【作者】 王新国；

【导师】 刘杞；

【作者基本信息】 重庆医科大学 ， 内科学， 2009， 博士

【摘要】 目的:人肝再生增强因子( human augmenter of liver regeneration, hALR)是一种非特异性的、具有热稳定性的、促肝细胞再生的细胞因子,能抑制单核细胞的增殖和细胞因子INF-γ、IL-2的产生,但抑制机制仍不明确。MAPK/ERK、PKC-NF-KB、钙离子信号通路是外周血单核细胞活化的主要通路,是很多免疫抑制剂作用的靶点,目前不清楚ALR是否抑制这几条通路发挥免疫抑制作用,因此,本研究拟从体外观察ALR对外周血单核细胞这三条信号通路的影响,明确ALR的作用机制。凋亡是免疫抑制的另一种重要方式,我们前期发现rALR能诱导大鼠单核细胞凋亡,但是机制仍不明确,是通过减少免疫营养因子IL-2抑或刺激凋亡信号通路?因此,本研究也观察人ALR对凋亡相关的信号通路Caspse-3及IL-2等细胞因子的影响,以期探明ALR免疫抑制机制。方法:1ALR对MAPK /ERK的影响1)观察ALR抑制ConA促PBMC增殖作用的最佳时间和浓度用MTT法观察ConA(5ug/ml)在16h、40h、60h对PBMC的促增殖作用,以P<0.01的时间作为ConA促PBMC增殖的最佳作用时间;选定60h时间点,观察系列浓度ALR(0.5ug/ml、1ug/ml、2ug/ml、7.5ug/ml、10 ug/ml、15 ug/ml、30 ug/ml)抑制ConA的促增殖作用,以P<0.01的浓度作为ALR的最佳浓度。2)观察MAPK/ERK的变化根据最佳时间(60h)和浓度(30 ug/ml),将细胞分成正常对照组(Normal control ,N)、ALR对照组、ConA和ALR+ConA组,利用Western Blot法观察ALR在60h对MAPK/ERK的影响。3)动态观察MAPK/ERK的变化在明确ERK变化的基础上,继续观察ALR在10min、30min、1h、2h、4h、8h、16h、32h、40h时间点对ERK的影响,以明确ALR抑制ERK的过程。2 ALR对Ras的影响利用Western Blot法观察ALR对Ras的影响及其影响是否与ERK同步,从而确定ALR是否经Ras-MAPK/ERK通路抑制细胞增殖。3 ALR对PKC-NF-KB通路的作用用Western blot法观察PKC、NF-KB的变化,以明确ALR在培养中期是否通过抑制PKC-NF-KB起免疫抑制作用。4 ALR对钙离子信号通路的作用在明确ALR对以上两条信号通路的作用后,ALR在4-8h的作用通路仍不明确,因此进一步观察钙离子信号通路变化。利用钙离子敏感探针装载,荧光分光光度计动态检测细胞内钙离子浓度的变化,利用甲基百里香酚蓝比色法检测细胞培养上清液中的钙离子浓度。5 ALR对凋亡的作用利用流式细胞仪检测ALR对PBMC凋亡的影响,用凝胶电泳法观察DNA是否降解,用Western Blot法观察ALR是否有活化Caspase-3的作用。6ALR对IL-2、IL-4、IL-10的影响用ELISA方法检测细胞培养上清中IL-2、IL-4、IL-10的动态变化。结果1 ALR对MAPK/ERK的影响1)ConA对单个核细胞的增殖作用随时间的增加而逐渐增强,16h和40h时增殖明显(p=0.0413,0.0479),60h时最显著(p<0.01)。ALR抑制增殖呈剂量依赖关系,剂量为30 ug/ml时抑制作用最明显(p<0.01)。2)ERK在60h的变化与正常组比较,ConA组磷酸化的ERK含量及非磷酸化的ERK含量均增加;ALR组磷酸化的ERK2含量减少。ALR+ConA组磷酸化及非磷酸化的ERK含量较ConA组均减少,并以磷酸化的ERK2减少为主。各组磷酸化ERK与非磷酸化ERK之比没有差异。3)ALR对ERK的动态影响磷酸化ERK含量变化: ConA组磷酸化ERK的含量在1h时较正常组明显增加,ALR组磷酸化ERK的含量在10min和1h时。ALR+ConA组磷酸化ERK的含量在10min、30min和1h时较ConA组明显减少,以ERK2为主;各组磷酸化ERK的含量在4h以后均无明显差别,且达到最低值。非磷酸化ERK含量变化:各组非磷酸化ERK的含量随时间逐渐增加,在4-16h达到较高水平,之后下降,40h最低。2 ALR对ras的影响在整个细胞培养过程中,各组Ras呈多次波动,其中N组(10min-1h、1-8h、8-40h)和ConA组(10min-1h、1-4h、4-40h)Ras呈现3次波动,而ALR+ConA组(1-8h、8-40h)和ALR组(1-8h、8-40h)出现2次。ALR+ConA组Ras在10min-1h之间明显被抑制,程度明显低于ConA组,以30min为著;之后变化与ConA组并行。ALR组Ras除1h低于N组外,之后均与N组并行。ConA组和ALR+ConA组细胞内Ras在16h均明显低于正常组。3 ALR对PKC-NF-KB通路的作用各组PKC、NF-KB表达的总体变化趋势一致,PKC-NF-KB系统在细胞培养8h之后发生改变,ConA组PKC、NF-KB表达的最高值在16h,ALR+ConA组PKC、NF-KB表达最高值明显后移,在32h。ALR+ConA组PKC、NF-KB抑制最强点在16h4 ALR对PBMC钙离子信号通路的作用除ALR+ ConA组钙离子在4h之前没有波动外,各组细胞内钙离子均有先升高后下降的现象,ALR组钙离子水平在30min时达到最高点;ConA组细胞内钙离子浓度最高点在1h,N组细胞内钙离子浓度最高点在2h。各组之间钙离子水平在4h时没有统计学差异。8h后ALR组出现两次波动。5 ALR对PBMC凋亡的作用60h之前,各组细胞没有凋亡;60h时,ALR组和ConA组细胞早期凋亡与正常组明显增加;而ALR+ConA组较ConA组细胞早期凋亡明显减少。60h时,ALR+ConA组Caspase-3含量较ConA组明显减少。6 ALR对细胞因子的影响ALR组IL-2的分泌最高峰在16h, IL-10峰值在32h,与ConA组IL-2和IL-10分泌峰值相同。ALR+ConA组IL-2峰值推迟,IL-10峰值提前。结论1 ALR通过抑制Ras-MAPK/ERK2通路从而抑制ConA的促人PBMC增殖作用。2 ALR对未经ConA刺激的人PBMC MAPK/ERK具有双向调节作用,早期促进ERK的磷酸化,晚期抑制ERK2的磷酸化。3 ALR通过抑制细胞内钙离子信号从而抑制人PBMC增殖。4 ALR对未经ConA刺激的人PBMC胞内钙离子有双向调节作用,早期启动钙离子信号通路,晚期抑制钙离子信号通路。5 ALR通过抑制PKC-NF-KB通路,从而影响细胞增殖和细胞因子分泌。6 ALR通过影响人PBMC凋亡发挥免疫调节作用。7 ALR通过IL-10抑制IL-2抑制免疫。更多还原

【Abstract】 Objective: Human augmenter of liver regeneration (hALR) is a non-specific, with thermal stability, and promoting cell regeneration factor, which inhibits the proliferation of mononuclear cells and production of cytokines INF-γ, IL-2. However, the mechanism is unclear. The signaling pathways of MAPK / ERK, PKC-NF-KB and calcium are involved in activation of peripheral blood mononuclear cells and also targets for many immunosuppressive agents. Whether ALR as an immunosuppressive agent inhibits these three main pathways or not is needed to be clarified. Therefore, this study was to observe the effect of ALR on these three signaling pathways.Apoptosis is another important way of immune suppression.we have noticed that rat ALR can promote monocyte apoptosis. It is unclear whether ALR induces apoptosis by inhibiting IL-2 production or stimulating the apoptosis signaling pathway? Therefore, in this study we also observe the effects of ALR on the signaling pathway of apoptosis-related Caspse-3 and cytokines production such as IL-2.Methods1 Role of ALR on MAPK / ERK1) Determination of the optimcal time and dose for ALR the optimal time was selected in the series of time 16h,40h and 60h at which PBMCs stimulated with ConA (5ug/ml) proliferated significantly (p<0.01) by the MTT method.The optimcal dose of ALR was determined among a series concentration of 0.5ug/ml, 1ug / ml, 2ug/ml, 7.5ug/ml, 10 ug / ml, 15 ug / ml and 30 ug / ml , at whith ALR inhibited the proliferation stimulated by ConA significantly at 60h.2) Alteration of MAPK/ERK according with the established dose and time to observe whether ALR 30ug/ml had effect on the MAPK / ERK at 60h. Cells were divided into N, ALR, ConA and ALR +ConA group . Western blot was intaked to detect expression of MAPK / ERK.3) Dynamic alteration of MAPK / ERK. Based on the changes of ERK at 60h, courses of ERK altreration were observed at a series time of 10 min, 30min、1h、2h、4h、8h、16h、32h and40h.2 Role of ALR on Ras The expression of Ras were observed at the same time point of ERK using Western Blot to further determine whether ALR suppression was through the pathway of Ras-MAPK/ERK.3 Role of ALR on PKC-NF-KB The PKC-NF-KB pathway function was measured by the expression in PBMC followed the observison that the ras didn’t manipulate the activation of ERK in the metaphase. 4 Role of ALR on calcium In the above two signaling pathway, no one was observed to work in the period of 4-8h. Therefore the intracellular calcium and supernatant calcium were studied by calcium-sensitive probe loading and methyl thymol blue colorimetric assay to assert the role of ALR.5 The role of ALR on apoptosis Flow cytometry was intake to appraise ALR effect on apoptosis in PBMC. Gel electrophoresis was used to observe whether there was DNA degradation. Western Blot used to dectect the activation of Caspase-3.6 Affect of ALR on IL-2、IL-4 and IL-10 The levels of IL-2, IL-4 and IL-10 were detected by ELISA in cell supernatants at the same time of ERK.Results1 Action for ALR on MAPK / ERK1) The proliferation of monocytes stimulated with ConA enhanced in time course-dependent manner, with the statistical significance at 16h ,40h (p = 0.0413,0.0479) and 60h (p <0.01). ALR inhibited cell proliferation in a dose-dependent maner and it attained the significant inhibitory effect (p <0.01) while the concentration reached to 30 ug / ml.2) Alteration of ERK at 60h compared with the normal group, the content of phosphorylated and non-phosphorylated ERK in ConA group were increased; with ConA group, ALR+ConA group non-phosphorylated ERK phosphorylation levels were reduced especially ERK2 phosphorylation; with the normal group, ALR group ERK2 phosphorylation decreased. In each group, there was no difference between the ratios of phosphorylated ERK to non-phosphorylated ERK.3) The role of ALR for ERK alteration The content of phosphorylated ERK in ALR group at 10 min and ConA group at 30min were significantly increased than that in normal group. The content of phosphorylated ERK in normal group peaked at 2h. The content of phosphorylated ERK,especially ERK2, in ALR+ConA group were significantly reduced at 10min、30min、1h and 2h.There was no difference of phosphorylated ERK among the groups at 4h.The total ERK content in each group Cells gradually increased, at 4-16h achieved a higher level, followed by decline, 40h reached the lowest level.The content of ERK in each group at the same time point showd no difference before 60h, while there were significant alteration between ERK contents at 60h.2 The role of ALR for Ras pathway In the whole culture process, Ras showed multiple fluctuations in each group. Ras in N and ConA group cells showed fluctuations 3 times (10min-1h、1-8h、8-40h) and (10min-1h、1-4h、4-40h) respectively , ALR and ALR+ConA group ras appeared 2 times (1-8h、8-40h). The content of Ras in ConA in the period of 10min-1h was significantly increased rather than that in normal group. Compared with the normal group the situation reversed in ALR+ConA and ConA groups at 16h. ALR+ConA cells’Ras in 10min-1h was significantly reduceed than ConA group especially at 30min.3 The role of ALR for PKC-NF-KB pathway In each group the expression of PKC and NF-KB shew the same trendency line.PKC-NF-KB system in the cell culture course altered after 8h and ALR made ConA-induced peak delay.4 The role of ALR for calcium alteration The level of calcium shew increasing and declining in turn besides of the group of ALR+ ConA. In ALR group, the concentration of calcium ion peaked at 30min, ConA group at 1h, and N group at 2h. Calcium concentration in each group at 4h was no significant difference, then rapid decline. After 8h calcium ion in ALR group fluctuate again. Compared with the ConA group, intracellular calcium ion concentration in ALR+ConA group was significant differences at 1h.5 Apoptosis of PBMC caused by ALR compared with the normal group, apoptosis in ALR and ConA group significantly increased at 60h, while apoptosis in ALR+ConA group significantly reduced than that in ConA group. Caspase-3 expression in ALR+ ConA group significantly decreased than ConA group at 60h time point. 6 Affect of ALR on IL-2、IL-4 and IL-10 IL -2 in ALR group’s supernantant peaked at 16h and IL-10 at 32h as well as in ConA group. Wihle the peaks of IL-2 was delayed and IL-10 was in advance in ALR+ConA group supernantant.Conclusion1 ALR inhibits PBMC proliferation by suppressing Ras-MAPK/ERK2 pathway.2 ALR has a role in two-way on MAPK / ERK in human PBMC without ConA stimulation, not only promote the phosphorylation of ERK, but also inhibite the phosphorylation of ERK2.3 ALR inhibits cell proliferation by inhibiting intracellular calcium signals.4 The role of ALR to modulate calcium is also two-way trip, activating the calcium signal and suppressing calcium signaling pathway.5 ALR inhibits the PKC-NF-KB pathway to affect cell proliferation and cytokine secretion.6 ALR affects PBMC apoptosis to regulate immune response.7 ALR inhibits IL-2 produce by IL-10 secreting.更多还原