PTEN在纤维化肝组织中的动态表达及其对肝星状细胞增殖与凋亡的影响
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摘要
肝纤维化(hepatic fibrosis, HF)是各种不同致病因子引起慢性肝病进而发展为肝硬化的共有病理改变和必经途径,是肝脏对各种慢性损伤产生的一种修复反应。其主要病理改变是细胞外间质(extracellular matrix, ECM)的过度合成与异常沉积。肝星状细胞(hepatic stellate cells, HSC)是参与该过程的最重要细胞,它的激活导致自身增殖和胶原合成增加被认为是肝纤维化形成的中心环节,而肝纤维化恢复期HSC凋亡明显增多。因此,抑制HSC活化、增殖或诱导其凋亡是逆转肝纤维化的关键所在。
第10号染色体缺失的磷酸酶张力蛋白同源物基因(phosphatase and tensin homology deleted on chromosome Ten, PTEN)是迄今发现的第一个具有磷酸酶活性的肿瘤抑制基因,可负性调控肿瘤细胞细胞周期、抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡,其缺失或表达异常与多种肿瘤的发生发展密切相关。
近年来,对PTEN的研究已从肿瘤领域逐渐延伸至非肿瘤领域。研究表明,在特发性肺纤维化患者的肺组织中,成纤维细胞的PTEN表达及其磷酸酶活力都低于正常肺成纤维细胞;并且其过表达或激活可抑制体外肺成纤维细胞增殖、诱导其凋亡。在有关PTEN与心肌纤维化的研究中也显示PTEN基因敲除的小鼠心脏与体重之比增加、心肌纤维化程度明显、心肌收缩性降低。这提示PTEN的低表达或失活参与了特发性肺纤维化及心肌纤维化的发生。而在PTEN与某些肝脏疾病的研究中,有研究发现特异性肝细胞PTEN缺失不仅可引起肝细胞癌而且还导致与肝纤维化密切相关的非酒精性脂肪性肝炎的发生。但迄今为止,PTEN在肝纤维化中的表达及作用,特别是对HSC增殖及凋亡的影响仍不清楚。为此,本研究应用胆总管结扎法建立胆汁淤积性大鼠肝纤维化模型,探讨PTEN在肝纤维化过程中的动态表达及其与在体HSC增殖、凋亡的关系;并进一步将携带野生型PTEN基因及其突变体G129E基因的重组腺病毒转染体外培养的活化HSC,观察过表达的PTEN对活化HSC增殖、凋亡及细胞周期的影响,同时探讨PTEN调控活化HSC增殖、凋亡及细胞周期的信号转导机制,旨在为深入揭示肝纤维化的病理生理机制,寻求有效预防和治疗肝纤维的新策略提供实验依据。实验内容主要包括以下四部分:
第一部分PTEN在大鼠纤维化肝组织中的动态表达及其与在体肝星状细胞增殖、凋亡的关系
目的:研究大鼠肝纤维化过程中肝组织的PTEN动态表达及其与在体HSC增殖、凋亡的关系。
方法:胆总管结扎法建立胆汁淤积性大鼠肝纤维化模型,HE、Masson三色染色观察肝脏病理组织学变化,免疫组织化学染色检测大鼠肝组织中PTEN及α-平滑肌肌动蛋白(alpha-smooth muscle actin,α-SMA)的分布;免疫荧光双标记共聚焦激光扫描显微镜技术测定大鼠肝组织中活化HSC的PTEN表达;末段脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末段标记(TUNEL)及α-SMA免疫组织化学双染检测在体活化HSC的凋亡;Western blot和Real-time Q-PCR检测大鼠肝组织中PTEN蛋白及mRNA表达。
结果:①HE及Masson三色染色显示,胆汁淤积性大鼠肝纤维化模型成功建立,随着造模时间延长肝纤维化程度逐渐加重。在造模不同时间均可见不同程度的肝细胞变性坏死而导致正常肝细胞逐渐减少。②α-SMA免疫组织化学染色显示,正常大鼠肝组织中仅在血管壁平滑肌细胞有弱阳性表达;随着肝纤维化的发展,大鼠肝组织中α-SMA阳性细胞逐渐增多,造模1 wk、2 wk、3 wk及4 wk不同时间大鼠肝组织α-SMA的阳性光密度值(0.16±0.01,0.17±0.01,0.21±0.01,0.26±0.02)均显著高于假手术组(0.07±0.01), P<0.01;且各组之间均有明显差异(P<0.01)。③TUNEL及α-SMA免疫组织化学双重染色显示,正常大鼠肝组织几乎看不见凋亡HSC,随着肝纤维化程度加重、活化HSC增多,凋亡HSC也增多,活化HSC与凋亡HSC共存。造模1 wk、2 wk、3 wk及4 wk不同时间大鼠肝组织HSC凋亡指数分别为4.57%±0.41%、4.02%±0.48%、3.45%±0.37%、2.88%±0.50%,即随着肝纤维化程度加重,活化HSC凋亡指数逐渐减少(P<0.01)。④PTEN免疫组织化学染色显示,正常大鼠肝组织中PTEN蛋白有广泛表达,主要表达于细胞浆,在胞核中也有表达;随着肝纤维化的进展,大鼠肝组织中PTEN阳性细胞逐渐减少,主要分布于汇管区、纤维间隔及增生的胆管周围细胞,PTEN蛋白的亚细胞定位没有明显变化。造模1 wk、2 wk、3 wk及4 wk大鼠肝组织PTEN的阳性光密度值(0.15±0.01,0.12±0.02,0.09±0.01,0.07±0.01)均显著低于假手术组(0.21±0.02),P<0.01;且各组之间均有明显差异(P<0.01)。⑤共聚焦激光扫描显微镜下观察PTEN和α-SMA免疫荧光双标记的大鼠肝组织切片,单通道扫描可见PTEN和α-SMA阳性反应产物分别呈绿色和红色荧光斑点状,将单通道扫描的图象混合后,在肝组织切片内除了绿色和红色荧光斑点外,还可见黄色荧光斑点,因为纤维化肝组织中只有活化HSC和少许血管平滑肌细胞表达α-SMA,故这些黄色斑点绝大部分可看做是PTEN和α-SMA在HSC的共表达产物,也就是活化HSC的PTEN阳性表达。共表达产物主要存在于活化HSC细胞浆,在部分细胞核中也有少许表达。图像分析结果显示,造模1 wk、2 wk、3 wk及4 wk大鼠肝组织中PTEN和α-SMA共存的HSC(PTEN表达阳性的活化HSC)占α-SMA表达阳性细胞(总的活化HSC)的比例分别为79.97%±5.49%、73.83%±5.04%、66.68%±4.58%、60.20%±4.65%。即随着肝纤维化的进展,表达PTEN的活化HSC比例逐渐减少(P<0.01)。⑥Western blot显示,造模1 wk、2 wk、3 wk及4 wk大鼠纤维化肝组织PTEN蛋白表达量(1.20±0.13,1.07±0.16, 0.88±0.08,0.73±0.07)均显著低于假手术组(1.37±0.14),P<0.01;且各组之间均有明显差异(P<0.01),即大鼠肝纤维化越重PTEN蛋白表达越低。⑦应用Real-time Q-PCR技术检测大鼠肝组织的PTEN mRNA表达,并采用相对定量2-△△Ct法比较PTEN mRNA在各组大鼠肝组织中的相对表达。以假手术组为对照组(其PTEN mRNA的表达量为1),则造模1 wk、2 wk、3 wk及4 wk大鼠纤维化肝组织中PTEN mRNA相对假手术组的表达倍数为0.66倍、0.53倍、0.44倍、0.37倍,均显著低于假手术组,且随着肝纤维化的发展逐渐下调(P<0.01)。⑧Pearson’s相关性分析发现,大鼠纤维化肝组织中PTEN表达与α-SMA表达呈显著负相关,与PTEN阳性的活化HSC比例呈显著正相关,与活化HSC凋亡指数呈显著正相关,r值分别为-0.92、0.78、0.76,P<0.01。
结论:胆总管结扎法成功建立胆汁淤积性大鼠肝纤维化模型;大鼠肝纤维化形成过程中HSC的活化、增殖逐渐加快,而活化HSC的凋亡指数却逐渐减少;随着肝纤维化进展,大鼠肝组织中PTEN蛋白及mRNA表达逐渐下调,在体HSC的PTEN表达亦降低,其动态表达与在体HSC的活化、增殖呈显著负相关,而与活化HSC凋亡指数呈显著正相关。
第二部分PTEN对体外活化肝星状细胞增殖与凋亡的影响
目的:探讨过表达的野生型PTEN及其突变体G129E(丧失了脂质磷酸酶活性仅保留蛋白磷酸酶活性)对体外活化的HSC增殖、凋亡的影响及可能的机制。
方法:利用AD293细胞扩增实验所需的腺病毒(Ad-PTEN、Ad-G129E、Ad-GFP),并测定滴度;以腺病毒为载体将野生型PTEN基因及其突变体G129E基因瞬时转染体外培养的活化的HSC;Western blot及Real-time Q-PCR检测HSC PTEN表达;MTT法检测HSC增殖;TUNEL及碘化丙啶(propidium iodide, PI)标记的流式细胞术测定HSC凋亡;Western blot检测HSC的凋亡调控基因Bcl-2及Bax表达。实验分组如下:①Control组,仅以含8%胎牛血清的DMEM细胞培养液培养细胞,在转染步骤入无血清无抗生素DMEM代替病毒液;②Ad-GFP组,转染表达绿色荧光蛋白(green fluorescent protein, GFP)的空病毒Ad-GFP;③Ad-PTEN组,转染携带野生型PTEN基因并表达GFP的重组腺病毒Ad-PTEN;④Ad-G129E组,转染携带PTEN的突变体G129E基因并表达GFP的重组腺病毒Ad-G129E。
结果:①通过反复感染AD293细胞的方法使病毒扩增获得了实验所需的病毒液(Ad-PTEN、Ad-G129E、Ad-GFP的滴度分别为1.2 x 109、1.5 x 109、1.8 x 109 pfu/ml)。②腺病毒感染HSC后72 h,应用Real time Q-PCR检测活化HSC PTEN mRNA表达,并采用相对定量2-△△Ct法比较PTEN mRNA在各组HSC中的表达。以Control组为对照组(其PTEN mRNA表达量为1),则Ad-GFP组、Ad-PTEN组和Ad-G129E组相对Control组的PTEN mRNA表达倍数分别为0.993倍、1.569倍和1.561倍。很明显,Ad-PTEN组及Ad-G129E组PTEN mRNA表达明显高于Control组及Ad-GFP组(P<0.01);而Ad-PTEN组与Ad-G129E组,以及Control组与Ad-GFP组之间无明显差异(P>0.05)。进一步应用Western blot分析各组HSC的PTEN蛋白表达,Ad-PTEN组(1.66±0.09)、Ad-G129E组(1.65±0.09)均显著高于Control组(1.10±0.07)及Ad-GFP组(1.09±0.07),P<0.01;而Control组与Ad-GFP组之间,以及Ad-PTEN组和Ad-G129E组之间均无明显差异(P>0.05)。证明野生型PTEN基因及其突变体G129E基因成功转染入体外活化的HSC。③MTT检测显示,野生型PTEN基因及G129E基因转染活化HSC后24 h对细胞增殖无明抑制(P>0.05)。但在48 h、72 h HSC增殖受到明显抑制,Ad-PTEN组增殖抑制率为14.03%、23.12%,而Ad-G129E组增殖抑制率是9.52%、12.63%(均以Control组为对照)。在各时间点,Ad-GFP组与Control组A值比较无明显差异(P<0.05)。显然,野生型PTEN对HSC增殖的抑制作用明显强于G129E。④TUNEL检测发现,在腺病毒感染HSC后72 h,Ad-PTEN组HSC凋亡率增至29.81%±2.52%,Ad-G129E组增至26.37%±1.97%均显著高于Control组(1.98%±0.25%)及Ad-GFP组(2.16%±0.28%),P<0.01;而Ad-PTEN组HSC凋亡率亦明显高于Ad-G129E组(P<0.01);Ad-GFP组与Control组比较无显著差异(P>0.05)。⑤PI标记的流式细胞术检测显示,Ad-PTEN组HSC凋亡率(20.84%±1.44%)、Ad-G129E组HSC凋亡率(17.54%±1.76%)均显著高于Control组(1.12%±0.57%)及Ad-GFP组(1.21%±0.22%) , P<0.01 ;而Ad-PTEN组亦明显高于Ad-G129E组(P<0.01);但Ad-GFP组与Control组比较无显著差异(P>0.05)。⑥Western blot检测腺病毒感染HSC后72 h,各组HSC Bcl-2蛋白表达,Ad-PTEN组(1.16±0.03)、Ad-G129E组(1.24±0.05)均显著低于Control组(1.37±0.06)及Ad-GFP组(1.34±0.08),P<0.01,并且Ad-PTEN组较Ad-G129E组也明显降低(P<0.05),但Control组与Ad-GFP组之间无明显差异(P>0.05);而各组HSC Bax蛋白表达,Ad-PTEN组(1.50±0.05)、Ad-G129E组(1.41±0.05)均较Control组(1.28±0.06)及Ad-GFP组(1.26±0.08)显著增高,P<0.01,同时Ad-PTEN组明显高于Ad-G129E组(P<0.05),Control组与Ad-GFP组之间无明显差异(P>0.05)。
结论:外源性野生型PTEN基因及G129E基因成功转染体外活化HSC;其过表达可明显抑制活化HSC增殖,诱导活化HSC凋亡,并引起HSC的凋亡调控基因Bax表达增加,Bcl-2表达下降。而且野生型PTEN的作用明显强于G129E。
第三部分PTEN对体外活化肝星状细胞细胞周期的调控作用
目的:探讨过表达的野生型PTEN及其突变体G129E对体外活化HSC细胞周期的调控作用。
方法:体外培养活化的HSC,以腺病毒为载体将野生型PTEN基因及其突变体G129E基因瞬时转染体外活化的HSC;流式细胞术测定HSC细胞周期时相;Western blot及Real-time Q-PCR检测HSC的PTEN、细胞周期素D1(CyclinD1)、细胞周期素依赖性激酶4(Cyclin dependent kinase4, CDK4)及细胞周期素依赖性激酶抑制因子(cyclin dependent kinase inhibitor, CDI)之一的P27kip1蛋白及mRNA表达。实验分组同第二部分。
结果:①Real-time Q-PCR及Western blot检测证实外源性野生型PTEN基因及其突变体G129E基因在体外活化HSC大量表达(结果同第二部分)。②流式细胞仪检测结果显示,G0/G1期HSC细胞数,Ad-PTEN组(67.68%±2.75%)、Ad-G129E组(61.17%±3.41%)均较Control组(53.01%±2.37%)及Ad-GFP组(53.85%±3.08%)明显增高(P<0.01) ,而Ad-PTEN组也明显高于Ad-G129E组(P<0.01);S期HSC细胞数,Ad-PTEN组(14.42%±1.81%)、Ad-G129E组(18.17%±2.43%)均较Control组(22.17%±1.99%)及Ad-GFP组(21.54%±1.74%)明显降低(P<0.01) ,而Ad-PTEN组亦明显低于Ad-G129E组(P<0.01);G2/M期HSC细胞数:Ad-PTEN组(17.90%±2.70%)、Ad-G129E组(20.66%±2.37%)均明显低于Control组(24.82%±3.81%)及Ad-GFP组(24.62%±3.15%),P<0.01、P<0.05,而Ad-PTEN组与Ad-G129E组之间差异无统计学意义(P>0.05)。此外,各期Control组与Ad-GFP组之间HSC细胞数差异均无统计学意义(P>0.05)。③腺病毒感染HSC后72 h,Western blot分析各组HSC cyclinD1蛋白表达,Ad-PTEN组(1.12±0.07)、Ad-G129E组(1.23±0.05)均较Control组(1.45±0.05)及Ad-GFP组(1.47±0.08)显著降低(P<0.01),Ad-PTEN组亦明显低于Ad-G129E组(P<0.01),而Control组与Ad-GFP组之间无明显差异(P>0.05)。进一步应用Real time Q-PCR检测各组HSC cyclinD1 mRNA表达,并应用相对定量2-△△Ct法比较cyclinD1 mRNA在各组HSC中的相对表达。以Control组为对照组(其cyclinD1 mRNA表达量为1),则Ad-GFP组、Ad-PTEN组及Ad-G129E组相对Control组的cyclinD1 mRNA表达倍数分别为1.011倍、0.773倍和0.838倍。显然,Ad-PTEN组及Ad-G129E组CyclinD1 mRNA表达明显低于Control组及Ad-GFP组(P<0.01),Ad-PTEN组较Ad-G129E组也显著降低,而Control组与Ad-GFP组之间无明显差异(P>0.05)。④腺病毒感染HSC后72 h,Western blot检测各组HSC CDK4蛋白表达,Ad-PTEN组(1.05±0.07)、Ad-G129E组(1.18±0.06)均较Control组(1.41±0.03)及Ad-GFP组(1.43±0.06)显著降低(P<0.01),而Ad-PTEN组亦明显低于Ad-G129E组(P<0.01),但Control组与Ad-GFP组之间差异无统计学意义(P>0.05)。进一步应用Real time Q-PCR检测各组HSC的CDK4 mRNA表达,并应用相对定量2-△△Ct法比较CDK4 mRNA在各组HSC中的相对表达。以Control组为对照组(其CDK4 mRNA表达量为1),则Ad-GFP组、Ad-PTEN组及Ad-G129E组相对Control组的CDK4 mRNA表达倍数为1.007倍、0.738倍和0.822倍。显然,Ad-PTEN组、Ad-G129E组CDK4 mRNA表达较Control组及Ad-GFP组明显降低(P<0.01),Ad-PTEN组也显著低于Ad-G129E组(P<0.01),但Control组与Ad-GFP组之间无明显差异(P>0.05)。⑤腺病毒感染HSC后72 h,Western blot分析各组HSC P27kip1蛋白表达,Ad-PTEN组(1.40±0.03)、Ad-G129E组(1.28±0.08)均显著高于Control组(1.11±0.04)及Ad-GFP组(1.12±0.04),P<0.01,Ad-PTEN组较Ad-G129E组亦明显升高(P<0.01),但Control组与Ad-GFP组之间无明显差异(P>0.05)。进一步应用Real time Q-PCR检测各组HSC P27kip1 mRNA表达,并应用相对定量2-△△Ct法比较P27kip1 mRNA在各组HSC中的表达。以Control组为对照组(其P27kip1 mRNA表达量为1),则Ad-GFP组、Ad-PTEN组和Ad-G129E组相对Control组的P27kip1 mRNA表达倍数为1.008倍、1.264倍和1.157倍。可以看出,Ad-PTEN组、Ad-G129E组P27kip1 mRNA表达明显高于Control组及Ad-GFP组(P<0.01),Ad-PTEN组也较Ad-G129E组显著升高(P<0.01),而Control组与Ad-GFP组之间无明显差异(P>0.05)。
结论:过表达的野生型PTEN及G129E显著抑制体外活化HSC细胞周期G1/S期转化,阻滞HSC细胞周期时相于G0/G1期;同时在转录和翻译水平上下调活化HSC的cyclinD1、CDK4表达,上调P27kip1表达,这可能是其阻滞活化HSC细胞周期进程的重要机制。并且,在上述作用中野生型PTEN明显强于G129E。
第四部分PTEN调控活化肝星状细胞行为的信号转导机制
目的:探讨PTEN调控活化肝星状细胞增殖、凋亡及细胞周期的信号转导机制。
方法:体外培养活化HSC,以腺病毒为载体将野生型PTEN基因及其突变体G129E基因瞬时转染体外活化HSC;Western blot检测HSC的丝氨酸-苏氨酸蛋白激酶B(serine-threonine protein kinase B, Akt)、p-Akt(Thr308)、细胞外信号调节激1(extracellular signal-regulated kinase1, ERK1)、p-ERK1蛋白表达;Real-time Q-PCR检测Akt、ERK1 mRNA表达。实验分组同第二部分。
结果:①Real-time Q-PCR及Western blot检测证实外源性野生型PTEN基因及其突变体G129E基因在体外活化HSC大量表达(结果同第二部分)。②腺病毒感染HSC后72 h,Western blot及Real-time Q-PCR显示野生型PTEN及G129E对HSC Akt蛋白及其mRNA表达均无明显影响(P>0.05);而Western blot显示Ad-PTEN组p-Akt(Thr308)蛋白表达(0.63±0.04)显著低于Ad-G129E组(0.93±0.03)、Control组(0.95±0.04)及Ad-GFP组(0.94±0.03),P<0.01,但Ad-G129E组、Control组及Ad-GFP组之间p-Akt(Thr308)表达无显著差别(P>0.05)。③腺病毒感染HSC后72 h,Western blot及Real-time Q-PCR显示野生型PTEN及G129E对HSC ERK1蛋白及mRNA表达均无明显影响(P>0.05);而Western blot显示Ad-PTEN组p-ERK1蛋白表达(0.65±0.04)、Ad-G129E组p-ERK1蛋白表达(0.68±0.07)均显著低于Control组(0.84±0.07)及Ad-GFP组(0.85±0.06),P<0.01,但Ad-PTEN组与Ad-G129E组,以及Control组与Ad-GFP组之间p-ERK1表达无显著差异(P>0.05)。
结论:PTEN通过其磷酸酶活性抑制Akt及ERK1的磷酸化;其负性调控活化HSC细胞周期、抑制活化HSC增殖及诱导活化HSC凋亡的作用与PI3K/Akt、ERk1/2信号通路的活性抑制有关。
Hepatic fibrosis, the liver’s wound healing response to virtually all forms of chronic liver injury, can result from several chronic liver diseases caused by many pathogenic factors. Given enough time, fibrosis will progress to cirrhosis. The main pathological characteristic of hepatic fibrosis is the increased irregular deposition of extracellular matrix (ECM). Currently, it is believed that the activation of hepatic stellate cells (HSC), which play a pivotal role in fibrosis process, into cells with spontaneous proliferation and strong fibrogenic activity appears to be the dominant driving force in fibrosis. While the apoptosis of HSC increases significantly during the reparative process following liver injury. So the inhibiting proliferation or inducing apoptosis of activated HSC play a key role in the process of reversion of hepatic fibrosis.
Phosphatase and tensin homolog deleted on chromosome ten (PTEN), the first tumor-suppressing gene found to exhibit phosphatase activity, negatively regulates cell cycle, inhibits the proliferation and promotes the apoptosis of tumor cells. As such, it is reasonable to conclude that dysfunction or absence of PTEN is intimately related to the formation and progression of human tumors.
Nevertheless, in recent years, PTEN research has gradually extended beyond cancer, focusing on its role in other disease states. Studies demonstrated that lowered expression and phosphatase activity of PTEN is found in lung fibroblasts of patients with idiopathic pulmonary fibrosis. Moreover, studies also documented that PTEN inhibits the proliferation and induces the apoptosis of lung fibroblasts cultured in vitro. A study on PTEN and myocardial fibrosis showed that knocking out PTEN in mice leads to an increased ratio of heart to body weight, decreased cardiac contractility and, eventually, interstitial fibrosis. This suggests that the low expression or deactivation of PTEN involves itself in the pathogenesis of lung fibrosis and myocardial fibrosis. In the liver, the absence of PTEN in specific hepatic cells may result not only in hepatocellular carcinoma, but also in non-alcoholic steatohepatitis, a condition closely related to hepatic fibrosis. Currently, though, the expression and function of PTEN in hepatic fibrosis, especially its effects on the proliferation and apoptosis of HSC, remain unclear. So, using the rat bile duct ligation (BDL) model, this study explored the dynamic expression of PTEN in the process of hepatic fibrosis in rats and its relation with the proliferation and apoptosis of HSC in vivo. And we set out to determine the effects of PTEN over-expression, via adenoviral transduction of wild type PTEN and its mutant G129E gene, on the proliferation and apoptosis as well as cell cycle of activated HSC cultured in vitro. Meanwhile, the signaling transduction pathways of PTEN on the proliferation, apoptosis and of cell cycle of activated HSC were studied. It was designed to provide new viewpoint and defined strategy for preventing and treating hepatic fibrosis.The project contain four parts as below:
Part 1:The dynamic expression of PTEN in fibrogenic liver tissues in rats and its relation with the proliferation and apoptosis of hepatic stellate cells in vivo
Objective: To investigate the dynamic expression of PTEN in liver tissues in the process of hepatic fibrosis in rats and its relation with the proliferation and apoptosis of HSC in vivo.
Methods: The rat model of hepatic fibrosis used in this study was established by means of common bile duct ligation (BDL). HE and Masson's trichrome staining were used to determine histopathology changes of liver tissues. At 4 time points, the expressions of PTEN in hepatic tissues and activated HSC of rats were measured by immunohistochemical staining, Western blot, Real-time Q-PCR and immunofluorescence confocal laser scanning microscopy, respectively. And alpha-smooth muscle actin (α-SMA), an activated HSC marker in rat liver tissues, was detected by immunohistochemical staining. Furthermore, apoptotic HSC in rat liver tissues were determined by dual staining both of the terminal deoxynucleotidy transferrase UTP-nick end labeling (TUNEL) and ofα-SMA immunohistochemistry.
Results: (1) HE and Masson's trichrome staining showed rat modes of hepatic fibrosis with BDL were established successfully. With each consecutive week after BDL, increased fibrosis, degeneration and necrosis were observed in rat liver cells. Not surprisingly, a disruption of normal architecture and a decrease in normal hepatic cells were concomitantly observed. (2)α-SMA immunohistochemistry showed that in hepatic tissues of normal rats, there was only weakly positive expression ofα-SMA in the smooth muscle cells of the vessel wall. With the development of hepatic fibrosis, though, theα-SMA positive cells in the hepatic tissues of rats increased significantly. At weekly time points after BDL, the optical density values ofα-SMA in rat liver tissues, 0.16±0.01, 0.17±0.01, 0.21±0.01, 0.26±0.02 (1, 2, 3 and 4 weeks, respectively) increased significantly with each passing week, P<0.01. Furthermore, optical density values from BDL rats were all significantly higher than those from the sham operation group, 0.07±0.01 (P<0.01). (3) Dual staining both of TUNEL and ofα-SMA immunohistochemistry showed that few apoptotic HSC in normal livers appeared, with the developing of liver fibrosis,increased activated HSC, the number of apoptotic HSC increased too. But at weekly time points after BDL, the apoptotic index of activated HSC in rat liver tissues, 4.57%±0.41%,4.02%±0.48%,3.45%±0.37%,2.88%±0.50% (1, 2, 3 and 4 weeks, respectively) decreased gradually with aggravation of liver fibrosis (P<0.01). (4) PTEN immunohistochemical examination of hepatic tissues from wild-type rats showed widespread staining of PTEN protein in the cytoplasm, and, to a lesser extent, in the nuclei. With the progression of hepatic fibrosis, PTEN signal in the rat liver correspondingly decreased in the portal area, the fibrous septa and the proliferated peripheral cells of the bile duct. Nevertheless, there were no significant changes in the intracellular localization of PTEN protein. At weekly time points after BDL, optical density values of PTEN in rat liver tissues, 0.15±0.01,0.12±0.02,0.09±0.01,0.07±0.01 (1, 2, 3, 4 weeks, respectively), decreased significantly with each subsequent week, P<0.01. Furthermore, optical density values from BDL rats were significantly lower than those from the the sham operation group, 0.21±0.02 (P<0.01). (5) PTEN andα-SMA immunofluorescence double labeled hepatic tissue slices were examined under a confocal laser scanning microscope. Single channel scanning displayed PTEN andα-SMA positive signal as green and red fluorescence foci, respectively. Alongside the green and red foci, after merging the images of single channel scanning, yellow foci corresponding to colocalized PTEN andα-SMA were found in the hepatic tissue slices. Since, in rat liver tissue, only activated HSC and a few vascular smooth muscle cells expressα-SMA during hepatic fibrosis, the yellow spots marked PTEN-positive, activated HSC. The yellow foci were observed primarily in the cytoplasm. Analysis of the images indicated that, at weekly time points after BDL, PTEN-positive, activated HSC, accounted for 79.97%±5.49%, 73.83%±5.04%, 66.68%±4.58%, 60.20%±4.65% (1, 2, 3 and 4 weeks, respectively) of theα-SMA positive expression cells (total activated HSC). Thus, with the development of hepatic fibrosis, the ratio of activated HSC of PTEN positive expression to total activated HSC significantly decreased (P<0.01). (6) Western blot at weekly time points after BDL showed that the expression levels of PTEN protein in fibrotic rat liver tissues, 1.20±0.13, 1.07±0.16, 0.88±0.08, 0.73±0.07 (1, 2, 3, and 4 weeks, respectively) decreased significantly with increasing severity of hepatic fibrosis, P<0.01. Furthermore, all values from BDL rats were significantly lower than those from the sham operation group, 1.37±0.14 (P<0.01). (7) Expression levels of PTEN mRNA in rat liver tissues were measured with Real-time Q-PCR. PTEN mRNA expressions in rat liver tissues were compared by using the method of fold increase (2-△△C t method). The expression level of PTEN gene in the sham operation group was assigned a reference value of 1. In the fibrotic liver tissues of BDL rats, the mRNA expression levels of PTEN were 0.66-, 0.53-, 0.44- and 0.37-fold (1, 2, 3 and 4 weeks, respectively), all were significantly lower than that in the sham operation group, and down-regulated gradually with the development of hepatic fibrosis (P<0.01). (8) Pearson’s correlation analysis showed the expression of PTEN had a significant negative correlation with the expression ofα-SMA, a significant positive correlation with the percentage of PTEN-positive activated HSC and the apoptotic index of activated HSC in fibrosis liver tissues in rats. r values were -0.92, 0.78, 0.76, respectively (P<0.01).
Conclusions: Rat models of hepatic fibrosis with bile duct ligation (BDL) are established succesfully. During liver fibrosis in rats, the activation and proliferation of HSC accelerate gradually, whereas the apoptotic index of activated HSC decreases gradually. The expressions of PTEN mRNA and protein are down-regulated gradually in fibrogenic rat liver tissues, PTEN expression in HSC in vivo also decreases with progression of fibrosis. Thus, the dynamic expression of PTEN in rat liver tissues has a significant negative correlation with the activation and proliferation of HSC in vivo, and a significant positive correlation with the apoptotic index of activated HSC in vivo.
Part 2:Effects of PTEN on the proliferation and apoptosis of activated hepatic stellate cell in vitro
Objective: To investigate effects of over-expression of wild type PTEN and its mutant G129E (only exhibit protein phosphatase and lose lipids phosphatase activity) on the proliferation and apoptosis of activated HSC culcured in vitro.
Methods: Amplifications of adenoviral vectors (Ad-PTEN, Ad-G129E and Ad-GFP) were performed in AD293 cells and viral titer estimates were conducted. The wild type PTEN and its mutant G129E gene were transduced into activated HSC in vitro via adenoviral vector, respectively. PTEN expression in HSC was then measured by Western blot and Real-time Q-PCR. Changes in Bcl-2 and Bax in HSC were monitored by Western blot. And MTT assay was used to determine cell proliferation and a TUNEL assay and propidium iodide (PI) labed flow cytometry (FCM) were used to detect cell apoptosis. Cells were grouped as follows: (1) Control group, cells were cultured under the same conditions, except DMEM (without FBS and antibiotics) was used in place of the adenovirus; (2) Ad-GFP group, HSC were infected with adenovirus expressing green fluorescent protein (GFP) alone; (3) Ad-PTEN group, HSC were infected with adenovirus harboring genes for both wild type PTEN and GFP; (4) Ad-G129E group, HSC were infected with adenovirus harboring genes for both PTEN mutant G129E and GFP.
Results: (1) Adenoviral vectors (viral titers of Ad-PTEN, Ad-G129E and Ad-GFP: 1.2 x 109, 1.5 x 109, 1.8 x 109 pfu/ml, respectively) for experiment were obtained via performing repeated amplifications of virus in AD293 cells. (2) PTEN expression in HSC was detected at 72 hours after adenoviral infection. Real time Q-PCR was used to extrapolate relative mRNA expression levels of PTEN in HSC. PTEN mRNA expression in HSC was compared by using the method of fold increase (2-△△C t method). PTEN mRNA expression levels in Ad-GFP group, Ad-PTEN group and Ad-G129E group were 0.993-, 1.569- and 1.561-fold, respectively (the expression value of the untreated control group was arbitrarily assigned an expression value of 1). Obviously, the expression of PTEN mRNA was significantly higher in the Ad-PTEN group and Ad-G129E group than those in both the control group and the Ad-GFP group, P<0.01. Western blot analysis recapitulated the Real time Q-PCR data by showing that expression levels of PTEN protein in Ad-PTEN group (1.66±0.09), Ad-G129E group (1.65±0.09) were significantly higher than those in control group (1.10±0.07) and Ad-GFP group (1.09±0.07), P<0.01. Furthermore, no significant differences were observed in the expressions of PTEN mRNA and protein between Ad-PTEN group and Ad-G129E group or between control group and Ad-GFP group (P>0.05). Overall, wild type PTEN gene and G129E gene were successfully transduced and expressed in HSC. (3)There was no significant difference in the proliferation of HSC at 24 hours after transduction of Ad-PTEN or Ad-G129E (P>0.05). At 48 and 72 hours after transduction, though, a precipitous time-dependent drop in proliferation was observed in the Ad-PTEN group or Ad-G129E group. Inhibition rates were 14.03% and 23.12% in Ad-PTEN group, 9.52% and 12.63% in Ad-G129E group, respectively, when compared with the control group (P<0.01). Obviously, the inhibitory effect of wild type PTEN on HSC proliferation was more powerful than that of G129E. Moreover, the A value between control and Ad-GFP groups showed no notable difference at various time points (P>0.05). (4) TUNEL assay showed that at 72 hours after adenoviral transduction, the apoptotic rates of HSC in Ad-PTEN group (29.81%±2.52%), Ad-G129E group (26.37%±1.97 %) were greatly higher than those in control group (1.98%±0.25%) and Ad-GFP group (2.16%±0.28%), P<0.01, and Ad-PTEN group increased notably in the apoptotic rates of HSC compared with Ad-G129E group too (P<0.01). In addition, no significant difference was found in the apoptotic rates of HSC between control group and Ad-GFP group (P>0.05). (5) At 72 hours after adenovirus transduction, the apoptotic rates of HSC analyzed by PI labeled FCM in Ad-PTEN group (20.84%±1.44%), Ad-G129E group (17.54%±1.76%) increased markedly compared with control group (1.12%±0.57%) and Ad-GFP group (1.21%±0.22%), P<0.01. And the action of wild type PTEN on HSC apoptosis was more powerful than that of Ad-G129E (P<0.01). Furthermore, no significant difference was observed in apoptotic rate of HSC between control group and Ad-GFP group (P>0.05). (6) Western blot analysis showed that the expression of Bcl-2 decreased significantly at 72 hours after Ad-PTEN or Ad-G129E infection compared to control group and Ad-GFP group (1.16±0.03 or 1.24±0.05vs 1.37±0.06 and 1.34±0.08, respectively, P<0.01), and Bcl-2 expression in Ad-PTEN group was significantly lower than that in Ad-G129E group too (P<0.01). In contrast, the expressions of Bax in HSC at 72 hours after infection of Ad-PTEN (1.50±0.05) or Ad-G129E (1.41±0.05) heightened markedly compared with those in control group (1.28±0.06) and Ad-GFP group (1.26±0.08), P<0.01. And Bax expression in Ad-PTEN group inceased notably compared with that in Ad-G129E group as well (P<0.01). Moreover, there were no significant differences in the expressions of Bcl-2 and Bax between control group and Ad-GFP group (P>0.05).
Conclusions: Exogenous wild type PTEN gene and G129E gene are successfully transduced and expressed in activated HSC in vitro respectively, inhibit the proliferation and induce apoptosis of them. At the same time, the expression of apoptosis regulating gene Bax is increased and Bcl-2 is decreased in activated HSC. Moreover, the action of wild type PTEN is more powerful than that of Ad-G129E.
Part 3:Regulatory effects of PTEN on cell cycle of activated hepatic stellate cells in vitro
Objective: To investigate regulatory effects of over-expression of wild type PTEN and its mutant G129E on cell cycle of activated HSC culcured in vitro. Methods: The wild type PTEN gene and its mutant G129E gene were transduced into activated HSC cultured in vitro mediated by adenoviral vector, respectively. PI labed FCM was then used to detect cell cycle phase of activated HSC. And the expressions of PTEN, cyclinD1, cyclin dependent kinase 4 (CDK4) and P27kip1 in HSC were measured by Western blot and Real-time Q-PCR, respectively. Cells were grouped in accordance with part 2.
Results: (1) Western blot and Real-time Q-PCR demonstrated that exogenous wild type PTEN gene and G129E gene were successfully transduced and expressed in activated HSC cultured in vitro (the result was clearly in accordance with the part 2). (2) At 72 hours after adenovirus infection, Cell cycle phase of HSC in each group was detected by PI labed FCM. At G0/G1 phase, the number of HSC in Ad-PTEN group (67.68%±2.75%), Ad-G129E group (61.17%±3.41%) increased greatly compared with those in control group (53.01%±2.37%) and Ad-GFP group (53.85%±3.08%), P<0.01, and the number of HSC in Ad-PTEN group was significantly higher than that in Ad-G129E group (P<0.01). At S phase, the number of HSC decreased notably in Ad-PTEN group (14.42%±1.81%), Ad-G129E group (18.17%±2.43%) compared with those in control group (22.17%±1.99%) and Ad-GFP group (21.54%±1.74%), P<0.01, and the number of HSC in Ad-PTEN group was lower than that in Ad-G129E group too (P<0.01). At G2/M phase, the number of HSC in Ad-PTEN group (17.90%±2.70%), Ad-G129E group (20.66%±2.37%) decreased significantly compared to those in control group (24.82%±3.81%) and Ad-GFP group (24.62%±3.15%), P<0.01, P<0.05, while there was no significant difference in the number of HSC between Ad-PTEN group and Ad-G129E group (P>0.50). Furthermore, at any phase, no significant difference was found in the number of HSC between control group and Ad-GFP group (P>0.50). (3) At 72 hours after adenoviral infection, the expression of cyclinD1 protein in HSC was analyzed by Western blot, the expression of cyclinD1 protein in Ad-PTEN group (1.12±0.07), Ad-G129E group (1.23±0.05) decreased greatly compared to those in control group (1.45±0.05) and Ad-GFP group (1.47±0.08), P<0.01, and cyclinD1 protein expressio in Ad-PTEN group was significantly lower than that in Ad-G129E group too (P<0.01). The Real time Q-PCR was further used to detect the expression level of cyclinD1 mRNA in HSC, the method of fold increase (2-△△Ct method) was used to calculate relative mRNA expression level of cyclinD1 in HSC in each group. If the mRNA expression level of cyclinD1 in control group was assigned a value of 1, then cyclinD1 mRNA expressions in Ad-GFP group, Ad-PTEN group and Ad-G129E group were 1.011-, 0.773- and 0.838-fold compared with control group, respectively. Obviously, the expressions of cyclinD1 mRNA decreased significantly in Ad-PTEN group and Ad-G129E group compared with those in both control group and Ad-GFP group (P<0.01), and the expression of cyclinD1 mRNA in Ad-PTEN group was markedly lower than that in Ad-G129E group (P<0.01). Moreover, no significant differences were observed in the protein and mRNA expressions of cyclinD1 between control group and Ad-GFP group (P>0.05). (4) At 72 hours after adenoviral infection, Western blot analysis showed the expressions of CDK4 protein in Ad-PTEN group (1.05±0.07), Ad-G129E group (1.18±0.06) were markedly lower than those in control group (1.41±0.03) and Ad-GFP group (1.43±0.06), P<0.01, and CDK4 protein expression in Ad-PTEN group reduced notably compared with that in Ad-G129E group as well (P<0.01). Real time Q-PCR was further used to detect the expression level of CDK4 mRNA in HSC, the method of fold increase (2-△△C t method) was used to calculate relative mRNA expression level of CDK4 in HSC in each group. If the mRNA expression level of CDK4 in control group was assigned a value of 1, then CDK4 mRNA expression levels in Ad-GFP group, Ad-PTEN group and Ad-G129E group were 1.007-, 0.738- and 0.822-fold, respectively. The expression of CDK4 mRNA down-regulated notably in Ad-PTEN group and Ad-G129E group compared with those in control group and Ad-GFP group (P<0.01), and the expression of CDK4 mRNA in Ad-PTEN group was significantly lower than that in Ad-G129E group too (P<0.01). In addition, no significant differences were observed in the protein and mRNA expressions of CDK4 between control group and Ad-GFP group (P>0.05). (5) At 72 hours after adenoviral infection, Western blot analysis showed that the protein expressions of P27kip1 in HSC in Ad-PTEN group (1.40±0.03), Ad-G129E group (1.28±0.08) increased significantly compared to those in control group (1.11±0.04) and Ad-GFP group (1.12±0.04), P<0.01, and the protein expression of P27kip1 in Ad-PTEN group was notably higher than that in Ad-G129E group as well (P<0.01). Real time Q-PCR was used to extrapolate relative mRNA expression level of P27kip1 in HSC. P27kip1 mRNA expression in HSC in each group was compared by using the method of fold increase (2-△△C t method). If the mRNA expression level of P27kip1 in control group was assigned a value of 1, then the mRNA expressions of P27kip1 in Ad-GFP group, Ad-PTEN group and Ad-G129E group were 1.008-, 1.264- and 1.157-fold compared with control group, respectively. Obviously, the expressions of P27kip1 mRNA increased greatly in Ad-PTEN group and Ad-G129E group compared with those in control group and Ad-GFP group (P<0.01), and the expression of P27kip1 mRNA in Ad-PTEN group was markedly higher than that in Ad-G129E group too (P<0.01). Moreover, no significant difference was found in expressions of P27kip1 protein and mRNA between control group and Ad-GFP group (P>0.05).
Conclusions: The over-expression of not only wild type PTEN but also G129E can inhibit transition of activated HSC in vitro from G1 to S phase, arrest cell cycle of them at G0/G1 phase, and thus negatively regulate cell cycle progression of them. Meanwhile, at both transcriptional and translational levels, the expressions of cyclinD1 and CDK4 are down-regulated and P27kip1 expression is up-regulated in activated HSC in vitro. In addition, wild type PTEN is more powerful than Ad-G129E for above-mentioned effects. Part 4: Signaling transduction mechanisms of PTEN on the activated hepatic stellate cell behaviors
Objective: To investigate the signaling transduction mechanisms of PTEN on the proliferation and apoptosis as well as cell cycle of activated HSC.
Methods: Using cell culture techniques in vitro, the wild type PTEN gene and its mutant G129E gene were transduced into activated HSC in vitro mediated by adenoviral vector, respectively. And the protein expressions of serine-threonine protein kinase B (Akt), p-Akt (Thr308), extracellular signal regulated kinase1 (ERK1) and p-ERK1 in activated HSC were measured by Western blot. And the mRNA expressions of Akt and ERK1 were detected by Real-time Q-PCR. Cells were grouped in accordance with part 2.
Results: ((1) Western blot and Real-time Q-PCR demonstrated that exogenous wild type PTEN gene and G129E gene were successfully transduced and expressed in activated HSC cultured in vitro (the result was clearly in accordance with the part 2). (2) Western blot and Real-time Q-PCR showed that there were no significant differences in the mRNA and protein expression of Akt in HSC at 72 hours after transduction of Ad-PTEN or Ad-G129E (P>0.05). While Western blot analysis documented the expression of p-Akt (Thr308) in HSC at 72 hours after adenoviral infection decreased greatly in Ad-PTEN group (0.63±0.04) compared to those in Ad-G129E group (0.93±0.03), control group (0.95±0.04) and Ad-GFP group (0.94±0.03), P<0.01. Moreover, no significant differences were observed in the expressions of p-Akt (Thr308) among Ad-G129E group, control group and Ad-GFP group (P>0.05). (3) At 72 hours after transduction of Ad-PTEN or Ad-G129E, there were no significant differences in the protein and mRNA expression of ERK1 in HSC (P>0.05). While Western blot analysis documented the protein expression of p-ERK1 in HSC at 72 hours after adenoviral infection reduced greatly in Ad-PTEN group (0.65±0.04), Ad-G129E group (0.68±0.07) compared to those in control group (0.84±0.07) and Ad-GFP control (0.85±0.06), P<0.01. In addition, no significant differences were found in the protein expressions of p-ERK1 between Ad-PTEN and Ad-G129E group or between control group and Ad-GFP group (P >0.05).
Conclusions: The PTEN phosphatase negatively regulates the phosphoinositol-3-kinase (PI3K)/Akt and ERK1/2 signal transduction pathways via inhibiting phosphorylation of Akt and ERK1 in activated HSC, and thus curbs the proliferation, induces apoptosis and negatively regulates cell cycle of activated HSC in vitro.
第10号染色体缺失的磷酸酶张力蛋白同源物基因(phosphatase and tensin homology deleted on chromosome Ten, PTEN)是迄今发现的第一个具有磷酸酶活性的肿瘤抑制基因,可负性调控肿瘤细胞细胞周期、抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡,其缺失或表达异常与多种肿瘤的发生发展密切相关。
近年来,对PTEN的研究已从肿瘤领域逐渐延伸至非肿瘤领域。研究表明,在特发性肺纤维化患者的肺组织中,成纤维细胞的PTEN表达及其磷酸酶活力都低于正常肺成纤维细胞;并且其过表达或激活可抑制体外肺成纤维细胞增殖、诱导其凋亡。在有关PTEN与心肌纤维化的研究中也显示PTEN基因敲除的小鼠心脏与体重之比增加、心肌纤维化程度明显、心肌收缩性降低。这提示PTEN的低表达或失活参与了特发性肺纤维化及心肌纤维化的发生。而在PTEN与某些肝脏疾病的研究中,有研究发现特异性肝细胞PTEN缺失不仅可引起肝细胞癌而且还导致与肝纤维化密切相关的非酒精性脂肪性肝炎的发生。但迄今为止,PTEN在肝纤维化中的表达及作用,特别是对HSC增殖及凋亡的影响仍不清楚。为此,本研究应用胆总管结扎法建立胆汁淤积性大鼠肝纤维化模型,探讨PTEN在肝纤维化过程中的动态表达及其与在体HSC增殖、凋亡的关系;并进一步将携带野生型PTEN基因及其突变体G129E基因的重组腺病毒转染体外培养的活化HSC,观察过表达的PTEN对活化HSC增殖、凋亡及细胞周期的影响,同时探讨PTEN调控活化HSC增殖、凋亡及细胞周期的信号转导机制,旨在为深入揭示肝纤维化的病理生理机制,寻求有效预防和治疗肝纤维的新策略提供实验依据。实验内容主要包括以下四部分:
第一部分PTEN在大鼠纤维化肝组织中的动态表达及其与在体肝星状细胞增殖、凋亡的关系
目的:研究大鼠肝纤维化过程中肝组织的PTEN动态表达及其与在体HSC增殖、凋亡的关系。
方法:胆总管结扎法建立胆汁淤积性大鼠肝纤维化模型,HE、Masson三色染色观察肝脏病理组织学变化,免疫组织化学染色检测大鼠肝组织中PTEN及α-平滑肌肌动蛋白(alpha-smooth muscle actin,α-SMA)的分布;免疫荧光双标记共聚焦激光扫描显微镜技术测定大鼠肝组织中活化HSC的PTEN表达;末段脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末段标记(TUNEL)及α-SMA免疫组织化学双染检测在体活化HSC的凋亡;Western blot和Real-time Q-PCR检测大鼠肝组织中PTEN蛋白及mRNA表达。
结果:①HE及Masson三色染色显示,胆汁淤积性大鼠肝纤维化模型成功建立,随着造模时间延长肝纤维化程度逐渐加重。在造模不同时间均可见不同程度的肝细胞变性坏死而导致正常肝细胞逐渐减少。②α-SMA免疫组织化学染色显示,正常大鼠肝组织中仅在血管壁平滑肌细胞有弱阳性表达;随着肝纤维化的发展,大鼠肝组织中α-SMA阳性细胞逐渐增多,造模1 wk、2 wk、3 wk及4 wk不同时间大鼠肝组织α-SMA的阳性光密度值(0.16±0.01,0.17±0.01,0.21±0.01,0.26±0.02)均显著高于假手术组(0.07±0.01), P<0.01;且各组之间均有明显差异(P<0.01)。③TUNEL及α-SMA免疫组织化学双重染色显示,正常大鼠肝组织几乎看不见凋亡HSC,随着肝纤维化程度加重、活化HSC增多,凋亡HSC也增多,活化HSC与凋亡HSC共存。造模1 wk、2 wk、3 wk及4 wk不同时间大鼠肝组织HSC凋亡指数分别为4.57%±0.41%、4.02%±0.48%、3.45%±0.37%、2.88%±0.50%,即随着肝纤维化程度加重,活化HSC凋亡指数逐渐减少(P<0.01)。④PTEN免疫组织化学染色显示,正常大鼠肝组织中PTEN蛋白有广泛表达,主要表达于细胞浆,在胞核中也有表达;随着肝纤维化的进展,大鼠肝组织中PTEN阳性细胞逐渐减少,主要分布于汇管区、纤维间隔及增生的胆管周围细胞,PTEN蛋白的亚细胞定位没有明显变化。造模1 wk、2 wk、3 wk及4 wk大鼠肝组织PTEN的阳性光密度值(0.15±0.01,0.12±0.02,0.09±0.01,0.07±0.01)均显著低于假手术组(0.21±0.02),P<0.01;且各组之间均有明显差异(P<0.01)。⑤共聚焦激光扫描显微镜下观察PTEN和α-SMA免疫荧光双标记的大鼠肝组织切片,单通道扫描可见PTEN和α-SMA阳性反应产物分别呈绿色和红色荧光斑点状,将单通道扫描的图象混合后,在肝组织切片内除了绿色和红色荧光斑点外,还可见黄色荧光斑点,因为纤维化肝组织中只有活化HSC和少许血管平滑肌细胞表达α-SMA,故这些黄色斑点绝大部分可看做是PTEN和α-SMA在HSC的共表达产物,也就是活化HSC的PTEN阳性表达。共表达产物主要存在于活化HSC细胞浆,在部分细胞核中也有少许表达。图像分析结果显示,造模1 wk、2 wk、3 wk及4 wk大鼠肝组织中PTEN和α-SMA共存的HSC(PTEN表达阳性的活化HSC)占α-SMA表达阳性细胞(总的活化HSC)的比例分别为79.97%±5.49%、73.83%±5.04%、66.68%±4.58%、60.20%±4.65%。即随着肝纤维化的进展,表达PTEN的活化HSC比例逐渐减少(P<0.01)。⑥Western blot显示,造模1 wk、2 wk、3 wk及4 wk大鼠纤维化肝组织PTEN蛋白表达量(1.20±0.13,1.07±0.16, 0.88±0.08,0.73±0.07)均显著低于假手术组(1.37±0.14),P<0.01;且各组之间均有明显差异(P<0.01),即大鼠肝纤维化越重PTEN蛋白表达越低。⑦应用Real-time Q-PCR技术检测大鼠肝组织的PTEN mRNA表达,并采用相对定量2-△△Ct法比较PTEN mRNA在各组大鼠肝组织中的相对表达。以假手术组为对照组(其PTEN mRNA的表达量为1),则造模1 wk、2 wk、3 wk及4 wk大鼠纤维化肝组织中PTEN mRNA相对假手术组的表达倍数为0.66倍、0.53倍、0.44倍、0.37倍,均显著低于假手术组,且随着肝纤维化的发展逐渐下调(P<0.01)。⑧Pearson’s相关性分析发现,大鼠纤维化肝组织中PTEN表达与α-SMA表达呈显著负相关,与PTEN阳性的活化HSC比例呈显著正相关,与活化HSC凋亡指数呈显著正相关,r值分别为-0.92、0.78、0.76,P<0.01。
结论:胆总管结扎法成功建立胆汁淤积性大鼠肝纤维化模型;大鼠肝纤维化形成过程中HSC的活化、增殖逐渐加快,而活化HSC的凋亡指数却逐渐减少;随着肝纤维化进展,大鼠肝组织中PTEN蛋白及mRNA表达逐渐下调,在体HSC的PTEN表达亦降低,其动态表达与在体HSC的活化、增殖呈显著负相关,而与活化HSC凋亡指数呈显著正相关。
第二部分PTEN对体外活化肝星状细胞增殖与凋亡的影响
目的:探讨过表达的野生型PTEN及其突变体G129E(丧失了脂质磷酸酶活性仅保留蛋白磷酸酶活性)对体外活化的HSC增殖、凋亡的影响及可能的机制。
方法:利用AD293细胞扩增实验所需的腺病毒(Ad-PTEN、Ad-G129E、Ad-GFP),并测定滴度;以腺病毒为载体将野生型PTEN基因及其突变体G129E基因瞬时转染体外培养的活化的HSC;Western blot及Real-time Q-PCR检测HSC PTEN表达;MTT法检测HSC增殖;TUNEL及碘化丙啶(propidium iodide, PI)标记的流式细胞术测定HSC凋亡;Western blot检测HSC的凋亡调控基因Bcl-2及Bax表达。实验分组如下:①Control组,仅以含8%胎牛血清的DMEM细胞培养液培养细胞,在转染步骤入无血清无抗生素DMEM代替病毒液;②Ad-GFP组,转染表达绿色荧光蛋白(green fluorescent protein, GFP)的空病毒Ad-GFP;③Ad-PTEN组,转染携带野生型PTEN基因并表达GFP的重组腺病毒Ad-PTEN;④Ad-G129E组,转染携带PTEN的突变体G129E基因并表达GFP的重组腺病毒Ad-G129E。
结果:①通过反复感染AD293细胞的方法使病毒扩增获得了实验所需的病毒液(Ad-PTEN、Ad-G129E、Ad-GFP的滴度分别为1.2 x 109、1.5 x 109、1.8 x 109 pfu/ml)。②腺病毒感染HSC后72 h,应用Real time Q-PCR检测活化HSC PTEN mRNA表达,并采用相对定量2-△△Ct法比较PTEN mRNA在各组HSC中的表达。以Control组为对照组(其PTEN mRNA表达量为1),则Ad-GFP组、Ad-PTEN组和Ad-G129E组相对Control组的PTEN mRNA表达倍数分别为0.993倍、1.569倍和1.561倍。很明显,Ad-PTEN组及Ad-G129E组PTEN mRNA表达明显高于Control组及Ad-GFP组(P<0.01);而Ad-PTEN组与Ad-G129E组,以及Control组与Ad-GFP组之间无明显差异(P>0.05)。进一步应用Western blot分析各组HSC的PTEN蛋白表达,Ad-PTEN组(1.66±0.09)、Ad-G129E组(1.65±0.09)均显著高于Control组(1.10±0.07)及Ad-GFP组(1.09±0.07),P<0.01;而Control组与Ad-GFP组之间,以及Ad-PTEN组和Ad-G129E组之间均无明显差异(P>0.05)。证明野生型PTEN基因及其突变体G129E基因成功转染入体外活化的HSC。③MTT检测显示,野生型PTEN基因及G129E基因转染活化HSC后24 h对细胞增殖无明抑制(P>0.05)。但在48 h、72 h HSC增殖受到明显抑制,Ad-PTEN组增殖抑制率为14.03%、23.12%,而Ad-G129E组增殖抑制率是9.52%、12.63%(均以Control组为对照)。在各时间点,Ad-GFP组与Control组A值比较无明显差异(P<0.05)。显然,野生型PTEN对HSC增殖的抑制作用明显强于G129E。④TUNEL检测发现,在腺病毒感染HSC后72 h,Ad-PTEN组HSC凋亡率增至29.81%±2.52%,Ad-G129E组增至26.37%±1.97%均显著高于Control组(1.98%±0.25%)及Ad-GFP组(2.16%±0.28%),P<0.01;而Ad-PTEN组HSC凋亡率亦明显高于Ad-G129E组(P<0.01);Ad-GFP组与Control组比较无显著差异(P>0.05)。⑤PI标记的流式细胞术检测显示,Ad-PTEN组HSC凋亡率(20.84%±1.44%)、Ad-G129E组HSC凋亡率(17.54%±1.76%)均显著高于Control组(1.12%±0.57%)及Ad-GFP组(1.21%±0.22%) , P<0.01 ;而Ad-PTEN组亦明显高于Ad-G129E组(P<0.01);但Ad-GFP组与Control组比较无显著差异(P>0.05)。⑥Western blot检测腺病毒感染HSC后72 h,各组HSC Bcl-2蛋白表达,Ad-PTEN组(1.16±0.03)、Ad-G129E组(1.24±0.05)均显著低于Control组(1.37±0.06)及Ad-GFP组(1.34±0.08),P<0.01,并且Ad-PTEN组较Ad-G129E组也明显降低(P<0.05),但Control组与Ad-GFP组之间无明显差异(P>0.05);而各组HSC Bax蛋白表达,Ad-PTEN组(1.50±0.05)、Ad-G129E组(1.41±0.05)均较Control组(1.28±0.06)及Ad-GFP组(1.26±0.08)显著增高,P<0.01,同时Ad-PTEN组明显高于Ad-G129E组(P<0.05),Control组与Ad-GFP组之间无明显差异(P>0.05)。
结论:外源性野生型PTEN基因及G129E基因成功转染体外活化HSC;其过表达可明显抑制活化HSC增殖,诱导活化HSC凋亡,并引起HSC的凋亡调控基因Bax表达增加,Bcl-2表达下降。而且野生型PTEN的作用明显强于G129E。
第三部分PTEN对体外活化肝星状细胞细胞周期的调控作用
目的:探讨过表达的野生型PTEN及其突变体G129E对体外活化HSC细胞周期的调控作用。
方法:体外培养活化的HSC,以腺病毒为载体将野生型PTEN基因及其突变体G129E基因瞬时转染体外活化的HSC;流式细胞术测定HSC细胞周期时相;Western blot及Real-time Q-PCR检测HSC的PTEN、细胞周期素D1(CyclinD1)、细胞周期素依赖性激酶4(Cyclin dependent kinase4, CDK4)及细胞周期素依赖性激酶抑制因子(cyclin dependent kinase inhibitor, CDI)之一的P27kip1蛋白及mRNA表达。实验分组同第二部分。
结果:①Real-time Q-PCR及Western blot检测证实外源性野生型PTEN基因及其突变体G129E基因在体外活化HSC大量表达(结果同第二部分)。②流式细胞仪检测结果显示,G0/G1期HSC细胞数,Ad-PTEN组(67.68%±2.75%)、Ad-G129E组(61.17%±3.41%)均较Control组(53.01%±2.37%)及Ad-GFP组(53.85%±3.08%)明显增高(P<0.01) ,而Ad-PTEN组也明显高于Ad-G129E组(P<0.01);S期HSC细胞数,Ad-PTEN组(14.42%±1.81%)、Ad-G129E组(18.17%±2.43%)均较Control组(22.17%±1.99%)及Ad-GFP组(21.54%±1.74%)明显降低(P<0.01) ,而Ad-PTEN组亦明显低于Ad-G129E组(P<0.01);G2/M期HSC细胞数:Ad-PTEN组(17.90%±2.70%)、Ad-G129E组(20.66%±2.37%)均明显低于Control组(24.82%±3.81%)及Ad-GFP组(24.62%±3.15%),P<0.01、P<0.05,而Ad-PTEN组与Ad-G129E组之间差异无统计学意义(P>0.05)。此外,各期Control组与Ad-GFP组之间HSC细胞数差异均无统计学意义(P>0.05)。③腺病毒感染HSC后72 h,Western blot分析各组HSC cyclinD1蛋白表达,Ad-PTEN组(1.12±0.07)、Ad-G129E组(1.23±0.05)均较Control组(1.45±0.05)及Ad-GFP组(1.47±0.08)显著降低(P<0.01),Ad-PTEN组亦明显低于Ad-G129E组(P<0.01),而Control组与Ad-GFP组之间无明显差异(P>0.05)。进一步应用Real time Q-PCR检测各组HSC cyclinD1 mRNA表达,并应用相对定量2-△△Ct法比较cyclinD1 mRNA在各组HSC中的相对表达。以Control组为对照组(其cyclinD1 mRNA表达量为1),则Ad-GFP组、Ad-PTEN组及Ad-G129E组相对Control组的cyclinD1 mRNA表达倍数分别为1.011倍、0.773倍和0.838倍。显然,Ad-PTEN组及Ad-G129E组CyclinD1 mRNA表达明显低于Control组及Ad-GFP组(P<0.01),Ad-PTEN组较Ad-G129E组也显著降低,而Control组与Ad-GFP组之间无明显差异(P>0.05)。④腺病毒感染HSC后72 h,Western blot检测各组HSC CDK4蛋白表达,Ad-PTEN组(1.05±0.07)、Ad-G129E组(1.18±0.06)均较Control组(1.41±0.03)及Ad-GFP组(1.43±0.06)显著降低(P<0.01),而Ad-PTEN组亦明显低于Ad-G129E组(P<0.01),但Control组与Ad-GFP组之间差异无统计学意义(P>0.05)。进一步应用Real time Q-PCR检测各组HSC的CDK4 mRNA表达,并应用相对定量2-△△Ct法比较CDK4 mRNA在各组HSC中的相对表达。以Control组为对照组(其CDK4 mRNA表达量为1),则Ad-GFP组、Ad-PTEN组及Ad-G129E组相对Control组的CDK4 mRNA表达倍数为1.007倍、0.738倍和0.822倍。显然,Ad-PTEN组、Ad-G129E组CDK4 mRNA表达较Control组及Ad-GFP组明显降低(P<0.01),Ad-PTEN组也显著低于Ad-G129E组(P<0.01),但Control组与Ad-GFP组之间无明显差异(P>0.05)。⑤腺病毒感染HSC后72 h,Western blot分析各组HSC P27kip1蛋白表达,Ad-PTEN组(1.40±0.03)、Ad-G129E组(1.28±0.08)均显著高于Control组(1.11±0.04)及Ad-GFP组(1.12±0.04),P<0.01,Ad-PTEN组较Ad-G129E组亦明显升高(P<0.01),但Control组与Ad-GFP组之间无明显差异(P>0.05)。进一步应用Real time Q-PCR检测各组HSC P27kip1 mRNA表达,并应用相对定量2-△△Ct法比较P27kip1 mRNA在各组HSC中的表达。以Control组为对照组(其P27kip1 mRNA表达量为1),则Ad-GFP组、Ad-PTEN组和Ad-G129E组相对Control组的P27kip1 mRNA表达倍数为1.008倍、1.264倍和1.157倍。可以看出,Ad-PTEN组、Ad-G129E组P27kip1 mRNA表达明显高于Control组及Ad-GFP组(P<0.01),Ad-PTEN组也较Ad-G129E组显著升高(P<0.01),而Control组与Ad-GFP组之间无明显差异(P>0.05)。
结论:过表达的野生型PTEN及G129E显著抑制体外活化HSC细胞周期G1/S期转化,阻滞HSC细胞周期时相于G0/G1期;同时在转录和翻译水平上下调活化HSC的cyclinD1、CDK4表达,上调P27kip1表达,这可能是其阻滞活化HSC细胞周期进程的重要机制。并且,在上述作用中野生型PTEN明显强于G129E。
第四部分PTEN调控活化肝星状细胞行为的信号转导机制
目的:探讨PTEN调控活化肝星状细胞增殖、凋亡及细胞周期的信号转导机制。
方法:体外培养活化HSC,以腺病毒为载体将野生型PTEN基因及其突变体G129E基因瞬时转染体外活化HSC;Western blot检测HSC的丝氨酸-苏氨酸蛋白激酶B(serine-threonine protein kinase B, Akt)、p-Akt(Thr308)、细胞外信号调节激1(extracellular signal-regulated kinase1, ERK1)、p-ERK1蛋白表达;Real-time Q-PCR检测Akt、ERK1 mRNA表达。实验分组同第二部分。
结果:①Real-time Q-PCR及Western blot检测证实外源性野生型PTEN基因及其突变体G129E基因在体外活化HSC大量表达(结果同第二部分)。②腺病毒感染HSC后72 h,Western blot及Real-time Q-PCR显示野生型PTEN及G129E对HSC Akt蛋白及其mRNA表达均无明显影响(P>0.05);而Western blot显示Ad-PTEN组p-Akt(Thr308)蛋白表达(0.63±0.04)显著低于Ad-G129E组(0.93±0.03)、Control组(0.95±0.04)及Ad-GFP组(0.94±0.03),P<0.01,但Ad-G129E组、Control组及Ad-GFP组之间p-Akt(Thr308)表达无显著差别(P>0.05)。③腺病毒感染HSC后72 h,Western blot及Real-time Q-PCR显示野生型PTEN及G129E对HSC ERK1蛋白及mRNA表达均无明显影响(P>0.05);而Western blot显示Ad-PTEN组p-ERK1蛋白表达(0.65±0.04)、Ad-G129E组p-ERK1蛋白表达(0.68±0.07)均显著低于Control组(0.84±0.07)及Ad-GFP组(0.85±0.06),P<0.01,但Ad-PTEN组与Ad-G129E组,以及Control组与Ad-GFP组之间p-ERK1表达无显著差异(P>0.05)。
结论:PTEN通过其磷酸酶活性抑制Akt及ERK1的磷酸化;其负性调控活化HSC细胞周期、抑制活化HSC增殖及诱导活化HSC凋亡的作用与PI3K/Akt、ERk1/2信号通路的活性抑制有关。
Hepatic fibrosis, the liver’s wound healing response to virtually all forms of chronic liver injury, can result from several chronic liver diseases caused by many pathogenic factors. Given enough time, fibrosis will progress to cirrhosis. The main pathological characteristic of hepatic fibrosis is the increased irregular deposition of extracellular matrix (ECM). Currently, it is believed that the activation of hepatic stellate cells (HSC), which play a pivotal role in fibrosis process, into cells with spontaneous proliferation and strong fibrogenic activity appears to be the dominant driving force in fibrosis. While the apoptosis of HSC increases significantly during the reparative process following liver injury. So the inhibiting proliferation or inducing apoptosis of activated HSC play a key role in the process of reversion of hepatic fibrosis.
Phosphatase and tensin homolog deleted on chromosome ten (PTEN), the first tumor-suppressing gene found to exhibit phosphatase activity, negatively regulates cell cycle, inhibits the proliferation and promotes the apoptosis of tumor cells. As such, it is reasonable to conclude that dysfunction or absence of PTEN is intimately related to the formation and progression of human tumors.
Nevertheless, in recent years, PTEN research has gradually extended beyond cancer, focusing on its role in other disease states. Studies demonstrated that lowered expression and phosphatase activity of PTEN is found in lung fibroblasts of patients with idiopathic pulmonary fibrosis. Moreover, studies also documented that PTEN inhibits the proliferation and induces the apoptosis of lung fibroblasts cultured in vitro. A study on PTEN and myocardial fibrosis showed that knocking out PTEN in mice leads to an increased ratio of heart to body weight, decreased cardiac contractility and, eventually, interstitial fibrosis. This suggests that the low expression or deactivation of PTEN involves itself in the pathogenesis of lung fibrosis and myocardial fibrosis. In the liver, the absence of PTEN in specific hepatic cells may result not only in hepatocellular carcinoma, but also in non-alcoholic steatohepatitis, a condition closely related to hepatic fibrosis. Currently, though, the expression and function of PTEN in hepatic fibrosis, especially its effects on the proliferation and apoptosis of HSC, remain unclear. So, using the rat bile duct ligation (BDL) model, this study explored the dynamic expression of PTEN in the process of hepatic fibrosis in rats and its relation with the proliferation and apoptosis of HSC in vivo. And we set out to determine the effects of PTEN over-expression, via adenoviral transduction of wild type PTEN and its mutant G129E gene, on the proliferation and apoptosis as well as cell cycle of activated HSC cultured in vitro. Meanwhile, the signaling transduction pathways of PTEN on the proliferation, apoptosis and of cell cycle of activated HSC were studied. It was designed to provide new viewpoint and defined strategy for preventing and treating hepatic fibrosis.The project contain four parts as below:
Part 1:The dynamic expression of PTEN in fibrogenic liver tissues in rats and its relation with the proliferation and apoptosis of hepatic stellate cells in vivo
Objective: To investigate the dynamic expression of PTEN in liver tissues in the process of hepatic fibrosis in rats and its relation with the proliferation and apoptosis of HSC in vivo.
Methods: The rat model of hepatic fibrosis used in this study was established by means of common bile duct ligation (BDL). HE and Masson's trichrome staining were used to determine histopathology changes of liver tissues. At 4 time points, the expressions of PTEN in hepatic tissues and activated HSC of rats were measured by immunohistochemical staining, Western blot, Real-time Q-PCR and immunofluorescence confocal laser scanning microscopy, respectively. And alpha-smooth muscle actin (α-SMA), an activated HSC marker in rat liver tissues, was detected by immunohistochemical staining. Furthermore, apoptotic HSC in rat liver tissues were determined by dual staining both of the terminal deoxynucleotidy transferrase UTP-nick end labeling (TUNEL) and ofα-SMA immunohistochemistry.
Results: (1) HE and Masson's trichrome staining showed rat modes of hepatic fibrosis with BDL were established successfully. With each consecutive week after BDL, increased fibrosis, degeneration and necrosis were observed in rat liver cells. Not surprisingly, a disruption of normal architecture and a decrease in normal hepatic cells were concomitantly observed. (2)α-SMA immunohistochemistry showed that in hepatic tissues of normal rats, there was only weakly positive expression ofα-SMA in the smooth muscle cells of the vessel wall. With the development of hepatic fibrosis, though, theα-SMA positive cells in the hepatic tissues of rats increased significantly. At weekly time points after BDL, the optical density values ofα-SMA in rat liver tissues, 0.16±0.01, 0.17±0.01, 0.21±0.01, 0.26±0.02 (1, 2, 3 and 4 weeks, respectively) increased significantly with each passing week, P<0.01. Furthermore, optical density values from BDL rats were all significantly higher than those from the sham operation group, 0.07±0.01 (P<0.01). (3) Dual staining both of TUNEL and ofα-SMA immunohistochemistry showed that few apoptotic HSC in normal livers appeared, with the developing of liver fibrosis,increased activated HSC, the number of apoptotic HSC increased too. But at weekly time points after BDL, the apoptotic index of activated HSC in rat liver tissues, 4.57%±0.41%,4.02%±0.48%,3.45%±0.37%,2.88%±0.50% (1, 2, 3 and 4 weeks, respectively) decreased gradually with aggravation of liver fibrosis (P<0.01). (4) PTEN immunohistochemical examination of hepatic tissues from wild-type rats showed widespread staining of PTEN protein in the cytoplasm, and, to a lesser extent, in the nuclei. With the progression of hepatic fibrosis, PTEN signal in the rat liver correspondingly decreased in the portal area, the fibrous septa and the proliferated peripheral cells of the bile duct. Nevertheless, there were no significant changes in the intracellular localization of PTEN protein. At weekly time points after BDL, optical density values of PTEN in rat liver tissues, 0.15±0.01,0.12±0.02,0.09±0.01,0.07±0.01 (1, 2, 3, 4 weeks, respectively), decreased significantly with each subsequent week, P<0.01. Furthermore, optical density values from BDL rats were significantly lower than those from the the sham operation group, 0.21±0.02 (P<0.01). (5) PTEN andα-SMA immunofluorescence double labeled hepatic tissue slices were examined under a confocal laser scanning microscope. Single channel scanning displayed PTEN andα-SMA positive signal as green and red fluorescence foci, respectively. Alongside the green and red foci, after merging the images of single channel scanning, yellow foci corresponding to colocalized PTEN andα-SMA were found in the hepatic tissue slices. Since, in rat liver tissue, only activated HSC and a few vascular smooth muscle cells expressα-SMA during hepatic fibrosis, the yellow spots marked PTEN-positive, activated HSC. The yellow foci were observed primarily in the cytoplasm. Analysis of the images indicated that, at weekly time points after BDL, PTEN-positive, activated HSC, accounted for 79.97%±5.49%, 73.83%±5.04%, 66.68%±4.58%, 60.20%±4.65% (1, 2, 3 and 4 weeks, respectively) of theα-SMA positive expression cells (total activated HSC). Thus, with the development of hepatic fibrosis, the ratio of activated HSC of PTEN positive expression to total activated HSC significantly decreased (P<0.01). (6) Western blot at weekly time points after BDL showed that the expression levels of PTEN protein in fibrotic rat liver tissues, 1.20±0.13, 1.07±0.16, 0.88±0.08, 0.73±0.07 (1, 2, 3, and 4 weeks, respectively) decreased significantly with increasing severity of hepatic fibrosis, P<0.01. Furthermore, all values from BDL rats were significantly lower than those from the sham operation group, 1.37±0.14 (P<0.01). (7) Expression levels of PTEN mRNA in rat liver tissues were measured with Real-time Q-PCR. PTEN mRNA expressions in rat liver tissues were compared by using the method of fold increase (2-△△C t method). The expression level of PTEN gene in the sham operation group was assigned a reference value of 1. In the fibrotic liver tissues of BDL rats, the mRNA expression levels of PTEN were 0.66-, 0.53-, 0.44- and 0.37-fold (1, 2, 3 and 4 weeks, respectively), all were significantly lower than that in the sham operation group, and down-regulated gradually with the development of hepatic fibrosis (P<0.01). (8) Pearson’s correlation analysis showed the expression of PTEN had a significant negative correlation with the expression ofα-SMA, a significant positive correlation with the percentage of PTEN-positive activated HSC and the apoptotic index of activated HSC in fibrosis liver tissues in rats. r values were -0.92, 0.78, 0.76, respectively (P<0.01).
Conclusions: Rat models of hepatic fibrosis with bile duct ligation (BDL) are established succesfully. During liver fibrosis in rats, the activation and proliferation of HSC accelerate gradually, whereas the apoptotic index of activated HSC decreases gradually. The expressions of PTEN mRNA and protein are down-regulated gradually in fibrogenic rat liver tissues, PTEN expression in HSC in vivo also decreases with progression of fibrosis. Thus, the dynamic expression of PTEN in rat liver tissues has a significant negative correlation with the activation and proliferation of HSC in vivo, and a significant positive correlation with the apoptotic index of activated HSC in vivo.
Part 2:Effects of PTEN on the proliferation and apoptosis of activated hepatic stellate cell in vitro
Objective: To investigate effects of over-expression of wild type PTEN and its mutant G129E (only exhibit protein phosphatase and lose lipids phosphatase activity) on the proliferation and apoptosis of activated HSC culcured in vitro.
Methods: Amplifications of adenoviral vectors (Ad-PTEN, Ad-G129E and Ad-GFP) were performed in AD293 cells and viral titer estimates were conducted. The wild type PTEN and its mutant G129E gene were transduced into activated HSC in vitro via adenoviral vector, respectively. PTEN expression in HSC was then measured by Western blot and Real-time Q-PCR. Changes in Bcl-2 and Bax in HSC were monitored by Western blot. And MTT assay was used to determine cell proliferation and a TUNEL assay and propidium iodide (PI) labed flow cytometry (FCM) were used to detect cell apoptosis. Cells were grouped as follows: (1) Control group, cells were cultured under the same conditions, except DMEM (without FBS and antibiotics) was used in place of the adenovirus; (2) Ad-GFP group, HSC were infected with adenovirus expressing green fluorescent protein (GFP) alone; (3) Ad-PTEN group, HSC were infected with adenovirus harboring genes for both wild type PTEN and GFP; (4) Ad-G129E group, HSC were infected with adenovirus harboring genes for both PTEN mutant G129E and GFP.
Results: (1) Adenoviral vectors (viral titers of Ad-PTEN, Ad-G129E and Ad-GFP: 1.2 x 109, 1.5 x 109, 1.8 x 109 pfu/ml, respectively) for experiment were obtained via performing repeated amplifications of virus in AD293 cells. (2) PTEN expression in HSC was detected at 72 hours after adenoviral infection. Real time Q-PCR was used to extrapolate relative mRNA expression levels of PTEN in HSC. PTEN mRNA expression in HSC was compared by using the method of fold increase (2-△△C t method). PTEN mRNA expression levels in Ad-GFP group, Ad-PTEN group and Ad-G129E group were 0.993-, 1.569- and 1.561-fold, respectively (the expression value of the untreated control group was arbitrarily assigned an expression value of 1). Obviously, the expression of PTEN mRNA was significantly higher in the Ad-PTEN group and Ad-G129E group than those in both the control group and the Ad-GFP group, P<0.01. Western blot analysis recapitulated the Real time Q-PCR data by showing that expression levels of PTEN protein in Ad-PTEN group (1.66±0.09), Ad-G129E group (1.65±0.09) were significantly higher than those in control group (1.10±0.07) and Ad-GFP group (1.09±0.07), P<0.01. Furthermore, no significant differences were observed in the expressions of PTEN mRNA and protein between Ad-PTEN group and Ad-G129E group or between control group and Ad-GFP group (P>0.05). Overall, wild type PTEN gene and G129E gene were successfully transduced and expressed in HSC. (3)There was no significant difference in the proliferation of HSC at 24 hours after transduction of Ad-PTEN or Ad-G129E (P>0.05). At 48 and 72 hours after transduction, though, a precipitous time-dependent drop in proliferation was observed in the Ad-PTEN group or Ad-G129E group. Inhibition rates were 14.03% and 23.12% in Ad-PTEN group, 9.52% and 12.63% in Ad-G129E group, respectively, when compared with the control group (P<0.01). Obviously, the inhibitory effect of wild type PTEN on HSC proliferation was more powerful than that of G129E. Moreover, the A value between control and Ad-GFP groups showed no notable difference at various time points (P>0.05). (4) TUNEL assay showed that at 72 hours after adenoviral transduction, the apoptotic rates of HSC in Ad-PTEN group (29.81%±2.52%), Ad-G129E group (26.37%±1.97 %) were greatly higher than those in control group (1.98%±0.25%) and Ad-GFP group (2.16%±0.28%), P<0.01, and Ad-PTEN group increased notably in the apoptotic rates of HSC compared with Ad-G129E group too (P<0.01). In addition, no significant difference was found in the apoptotic rates of HSC between control group and Ad-GFP group (P>0.05). (5) At 72 hours after adenovirus transduction, the apoptotic rates of HSC analyzed by PI labeled FCM in Ad-PTEN group (20.84%±1.44%), Ad-G129E group (17.54%±1.76%) increased markedly compared with control group (1.12%±0.57%) and Ad-GFP group (1.21%±0.22%), P<0.01. And the action of wild type PTEN on HSC apoptosis was more powerful than that of Ad-G129E (P<0.01). Furthermore, no significant difference was observed in apoptotic rate of HSC between control group and Ad-GFP group (P>0.05). (6) Western blot analysis showed that the expression of Bcl-2 decreased significantly at 72 hours after Ad-PTEN or Ad-G129E infection compared to control group and Ad-GFP group (1.16±0.03 or 1.24±0.05vs 1.37±0.06 and 1.34±0.08, respectively, P<0.01), and Bcl-2 expression in Ad-PTEN group was significantly lower than that in Ad-G129E group too (P<0.01). In contrast, the expressions of Bax in HSC at 72 hours after infection of Ad-PTEN (1.50±0.05) or Ad-G129E (1.41±0.05) heightened markedly compared with those in control group (1.28±0.06) and Ad-GFP group (1.26±0.08), P<0.01. And Bax expression in Ad-PTEN group inceased notably compared with that in Ad-G129E group as well (P<0.01). Moreover, there were no significant differences in the expressions of Bcl-2 and Bax between control group and Ad-GFP group (P>0.05).
Conclusions: Exogenous wild type PTEN gene and G129E gene are successfully transduced and expressed in activated HSC in vitro respectively, inhibit the proliferation and induce apoptosis of them. At the same time, the expression of apoptosis regulating gene Bax is increased and Bcl-2 is decreased in activated HSC. Moreover, the action of wild type PTEN is more powerful than that of Ad-G129E.
Part 3:Regulatory effects of PTEN on cell cycle of activated hepatic stellate cells in vitro
Objective: To investigate regulatory effects of over-expression of wild type PTEN and its mutant G129E on cell cycle of activated HSC culcured in vitro. Methods: The wild type PTEN gene and its mutant G129E gene were transduced into activated HSC cultured in vitro mediated by adenoviral vector, respectively. PI labed FCM was then used to detect cell cycle phase of activated HSC. And the expressions of PTEN, cyclinD1, cyclin dependent kinase 4 (CDK4) and P27kip1 in HSC were measured by Western blot and Real-time Q-PCR, respectively. Cells were grouped in accordance with part 2.
Results: (1) Western blot and Real-time Q-PCR demonstrated that exogenous wild type PTEN gene and G129E gene were successfully transduced and expressed in activated HSC cultured in vitro (the result was clearly in accordance with the part 2). (2) At 72 hours after adenovirus infection, Cell cycle phase of HSC in each group was detected by PI labed FCM. At G0/G1 phase, the number of HSC in Ad-PTEN group (67.68%±2.75%), Ad-G129E group (61.17%±3.41%) increased greatly compared with those in control group (53.01%±2.37%) and Ad-GFP group (53.85%±3.08%), P<0.01, and the number of HSC in Ad-PTEN group was significantly higher than that in Ad-G129E group (P<0.01). At S phase, the number of HSC decreased notably in Ad-PTEN group (14.42%±1.81%), Ad-G129E group (18.17%±2.43%) compared with those in control group (22.17%±1.99%) and Ad-GFP group (21.54%±1.74%), P<0.01, and the number of HSC in Ad-PTEN group was lower than that in Ad-G129E group too (P<0.01). At G2/M phase, the number of HSC in Ad-PTEN group (17.90%±2.70%), Ad-G129E group (20.66%±2.37%) decreased significantly compared to those in control group (24.82%±3.81%) and Ad-GFP group (24.62%±3.15%), P<0.01, P<0.05, while there was no significant difference in the number of HSC between Ad-PTEN group and Ad-G129E group (P>0.50). Furthermore, at any phase, no significant difference was found in the number of HSC between control group and Ad-GFP group (P>0.50). (3) At 72 hours after adenoviral infection, the expression of cyclinD1 protein in HSC was analyzed by Western blot, the expression of cyclinD1 protein in Ad-PTEN group (1.12±0.07), Ad-G129E group (1.23±0.05) decreased greatly compared to those in control group (1.45±0.05) and Ad-GFP group (1.47±0.08), P<0.01, and cyclinD1 protein expressio in Ad-PTEN group was significantly lower than that in Ad-G129E group too (P<0.01). The Real time Q-PCR was further used to detect the expression level of cyclinD1 mRNA in HSC, the method of fold increase (2-△△Ct method) was used to calculate relative mRNA expression level of cyclinD1 in HSC in each group. If the mRNA expression level of cyclinD1 in control group was assigned a value of 1, then cyclinD1 mRNA expressions in Ad-GFP group, Ad-PTEN group and Ad-G129E group were 1.011-, 0.773- and 0.838-fold compared with control group, respectively. Obviously, the expressions of cyclinD1 mRNA decreased significantly in Ad-PTEN group and Ad-G129E group compared with those in both control group and Ad-GFP group (P<0.01), and the expression of cyclinD1 mRNA in Ad-PTEN group was markedly lower than that in Ad-G129E group (P<0.01). Moreover, no significant differences were observed in the protein and mRNA expressions of cyclinD1 between control group and Ad-GFP group (P>0.05). (4) At 72 hours after adenoviral infection, Western blot analysis showed the expressions of CDK4 protein in Ad-PTEN group (1.05±0.07), Ad-G129E group (1.18±0.06) were markedly lower than those in control group (1.41±0.03) and Ad-GFP group (1.43±0.06), P<0.01, and CDK4 protein expression in Ad-PTEN group reduced notably compared with that in Ad-G129E group as well (P<0.01). Real time Q-PCR was further used to detect the expression level of CDK4 mRNA in HSC, the method of fold increase (2-△△C t method) was used to calculate relative mRNA expression level of CDK4 in HSC in each group. If the mRNA expression level of CDK4 in control group was assigned a value of 1, then CDK4 mRNA expression levels in Ad-GFP group, Ad-PTEN group and Ad-G129E group were 1.007-, 0.738- and 0.822-fold, respectively. The expression of CDK4 mRNA down-regulated notably in Ad-PTEN group and Ad-G129E group compared with those in control group and Ad-GFP group (P<0.01), and the expression of CDK4 mRNA in Ad-PTEN group was significantly lower than that in Ad-G129E group too (P<0.01). In addition, no significant differences were observed in the protein and mRNA expressions of CDK4 between control group and Ad-GFP group (P>0.05). (5) At 72 hours after adenoviral infection, Western blot analysis showed that the protein expressions of P27kip1 in HSC in Ad-PTEN group (1.40±0.03), Ad-G129E group (1.28±0.08) increased significantly compared to those in control group (1.11±0.04) and Ad-GFP group (1.12±0.04), P<0.01, and the protein expression of P27kip1 in Ad-PTEN group was notably higher than that in Ad-G129E group as well (P<0.01). Real time Q-PCR was used to extrapolate relative mRNA expression level of P27kip1 in HSC. P27kip1 mRNA expression in HSC in each group was compared by using the method of fold increase (2-△△C t method). If the mRNA expression level of P27kip1 in control group was assigned a value of 1, then the mRNA expressions of P27kip1 in Ad-GFP group, Ad-PTEN group and Ad-G129E group were 1.008-, 1.264- and 1.157-fold compared with control group, respectively. Obviously, the expressions of P27kip1 mRNA increased greatly in Ad-PTEN group and Ad-G129E group compared with those in control group and Ad-GFP group (P<0.01), and the expression of P27kip1 mRNA in Ad-PTEN group was markedly higher than that in Ad-G129E group too (P<0.01). Moreover, no significant difference was found in expressions of P27kip1 protein and mRNA between control group and Ad-GFP group (P>0.05).
Conclusions: The over-expression of not only wild type PTEN but also G129E can inhibit transition of activated HSC in vitro from G1 to S phase, arrest cell cycle of them at G0/G1 phase, and thus negatively regulate cell cycle progression of them. Meanwhile, at both transcriptional and translational levels, the expressions of cyclinD1 and CDK4 are down-regulated and P27kip1 expression is up-regulated in activated HSC in vitro. In addition, wild type PTEN is more powerful than Ad-G129E for above-mentioned effects. Part 4: Signaling transduction mechanisms of PTEN on the activated hepatic stellate cell behaviors
Objective: To investigate the signaling transduction mechanisms of PTEN on the proliferation and apoptosis as well as cell cycle of activated HSC.
Methods: Using cell culture techniques in vitro, the wild type PTEN gene and its mutant G129E gene were transduced into activated HSC in vitro mediated by adenoviral vector, respectively. And the protein expressions of serine-threonine protein kinase B (Akt), p-Akt (Thr308), extracellular signal regulated kinase1 (ERK1) and p-ERK1 in activated HSC were measured by Western blot. And the mRNA expressions of Akt and ERK1 were detected by Real-time Q-PCR. Cells were grouped in accordance with part 2.
Results: ((1) Western blot and Real-time Q-PCR demonstrated that exogenous wild type PTEN gene and G129E gene were successfully transduced and expressed in activated HSC cultured in vitro (the result was clearly in accordance with the part 2). (2) Western blot and Real-time Q-PCR showed that there were no significant differences in the mRNA and protein expression of Akt in HSC at 72 hours after transduction of Ad-PTEN or Ad-G129E (P>0.05). While Western blot analysis documented the expression of p-Akt (Thr308) in HSC at 72 hours after adenoviral infection decreased greatly in Ad-PTEN group (0.63±0.04) compared to those in Ad-G129E group (0.93±0.03), control group (0.95±0.04) and Ad-GFP group (0.94±0.03), P<0.01. Moreover, no significant differences were observed in the expressions of p-Akt (Thr308) among Ad-G129E group, control group and Ad-GFP group (P>0.05). (3) At 72 hours after transduction of Ad-PTEN or Ad-G129E, there were no significant differences in the protein and mRNA expression of ERK1 in HSC (P>0.05). While Western blot analysis documented the protein expression of p-ERK1 in HSC at 72 hours after adenoviral infection reduced greatly in Ad-PTEN group (0.65±0.04), Ad-G129E group (0.68±0.07) compared to those in control group (0.84±0.07) and Ad-GFP control (0.85±0.06), P<0.01. In addition, no significant differences were found in the protein expressions of p-ERK1 between Ad-PTEN and Ad-G129E group or between control group and Ad-GFP group (P >0.05).
Conclusions: The PTEN phosphatase negatively regulates the phosphoinositol-3-kinase (PI3K)/Akt and ERK1/2 signal transduction pathways via inhibiting phosphorylation of Akt and ERK1 in activated HSC, and thus curbs the proliferation, induces apoptosis and negatively regulates cell cycle of activated HSC in vitro.
引文
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