本文選題：人肝再生增強(qiáng)因子 + PBMC��； 參考：《重慶醫(yī)科大學(xué)》2009年博士論文

【摘要】： 目的:人肝再生增強(qiáng)因子( human augmenter of liver regeneration, hALR)是一種非特異性的、具有熱穩(wěn)定性的、促肝細(xì)胞再生的細(xì)胞因子,能抑制單核細(xì)胞的增殖和細(xì)胞因子INF-γ、IL-2的產(chǎn)生,但抑制機(jī)制仍不明確。MAPK/ERK、PKC-NF-KB、鈣離子信號(hào)通路是外周血單核細(xì)胞活化的主要通路,是很多免疫抑制劑作用的靶點(diǎn),目前不清楚ALR是否抑制這幾條通路發(fā)揮免疫抑制作用,因此,本研究擬從體外觀察ALR對(duì)外周血單核細(xì)胞這三條信號(hào)通路的影響,明確ALR的作用機(jī)制。 凋亡是免疫抑制的另一種重要方式,我們前期發(fā)現(xiàn)rALR能誘導(dǎo)大鼠單核細(xì)胞凋亡,但是機(jī)制仍不明確,是通過(guò)減少免疫營(yíng)養(yǎng)因子IL-2抑或刺激凋亡信號(hào)通路?因此,本研究也觀察人ALR對(duì)凋亡相關(guān)的信號(hào)通路Caspse-3及IL-2等細(xì)胞因子的影響,以期探明ALR免疫抑制機(jī)制。 方法: 1ALR對(duì)MAPK /ERK的影響 1)觀察ALR抑制ConA促PBMC增殖作用的最佳時(shí)間和濃度用MTT法觀察ConA(5ug/ml)在16h、40h、60h對(duì)PBMC的促增殖作用,以P0.01的時(shí)間作為ConA促PBMC增殖的最佳作用時(shí)間;選定60h時(shí)間點(diǎn),觀察系列濃度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的濃度作為ALR的最佳濃度。 2)觀察MAPK/ERK的變化根據(jù)最佳時(shí)間(60h)和濃度(30 ug/ml),將細(xì)胞分成正常對(duì)照組(Normal control ,N)、ALR對(duì)照組、ConA和ALR+ConA組,利用Western Blot法觀察ALR在60h對(duì)MAPK/ERK的影響。 3)動(dòng)態(tài)觀察MAPK/ERK的變化在明確ERK變化的基礎(chǔ)上,繼續(xù)觀察ALR在10min、30min、1h、2h、4h、8h、16h、32h、40h時(shí)間點(diǎn)對(duì)ERK的影響,以明確ALR抑制ERK的過(guò)程。 2 ALR對(duì)Ras的影響利用Western Blot法觀察ALR對(duì)Ras的影響及其影響是否與ERK同步,從而確定ALR是否經(jīng)Ras-MAPK/ERK通路抑制細(xì)胞增殖。 3 ALR對(duì)PKC-NF-KB通路的作用用Western blot法觀察PKC、NF-KB的變化,以明確ALR在培養(yǎng)中期是否通過(guò)抑制PKC-NF-KB起免疫抑制作用。 4 ALR對(duì)鈣離子信號(hào)通路的作用在明確ALR對(duì)以上兩條信號(hào)通路的作用后,ALR在4-8h的作用通路仍不明確,因此進(jìn)一步觀察鈣離子信號(hào)通路變化。利用鈣離子敏感探針裝載,熒光分光光度計(jì)動(dòng)態(tài)檢測(cè)細(xì)胞內(nèi)鈣離子濃度的變化,利用甲基百里香酚藍(lán)比色法檢測(cè)細(xì)胞培養(yǎng)上清液中的鈣離子濃度。 5 ALR對(duì)凋亡的作用利用流式細(xì)胞儀檢測(cè)ALR對(duì)PBMC凋亡的影響,用凝膠電泳法觀察DNA是否降解,用Western Blot法觀察ALR是否有活化Caspase-3的作用。 6ALR對(duì)IL-2、IL-4、IL-10的影響用ELISA方法檢測(cè)細(xì)胞培養(yǎng)上清中IL-2、IL-4、IL-10的動(dòng)態(tài)變化。 結(jié)果 1 ALR對(duì)MAPK/ERK的影響 1)ConA對(duì)單個(gè)核細(xì)胞的增殖作用隨時(shí)間的增加而逐漸增強(qiáng),16h和40h時(shí)增殖明顯(p=0.0413,0.0479),60h時(shí)最顯著(p0.01)。ALR抑制增殖呈劑量依賴(lài)關(guān)系,劑量為30 ug/ml時(shí)抑制作用最明顯(p0.01)。 2)ERK在60h的變化與正常組比較,ConA組磷酸化的ERK含量及非磷酸化的ERK含量均增加;ALR組磷酸化的ERK2含量減少。ALR+ConA組磷酸化及非磷酸化的ERK含量較ConA組均減少,并以磷酸化的ERK2減少為主。各組磷酸化ERK與非磷酸化ERK之比沒(méi)有差異。 3)ALR對(duì)ERK的動(dòng)態(tài)影響磷酸化ERK含量變化: ConA組磷酸化ERK的含量在1h時(shí)較正常組明顯增加,ALR組磷酸化ERK的含量在10min和1h時(shí)。ALR+ConA組磷酸化ERK的含量在10min、30min和1h時(shí)較ConA組明顯減少,以ERK2為主;各組磷酸化ERK的含量在4h以后均無(wú)明顯差別,且達(dá)到最低值。 非磷酸化ERK含量變化:各組非磷酸化ERK的含量隨時(shí)間逐漸增加,在4-16h達(dá)到較高水平,之后下降,40h最低。 2 ALR對(duì)ras的影響在整個(gè)細(xì)胞培養(yǎng)過(guò)程中,各組Ras呈多次波動(dòng),其中N組(10min-1h、1-8h、8-40h)和ConA組(10min-1h、1-4h、4-40h)Ras呈現(xiàn)3次波動(dòng),而ALR+ConA組(1-8h、8-40h)和ALR組(1-8h、8-40h)出現(xiàn)2次。ALR+ConA組Ras在10min-1h之間明顯被抑制,程度明顯低于ConA組,以30min為著;之后變化與ConA組并行。ALR組Ras除1h低于N組外,之后均與N組并行。ConA組和ALR+ConA組細(xì)胞內(nèi)Ras在16h均明顯低于正常組。 3 ALR對(duì)PKC-NF-KB通路的作用各組PKC、NF-KB表達(dá)的總體變化趨勢(shì)一致,PKC-NF-KB系統(tǒng)在細(xì)胞培養(yǎng)8h之后發(fā)生改變,ConA組PKC、NF-KB表達(dá)的最高值在16h,ALR+ConA組PKC、NF-KB表達(dá)最高值明顯后移,在32h。ALR+ConA組PKC、NF-KB抑制最強(qiáng)點(diǎn)在16h 4 ALR對(duì)PBMC鈣離子信號(hào)通路的作用除ALR+ ConA組鈣離子在4h之前沒(méi)有波動(dòng)外,各組細(xì)胞內(nèi)鈣離子均有先升高后下降的現(xiàn)象,ALR組鈣離子水平在30min時(shí)達(dá)到最高點(diǎn);ConA組細(xì)胞內(nèi)鈣離子濃度最高點(diǎn)在1h,N組細(xì)胞內(nèi)鈣離子濃度最高點(diǎn)在2h。各組之間鈣離子水平在4h時(shí)沒(méi)有統(tǒng)計(jì)學(xué)差異。8h后ALR組出現(xiàn)兩次波動(dòng)。 5 ALR對(duì)PBMC凋亡的作用60h之前,各組細(xì)胞沒(méi)有凋亡;60h時(shí),ALR組和ConA組細(xì)胞早期凋亡與正常組明顯增加;而ALR+ConA組較ConA組細(xì)胞早期凋亡明顯減少。60h時(shí),ALR+ConA組Caspase-3含量較ConA組明顯減少。 6 ALR對(duì)細(xì)胞因子的影響ALR組IL-2的分泌最高峰在16h, IL-10峰值在32h,與ConA組IL-2和IL-10分泌峰值相同。ALR+ConA組IL-2峰值推遲,IL-10峰值提前。 結(jié)論 1 ALR通過(guò)抑制Ras-MAPK/ERK2通路從而抑制ConA的促人PBMC增殖作用。 2 ALR對(duì)未經(jīng)ConA刺激的人PBMC MAPK/ERK具有雙向調(diào)節(jié)作用,早期促進(jìn)ERK的磷酸化,晚期抑制ERK2的磷酸化。 3 ALR通過(guò)抑制細(xì)胞內(nèi)鈣離子信號(hào)從而抑制人PBMC增殖。 4 ALR對(duì)未經(jīng)ConA刺激的人PBMC胞內(nèi)鈣離子有雙向調(diào)節(jié)作用,早期啟動(dòng)鈣離子信號(hào)通路,晚期抑制鈣離子信號(hào)通路。 5 ALR通過(guò)抑制PKC-NF-KB通路,從而影響細(xì)胞增殖和細(xì)胞因子分泌。 6 ALR通過(guò)影響人PBMC凋亡發(fā)揮免疫調(diào)節(jié)作用。 7 ALR通過(guò)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 楊長(zhǎng)永;謝大興;周毅;黃丹;龔建平;;膜受體介導(dǎo)的細(xì)胞凋亡與細(xì)胞周期的關(guān)系[J];癌癥;2006年05期

2 唐琳;孫航;張林;郭暉;張玲;劉杞;;阻斷肝再生增強(qiáng)因子的表達(dá)對(duì)人肝癌細(xì)胞株HepG2增殖的抑制作用[J];癌癥;2006年06期

3 張玲,廖曉輝,劉杞,宋曉英;肝再生增強(qiáng)因子在急性腎衰大鼠模型腎組織中的表達(dá)及意義[J];重慶醫(yī)學(xué);2003年06期

4 王志毅;張艷;石小楓;劉杞;;肝細(xì)胞移植聯(lián)合肝再生增強(qiáng)因子治療大鼠急性肝功能衰竭的研究[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2007年10期

5 林瑩;佟明華;汪嶸;孔祥平;梁世中;;Na~+,K~+-ATPase調(diào)節(jié)肝再生增強(qiáng)因子促HepG2細(xì)胞增殖[J];廣西農(nóng)業(yè)生物科學(xué);2006年01期

6 楊曉明,謝玲,賀福初,吳祖澤;PCR介導(dǎo)的大鼠再生肝cDNA文庫(kù)構(gòu)建[J];軍事醫(yī)學(xué)科學(xué)院院刊;1996年03期

7 譚余慶,張永祥;蛋白激酶C和I_kΒNF_kΒ在免疫調(diào)節(jié)中的作用[J];免疫學(xué)雜志;2000年06期

8 石小楓;孫航;唐琳;陳學(xué)華;劉杞;;肝再生增強(qiáng)因子對(duì)外原性抗原引起機(jī)體免疫應(yīng)答影響的研究[J];免疫學(xué)雜志;2007年06期

9 張艷;王志毅;石小楓;孫航;李紅;劉杞;;同種肝細(xì)胞移植細(xì)胞排斥反應(yīng)機(jī)理及肝再生增強(qiáng)因子的作用[J];免疫學(xué)雜志;2007年06期

10 閭軍,許望翔,詹軼群,崔曉林,蔡衛(wèi)民,賀福初,楊曉明;一種新的肝細(xì)胞生成素(HPO)轉(zhuǎn)錄本及其生物學(xué)活性[J];生物化學(xué)與生物物理學(xué)報(bào);2002年02期

相關(guān)碩士學(xué)位論文 前1條

1 謝華;rALR對(duì)大鼠單個(gè)核細(xì)胞作用的初步研究[D];重慶醫(yī)科大學(xué);2004年

，

本文編號(hào)：1967661