BMSCs通过PGE2抑制Kupffer细胞中NLRP3炎症小体活化减轻内毒素导致的急性肝损伤

发布时间：2018-05-04 19:10

本文选题：Kupffer细胞 + 骨髓间充质干细胞　；参考：《重庆医科大学》2017年博士论文

【摘要】：第一部分:BMSCs在内毒素导致的小鼠急性肝损伤中的保护作用目的:观察静脉给予外源性BMSCs能否抑制内毒素(LPS)诱导的小鼠急性肝损伤以及肝脏KCs中的NLRP3炎症小体活化及血清中的炎症介质表达水平,阐明BMSCs在脓毒症导致的急性肝损伤中的意义及潜在机制。方法:全骨髓细胞选择性贴壁法从Balb/C小鼠胫骨、股骨中分离BMSCs,采用慢病毒转染ptges及ptges sh RNA至BMSCs,建立稳定的ptges过表达BMSCs细胞株(BMSCs-PGE2(+))及ptges沉默的BMSCs细胞株(BMSCs-PGE2(-));30只Balb/C小鼠随机分为5组:(1)对照组,(2)LPS组,(3)LPS+BMSCs组,(4)LPS+BMSCs-PGE2(+)组,(5)LPS+BMSCs-PGE2(-)。对照组小鼠腹膜腔及尾静脉均注射PBS,另外四组小鼠先予以腹膜腔注射LPS(10 mg/kg)处理12小时,然后分别尾静脉注射PBS、BMSCs、BMSCs-PGE2(+)及BMSCs-PGE2(-)(1×106个)处理12小时;获取小鼠肝脏,采用HE染色评估肝脏炎症程度,以TUNEL染色评估肝脏细胞凋亡情况;分离肝脏KCs,通过Western blotting检测KCs中NLRP3,ASC,Pro-Casp1,Caspase-1及Pro-IL-1β蛋白表达水平;荧光实时定量PCR检测KCs中NLRP3,ASC,Pro-Casp1及Caspase-1 m RNA表达水平;收集小鼠血液,ELISA法测定血清中ALT、AST、IL-1β、IL-10及PGE2水平。结果:(1)LPS组肝细胞明显水肿,肝脏内大量炎细胞浸润;LPS+BMSCs组肝细胞水肿减轻,炎细胞浸润减少,LPS+BMSCs-PGE2(+)处理组,肝脏炎症程度进一步减轻,而LPS+BMSCs-PGE2(-)组较LPS组无明显变化。肝脏炎细胞浸润数量于LPS组最多,LPS+BMSCs组明显下降,LPS+BMSCs-PGE2(+)处理后炎细胞数量进一步减少,差异具有统计学意义(P0.05);而LPS+BMSCs-PGE2(-)组较LPS组无明显变化,差异无统计学意义(P0.05)。(2)LPS组中TUNEL染色阳性表现的肝脏凋亡细胞较对照组明显增加,LPS+BMSCs组肝脏凋亡细胞较LPS组明显减少(P0.05),LPS+BMSC-PGE2(+)组肝脏内仅见零星分布的凋亡细胞(P0.05),差异具有统计学意义;而LPS+BMSC-PGE2(-)组,肝脏内凋亡细胞较LPS组无明显变化,差异不具有统计学意义(P0.05)。(3)KCs中NLRP3,ASC及caspase-1 m RNA相对表达量在LPS组最高,LPS+BMSC组较LPS组明显下降,LPS+BMSC-PGE2(+)组较LPS+BMSC组进一步下降,差异具有统计学意义(P0.05);与LPS组比较,LPS+BMSC-PGE2(-)组无明显下降,差别不具有统计学意义(P0.05)。(4)KCs中NLRP3,ASC,Pro-Casp1,Caspase-1及Pro-IL-1β蛋白表达量在LPS组最高,LPS+BMSC组较LPS组明显减低,LPS+BMSC-PGE2(+)组较LPS+BMSC组进一步下降,差异具有统计学意义;而LPS+BMSC-PGE2(-)组较LPS组无明显变化(P0.05)。(5)血清PGE2水平在对照组最低,LPS组略有升高;LPS+BMSC组较LPS组明显升高,差异具有统计学意义(P0.05);LPS+BMSC-PGE2(+)组最高,明显高于LPS+BMSCs组,差异具有统计学意义(P0.05);而在LPS+BMSC-PGE2(-)组与LPS组及空白对照组无明显差异(P0.05)。(6)血清ALT,AST及IL-1β浓度在LPS组明显最高;LPS+BMSC组较LPS组明显降低,LPS+BMSC-PGE2(+)组较LPS+BMSC组进一步下降,差异均具有统计学意义(P0.05);而LPS+BMSC-PGE2(-)组较LPS组无明显下降,差异无统计学意义(P0.05)。血清IL-10浓度在LPS组较空白对照组轻度上升;LPS+BMSC组明显高于LPS组;LPA+BMSC-PGE2(+)组较BMSCs处理组进一步升高,差异具有统计学意义(P0.05);而在LPS+BMSC-PGE2(-)组,IL-10水平较LPS组无明显变化(P0.05)。结论:尾静脉注射BMSCs可以减轻腹膜腔注射LPS诱导的小鼠急性肝损伤,并可抑制LPS诱导的小鼠肝脏KCs中NLRP3炎症小体的表达;过表达ptges增加了BMSCs分泌PGE2的能力,并加强了BMSCs的肝脏保护作用及对肝脏KCs中NLRP3炎症小体的抑制。第二部分:观察BMSCs对Kupffer细胞中NLRP3炎症小体活化的影响目的:进一步证实BMSCs可以直接抑制的KCs中NLRP3炎症小体的活化,观察BMSCs是否通过分泌PGE2达到抑制KCs中NLRP3炎症小体的活化。方法:KCs以2×106/皿接种在Transwell上室中,随机分为5组,对照组,LPS处理组,LPS+BMSC组,LPS+BMSC-PGE2(+)组及LPS+BMSC-PGE2(-)组,共培养三组中将BMSC/BMSC-PGE2(+)/BMSC-PGE2(-)以1×106/皿种于Transwell下室。根据分组将KCs与BMSC/BMSC-PGE2(+)/BMSC-PGE2(-)置入Tranwell共培养体系,LPS组及共培养体系组予以终浓度为10 ng/ml LPS的无血清培养基共培养5 h,然后以终浓度为5m M的ATP的无血清培养基共培养1h。以Western blotting检测KCs中NLRP3,ASC,Pro-Casp1,Caspase-1及Pro-IL-1β蛋白表达水平,RT-q PCR检测KCs中NLRP3,ASC及Caspase-1 m RNA表达水平,ELISA检测培养上清中PGE2、IL-1β、IL-18、IL-10及TNF-α。结果:(1)NLRP3,ASC及caspase-1 m RNA相对表达量在LPS组中较对照组明显上升(P0.05);与LPS组相比较,LPS+BMSC组明显下降,并在LPS+BMSC-PGE2(+)组进一步下降;然而与LPS组比较,LPS+BMSC-PGE2(-)组中NLRP3,ASC及caspase-1 m RNA相对表达量并未明显下降(P0.05)。(2)NLRP3,ASC,Pro-Casp1,Caspase-1及Pro-IL-1β蛋白表达量在LPS组中较空白对照组明显升高(P0.05);与LPS组相比较,在LPS+BMSC组中的表达明显减低,且在LPS+BMSC-PGE2(+)组进一步下降(P0.05);而LPS+BMSC-PGE2(-)组较LPS组无明显变化(P0.05)。(3)培养上清液中PGE2水平LPS组较空白对照组略有上升;LPS+BMSC组较LPS组明显升高(P0.05);LPS+BMSC-PGE2(+)组最高,明显高于LPS+BMSCs组(P0.05);而在LPS+BMSC-PGE2(-)组显著低于LPS+BMSC-PGE2(+)组(P0.05),且与LPS组及空白对照组无明显差异(P0.05)。(4)培养上清液IL-1β,IL-18及TNF-α浓度在LPS组最高;在LPS+BMSCs组较LPS组明显降低,在LPS+BMSC-PGE2(+)组进一步下降(P0.05);而LPS+BMSC-PGE2(-)组较LPS组无明显下降(P0.05)。培养上清液中IL-10水平在LPS组较空白对照组轻度升高,在给予BMSCs处理后,IL-10明显增加,给予BMSC-PGE2(+)处理后,IL-10水平进一步升高(P0.05);而在LPS+BMSC-PGE2(-)组,IL-10水平较LPS组无明显变化(P0.05)。结论:共培养实验进一步证实BMSCs拥有抑制LPS+ATP诱导的KCs中NLRP3炎症小体活化的作用;过表达PGE2加强了BMSCs对NLRP3炎症小体的抑制作用,沉默PGE2抑制了BMSCs对NLRP3炎症小体的抑制作用。第三部分:PGE2抑制Kupffer细胞中NLRP3炎症小体活化的分子机制目的:为探索PGE2是直接抑制了KCs中NLRP3炎症小体,还是通过促使KCs增加IL-10的分泌而间接抑制了NLRP3炎症小体的活化。方法:KCs随机分为4组:Group A为对照组;Group B予以LPS(10ng/ml)处理5小时,然后在予以ATP(5m M)处理1小时;Group C在Group B组处理因素基础上,于最后30分钟加入PGE2(终浓度为0.1m M);Group D在C组处理因素基础上,于PGE2加入前30分钟加入FR180204)。Western blotting检测KCs中NLRP3,ASC,Caspase-1及p-ERK1蛋白表达水平。RT-q PCR检测KCs中NLRP3,ASC及Caspase-1 m RNA。ELISA检测培养上清中IL-1β,IL-18,TNF-α及IL-10水平。结果:(1)在B组中给予LPS+ATP处理后,NLRP3,ASC及Caspase-1蛋白表达水平较A组明显升高(P0.05);p-ERK1蛋白表达水平较A组无明显变化;C组NLRP3,ASC及Caspase-1蛋白表达水平较B组明显降低,p-ERK1蛋白表达水平较及B组明显增高;D组,NLRP3,ASC及Caspase-1蛋白表达水平较C组升高(P0.05),但仍略低于B组(P0.05);p-ERK1蛋白表达水平较C组显著降低,(P0.05),与B组差异不明显(P0.05)。(2)NLRP3,ASC及caspase-1m RNA相对表达量与蛋白表达趋势基本一致。(3)培养上清液中IL-1β,IL-18及TNF-α浓度在LPS+ATP处理的B组中明显最高,显著高于A组及C组(P0.05);C组中给予PGE2处理后以上促炎因子较B组明显降低(P0.05);D组中给予FR180204处理后IL-1β,IL-18及TNF-α水平明显升高,与B组相当(P0.05),显著高于C组(P0.05)。(4)培养上清液中IL-10 LPS+ATP处理的B组较A组略有升高,C组中给予PGE2处理后IL-10较B组明显升高(P0.05);D组中给予FR180204处理后IL-10水平显著底于C组(P0.05),恢复至B组水平(P0.05)。结论:KCs中ERK1信号通路介导的IL-10分泌可能是PGE2抑制NLRP3炎症小体活化的关键点。
[Abstract]:Part one: the protective effect of BMSCs in acute liver injury induced by endotoxin: whether intravenous administration of exogenous BMSCs can inhibit acute liver injury induced by endotoxin (LPS) in mice and the activation of NLRP3 inflammatory corpuscles in the liver KCs and the level of inflammatory mediators in serum, and to clarify the acute liver caused by BMSCs in sepsis Methods: all bone marrow cells were selectively attached to the bone marrow cells from the tibia and the femur of Balb/C mice. BMSCs was isolated from the tibia and femur of the Balb/C mice. The stable ptges and ptges sh RNA to BMSCs were used to establish a stable ptges overexpressed BMSCs cell line (BMSCs-PGE2 (+)) and ptges silent BMSCs cell line. 5 groups: (1) control group, (2) LPS group, (3) LPS+BMSCs group, (4) LPS+BMSCs-PGE2 (+) group, (5) LPS+BMSCs-PGE2 (-). PBS was injected into the peritoneal cavity and tail vein of the control group, the other four groups were injected with LPS (10 mg/kg) for 12 hours, then the tail vein was injected with PBS, BMSCs, BMSCs-PGE2 (+) and BMSCs-PGE2 (1 * 106) treatment 12 HE staining was used to evaluate the degree of liver inflammation, TUNEL staining was used to evaluate the liver cell apoptosis, and the liver KCs was isolated and the expression level of NLRP3, ASC, Pro-Casp1, Caspase-1 and Pro-IL-1 beta protein in KCs was detected by Western blotting. The blood of mice was collected and the serum levels of ALT, AST, IL-1 beta, IL-10 and PGE2 were measured by ELISA. Results: (1) there were obvious edema of the liver cells in the LPS group and the infiltration of large number of inflammatory cells in the liver; the edema of the liver cells in the group LPS+BMSCs, the decrease of inflammatory cell infiltration, the group of LPS+BMSCs-PGE2 (+), the degree of liver inflammation were further reduced, and the LPS+BMSCs-PGE2 (-) group was more than the LPS group. There was no obvious change in the number of liver inflammatory cells in the LPS group, the LPS+BMSCs group decreased significantly, and the number of inflammatory cells decreased further after LPS+BMSCs-PGE2 (+) treatment, and the difference was statistically significant (P0.05), but there was no significant difference between the LPS+BMSCs-PGE2 (-) group and the LPS group (P0.05). (2) the positive expression of the liver in LPS group was positive. The apoptotic cells in the LPS+BMSCs group were significantly lower than those in the control group (P0.05). The apoptotic cells in the LPS+BMSC-PGE2 (+) group had only sporadic apoptotic cells (P0.05) in the liver of LPS+BMSC-PGE2 (+) group, and the difference was statistically significant in the LPS+BMSC-PGE2 (-) group, and there was no significant difference in the number of apoptotic cells in the liver (P0.), and the difference was not statistically significant (P0.). 05) (3) the relative expression of NLRP3, ASC and caspase-1 m RNA in the KCs group was the highest in the LPS group, and the LPS+BMSC group was significantly lower than the LPS group. The LPS+BMSC-PGE2 (+) group was further decreased than the LPS+BMSC group, and the difference was statistically significant (P0.05). The expression of -Casp1, Caspase-1 and Pro-IL-1 beta protein in the LPS group was the highest, the LPS+BMSC group was significantly lower than the LPS group, and the LPS+BMSC-PGE2 (+) group was further decreased than that in the LPS+BMSC group. The LPS+BMSC-PGE2 (-) group had no significant difference compared with the LPS group (P0.05). (5) the level of the serum PGE2 was the lowest in the control group and a slight increase in the control group. The difference was statistically significant (P0.05), and LPS+BMSC-PGE2 (+) group was the highest, obviously higher than group LPS+BMSCs (P0.05), but there was no significant difference between LPS+BMSC-PGE2 (-) group and LPS group and blank control group (P0.05). (6) the concentration of serum ALT, AST and IL-1 beta in LPS group was the highest, and the LPS+BMSC group was significantly lower than those in the LPS+BMSC group. The PS+BMSC-PGE2 (+) group was further lower than the LPS+BMSC group, and the difference was statistically significant (P0.05), while the LPS+BMSC-PGE2 (-) group had no significant decrease compared with the LPS group (P0.05). The serum IL-10 concentration was slightly higher in the LPS group than that in the blank control group; the LPS+BMSC group was significantly higher than the LPS group; LPA+BMSC-PGE2 (+) group was further than the BMSCs treatment group. The difference was statistically significant (P0.05), but in the LPS+BMSC-PGE2 (-) group, there was no significant change in the level of IL-10 in the group LPS (P0.05). Conclusion: the tail vein injection of BMSCs could reduce the acute liver injury induced by peritoneal injection of LPS in mice and inhibit the expression of NLRP3 inflammatory corpuscles in LPS induced mice liver KCs, and the ptges increased the BMSCs fraction. The ability to secrete PGE2 and enhance the protective effect of BMSCs liver and the inhibition of NLRP3 inflammatory bodies in the liver KCs. Second: To observe the effect of BMSCs on the activation of NLRP3 inflammatory corpuscles in Kupffer cells: to further confirm the activation of BMSCs that can directly inhibit the inflammatory corpuscle of NLRP3 in KCs, and to observe whether BMSCs is suppressed through secretory PGE2. Methods: the activation of NLRP3 inflammatory body in KCs. Methods: KCs was inoculated in the upper room of Transwell with 2 x 106/. It was randomly divided into 5 groups, the control group, LPS treatment group, LPS+BMSC group, LPS+BMSC-PGE2 (+) group and LPS+BMSC-PGE2 (-) group. The BMSC/BMSC-PGE2 (+) /BMSC-PGE2 (-) was planted in the lower chamber in the three groups. GE2 (+) /BMSC-PGE2 (+) was implanted into the Tranwell co culture system. The LPS group and the co culture system group gave a total concentration of 10 ng/ml LPS in a serum-free medium. Q PCR detected the expression level of NLRP3, ASC and Caspase-1 m RNA in KCs, and PGE2, IL-1 beta, IL-18, and alpha in the ELISA detection culture. Compared with the LPS group, the relative expression of NLRP3, ASC and caspase-1 m RNA in the LPS+BMSC-PGE2 (-) group was not significantly decreased (P0.05). (2) NLRP3, ASC, Pro-Casp1, the expression of Caspase-1 and beta protein in the group was significantly higher than that in the blank control group. The group was further decreased (P0.05), while the LPS+BMSC-PGE2 (-) group had no obvious changes in the group LPS (P0.05). (3) the PGE2 level in the culture supernatant was slightly higher than that in the blank control group; the LPS+BMSC group was significantly higher than that in the LPS group (P0.05), and the LPS+BMSC-PGE2 (+) group was higher than the LPS+BMSCs group (P0.05), and the group (+) was significantly lower than that in the LPS+BMSCs group (+). Group (P0.05), and no significant difference between group LPS and blank control group (P0.05). (4) the concentration of IL-1 beta, IL-18 and TNF- alpha in the culture supernatant was the highest in LPS group, and decreased in LPS+BMSCs group compared with LPS group, and decreased in LPS+BMSC-PGE2 (+) group (P0.05), while LPS+BMSC-PGE2 (-) group had no obvious decline. Compared with the blank control group, the IL-10 increased significantly after BMSCs treatment, and the level of IL-10 increased further after BMSC-PGE2 (+) treatment (P0.05). In LPS+BMSC-PGE2 (-) group, there was no significant change in IL-10 level compared with that of LPS group (P0.05). Conclusion: co culture tests further confirmed that BMSCs has the inhibition of inflammatory corpuscles in LPS+ATP induced KCs. Activation effect; overexpression of PGE2 enhanced the inhibitory effect of BMSCs on NLRP3 inflammatory bodies. Silence PGE2 inhibited the inhibitory effect of BMSCs on NLRP3 inflammatory bodies. The third part: PGE2 inhibits the molecular mechanism of NLRP3 inflammatory corpuscle activation in Kupffer cells: To explore PGE2 is to directly inhibit KCs NLRP3 inflammatory corpuscles, or by prompting them. Increase the secretion of IL-10 and indirectly inhibit the activation of NLRP3 inflammatory bodies. Methods: KCs was randomly divided into 4 groups: Group A as the control group; Group B was treated with LPS (10ng/ml) for 5 hours, and then treated with ATP (5m M) for 1 hours. On the basis of the factors,.Western blotting was used to detect NLRP3, ASC, Caspase-1 and p-ERK1 protein expression level in KCs 30 minutes before the addition of FR180204), and.RT-q PCR detection of the protein expression level in KCs. The expression level of SE-1 protein was significantly higher than that in the A group (P0.05), and the expression level of p-ERK1 protein was not significantly higher than that in the A group; the expression level of NLRP3, ASC and Caspase-1 protein in the C group was significantly lower than that in the B group, and the expression level of p-ERK1 protein was significantly higher than that of the B group. The expression level of -ERK1 protein was significantly lower than that in the C group (P0.05), and there was no significant difference with the B group (P0.05). (2) NLRP3, the RNA relative expression of ASC and caspase-1m was the same as that of protein expression. (3) the concentration of IL-1 beta, IL-18 and TNF- alpha in the culture supernatant was the highest, significantly higher than that in the group and the group. The above proinflammatory factors were significantly lower than those in the B group (P0.05), and the level of IL-1 beta, IL-18 and TNF- alpha in the group D was significantly higher than that in the B group (P0.05), which was significantly higher than that of the C group (P0.05). (4) the groups in the culture supernatant were slightly higher than those in the group. After treatment with FR180204, the level of IL-10 was significantly lower than that of group C (P0.05) and recovered to B level (P0.05). Conclusion: IL-10 secretion mediated by ERK1 signaling pathway in KCs may be the key point for PGE2 to inhibit the activation of NLRP3 inflammatory corpuscle.

【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】：R459.7

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