), but there was no significant difference between group sh RNA-NC (P0.05).HMGB1 group (P0.05).HMGB1 group (528.6 +). (P0.05) (3) (3) the migration force and invasion of co culture of group HMGB1 were significantly higher than that in group LP17+HMGB1 and sh RNA-TREM1 group (P0.05), but there was no significant difference from sh RNA-NC group (P0.05). Conclusion: HMGB1 can cause KCs to polarization polarization; silence or specific antagonism can prevent this effect. The third part: the third part: the role of JAK-STAT signaling pathway in HMGB1/TREM-1 activation of Kupffer cells to M2 polarization: the study of the role of JAK-STAT signaling pathway in HMGB1/TREM-1 induced KCs to M2 polarization. Method: HCC line HIFU treatment from June 2014 to February 2015 According to the inclusion and exclusion criteria, according to the inclusion and exclusion criteria, 15 patients were treated with HCC after HIFU treatment, and 15 healthy controls were set up to determine the level of HMGB1 and s TREM1 in the peripheral blood. 3 cases of HCC were examined for HMGB1, TREM-1, DAP12, STAT6, PPARg expression in the tumor tissues and adjacent tissues after HIFU treatment. The rat KCs was inoculated with 3-4 * 106 per pore in the culture plate, randomly divided into four groups: (1) group HMGB1: (500ng/ml, HMGB1); (2) group LP17+HMGB1: (100 mol/L, LP17+500ng/ml, HMGB1); (3) HMGB1+GW9662 group: (25 micron mol/L, GW9662+HMGB1); (4) the control group (Control group). Flow cytometry was used to detect the surface protein of membrane protein. F4/80, CD206 expression, Western blot detection of DAP12, STAT6, VEGF, TGF- beta, and MMP-9 protein expression levels; ELISA detection of IL-6, IL-8, and concentrations in cell supernatants. Repeat the above tests. (1) the level of peripheral blood and the levels of the peripheral blood were significantly higher than those in the healthy group. (2) compared with the paracancerous tissue, the tumor group was compared. The expression of HMGB1, TREM1, DAP12, STAT6 and PPAR increased significantly in the weave (P0.05). (3) the level of IL-10 in the cell culture supernatant was significantly higher than that in the LP17+HMGB1 group, Control and GW9662+HMGB1 group (P0.05) in the HMGB1 group in vitro, and the level of flow cytometry showed that the level of cell culture was significantly lower than that in the group and GW9662+HMGB1 group. The ratio of KCs membrane protein CD206 was significantly higher than that in group LP17+HMGB1, and the expression of DAP12 protein in group GW9662+HMGB1 and Control group (P0.05) was significantly higher than that in Control group and LP17+HMGB1 group (P0.05). In group GW9662+HMGB1, the expression of STAT6 protein in group GW9662+HMGB1 was significantly higher than that in group Control and group LP17+HMGB1 (P0.05), and the expression of STAT6 protein in HMGB1 group was higher than that of STAT6 protein in HMGB1 group, but the difference was not obvious, but the difference was not obvious. 5) MMP-9, TGF- beta protein expression in group.HMGB1 was significantly higher than that in group Control, LP17+HMGB1 and GW9662+HMGB1 group (P0.05); Control, LP17+HMGB1 and GW9662+HMGB1 groups were MMP-9, TGF- beta protein expression, but the difference was not obvious. The expression of HMGB1, TREM1, DAP12, STAT6 and PPAR in the tissue was significantly higher than that in the para cancerous tissues. (2) after 24 hours of KCs stimulation, KCS was polarized to M2 type; LP17, GW9662 could obviously inhibit HMGB1 stimulation. (3) the combination of HMGB1 and HMGB1 is activated by the pathway.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.5

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