【作者】 朱艳凌；

【导师】 孙黎光；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2009， 博士

【摘要】 目的碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)属于有丝分裂原,通过与成纤维细胞生长因子受体(fibroblast growth factor receptor,FGFR)结合,激活磷脂酰肌醇-3-激酶(Phosphoinositide 3-kinase,PI3K)/蛋白激酶B(Protein kinase B,PKB)等信号通路,在影响细胞增殖、分化及生存方面起关键作用。肿瘤是细胞增殖、分化和凋亡异常引起的以细胞失控性生长和增殖为主要特征的疾病,对细胞增殖和分裂至关重要的信号转导途径和细胞周期调控机制是肿瘤研究的焦点。细胞周期调控异常是肿瘤发病的主要机制,肿瘤转化大部分是G1、G2期机制失调的结果。PI3K/PKB信号转导途径在癌细胞中上调,经常通过促进G1-S期细胞周期进程,刺激肿瘤细胞增殖。研究表明,肝癌过度表达bFGF和FGFR1,可能通过自分泌或旁分泌等方式发挥作用,由bFGF激活的PI3K/PKB信号转导途径,与肝癌的发生发展密切相关。FOXO1转录因子是PI3K/PKB直接的下游信号分子,通过调节靶基因的表达在控制细胞周期进程、DNA修复、抵御氧化损伤和凋亡中起重要作用,FOXO活性降低或功能缺失可能导致细胞的无限增殖和瘤的转化,被看作是肿瘤抑制因子。p27是细胞周期素依赖激酶(cyclin dependent kinase,CDK)的抑制蛋白,主要通过抑制CDK或Cyclin/CDK复合物活性,阻断G1-S转换,对细胞周期进行负调控,是关键的闸门抑制因子,其表达及代谢发生异常可导致细胞周期失控并促进细胞异常增殖。在肿瘤中p27的调节主要发生在翻译后水平,最主要的是通过泛素一蛋白酶体系统(ubiquitin-proteasome system,UPS)降解。在泛素化过程中,S期激酶相关蛋白2(S-phase kinase-associated protein 2,Skp2)能特异性识别磷酸化的底物,是介导p27泛素化和降解的重要蛋白。Skp2是一种癌蛋白,在人类很多恶性肿瘤中,Skp2的蛋白表达通常很高,与p27的蛋白表达呈负相关,而且,Skp2表达水平越高,肿瘤的恶性程度越高,侵袭性越强,表明Skp2依赖的p27蛋白质降解在肿瘤的发生发展中有重要作用。目前关于bFGF调控肿瘤细胞增殖的PI3K/PKB通路与FOXO1、p27以及Skp2之间的关系尚不十分清楚。本研究利用无血清培养使人肝癌Bel-7402细胞同步化,观察外源性bFGF对细胞增殖、PKB、FOXO1活性以及p27、Skp2表达的影响,以探讨bFGF对人肝癌Bel-7402细胞增殖的调控作用及可能的信号转导机制。方法1、肝癌Bel-7402细胞同步化后无血清饥饿培养,分别加入bFGF、Wortmannin(PKB上游激酶PI3K的特异性抑制剂),继续饥饿培养至不同时间。2、MTT、流式细胞术等观察bFGF、Wortmannin对饥饿培养诱导的Bel-7402细胞增殖的影响。3、Western blot法检测bFGF、Wortmannin对饥饿培养的Bel-7402细胞蛋白激酶PKB、FOXO1的激活作用,对p27、Skp2表达的影响及其可能的信号转导通路。免疫荧光检测bFGF、Wortmannin对饥饿培养的Bel-7402细胞p-FOXO1分布的影响。4、RT-PCR法检测bFGF、Wortmannin对饥饿培养的Bel-7402细胞Skp2、p27 mRNA表达的影响及其可能的信号转导通路。结果1、外源性bFGF呈剂量、时间依赖性迅速激活肝癌Bel-7402细胞PKB活性,PKB蛋白总量表达无改变。Wortmannin(PKB上游激酶PI3K抑制剂)可部分抑制bFGF的上述作用。与bFGF组相比,Wortmannin组PKB活性约下降36.80%(P＜0.01)。bFGF呈时间依赖性磷酸化FOXO1,免疫荧光显示bFGF可诱导磷酸化FOXO1出胞核进入胞浆,Wortmannin可部分阻断FOXO1的磷酸化。2、MTT结果显示:Bel-7402细胞经bFGF处理细胞增殖比明显增加,且与bFGF水平成剂量依赖关系,bFGF浓度为25 ng/ml时细胞增殖比最高为145%,各bFGF处理组与对照组相比有统计学意义(P＜0.05),经Wortmannin预处理后可抑制细胞增殖(P＜0.01)。3、流式细胞术分析显示:Bel-7402细胞经25 ng/ml bFGF孵育16 h后,与对照组相比G1期细胞减少(75.56±4.12)%→(57.50±3.75)%,S期细胞增多(16.53±1.95)%→(30.13±4.03)%,bFGF促进细胞由G1期进入S期(P＜0.01)。Wortmannin使bFGF促进细胞周期的作用受到抑制[G1期:(67.53±2.30)%,S期:(22.63±2.54)%,P＜0.05]。4、bFGF呈时间依赖性诱导饥饿培养的Bel-7402细胞Skp2的mRNA及蛋白表达增加。bFGF处理6 h时Skp2 mRNA达峰值,8 h时Skp2蛋白表达最高,Wortmannin可抑制bFGF对Skp2的诱导作用(P＜0.01)。5、bFGF对饥饿培养的Bel-7402细胞p27 mRNA的表达无影响,但可抑制p27蛋白表达。bFGF处理10 h时p27蛋白表达最低,Wortmannin和蛋白酶体抑制剂MG-132可抑制此诱导作用(P＜0.05)。结论1、bFGF经PI3K途径迅速激活Bel-7402细胞PKB、磷酸化FOXO1。2、bFGF对PKB、FOXO1活性的改变是通过快速磷酸化而实现的。3、bFGF可能通过PI3K/PKB/FOXO1信号通路促进Bel-7402细胞增殖。4、bFGF可能通过激活PI3K途径下调p27的蛋白表达、上调Skp2 mRNA及蛋白表达,促进肝癌Bel-7402细胞增殖。更多还原

【Abstract】 ObjectiveBasic fibroblast growth factor(bFGF) is a potent mitogen that plays an important role in cell proliferation,differentiation and survival by phosphoinositide 3-kinase (PI3K)/Protein kinase B(PKB) after binding to fibroblast growth factor receptor (FGFR).Because the tumors are characterized of overgrowth and abnormal proliferation,the signal pathways underlying regualtion of cell cycle becomes the focus of medical research.Most of tumors result from dysregulated phase G1 and G2 in cell cycle.It is reported that bFGF and FGFR are overexpressed in hepatic cancer which may involve in carcinogenesis through activating PI3K/PKB signal pathway,promoting phase G1-S and strengthening the cell growth.FOXO1,a transcription factor,is an important downstream of PI3K/PKB pathway in the regulation of metabolism,cell cycle, proliferation and survival.As a tumor inhibitor,either low activity or dysfunction of FOXO1 will lead to the cell overgrowth and transformation of tumors.Differently,p27,the inhibitor of cyclin dependent kinase(CDK) and Cyclin/CDK, supresses the cell cycle and disrupt G1-S.In the tumor,S-phase kinase-associated protein 2(Skp2)-mediating ubiquitin-proteasome system(UPS) is the important pathway to degrade p27 and results in overgrowth.Most of human cancers overexpress Skp2,downexpress p27 and exhibit the abnormal proliferation.It indicates that degradation of p27 depedenting on Skp2 may paly an critical role in the carcinogenesis and development of tumors.However,the relationship between bFGF-mediating PI3K/PKB signal pathway and its downstream FOXO1,p27 and Skp2 has not been well defined.Hepatic cancer is one of the popular tumors and has the highest mortality among malignant tumors associated to the unbalance of proliferation and apoptosis.To evaluate the impact of bFGF-mediated activation on the cell proliferation in the hepatic cancer,we investigate the effects of bFGF on proliferation,activity of PKB,FOXO1,p27 and Skp2 expression in hepatic cancer Bel-7402 cells,and explore the relationship between these effects and PI3K/PKB signal pathway.Methods1.Bel-7402 cells were cultured in serum-free DMEM with bFGF which blocked by Wortmannin(PI3K inhibitor).2.The effects of bFGF and Wortmannin on the proliferation in starvated Bel-7402 cells were estimated by MTT and FCM analysis.3.The changes of the activity of PKB and FOXO1 induced by bFGF and Wortmannin were accessed by western blotting.p-FOXO1 distribution induced by bFGF was detected by immunofluorescence technique.4.The changes of the activity of the protein expression of p27 and Skp2 induced by bFGF,Wortmannin and MG132 were accessed by western blotting.5.The mRNA expression of p27 and Skp2 induced by bFGF and Wortmannin were determined by reverse transcription PCR(RT-PCR).Results1.As compared to starvation group,bFGF treatment activated PKB and phosphorylated FOXO1 dose-and time-dependently which were prevented by Wortmannin effectively P＜0.01).The phosphoralation of FOXO1 induced by bFGF occurred in cytoplasm.2.MTT and FCM analysis display that Bel-7402 cells were still viable after bFGF treatment,had increased proliferation rate and cell distribution from phase G1 to phase S,dose-dependently.Wortmannin would prevent the roles of bFGF all above effectively(P＜0.01).3.As compared to starvation group,bFGF treatment restraind the expression of p27,but accelerated the mRNA and protein expression of Skp2 time-dependently which peak at 6 h and 8 h after initiation of bFGF.Wortmannin would inhibit this process effectively(P＜0.01).bFGF treatment has no effect on the mRNA expression of p27.Proteasome inhibitor(MG132) could enhance the protein expression of p27. Conclusion1.bFGF could phosphorylate PKB and FOXO1.2.The activation of PKB and FOXO1 mediated by bFGF resulted from phosphorylating the kinases.3.bFGF could promote the proliferation of Bel-7402 cells by PI3K/PKB /FOXO1 signal pathway.4.bFGF could downregulate p27 protein expression and increase Skp2 mRNA and protein expression by PI3K signal transduction pathway that maybe contributes to the proliferation in Bel-7402 cells.更多还原