Akt-Mdm2-p53信号通路在幽门螺杆菌致病中的作用
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摘要
研究背景和目的：
幽门螺杆菌(Helicobacter pylori,Hp)感染约占全球人口的一半,但只有少部分的感染者发病。现已明确,Hp是慢性胃炎、消化性溃疡、胃MALT淋巴瘤和胃癌的致病因子。在正常胃黏膜中,胃上皮细胞增殖和凋亡保持动态平衡,而Hp感染能打破这一平衡,并与感染结局及致病多样性密切相关。目前有关Hp导致胃上皮细胞凋亡增殖的研究中,结论很不一致,甚至相互矛盾。许多体内研究表明Hp感染既可促进胃上皮细胞凋亡,又可诱导细胞增殖,从而加速了细胞的更新,认为和其诱导的免疫炎症反应和代偿性增生有关。许多体外研究报道Hp能直接诱导胃上皮细胞凋亡,但也有研究认为Hp主要是刺激胃上皮细胞增殖,并能在早期直接激活细胞增殖信号通路,促进DNA合成增加,从而使细胞恶变风险增加。目前体外研究所选用的大多是不同类型的胃癌上皮细胞,而这些胃癌细胞本身在凋亡、增殖信号传导等方面已存在异常。不同浓度的Hp和作用时间长短的影响也不同,这可能是结果不一致的原因之一。
在细胞凋亡增殖的调控中,Akt-Mdm2-p53通路发挥重要作用。Akt促进细胞增殖和抗细胞凋亡,它磷酸化Mdm2,使Mdm2稳定和可更有效地转位到核内,通过泛素化途径降解p53蛋白而下调p53功能,使细胞能抗衡p53介导的凋亡。而Mdm2也是p53的一个靶基因,可被p53负反馈调控。同时,p53可通过上调PTEN,而发挥间接抑制Akt的作用。因此,Akt-Mdm2-p53信号通路在决定细胞凋亡或增殖中扮演重要角色,而它在Hp致病中的作用尚不清楚。
因此,针对以上研究现状,本课题研究不同阶段胃黏膜病变中Akt-Mdm2-p53信号通路相关蛋白的表达和Hp感染的关系；同时进行体外实验研究,选用来源于人正常胃上皮细胞的GES-1细胞为实验对象(预试验提示能表达野生型p53),研究Hp培养滤液对GES-1细胞凋亡增殖的直接影响及和Akt-Mdm2-p53信号通路的关系,为探讨Hp的致病机制提供理论和实验依据。方法：
1、收集Hp阳性和阴性的慢性非萎缩性胃炎(CNAG)、化生性萎缩(MA)、异型增生(Dys)和胃癌(GC)胃黏膜病理标本,行Akt-Mdm2-p53信号通路相关蛋白的检测。
2、制备NCTC11637Hp标准菌株培养滤液,测定浓度为11.1mg/ml,以此为初浓度,稀释不同的浓度,和GES-1细胞共培养,设立只加Hp培养液的对照组,根据实验在不同时间点收集细胞行相应检测。
3、Akt信号通路的阻断：加40umol/L的LY294002预处理GES-1细胞1h,设立加40umol/L的DMSO为对照组。
4、相应指标的检测：
(1)病理标本Hp感染检测：采用Giemsa染色法检测。
(2)病理标本蛋白的检测：应用免疫组化PV-9000法检测。
(3)细胞形态学检测：倒置显微镜和电镜观察。
(4)细胞活力的检测：采用四唑蓝(MTT)比色试验检测。
(5)细胞周期的检测：采用PI染色流式细胞术方法检测。
(6)细胞凋亡的检测：采用DNA电泳和Annexinⅴ/PI双染色流式细胞术检测。
(7)DNA损伤检测：采用单细胞凝胶电泳检测。
(8)细胞mRNA的检测：采用逆转录聚合酶链反应(RT-PCR)法检测。
(9)细胞蛋白的检测：采用Western blotting方法检测。结果：
1、不同胃粘膜病变中Hp感染和Akt-Mdm2-p53信号通路表达的关系
(1)在CNAG、MA、DYS和GC各粘膜病变中分别行Hp阳性者和Hp阴性者比较,pAkt在CNAG组的Hp阳性者显著高于Hp阴性者(P<0.05)；Akt在各粘膜病变中均无显著性差异(P>0.05);Mdm2在Dys组Hp阳性者显著高于阴性者(P<0.05)；突变型p53在MA组Hp阳性者显著高于阴性者(P<0.05)；PCNA在Dys组Hp阳性者显著高于阴性者(P<0.05)；Bax在CNAG、MA组阳性者显著高于阴性者(P<0.05),其余各组无显著性差异(P>0.05)。
(2)在Hp阳性的CNAG、MA、DYS和GC各粘膜病变中,Akt、pAkt在各病变组的表达无显著差异(P>0.05)。Mdm2、PCNA蛋白的表达在GC、Dys组显著高于CNAG、MA组(P<0.05),Bax蛋白的表达在MA组最高,显著高于CNAG、Dys、GC组(P<0.05)
(3)在Hp阳性的CNAG、MA、Dys胃粘膜病变中,对pAkt、Mdm2、突变型p53、Bax和PCNA蛋白的表达行相关性分析,pAkt和Mdm2,Mdm2和PCNA的表达呈正相关(P<0.05)。
2、Hp培养滤液对GES-1细胞生物学行为的影响
(1)形态学变化：Hp培养滤液作用GES-1细胞后48h内电镜和倒置显微镜下观察,细胞呈现典型的空泡化、凋亡改变,且随Hp培养滤液浓度的增加和作用时间的延长而加重。
(2)1：4浓度Hp培养滤液作用GES-1细胞48h后行DNA电泳,结果DNA条带呈凋亡特征性梯状改变,而对照组无此改变。
(3)MTT检测Hp培养滤液作用GES-1细胞后的增殖活性：在3h、6h和12h时间点,各浓度组的细胞存活率和照组比较均无显著性差异(P>0.05)。作用24h,各浓度组的细胞存活率显著低于对照组(P<0.05),但各浓度组间无显著性差异(P>0.05)。作用48h,1：4、1：2浓度组的细胞存活率(74.7±12.1%,62.2±12.6%)均显著低于对照组(100.0±8.3%)(P<0.05),且呈浓度依赖性。但对照组与1：8组之间无显著性差异(P>0.05)；作用72h时,1：8、1：4、1：2、1：1组的细胞存活率显著低于对照组(P<0.05),且呈浓度依赖性,均有显著性差异(P<0.05)。行1：·8浓度的Hp培养滤液作用GES-1细胞80天后,和平行传代的对照组比较,细胞存活率显著增加。(P<0.05)
(4)流式细胞仪分析显示：1：4浓度的Hp培养滤液作用GES-1细胞48h后,细胞周期主要阻滞在G0-G1期(98.73±1.12%vs45.37±4.89%),凋亡率显著上升(26.93±5.34%vsl.65±0.44%)(P<0.05)。
(5)单细胞凝胶电泳检测DNA损伤：1：4浓度的Hp培养滤液作用GES-1细胞3h后,和对照组比较,彗星尾部DNA量占全部DNA量的比例无显著性差异(P>0.05)；作用48h后,彗星尾部DNA量显著增加。(P<0.05)。
3、Akt-Mdm2-p53信号通路在Hp培养滤液对GES-1细胞影响中的作用
(1)Hp培养滤液作用GES-1细胞后p53、Mdm2、Bax mRNA的表达：1：4浓度的Hp培养滤液作用GES-1细胞48h,p53 mRNA无显著变化(P>0.05)；Mdm2 mRNA、Bax mRNA的表达显著上调(P<0.05)；
(2)Hp培养滤液作用GES-1细胞后Akt-Mdm2-p53通路相关蛋白水平及活性的变化：1：4浓度的Hp培养滤液作用GES-1细胞不同时间后,Akt蛋白无明显变化,pAkt在1h后上升,在3h达最高峰,在48h内持续升高,Mdm2蛋白具有类似变化趋势；p53蛋白12h内无明显变化,在24、48h上升；Bax蛋白在1h后上升,48h内持续升高。
(3)加入Akt抑制剂LY294002处理GES-1细胞后,用1：4浓度的Hp培养滤液作用细胞,与未加入抑制剂比较,12h和24h的细胞存活率显著下降(P<0.05)。在作用24h后pAkt、Mdm2蛋白表达显著下降(P<0.05),而p53蛋白表达显著上调(P<0.05)。结论：
1、Akt-Mdm2-p53信号通路参与了Hp感染相关的胃黏膜病变发生、发展,在Hp感染导致的CNAG、MA、Dys病变阶段中起作用,而在胃癌病变阶段中与Hp感染无明显关系,提示Akt-Mdm2-p53信号通路主要在Hp致癌的早期阶段起作用。
2、在短期作用下,Hp培养滤液可导致胃上皮细胞凋亡,活力下降,DNA损伤,且呈浓度和时间依赖关系；在低浓度培养滤液长期作用下,Hp能诱导胃上皮细胞增殖活性升高。
3、Hp可同时激活凋亡和增殖信号通路。在早期,Hp通过激活Akt和诱导Mdm2的表达对抗Hp诱导的凋亡作用,使p53水平无明显变化。但在细胞DNA损害加重时,Hp激活p53通路促进细胞凋亡和使细胞阻滞在G0/G1期。在Hp感染时阻断Akt通路可使Hp对细胞的抑制作用加重,Mdm2蛋白下调,p53蛋白上调。因此,在Hp作用的不同时期,Akt-Mdm2-p53信号通路根据细胞的损害程度共同调控细胞的存活或死亡,在Hp致病中起重要作用。
Background and purposes:
Although half of the world's population is infected by Helicobacter pylori(H.pylori), few fall ill. Now it is accepted that H.pylori is the causative agent of chronic gastritis, peptic ulcer, gastric MALT lymphoma and gastric cancer. The balance of human gastric epithelial cell proliferation and apoptosis will be broken by the infection of H.pylori, and the infection is closely related to the diversity of outcome. however it is still very controversial about the effect of H.pylori on gastric epithelial cell proliferation and apoptosis. It has been proven in vivo that the infection of H.pylori could simultaneously accelerate the apoptosis and induce the proliferation, promoting the cell renewal and this effect is related to immuno- inflammatory response and compensatory hyperplasia caused by the infection of H.pylori. Meanwhile some in vitro studies show that H.pylori could induce the epithelial cell apoptosis directly, and some others show that H.pylori could promote the epithelial cell proliferation, activate the signal pathway of early proliferation directly, increase DNA synthesis and increase the risk of malignant transformation. At present most in vitro studies choose different styles of gastric cancer epithelial cell, however these cells themselves may be different in the signal transductions of apoptosis and proliferation, and the reactions to different concentrations of H.pylori and action time may also be different, which might be one of the reasons for the diversity of results.
Akt-Mdm2-p53 signaling axis plays an important role in the regulation of proliferation and apoptosis. Akt promotes proliferation and restrains apoptosis. Akt phosphorylates Mdm2, makes Mdm2 stable and nuclear translocation more effective, down regulate the function of p53 by the ubiquitination of p53, then help cells resist the p53- induced apoptosis.Mdm2 is a target gene of p53 which also receive the negative-feedback regulation of p53. At the same time, p53 can restrain the effect of Akt by up-regulating PTEN. However the role of Akt-Mdm2-p53 signaling axis in the pathopoiesis of H.pylori is still unkown.
Therefore, we study the relationship between the Akt-Mdm2-p53 signaling axis related protein expression in different stages of gastric mucosal lesions and the infection of H.pylori; at the same time in vitro study we choose normal human gastric epithelial GES-1 cells as our experimental subject (in pretest wild type p53 expressed) to research the effect of culture filtrate of H.pylori on GES-1 cell proliferation and apoptosis and the relationship between it and Akt-Mdm2-p53 signaling axis; Finally find out the possible explanations to the machnism of the pathopoiesis of H.pylori.
Methods:
1. Collect pathologic specimens of gastric mucosa (H.pylori related or not)with chronic no-atrophic gastritis (CNAG), metaplastic atrophy (MA), dysplasia (Dys) or gastric cancer(GC) and detect the Akt-Mdm2-p53 signaling axis related proteins.
2. Prepare the culture filtrates of type culture strain (NCTC11637H.pylori), concentration is set to be 11mg/ml, and then dilute it to different concentrations, co-cultured with GES-1 cells. In control group only H.pylori culture medium is added, cells are collected and detected at different points.
3. Block the Akt-Mdm2-p53 signaling axis:GES-1 cells are pretreated with 40μmol/l LY294002, set up a control group only with 40μmol/l DMSO.
4. Detections of related parameters:
(1)Test for H.pylori infection in pathologic specimens:Giemsa's staining
(2) Test for proteins in pathologic specimens:PV9000 immunohistochemical method
(3) Cytomorphological examination:invert microscope and electron microscopy
(4) Cytoactive test:MTT method
(5) Test for cell cycle:PI flow cytometry
(6) Apoptosis test:DNA electrophoresis and Annexinⅴ/PI flow cytometry
(7) Detection of DNA damage:single cell gel electrophoresis
(8) mRNA test:reverse transcriptase PCR (RT-PCR)
(9) Test for cell protein:Western blotting
Results:
1. The relationship between the expression of Akt-Mdm2-p53 signaling axis and the infection of H.pylori in different gastric mucosal lesions.
(1) In gastric mucosal lesions with CNAG or MA, DYS or GC, pAkt expression in Group CNAG was much higher in H.pylori-positive specimens compared to H.pylori-negative specimens (P<0.05); Akt expression showed no significant difference among all the groups (P>0.05);Mdm2 expression in Group Dys was also significantly higher in H.pylori-positive specimens compared to H.pylori-nagative specimens (P<0.05); Mutant p53 of Group MA was much higher in H.pylori-positive specimens compared to H.pylori-negative specimens (P<0.05); PCNA of Group Dys was significantly higher in H.pylori-positive specimens compared to H.pylori-negative specimens (P<0.05); Bax expressions of Group CNAG and MA were also much higher in H.pylori-positive specimens compared to H.pylori-negative specimens (P<0.05); Among the other groups no significant difference was found (P>0.05).
(2) In H.pylori related gastric mucosal lesions with CNAG or MA, DYS or GC, Akt, pAkt and mutant p53 expression showed no significant difference among all the groups (P>0.05). Mdm2 and PCNA protein expressions in Group GC and Dys were significantly higher than in Group CNAG and MA (P<0.05). Bax protein expression showed much higher in Group MA than those in Group CNAG, Dys and Group GC (P<0.05)
(3) In gastric mucosal lesions with CNAG or MA or DYS, between pAkt and Mdm2 expressions, a positive correlation was found (P<0.05). Similar results were also found between Mdm2 and PCNA expressions (P<0.05).
2. The effect of culture filtrate of H.pylori on the biologic behavior of GES-1 cells
(1) morphological changes:during 48hrs after the introduction of culture filtrate of H.pylori to GES-1 cells, with invert microscope and electron microscopy typical vacuolization and apoptosis were observed in those cells, and getting worse with the increasing of culture filtrate concentration and action time.
(2) 48hrs after the introduction of 1:4 (dilution ratio) culture filtrate of H.pylori to GES-1 cells, in DNA electrophoresis clear DNA ladder were observed, but in control group similar changes were not found.
(3) Proliferative activity of GES-1 cells after the introduction of culture filtrate of H.pylori (MTT method):at the 3rd hr,6th hr and 12th hr survival rates were found similar among all the groups (compared to control, P>0.05). At the 24th hr survival rates of all the concentrations were found significantly lower compared to control (P<0.05), but among those concentrations no significant difference was found (P>0.05). At the 48th hr survival rates of 1:4 and 1:2 (dilution ratio) culture filtrate (74.7±12.1%,62.2±12.6%) were found significantly lower than those of control (100.0±8.3%,P<0.05) in a concentration-dependent way; but no significant difference was found in that of 1:8 culture filtrate compared to control (P>0.05); at 72nd hr survival rates of 1:8、1:4、1:2、1:1(dilution ratio) groups were found significantly lower compared to control (P<0.05) in a concentration-dependent way, among them significant differences were found (P<0.05); at the 80th day after the introduction of 1:8 culture filtrate of H.pylori, compared to control (Parallel Passage) survival rate increased (P<0.05).
(4) Flow cytometry:3hrs after the introduction of 1:4 (dilution ratio) culture filtrate of H.pylori, cell cycles and apoptosis rate did not change (P<0.05); 48hrs after the introduction G0/G1 cell cycle arrest was found (98.73±1.12%vs45.37±4.89%)and apoptosis rate increased (26.93±5.34vs1.65±0.44) (P<0.05).
(5) Detection of DNA damage with single cell gel electrophoresis:3hrs after the introduction of 1:4 (dilution ratio) culture filtrate of H.pylori to GES-1 cells, the percentage of DNA in the comet tail were similar to those of control (P>0.05); 48hrs after the introduction,the percentage of DNA in the comet tail were increased significantly (P<0.05).
3. The role of Akt-Mdm2-p53 signaling axis in the effect of culture filtrate of H.pylori on GES-1 cells.
(1) Expressions of p53, Mdm2 and Bax mRNA after the introduction of culture filtrate of H.pylori to GES-1 cells:48hrs after the introduction of 1:4 dilution of culture filtrate of H.pylori, p53 mRNA changed little(P>0.05); Mdm2 mRNA, Bax mRNA expressions increased (P<0.05).
(2) Akt-Mdm2-p53 signaling axis related protein after the introduction of culture filtrate of H.pylori to GES-1 cells:After the introduction of 1:4 dilution of culture filtrate of H.pylori, Akt changed little (P>0.05). pAkt increased 1hr after the introduction,reached the peak at 3rd hr and kept increasing during 48hrs after the introduction. Mdm2 showed similar changes. p53 did not change during 12hrs but increased at 24th and 48th hr. Bax increased lhr after the introduction and kept increasing during 48hrs.
(3) After the treatment with Akt inhibitor, LY294002 introduce 1:4 dilution of culture filtrate of H.pylori to GES-1 cells at 12th,24th hr the survival rate decreased significantly and apoptosis rate increased significantly compared to the group without inhibitor treatment (P<0.05).24hrs after the introduction phosphorylated-Akt and Mdm2 expressions decreased significantly (p<0.05), but p53 protein increased significantly. (P>0.05).
Conclusions:
1.Akt-Mdm2-p53 signaling axis was related to the occurrence and development of the H.pylori related gastric mucosal lesions, acting at the stage of CNAG, MA and Dys caused by H.pylori infection, No relationship was found between Akt-Mdm2-p53 signaling axis and H.pylori infection in gastric cancer stages, which suggested Akt-Mdm2-p53 signaling axis mainly played a role in the early stage of H.pylori-related gastric cancer.
2.In a short-term effect, culture filtrate of H.pylori caused the apoptosis of human gastric epithelial cell, decay of activity and DNA damage, and this kind of effect was related to concentration and action time; with a long-term introduction of low concentration of culture filtrate, H.pylori could induce the increase of cytoactive of gastric epithelial cell.
3. H.pylori could simultaneously activate the signaling pathways of apoptosis and proliferation. In the early stage H.pylori restrained apoptosis by activating Akt and promoting Mdm2 expression, which made p53 changed little. When DNA damage became worse, H.pylori promoted apoptosis by activating p53 and caused G0/G1 cell cycle arrest. The blockade of Akt pathway could cause the inhibition effect of H.pylori to GES-1,the decline of Mdm2 and upgrade of p53. Therefore, at different stages of H.pylori action Akt-Mdm2-p53 signaling axis could co-regulate the apoptosis and proliferation according to the damage of cells, playing an important role in the pathopoiesis of H.pylori.
幽门螺杆菌(Helicobacter pylori,Hp)感染约占全球人口的一半,但只有少部分的感染者发病。现已明确,Hp是慢性胃炎、消化性溃疡、胃MALT淋巴瘤和胃癌的致病因子。在正常胃黏膜中,胃上皮细胞增殖和凋亡保持动态平衡,而Hp感染能打破这一平衡,并与感染结局及致病多样性密切相关。目前有关Hp导致胃上皮细胞凋亡增殖的研究中,结论很不一致,甚至相互矛盾。许多体内研究表明Hp感染既可促进胃上皮细胞凋亡,又可诱导细胞增殖,从而加速了细胞的更新,认为和其诱导的免疫炎症反应和代偿性增生有关。许多体外研究报道Hp能直接诱导胃上皮细胞凋亡,但也有研究认为Hp主要是刺激胃上皮细胞增殖,并能在早期直接激活细胞增殖信号通路,促进DNA合成增加,从而使细胞恶变风险增加。目前体外研究所选用的大多是不同类型的胃癌上皮细胞,而这些胃癌细胞本身在凋亡、增殖信号传导等方面已存在异常。不同浓度的Hp和作用时间长短的影响也不同,这可能是结果不一致的原因之一。
在细胞凋亡增殖的调控中,Akt-Mdm2-p53通路发挥重要作用。Akt促进细胞增殖和抗细胞凋亡,它磷酸化Mdm2,使Mdm2稳定和可更有效地转位到核内,通过泛素化途径降解p53蛋白而下调p53功能,使细胞能抗衡p53介导的凋亡。而Mdm2也是p53的一个靶基因,可被p53负反馈调控。同时,p53可通过上调PTEN,而发挥间接抑制Akt的作用。因此,Akt-Mdm2-p53信号通路在决定细胞凋亡或增殖中扮演重要角色,而它在Hp致病中的作用尚不清楚。
因此,针对以上研究现状,本课题研究不同阶段胃黏膜病变中Akt-Mdm2-p53信号通路相关蛋白的表达和Hp感染的关系；同时进行体外实验研究,选用来源于人正常胃上皮细胞的GES-1细胞为实验对象(预试验提示能表达野生型p53),研究Hp培养滤液对GES-1细胞凋亡增殖的直接影响及和Akt-Mdm2-p53信号通路的关系,为探讨Hp的致病机制提供理论和实验依据。方法：
1、收集Hp阳性和阴性的慢性非萎缩性胃炎(CNAG)、化生性萎缩(MA)、异型增生(Dys)和胃癌(GC)胃黏膜病理标本,行Akt-Mdm2-p53信号通路相关蛋白的检测。
2、制备NCTC11637Hp标准菌株培养滤液,测定浓度为11.1mg/ml,以此为初浓度,稀释不同的浓度,和GES-1细胞共培养,设立只加Hp培养液的对照组,根据实验在不同时间点收集细胞行相应检测。
3、Akt信号通路的阻断：加40umol/L的LY294002预处理GES-1细胞1h,设立加40umol/L的DMSO为对照组。
4、相应指标的检测：
(1)病理标本Hp感染检测：采用Giemsa染色法检测。
(2)病理标本蛋白的检测：应用免疫组化PV-9000法检测。
(3)细胞形态学检测：倒置显微镜和电镜观察。
(4)细胞活力的检测：采用四唑蓝(MTT)比色试验检测。
(5)细胞周期的检测：采用PI染色流式细胞术方法检测。
(6)细胞凋亡的检测：采用DNA电泳和Annexinⅴ/PI双染色流式细胞术检测。
(7)DNA损伤检测：采用单细胞凝胶电泳检测。
(8)细胞mRNA的检测：采用逆转录聚合酶链反应(RT-PCR)法检测。
(9)细胞蛋白的检测：采用Western blotting方法检测。结果：
1、不同胃粘膜病变中Hp感染和Akt-Mdm2-p53信号通路表达的关系
(1)在CNAG、MA、DYS和GC各粘膜病变中分别行Hp阳性者和Hp阴性者比较,pAkt在CNAG组的Hp阳性者显著高于Hp阴性者(P<0.05)；Akt在各粘膜病变中均无显著性差异(P>0.05);Mdm2在Dys组Hp阳性者显著高于阴性者(P<0.05)；突变型p53在MA组Hp阳性者显著高于阴性者(P<0.05)；PCNA在Dys组Hp阳性者显著高于阴性者(P<0.05)；Bax在CNAG、MA组阳性者显著高于阴性者(P<0.05),其余各组无显著性差异(P>0.05)。
(2)在Hp阳性的CNAG、MA、DYS和GC各粘膜病变中,Akt、pAkt在各病变组的表达无显著差异(P>0.05)。Mdm2、PCNA蛋白的表达在GC、Dys组显著高于CNAG、MA组(P<0.05),Bax蛋白的表达在MA组最高,显著高于CNAG、Dys、GC组(P<0.05)
(3)在Hp阳性的CNAG、MA、Dys胃粘膜病变中,对pAkt、Mdm2、突变型p53、Bax和PCNA蛋白的表达行相关性分析,pAkt和Mdm2,Mdm2和PCNA的表达呈正相关(P<0.05)。
2、Hp培养滤液对GES-1细胞生物学行为的影响
(1)形态学变化：Hp培养滤液作用GES-1细胞后48h内电镜和倒置显微镜下观察,细胞呈现典型的空泡化、凋亡改变,且随Hp培养滤液浓度的增加和作用时间的延长而加重。
(2)1：4浓度Hp培养滤液作用GES-1细胞48h后行DNA电泳,结果DNA条带呈凋亡特征性梯状改变,而对照组无此改变。
(3)MTT检测Hp培养滤液作用GES-1细胞后的增殖活性：在3h、6h和12h时间点,各浓度组的细胞存活率和照组比较均无显著性差异(P>0.05)。作用24h,各浓度组的细胞存活率显著低于对照组(P<0.05),但各浓度组间无显著性差异(P>0.05)。作用48h,1：4、1：2浓度组的细胞存活率(74.7±12.1%,62.2±12.6%)均显著低于对照组(100.0±8.3%)(P<0.05),且呈浓度依赖性。但对照组与1：8组之间无显著性差异(P>0.05)；作用72h时,1：8、1：4、1：2、1：1组的细胞存活率显著低于对照组(P<0.05),且呈浓度依赖性,均有显著性差异(P<0.05)。行1：·8浓度的Hp培养滤液作用GES-1细胞80天后,和平行传代的对照组比较,细胞存活率显著增加。(P<0.05)
(4)流式细胞仪分析显示：1：4浓度的Hp培养滤液作用GES-1细胞48h后,细胞周期主要阻滞在G0-G1期(98.73±1.12%vs45.37±4.89%),凋亡率显著上升(26.93±5.34%vsl.65±0.44%)(P<0.05)。
(5)单细胞凝胶电泳检测DNA损伤：1：4浓度的Hp培养滤液作用GES-1细胞3h后,和对照组比较,彗星尾部DNA量占全部DNA量的比例无显著性差异(P>0.05)；作用48h后,彗星尾部DNA量显著增加。(P<0.05)。
3、Akt-Mdm2-p53信号通路在Hp培养滤液对GES-1细胞影响中的作用
(1)Hp培养滤液作用GES-1细胞后p53、Mdm2、Bax mRNA的表达：1：4浓度的Hp培养滤液作用GES-1细胞48h,p53 mRNA无显著变化(P>0.05)；Mdm2 mRNA、Bax mRNA的表达显著上调(P<0.05)；
(2)Hp培养滤液作用GES-1细胞后Akt-Mdm2-p53通路相关蛋白水平及活性的变化：1：4浓度的Hp培养滤液作用GES-1细胞不同时间后,Akt蛋白无明显变化,pAkt在1h后上升,在3h达最高峰,在48h内持续升高,Mdm2蛋白具有类似变化趋势；p53蛋白12h内无明显变化,在24、48h上升；Bax蛋白在1h后上升,48h内持续升高。
(3)加入Akt抑制剂LY294002处理GES-1细胞后,用1：4浓度的Hp培养滤液作用细胞,与未加入抑制剂比较,12h和24h的细胞存活率显著下降(P<0.05)。在作用24h后pAkt、Mdm2蛋白表达显著下降(P<0.05),而p53蛋白表达显著上调(P<0.05)。结论：
1、Akt-Mdm2-p53信号通路参与了Hp感染相关的胃黏膜病变发生、发展,在Hp感染导致的CNAG、MA、Dys病变阶段中起作用,而在胃癌病变阶段中与Hp感染无明显关系,提示Akt-Mdm2-p53信号通路主要在Hp致癌的早期阶段起作用。
2、在短期作用下,Hp培养滤液可导致胃上皮细胞凋亡,活力下降,DNA损伤,且呈浓度和时间依赖关系；在低浓度培养滤液长期作用下,Hp能诱导胃上皮细胞增殖活性升高。
3、Hp可同时激活凋亡和增殖信号通路。在早期,Hp通过激活Akt和诱导Mdm2的表达对抗Hp诱导的凋亡作用,使p53水平无明显变化。但在细胞DNA损害加重时,Hp激活p53通路促进细胞凋亡和使细胞阻滞在G0/G1期。在Hp感染时阻断Akt通路可使Hp对细胞的抑制作用加重,Mdm2蛋白下调,p53蛋白上调。因此,在Hp作用的不同时期,Akt-Mdm2-p53信号通路根据细胞的损害程度共同调控细胞的存活或死亡,在Hp致病中起重要作用。
Background and purposes:
Although half of the world's population is infected by Helicobacter pylori(H.pylori), few fall ill. Now it is accepted that H.pylori is the causative agent of chronic gastritis, peptic ulcer, gastric MALT lymphoma and gastric cancer. The balance of human gastric epithelial cell proliferation and apoptosis will be broken by the infection of H.pylori, and the infection is closely related to the diversity of outcome. however it is still very controversial about the effect of H.pylori on gastric epithelial cell proliferation and apoptosis. It has been proven in vivo that the infection of H.pylori could simultaneously accelerate the apoptosis and induce the proliferation, promoting the cell renewal and this effect is related to immuno- inflammatory response and compensatory hyperplasia caused by the infection of H.pylori. Meanwhile some in vitro studies show that H.pylori could induce the epithelial cell apoptosis directly, and some others show that H.pylori could promote the epithelial cell proliferation, activate the signal pathway of early proliferation directly, increase DNA synthesis and increase the risk of malignant transformation. At present most in vitro studies choose different styles of gastric cancer epithelial cell, however these cells themselves may be different in the signal transductions of apoptosis and proliferation, and the reactions to different concentrations of H.pylori and action time may also be different, which might be one of the reasons for the diversity of results.
Akt-Mdm2-p53 signaling axis plays an important role in the regulation of proliferation and apoptosis. Akt promotes proliferation and restrains apoptosis. Akt phosphorylates Mdm2, makes Mdm2 stable and nuclear translocation more effective, down regulate the function of p53 by the ubiquitination of p53, then help cells resist the p53- induced apoptosis.Mdm2 is a target gene of p53 which also receive the negative-feedback regulation of p53. At the same time, p53 can restrain the effect of Akt by up-regulating PTEN. However the role of Akt-Mdm2-p53 signaling axis in the pathopoiesis of H.pylori is still unkown.
Therefore, we study the relationship between the Akt-Mdm2-p53 signaling axis related protein expression in different stages of gastric mucosal lesions and the infection of H.pylori; at the same time in vitro study we choose normal human gastric epithelial GES-1 cells as our experimental subject (in pretest wild type p53 expressed) to research the effect of culture filtrate of H.pylori on GES-1 cell proliferation and apoptosis and the relationship between it and Akt-Mdm2-p53 signaling axis; Finally find out the possible explanations to the machnism of the pathopoiesis of H.pylori.
Methods:
1. Collect pathologic specimens of gastric mucosa (H.pylori related or not)with chronic no-atrophic gastritis (CNAG), metaplastic atrophy (MA), dysplasia (Dys) or gastric cancer(GC) and detect the Akt-Mdm2-p53 signaling axis related proteins.
2. Prepare the culture filtrates of type culture strain (NCTC11637H.pylori), concentration is set to be 11mg/ml, and then dilute it to different concentrations, co-cultured with GES-1 cells. In control group only H.pylori culture medium is added, cells are collected and detected at different points.
3. Block the Akt-Mdm2-p53 signaling axis:GES-1 cells are pretreated with 40μmol/l LY294002, set up a control group only with 40μmol/l DMSO.
4. Detections of related parameters:
(1)Test for H.pylori infection in pathologic specimens:Giemsa's staining
(2) Test for proteins in pathologic specimens:PV9000 immunohistochemical method
(3) Cytomorphological examination:invert microscope and electron microscopy
(4) Cytoactive test:MTT method
(5) Test for cell cycle:PI flow cytometry
(6) Apoptosis test:DNA electrophoresis and Annexinⅴ/PI flow cytometry
(7) Detection of DNA damage:single cell gel electrophoresis
(8) mRNA test:reverse transcriptase PCR (RT-PCR)
(9) Test for cell protein:Western blotting
Results:
1. The relationship between the expression of Akt-Mdm2-p53 signaling axis and the infection of H.pylori in different gastric mucosal lesions.
(1) In gastric mucosal lesions with CNAG or MA, DYS or GC, pAkt expression in Group CNAG was much higher in H.pylori-positive specimens compared to H.pylori-negative specimens (P<0.05); Akt expression showed no significant difference among all the groups (P>0.05);Mdm2 expression in Group Dys was also significantly higher in H.pylori-positive specimens compared to H.pylori-nagative specimens (P<0.05); Mutant p53 of Group MA was much higher in H.pylori-positive specimens compared to H.pylori-negative specimens (P<0.05); PCNA of Group Dys was significantly higher in H.pylori-positive specimens compared to H.pylori-negative specimens (P<0.05); Bax expressions of Group CNAG and MA were also much higher in H.pylori-positive specimens compared to H.pylori-negative specimens (P<0.05); Among the other groups no significant difference was found (P>0.05).
(2) In H.pylori related gastric mucosal lesions with CNAG or MA, DYS or GC, Akt, pAkt and mutant p53 expression showed no significant difference among all the groups (P>0.05). Mdm2 and PCNA protein expressions in Group GC and Dys were significantly higher than in Group CNAG and MA (P<0.05). Bax protein expression showed much higher in Group MA than those in Group CNAG, Dys and Group GC (P<0.05)
(3) In gastric mucosal lesions with CNAG or MA or DYS, between pAkt and Mdm2 expressions, a positive correlation was found (P<0.05). Similar results were also found between Mdm2 and PCNA expressions (P<0.05).
2. The effect of culture filtrate of H.pylori on the biologic behavior of GES-1 cells
(1) morphological changes:during 48hrs after the introduction of culture filtrate of H.pylori to GES-1 cells, with invert microscope and electron microscopy typical vacuolization and apoptosis were observed in those cells, and getting worse with the increasing of culture filtrate concentration and action time.
(2) 48hrs after the introduction of 1:4 (dilution ratio) culture filtrate of H.pylori to GES-1 cells, in DNA electrophoresis clear DNA ladder were observed, but in control group similar changes were not found.
(3) Proliferative activity of GES-1 cells after the introduction of culture filtrate of H.pylori (MTT method):at the 3rd hr,6th hr and 12th hr survival rates were found similar among all the groups (compared to control, P>0.05). At the 24th hr survival rates of all the concentrations were found significantly lower compared to control (P<0.05), but among those concentrations no significant difference was found (P>0.05). At the 48th hr survival rates of 1:4 and 1:2 (dilution ratio) culture filtrate (74.7±12.1%,62.2±12.6%) were found significantly lower than those of control (100.0±8.3%,P<0.05) in a concentration-dependent way; but no significant difference was found in that of 1:8 culture filtrate compared to control (P>0.05); at 72nd hr survival rates of 1:8、1:4、1:2、1:1(dilution ratio) groups were found significantly lower compared to control (P<0.05) in a concentration-dependent way, among them significant differences were found (P<0.05); at the 80th day after the introduction of 1:8 culture filtrate of H.pylori, compared to control (Parallel Passage) survival rate increased (P<0.05).
(4) Flow cytometry:3hrs after the introduction of 1:4 (dilution ratio) culture filtrate of H.pylori, cell cycles and apoptosis rate did not change (P<0.05); 48hrs after the introduction G0/G1 cell cycle arrest was found (98.73±1.12%vs45.37±4.89%)and apoptosis rate increased (26.93±5.34vs1.65±0.44) (P<0.05).
(5) Detection of DNA damage with single cell gel electrophoresis:3hrs after the introduction of 1:4 (dilution ratio) culture filtrate of H.pylori to GES-1 cells, the percentage of DNA in the comet tail were similar to those of control (P>0.05); 48hrs after the introduction,the percentage of DNA in the comet tail were increased significantly (P<0.05).
3. The role of Akt-Mdm2-p53 signaling axis in the effect of culture filtrate of H.pylori on GES-1 cells.
(1) Expressions of p53, Mdm2 and Bax mRNA after the introduction of culture filtrate of H.pylori to GES-1 cells:48hrs after the introduction of 1:4 dilution of culture filtrate of H.pylori, p53 mRNA changed little(P>0.05); Mdm2 mRNA, Bax mRNA expressions increased (P<0.05).
(2) Akt-Mdm2-p53 signaling axis related protein after the introduction of culture filtrate of H.pylori to GES-1 cells:After the introduction of 1:4 dilution of culture filtrate of H.pylori, Akt changed little (P>0.05). pAkt increased 1hr after the introduction,reached the peak at 3rd hr and kept increasing during 48hrs after the introduction. Mdm2 showed similar changes. p53 did not change during 12hrs but increased at 24th and 48th hr. Bax increased lhr after the introduction and kept increasing during 48hrs.
(3) After the treatment with Akt inhibitor, LY294002 introduce 1:4 dilution of culture filtrate of H.pylori to GES-1 cells at 12th,24th hr the survival rate decreased significantly and apoptosis rate increased significantly compared to the group without inhibitor treatment (P<0.05).24hrs after the introduction phosphorylated-Akt and Mdm2 expressions decreased significantly (p<0.05), but p53 protein increased significantly. (P>0.05).
Conclusions:
1.Akt-Mdm2-p53 signaling axis was related to the occurrence and development of the H.pylori related gastric mucosal lesions, acting at the stage of CNAG, MA and Dys caused by H.pylori infection, No relationship was found between Akt-Mdm2-p53 signaling axis and H.pylori infection in gastric cancer stages, which suggested Akt-Mdm2-p53 signaling axis mainly played a role in the early stage of H.pylori-related gastric cancer.
2.In a short-term effect, culture filtrate of H.pylori caused the apoptosis of human gastric epithelial cell, decay of activity and DNA damage, and this kind of effect was related to concentration and action time; with a long-term introduction of low concentration of culture filtrate, H.pylori could induce the increase of cytoactive of gastric epithelial cell.
3. H.pylori could simultaneously activate the signaling pathways of apoptosis and proliferation. In the early stage H.pylori restrained apoptosis by activating Akt and promoting Mdm2 expression, which made p53 changed little. When DNA damage became worse, H.pylori promoted apoptosis by activating p53 and caused G0/G1 cell cycle arrest. The blockade of Akt pathway could cause the inhibition effect of H.pylori to GES-1,the decline of Mdm2 and upgrade of p53. Therefore, at different stages of H.pylori action Akt-Mdm2-p53 signaling axis could co-regulate the apoptosis and proliferation according to the damage of cells, playing an important role in the pathopoiesis of H.pylori.
引文
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