泛素化对多器官功能障碍综合征脾脏树突状细胞功能的调节作用与机制研究
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摘要
目的：研究多器官功能障碍综合征（multiple organs dysfunction syndrome，MODS）病程中脾脏树突状细胞（dendritic cells，DC）的变化及MHC-Ⅱ蛋白的泛素依赖性作用，证实泛素-蛋白酶体途径在MODS病程中对脾脏DC MHC-Ⅱ变化及DC功能的影响，探讨E3泛素连接酶MARCH1在MODS第一次打击期对脾脏DC MHC-Ⅱ泛素化调节的作用机制，并观察干预MARCH1对MODS第一次打击期脾脏DC免疫功能的影响，进而为MODS的免疫调节与防治寻找新的靶点。
方法：采用腹腔注射酵母多糖复制MODS小鼠模型。120只小鼠随机分为正常对照组和MODS1d、3d、5d、7d和10d组。各组实验小鼠分别分离脾脏和胸腺组织，一部分脾脏应用免疫荧光方法检测CD11c+DC数量的变化，剩余组织应用MiniMACS免疫磁珠分别分离DC和T细胞用于体外细胞学实验。运用流式细胞术、Real-Time PCR、Western Blot和MTT法等检测DC对CD86、MHC-Ⅱ、泛素、E3泛素连接酶（MARCH1-8）的表达，DC分泌IL-12、TNF-α的水平以及诱导T细胞增殖和分泌IL-2、IFN-γ的水平。接着运用免疫共沉淀及WesternBlot检测正常对照组和MODS1d组DC MHC-Ⅱ蛋白泛素化水平，并用MG132分别处理两组DC各6h，运用PI染色法、Real-Time PCR、Western Blot和MTT法等检测处理前后各组DC的凋亡率，DC对MHC-Ⅱ蛋白的表达以及诱导T细胞增殖和分泌IL-2、IFN-γ、TNF-α的水平。通过从小鼠尾部提取cDNA作为模板，采用特异的引物，运用聚合酶链式反应（polymerase chain reaction，PCR）技术扩增出MARCH1cDNA片段，EcoRⅠ和BamHⅠ双酶切后，连接于载体pMB-HA质粒上，构建pMB-HA-MARCH1重组载体，经双酶切和DNA测序分析检测重组载体构建结果，转化大肠埃希菌DH5α感受态菌、筛选阳性克隆、抽提质粒并进行纯化。将pMB-HA-MARCH1表达载体和SiRNA分别转染进MODS1d组DC，运用MTT法、PI染色法、流式细胞术、Real-Time PCR、免疫共沉淀及Western Blot等比较MODS1d组、MARCH1siRNA敲低组和MARCH1过表达组DC的形态、活力和凋亡率，DC对MHC-Ⅱ蛋白的表达和泛素化水平以及诱导T细胞增殖和分泌IL-2、IFN-γ、TNF-α的水平。
结果：MODS病程中CD11c+DC数量逐渐减少，DC表面MHC-Ⅱ和CD86蛋白，DC分泌的IL-12、TNF-α和其介导的T细胞增殖活性以及T细胞分泌的IL-2和IFN-γ的表达均为先升高，在MODS1d组达到峰值，此后下降，至MODS期降到最低点，但MHC-Ⅱ和泛素的mRNA水平变化均不明显。而E3泛素连接酶中MARCH1的变化最显著，mRNA和蛋白水平表达均为先下降，在MODS1d组达到最低值，后略有升高趋势，直至MODS10d组又有所下降。在MODS病程中MHC-Ⅱ与MARCH1的变化趋势大致相反。MODS1d组脾脏DC MHC-Ⅱ蛋白泛素化程度较正常对照组显著降低，MG132处理前后两组DC凋亡率无明显变化，而DC MHC-Ⅱ蛋白的表达以及诱导T细胞增殖和分泌IL-2、IFN-γ、TNF-α的水平均为MODS1d组与MG132处理后的正常对照组比正常对照组高，其中前者比后者稍高，MG132处理后的MODS1d组为最高。成功构建了E3泛素连接酶MARCH1的真核表达载体pMB-HA-MARCH1，并合成了3条效果比较理想的MARCH1的siRNA。对MODS1d组DC进行MARCH1过表达和siRNA敲低干预，发现三组DC的形态、活力和凋亡率均无明显变化，而MARCH1过表达干预可以增加MHC-Ⅱ的泛素化程度，减少MHC-Ⅱ的蛋白表达水平，并降低DC诱导T细胞增殖和分泌IL-2、IFN-γ、TNF-α的功能；而MARCH1siRNA敲低干预则相反可以降低MHC-Ⅱ的泛素化程度，增加MHC-Ⅱ的蛋白表达水平，并增强DC诱导T细胞增殖和分泌IL-2、IFN-γ、TNF-α的功能。
结论：1、在MODS第一次打击期DC的数量虽有下降，但其抗原提呈功能与激活T细胞介导免疫应答反应的能力却是增强的；而到了MODS期，DC的数量及功能均降至最低，机体处于免疫抑制状态。2、在MODS病程中，是MARCH1通过泛素化降解MHC-Ⅱ减少了小鼠脾脏DC MHC-Ⅱ的表达。3、在MODS中脾脏DC MHC-Ⅱ的变化确实是具有泛素依赖性。4、成功构建了E3泛素连接酶MARCH1的真核表达载体pMB-HA-MARCH1，并合成了3条效果较理想的MARCH1的siRNA。5、在MODS病程第一次打击期，脾脏DC MHC-Ⅱ过表达的原因与MARCH1对MHC-Ⅱ的泛素化降解程度较低有关。提示病程初期用MARCH1过表达干预，可以作为防止MODS免疫机能过亢和炎症反应过度的一个潜在的治疗靶点。
Objective:To study the changes of spleen dendritic cells(DC) and the ubiquitin-dependent effects of MHC-Ⅱ protein in the course of multiple organ dysfunctionsyndrome(MODS). To confirm the influences of MHC-Ⅱ and spleen DC in the courseof MODS by the ubiquitin-proteasome pathway. To explore the ubiquitinationregulatory mechanisms of MHC-Ⅱ in spleen DC during the first strike period ofMODS by the E3ubiquitin ligase enzyme MARCH1. To observe the influences ofsplenic DC immune function during the first strike period of MODS by theintervention of MARCH1, and to find a new target for the immune regulation andprevention of MODS.
Methods: MODS mice model were established by intraperitoneal injection ofzymosan.120mice were randomly divided into normal control group and the MODS1d,3d,5d,7d and10d groups. Spleen and thymus tissue samples of each group wereisolated, part of the spleen tissue samples were used to detect the changes of thenumber of CD11c+DC by immunofluorescence method, and the rest tissue sampleswere used to separate Spleen DC and T cells for cytological experiments in vitro byMiniMACS microbeads. The expression of CD86, MHC-Ⅱ, ubiquitin and E3ubiquitin ligase (MARCH1-8) on DC, the levels of IL-12and TNF-α secreted by DC,the proliferative activity of T cells and the levels of IL-2and IFN-γ secreted by T cellswere detected by flow cytometry, Real-Time PCR, Western blot and MTT method.Then the ubiquitination levels of MHC-Ⅱ proteins in DC of the normal control groupand MODS1d group were detected by co-immunoprecipitation and western Blot.After treating the two groups with MG132respectively for6h, we detected theapoptosis rate of DC, the expression of MHC-Ⅱ protein on DC, the proliferative activity of T cells and the levels of IL-2, IFN-γ and TNF-α secreted by T cells by PIstaining, Real-Time PCR, Western blot and MTT method. Cellular cDNA wasextracted from the tail of mouse as a template. The MARCH1cDNA was synthesizedby PCR with the specific primers. After restriction endonuclease EcoRⅠand BamHⅠdouble digested it，the MARCH1cDNA was cloned in pMB-HA vector to constructrecombinant vectors. The result of recombinant vector was identified usingdouble-digestion and DNA sequencing. Then we transformed the recombinant vectorinto competent E.coli DH5α cells, screened positive clones, extracted plasmid, andpurified it. The pMB-HA-MARCH1expression vectors and siRNA were transfectedinto DC of MODS1d group. The morphology, viability and apoptosis rate of DC, theexpression of MHC-Ⅱ proteins on DC, the proliferative activity of T cells and thelevels of IL-2, IFN-γ and TNF-α secreted by T cells were detected by MTT, PIstaining, flow cytometry, Real-Time PCR, co-immunoprecipitation and Western Blotin MODS1d group, MARCH1siRNA knock down group and MARCH1overexpression group.
Result:The number of CD11c+DC in the course of MODS was graduallyreduced. The expression of CD86and MHC-Ⅱ proteins on DC, the levels of IL-12and TNF-α secreted by DC, the proliferative activity of T cells and the levels of IL-2and IFN-γ secreted by T cells were increased first, and reached a maximum value inthe MODS1d group, thereafter decreased to the lowest point in the MODS period, butthe mRNA levels of MHC-Ⅱ and ubiquitin were not significant. The changes of E3ubiquitin ligase enzyme MARCH1were extremely significant. The levels of mRNAand protein expression of MARCH1were decreased first and reached a minimumvalue in the MODS1d group, then after a slight increasing trend they were decreasedagain in the MODS10d group. The trend of MHC-Ⅱ and MARCH1was roughly theopposite in the course of MODS. Compared with normal control group the degree ofspleen DC MHC-Ⅱ protein ubiquitination in the MODS1d group was significantlylower. The change of apoptosis rate of DC in the groups treated by MG132was notsignificant. The expression of MHC-Ⅱ proteins on DC, the proliferative activity of Tcells and the levels of IL-2, IFN-γ and TNF-α secreted by T cells in the MODS1dgroup and MG132treated normal control group were higher than the normal controlgroup, where the former is slightly higher than the latter, and the MODS1d grouptreated by MG132was the highest. We successfully constructed the eukaryoticexpression vector pMB HA-MARCH1of the E3ubiquitin ligase MARCH1, and synthesized three effective siRNA of MARCH1. We interfered DC in the MODS1dgroup with overexpression MARCH1and siRNA, and found that the morphology,vitality, and apoptosis rates of DC in the three groups had no significant changes.Thedegree of ubiquitination of MHC-Ⅱ was increased, and the expression of MHC-Ⅱproteins on DC, the proliferative activity of T cells and the levels of IL-2, IFN-γ andTNF-α secreted by T cells were reduced by interfered DC in the MODS1d group withoverexpression MARCH1. While the degree of ubiquitination of MHC-Ⅱ was reduced,and the expression of MHC-Ⅱ proteins on DC, the proliferative activity of T cells andthe levels of IL-2, IFN-γ and TNF-α secreted by T cells were increased by interferedDC in the MODS1d group with MARCH1siRNA.
Conclusion:1、The number of DC was decreased, but the antigen-presentingability and the capability to activate T cell-mediated immune response of DC wereenhanced during the first strike period of MODS; the number and the immunefunctions of DC dropped to the lowest and the organism was in a state of immunesuppression during the MODS period.2、MARCH1can degrade the expression ofMHC-Ⅱ on spleen DC by ubiquitination in the course of MODS.3、The change ofMHC-Ⅱ on spleen DC in MODS was really ubiquitin-dependent.4、We successfullyconstructed the eukaryotic expression vector pMB HA-MARCH1of the E3ubiquitinligase MARCH1, and synthesized three effective siRNA of MARCH1.5、Interferingthe expression of MARCH1can change the expression of MHC-Ⅱ on DC and theimmune function of DC by degrading MHC-Ⅱ through ubiquitination in the firststrike period of MODS. It suggested that we can use MARCH1as a potentialtherapeutic target for MODS.
方法：采用腹腔注射酵母多糖复制MODS小鼠模型。120只小鼠随机分为正常对照组和MODS1d、3d、5d、7d和10d组。各组实验小鼠分别分离脾脏和胸腺组织，一部分脾脏应用免疫荧光方法检测CD11c+DC数量的变化，剩余组织应用MiniMACS免疫磁珠分别分离DC和T细胞用于体外细胞学实验。运用流式细胞术、Real-Time PCR、Western Blot和MTT法等检测DC对CD86、MHC-Ⅱ、泛素、E3泛素连接酶（MARCH1-8）的表达，DC分泌IL-12、TNF-α的水平以及诱导T细胞增殖和分泌IL-2、IFN-γ的水平。接着运用免疫共沉淀及WesternBlot检测正常对照组和MODS1d组DC MHC-Ⅱ蛋白泛素化水平，并用MG132分别处理两组DC各6h，运用PI染色法、Real-Time PCR、Western Blot和MTT法等检测处理前后各组DC的凋亡率，DC对MHC-Ⅱ蛋白的表达以及诱导T细胞增殖和分泌IL-2、IFN-γ、TNF-α的水平。通过从小鼠尾部提取cDNA作为模板，采用特异的引物，运用聚合酶链式反应（polymerase chain reaction，PCR）技术扩增出MARCH1cDNA片段，EcoRⅠ和BamHⅠ双酶切后，连接于载体pMB-HA质粒上，构建pMB-HA-MARCH1重组载体，经双酶切和DNA测序分析检测重组载体构建结果，转化大肠埃希菌DH5α感受态菌、筛选阳性克隆、抽提质粒并进行纯化。将pMB-HA-MARCH1表达载体和SiRNA分别转染进MODS1d组DC，运用MTT法、PI染色法、流式细胞术、Real-Time PCR、免疫共沉淀及Western Blot等比较MODS1d组、MARCH1siRNA敲低组和MARCH1过表达组DC的形态、活力和凋亡率，DC对MHC-Ⅱ蛋白的表达和泛素化水平以及诱导T细胞增殖和分泌IL-2、IFN-γ、TNF-α的水平。
结果：MODS病程中CD11c+DC数量逐渐减少，DC表面MHC-Ⅱ和CD86蛋白，DC分泌的IL-12、TNF-α和其介导的T细胞增殖活性以及T细胞分泌的IL-2和IFN-γ的表达均为先升高，在MODS1d组达到峰值，此后下降，至MODS期降到最低点，但MHC-Ⅱ和泛素的mRNA水平变化均不明显。而E3泛素连接酶中MARCH1的变化最显著，mRNA和蛋白水平表达均为先下降，在MODS1d组达到最低值，后略有升高趋势，直至MODS10d组又有所下降。在MODS病程中MHC-Ⅱ与MARCH1的变化趋势大致相反。MODS1d组脾脏DC MHC-Ⅱ蛋白泛素化程度较正常对照组显著降低，MG132处理前后两组DC凋亡率无明显变化，而DC MHC-Ⅱ蛋白的表达以及诱导T细胞增殖和分泌IL-2、IFN-γ、TNF-α的水平均为MODS1d组与MG132处理后的正常对照组比正常对照组高，其中前者比后者稍高，MG132处理后的MODS1d组为最高。成功构建了E3泛素连接酶MARCH1的真核表达载体pMB-HA-MARCH1，并合成了3条效果比较理想的MARCH1的siRNA。对MODS1d组DC进行MARCH1过表达和siRNA敲低干预，发现三组DC的形态、活力和凋亡率均无明显变化，而MARCH1过表达干预可以增加MHC-Ⅱ的泛素化程度，减少MHC-Ⅱ的蛋白表达水平，并降低DC诱导T细胞增殖和分泌IL-2、IFN-γ、TNF-α的功能；而MARCH1siRNA敲低干预则相反可以降低MHC-Ⅱ的泛素化程度，增加MHC-Ⅱ的蛋白表达水平，并增强DC诱导T细胞增殖和分泌IL-2、IFN-γ、TNF-α的功能。
结论：1、在MODS第一次打击期DC的数量虽有下降，但其抗原提呈功能与激活T细胞介导免疫应答反应的能力却是增强的；而到了MODS期，DC的数量及功能均降至最低，机体处于免疫抑制状态。2、在MODS病程中，是MARCH1通过泛素化降解MHC-Ⅱ减少了小鼠脾脏DC MHC-Ⅱ的表达。3、在MODS中脾脏DC MHC-Ⅱ的变化确实是具有泛素依赖性。4、成功构建了E3泛素连接酶MARCH1的真核表达载体pMB-HA-MARCH1，并合成了3条效果较理想的MARCH1的siRNA。5、在MODS病程第一次打击期，脾脏DC MHC-Ⅱ过表达的原因与MARCH1对MHC-Ⅱ的泛素化降解程度较低有关。提示病程初期用MARCH1过表达干预，可以作为防止MODS免疫机能过亢和炎症反应过度的一个潜在的治疗靶点。
Objective:To study the changes of spleen dendritic cells(DC) and the ubiquitin-dependent effects of MHC-Ⅱ protein in the course of multiple organ dysfunctionsyndrome(MODS). To confirm the influences of MHC-Ⅱ and spleen DC in the courseof MODS by the ubiquitin-proteasome pathway. To explore the ubiquitinationregulatory mechanisms of MHC-Ⅱ in spleen DC during the first strike period ofMODS by the E3ubiquitin ligase enzyme MARCH1. To observe the influences ofsplenic DC immune function during the first strike period of MODS by theintervention of MARCH1, and to find a new target for the immune regulation andprevention of MODS.
Methods: MODS mice model were established by intraperitoneal injection ofzymosan.120mice were randomly divided into normal control group and the MODS1d,3d,5d,7d and10d groups. Spleen and thymus tissue samples of each group wereisolated, part of the spleen tissue samples were used to detect the changes of thenumber of CD11c+DC by immunofluorescence method, and the rest tissue sampleswere used to separate Spleen DC and T cells for cytological experiments in vitro byMiniMACS microbeads. The expression of CD86, MHC-Ⅱ, ubiquitin and E3ubiquitin ligase (MARCH1-8) on DC, the levels of IL-12and TNF-α secreted by DC,the proliferative activity of T cells and the levels of IL-2and IFN-γ secreted by T cellswere detected by flow cytometry, Real-Time PCR, Western blot and MTT method.Then the ubiquitination levels of MHC-Ⅱ proteins in DC of the normal control groupand MODS1d group were detected by co-immunoprecipitation and western Blot.After treating the two groups with MG132respectively for6h, we detected theapoptosis rate of DC, the expression of MHC-Ⅱ protein on DC, the proliferative activity of T cells and the levels of IL-2, IFN-γ and TNF-α secreted by T cells by PIstaining, Real-Time PCR, Western blot and MTT method. Cellular cDNA wasextracted from the tail of mouse as a template. The MARCH1cDNA was synthesizedby PCR with the specific primers. After restriction endonuclease EcoRⅠand BamHⅠdouble digested it，the MARCH1cDNA was cloned in pMB-HA vector to constructrecombinant vectors. The result of recombinant vector was identified usingdouble-digestion and DNA sequencing. Then we transformed the recombinant vectorinto competent E.coli DH5α cells, screened positive clones, extracted plasmid, andpurified it. The pMB-HA-MARCH1expression vectors and siRNA were transfectedinto DC of MODS1d group. The morphology, viability and apoptosis rate of DC, theexpression of MHC-Ⅱ proteins on DC, the proliferative activity of T cells and thelevels of IL-2, IFN-γ and TNF-α secreted by T cells were detected by MTT, PIstaining, flow cytometry, Real-Time PCR, co-immunoprecipitation and Western Blotin MODS1d group, MARCH1siRNA knock down group and MARCH1overexpression group.
Result:The number of CD11c+DC in the course of MODS was graduallyreduced. The expression of CD86and MHC-Ⅱ proteins on DC, the levels of IL-12and TNF-α secreted by DC, the proliferative activity of T cells and the levels of IL-2and IFN-γ secreted by T cells were increased first, and reached a maximum value inthe MODS1d group, thereafter decreased to the lowest point in the MODS period, butthe mRNA levels of MHC-Ⅱ and ubiquitin were not significant. The changes of E3ubiquitin ligase enzyme MARCH1were extremely significant. The levels of mRNAand protein expression of MARCH1were decreased first and reached a minimumvalue in the MODS1d group, then after a slight increasing trend they were decreasedagain in the MODS10d group. The trend of MHC-Ⅱ and MARCH1was roughly theopposite in the course of MODS. Compared with normal control group the degree ofspleen DC MHC-Ⅱ protein ubiquitination in the MODS1d group was significantlylower. The change of apoptosis rate of DC in the groups treated by MG132was notsignificant. The expression of MHC-Ⅱ proteins on DC, the proliferative activity of Tcells and the levels of IL-2, IFN-γ and TNF-α secreted by T cells in the MODS1dgroup and MG132treated normal control group were higher than the normal controlgroup, where the former is slightly higher than the latter, and the MODS1d grouptreated by MG132was the highest. We successfully constructed the eukaryoticexpression vector pMB HA-MARCH1of the E3ubiquitin ligase MARCH1, and synthesized three effective siRNA of MARCH1. We interfered DC in the MODS1dgroup with overexpression MARCH1and siRNA, and found that the morphology,vitality, and apoptosis rates of DC in the three groups had no significant changes.Thedegree of ubiquitination of MHC-Ⅱ was increased, and the expression of MHC-Ⅱproteins on DC, the proliferative activity of T cells and the levels of IL-2, IFN-γ andTNF-α secreted by T cells were reduced by interfered DC in the MODS1d group withoverexpression MARCH1. While the degree of ubiquitination of MHC-Ⅱ was reduced,and the expression of MHC-Ⅱ proteins on DC, the proliferative activity of T cells andthe levels of IL-2, IFN-γ and TNF-α secreted by T cells were increased by interferedDC in the MODS1d group with MARCH1siRNA.
Conclusion:1、The number of DC was decreased, but the antigen-presentingability and the capability to activate T cell-mediated immune response of DC wereenhanced during the first strike period of MODS; the number and the immunefunctions of DC dropped to the lowest and the organism was in a state of immunesuppression during the MODS period.2、MARCH1can degrade the expression ofMHC-Ⅱ on spleen DC by ubiquitination in the course of MODS.3、The change ofMHC-Ⅱ on spleen DC in MODS was really ubiquitin-dependent.4、We successfullyconstructed the eukaryotic expression vector pMB HA-MARCH1of the E3ubiquitinligase MARCH1, and synthesized three effective siRNA of MARCH1.5、Interferingthe expression of MARCH1can change the expression of MHC-Ⅱ on DC and theimmune function of DC by degrading MHC-Ⅱ through ubiquitination in the firststrike period of MODS. It suggested that we can use MARCH1as a potentialtherapeutic target for MODS.
引文
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