人肝再生增强因子免疫抑制机制研究

发布时间：2018-06-02 05:52

  本文选题：人肝再生增强因子 + PBMC　； 参考：《重庆医科大学》2009年博士论文

【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P0.01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P0.01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(p0.01)。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(p0.01)。 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16h 4 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。
[Abstract]:Objective: human augmenter of liver regeneration (hALR) is a nonspecific, thermal stable cytokine that promotes hepatocyte regeneration and can inhibit the proliferation of mononuclear cells and the production of cytokine INF- gamma and IL-2, but the inhibition mechanism is still not clear.MAPK/ERK, PKC-NF-KB, and calcium signaling pathway is outside. The main pathway of monocyte activation in peripheral blood is the target of many immunosuppressive agents. It is not clear whether ALR inhibits the immunosuppressive effects of these pathways. Therefore, this study intends to observe the effect of the three signal pathways in peripheral blood mononuclear cells from ALR in vitro, and the mechanism of the action of ALR is clear.
Apoptosis is another important way of immunosuppression. We have found that rALR can induce apoptosis in rat mononuclear cells, but the mechanism is still not clear, which is by reducing IL-2 or stimulating apoptosis signaling pathway? Therefore, this study also observed the effect of human ALR on apoptosis related signaling pathways such as Caspse-3 and IL-2. The immunosuppressive mechanism of ALR was explored.
Method:
The effect of 1ALR on MAPK /ERK
1) to observe the best time and concentration of ALR inhibiting the proliferation of PBMC by ConA, and to observe the proliferation of ConA (5ug/ml) in 16h, 40H, 60H to PBMC by MTT method, and the best time to promote the proliferation of PBMC in P0.01 time. The promoting effect of ConA was P0.01 concentration as the best concentration of ALR.
2) the changes of MAPK/ERK were observed on the basis of the optimal time (60H) and concentration (30 ug/ml), and the cells were divided into normal control group (Normal control, N), ALR control group, ConA and ALR+ConA group, and Western Blot method was used to observe the effect of ALR.
3) dynamically observe the changes of MAPK/ERK on the basis of clear ERK changes, and continue to observe the effect of ALR on 10min, 30min, 1H, 2h, 4h, 8h, 16h, 32H, 40H time points.
The effect of 2 ALR on Ras was observed by Western Blot method to observe the effect of ALR on Ras and whether the effect was synchronized with ERK, thus determining whether ALR could inhibit cell proliferation through the Ras-MAPK/ERK pathway.
The effect of 3 ALR on the PKC-NF-KB pathway was observed by Western blot method to observe the changes of PKC and NF-KB in order to determine whether ALR could inhibit the immune suppression by inhibiting PKC-NF-KB in the medium period of culture.
The effect of 4 ALR on the calcium signal pathway is clear after the action of ALR on the above two signaling pathways. The pathway of ALR in 4-8h is still unclear. Therefore, the calcium ion signal pathway is further observed. The calcium concentration in cell culture supernatant was detected by phenol blue colorimetry.
The effect of 5 ALR on apoptosis was detected by flow cytometry. The effect of ALR on the apoptosis of PBMC was detected. The degradation of DNA was observed by gel electrophoresis. The effect of ALR on the activation of Caspase-3 was observed by Western Blot method.
The effects of 6ALR on IL-2, IL-4 and IL-10 were detected by ELISA. The dynamic changes of IL-2, IL-4 and IL-10 in cell culture supernatant were detected.
Result
The influence of 1 ALR on MAPK/ERK
1) the proliferation of ConA to mononuclear cells increased with time, and the proliferation of 16h and 40H was obvious (p=0.0413,0.0479). The most significant (P0.01).ALR inhibition proliferation in 60H was dose-dependent, and the inhibitory effect was most obvious at the dose of 30 ug/ml (P0.01).
2) compared with the normal group, the ERK content of phosphorylation in ConA group and the content of non phosphorylated ERK increased in the 60H group, and the content of phosphorylation ERK2 in group ALR reduced.ALR+ConA group phosphorylation and non phosphorylation of ERK content compared with ConA group, and the ERK2 decrease of phosphorylation was mainly. The ratio of phosphorylation ERK and non phosphorylation was not different in each group.
3) the dynamic effect of ALR on the dynamic effects of ERK on the phosphorylation of ERK content: the content of phosphorylated ERK in the ConA group increased obviously in 1H than in the normal group. The content of phosphorylated ERK in the ALR group was at 10min and 1H in the.ALR+ConA group. Difference and reach the lowest value.
Non phosphorylated ERK content changes: the content of non phosphorylated ERK increased gradually with time, reached a higher level at 4-16h, then decreased, and 40H was lowest.
The effect of 2 ALR on RAS in the whole cell culture process, each group of Ras fluctuates many times, of which group N (10min-1h, 1-8h, 8-40h) and ConA group (10min-1h, 1-4h, 4-40h) Ras presents 3 fluctuations. In the subsequent changes and group ConA parallel.ALR group Ras except 1H was lower than the N group, then the Ras in the.ConA group and the ALR+ConA group in the.ConA group and the ALR+ConA group were all significantly lower than the normal group.
The overall change trend of PKC and NF-KB expression in 3 ALR PKC-NF-KB pathway is consistent. PKC-NF-KB system changes after cell culture 8h. The highest value of PKC, NF-KB expression in ConA group is in 16h, ALR+ConA group PKC.
The effect of 4 ALR on the signal pathway of PBMC calcium ion was not fluctuated before 4H in group ALR+ ConA, and the calcium ion in each group increased first and then decreased. The level of calcium ion in ALR group reached the highest point in 30min; the highest point of intracellular calcium concentration in ConA group was in 1H, and the highest point of intracellular calcium concentration in N group was in 2h. groups. There was no significant difference in the level of calcium between 4H group and.8h group. There was a two fluctuation after ALR.
5 ALR on PBMC apoptosis before 60H, no cell apoptosis in each group, 60H, ALR group and ConA group early apoptosis and normal group increased obviously, while ALR+ConA group compared with ConA group early apoptosis significantly decreased.60h, Caspase-3 content of ALR+ConA group was significantly lower than that of the ConA group.
The effect of 6 ALR on cytokine was the peak of IL-2 secretion in group ALR, the peak value of IL-10 was 32H, and the peak value of.ALR+ConA group of IL-2 and IL-10 secreted in ConA group was delayed, and the peak of IL-10 was ahead of time.
conclusion
1 ALR inhibits the proliferation of human PBMC induced by ConA by inhibiting the Ras-MAPK/ERK2 pathway.
2 ALR has a bidirectional regulatory effect on human PBMC MAPK/ERK without ConA stimulation, which promotes early phosphorylation of ERK and inhibits ERK2 phosphorylation at an advanced stage.
3 ALR inhibited the proliferation of human PBMC by inhibiting intracellular calcium signaling.
4 ALR has bidirectional regulation effect on intracellular calcium in human PBMC without ConA stimulation, initiating calcium signaling pathway at early stage, and inhibiting calcium signaling pathway at an advanced stage.
5 ALR inhibits PKC-NF-KB pathway, thereby affecting cell proliferation and cytokine secretion.
6 ALR exerts an immunomodulatory effect by affecting the apoptosis of human PBMC.
7 ALR inhibits immunization by inhibiting IL-2 through IL-10.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392

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