【作者】 叶丽平；

【导师】 孙黎光；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2007， 博士

【摘要】 碱性成纤维细胞生长因子抑制卵巢癌CAOV3细胞凋亡的信号转导机制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，激活有丝分裂原活化蛋白激酶的激酶（Mitogen-activated protein kinase kinase，MEK）／细胞外信号调节激酶（Extracellullar signal-regulated protein kinase，ERK），磷脂酰肌醇-3-激酶（Phosphoinositide 3-kinase，PI3K）／蛋白激酶B（Protein kinase B，PKB）和蛋白激酶C（Protein kinase C，PKC）等信号通路，在促进细胞增殖、阻止细胞凋亡的过程中起关键作用。在恶性肿瘤的发生、发展中，细胞不仅发生异常增殖，更重要的是凋亡的能力和倾向降低了。细胞凋亡是有核细胞在凋亡信号的刺激下启动的细胞内死亡机制，经过一系列信号转导途径，最终发生程序性死亡的过程。细胞凋亡能力的降低不仅在肿瘤发生、发展中起重要作用，也是肿瘤细胞产生耐药的主要原因。因此，利用化疗、放疗和生物治疗等方法诱导肿瘤细胞凋亡成为治疗肿瘤的主要方法。卵巢癌是女性生殖器官三大恶性肿瘤之一，死亡率极高，其发生发展与细胞增殖及凋亡的失控密切相关。研究表明，卵巢癌过度表达bFGF和FGFR1，可能通过自分泌或旁分泌等方式在卵巢癌的发生、发展中发挥重要作用。研究还发现，Bcl-2家族是调控线粒体及绌胞色素C介导的细胞凋亡的重要分子。主要包括凋亡抑制蛋白（Bcl-2、Bcl-x1）和促凋亡的蛋白（Bax、Bak、Bad）等。Bcl-2家族的作用位点主要在线粒体膜上，除线粒体外，还可结合于内质网（endoplasmic reticulum，ER）膜上。ER是蛋白合成、维持细胞正常功能的重要场所。在缺氧，低糖等应激环境下，ER内错误折叠蛋白增多，可引发未折叠蛋白反应（unfolded Protein Reaction，UPR），通过上调分子伴侣——葡萄糖调节蛋白78（glucose regulated proteins，Grp78）等帮助蛋白正确折叠以减轻细胞损伤。当应激强度过大，ER功能严重受损时细胞将启动生长抑制DNA损伤诱导因子（growth arrest and DNA damage inducible gene，GADD153）以及Caspase-12等信号，触发细胞凋亡。Bcl-2家族可在ER水平调节GADD153及Caspase-12诱导的细胞凋亡。因此，线粒体与ER介导的死亡通路可能有着共同的信号调节分子。目前，关于bFGF抑制肿瘤细胞凋亡的信号通路，以及与Bcl-2、ER蛋白之间的关系尚不十分清楚。本研究利用无血清培养模拟人卵巢癌CAOV3细胞ER应激并诱导细胞凋亡，观察外源性bFGF对细胞生存，对Bcl-2家族蛋白（Bcl-2、Bcl-x1、Bax、Bad）、Bcl-2转录因子CREB、Grp78、GADD153表达的影响，以及MEK／ERK、PI3K／PKB信号通路在此过程中的调控作用，以探讨bFGF对无血清诱导的人卵巢癌CAOV3细胞凋亡的调控作用及可能的信号转导机制。方法1、卵巢癌CAOV3细胞同步化后无血清饥饿培养，分别加入bFGF、PD98059（ERK上游激酶MEK₁的特异性抑制剂）及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、H E染色、考马斯亮蓝染色、Annexin-EGFP／PI双荧光染色、DNA梯度电泳、流式细胞术等观察bFGF、PD98059、Wortmannin对饥饿培养诱导的CAOV3细胞凋亡的影响。3、Western blot法检测bFGF、PD98059及Wortmannin对饥饿培养的CAOV3细胞蛋白激酶ERK、PKB的激活作用，对Bcl-2、Bax、Bad、CREB、Grp78、GADD153表达的影响及其可能的信号转导通路。4、RT-PCR法检测bFGF、PD98059及Wortmannin对饥饿培养的CAOV3细胞Bcl-2、Bcl-xl、Grp78 mRNA表达的影响及其可能的信号转导通路。结果1、外源性bFGF可迅速激活卵巢癌CAOV3细胞ERK、PKB活性，明显抑制无血清饥饿诱导的细胞凋亡，PD98059（ERK上游激酶MEK₁抑制剂）和Wortmannin（PKB上游激酶P13K抑制剂）可部分抑制bFGF的上述作用。H E染色、考马斯亮蓝染色结果显示：bFGF组细胞生长良好。饥饿对照组、PD98059组及Wortmannin组出现明显的凋亡细胞。Annexin-EGFP／PI双荧光染色结果显示：bFGF组几乎均为胞核、胞膜无荧光着染的活细胞；饥饿对照组、PD98059组及Wortmannin组可见胞核无色、胞膜绿色着染的早期凋亡细胞，以及胞膜绿色，胞核红色着染的坏死和晚期凋亡细胞。DNA梯度电泳显示：bFGF组无明显DNA梯状带，饥饿对照组、PD98059及Wortmannin组均出现明显的梯状带，说明出现较多的凋亡细胞。流式细胞术分析显示：应用无血清饥饿成功地建立了CAOV3细胞的凋亡模型。饥饿对照组细胞经无血清饥饿培养24h即可诱导细胞凋亡（33.767±4.954％），bFGF组细胞同样饥饿24h几乎没有凋亡细胞（7.300±1.510％）。PD98059及Wortmannin组均可部分抑制bFGF的抗凋亡作用，凋亡百分比分别为23.700±3.2055％，19.667±2.701％。Western blot结果显示：bFGF呈剂量、时间依赖性诱导饥饿培养的CAOV3细胞ERK、PKB活性增高。饥饿对照组ERK、PKB活性均无明显变化。与bFGF组相比，PD98059组ERK活性约下降58.45％，Wortmannin组PKB活性约下降51.82％（P＜0.01）。ERK、PKB蛋白总量表达无改变。2、bFGF呈时间依赖性诱导饥饿培养的CAOV3细胞CREB^ser133活性增高，刺激Bcl-2的mRNA及蛋白表达增加。bFGF处理45 min时CREB活性达高峰，4h时Bcl-2mRNA达峰值，8h时Bcl-2蛋白表达最高，PD98059可抑制此诱导作用（P＜0.01）。Wortmannin对bFGF的上述作用无影响。饥饿对照组细胞CREB、Bcl-2的mRNA及蛋白表达无变化。3、饥饿培养及bFGF处理对CAOV3细胞Bcl-xl的mRNA表达均无影响。4、饥饿培养可诱导CAOV3细胞Bax及GADD153蛋白表达增高。饥饿8h时GADD153表达达高峰，24h时Bax表达最高。bFGF可抑制饥饿诱导的Bax及GADD153表达的上调。PD98059及Wortmannin均可阻断bFGF的上述作用（P＜0.01）。5、bFGF组呈时间依赖性诱导饥饿培养的CAOV3细胞Bad^ser136活性增高。bFGF处理15 min时Bad活性最高。Wortmannin可阻断此诱导作用（P＜0.01），PD98059对bFGF的上述作用无影响。饥饿对照组细胞Bad活性无改变。6、饥饿培养可诱导CAOV3细胞ER应激，Grp78 mRNA及蛋白表达短暂升高，饥饿2 h时Grp78 mRNA、蛋白表达最高。与饥饿对照组相比，bFGF呈时间依赖性诱导饥饿培养的CAOV3细胞Grp78 mRNA及蛋白持续高表达。bFGF处理4 h时Grp78 mRNA达峰值、8 h时Grp78蛋白表达最高。Wortmannin可阻断此诱导作用（P＜0.01），PD98059对bFGF的上述作用无影响。结论1、bFGF经MEK／ERK、PI3K／PKB信号通路抑制无血清饥饿诱导的卵巢癌CAOV3细胞凋亡。2、bFGF经MEK途径迅速激活无血清饥饿培养的卵巢癌CAOV3细胞ERK活性，经PI3K途径迅速激活PKB活性。bFGF对ERK、PKB活性的改变是通过快速磷酸化激酶蛋白实现的，并不是促进蛋白合成的增加。3、bFGF可能经MEK／ERK／CREB^ser133通路促进无血清饥饿培养的卵巢癌CAOV3细胞Bcl-2转录及蛋白表达，经MEK／ERK、PI3K／PKB信号通路抑制Bax蛋白表达。4、bFGF经PI3K／PKB通路促进无血清饥饿培养的卵巢癌CAOV3细胞Bad^ser136磷酸化，对Bcl-xl表达无影响。5、bFGF经PI3K／PKB通路促进无血清饥饿培养的卵巢癌CAOV3细胞Grp78转录及蛋白表达，经MEK／ERK、PI3K／PKB信号通路抑制GADD153蛋白表达。更多还原

【Abstract】 The signal mechanism underlying basic fibroblast growth factor-mediated anti-apoptosis in ovarian cancer CAOV3 cellsObjectiveAs a potent mitogen, basic fibroblast growth factor（bFGF）, plays an important role in cell proliferation, differentiation, angiogenesis and survival through several signal pathways including Mitogen-activated protein kinase kinase （MEK）/Extracellullar signal regulated protein kinase （ERK）, Phosphoinositide 3-kinase （PI3K）/Protein kinase B （PKB）,Protein kinase C （PKC） by binding to fibroblast growth factor receptor （FGFR）. However, its downstream mechanism has not been well defined.Ovarian cancer is one of the popular gynecology tumors and has the highest mortality among malignant gynecology tumors associated to the unbalance of proliferation and apoptosis. It is reported that bFGF and FGFR are overexpressed in ovarian cancer which may involve in carcinogenesis by autocrine or paracine mechanism.The tumor is characterized by increasing proliferation and decreasing apoptosis. Apoptosis involves a subset of effector caspase proteases .Activation of these caspases occurs via mitochondria-independent and -dependent mechanisms. It is well known that mitochondria-dependent apoptotic signal can be effectively blocked by anti-apoptotic members of Bcl-2 family such as Bcl-2 or Bcl-x1 and promoted by pro-apoptotic members such as Bax or Bad. Although Bcl-2 may have a direct action on the mitochondria membrance, it also resides and functions on the endoplasmic recticulum（ER）,and there is increasing evidence for a role of the ER in apoptosis regulation as well. ER is an organelle responsible for the protein synthesis and homeostasis. Stress can bring about alterations of ER homeostasis which cause ER stress. Unfolded Protein Reaction（UPR） is an important genomic response to ER stress by synthesis ER chaperones proteins such as glucose regulated proteins 78（Grp78）, which plays critical roles in cell survival as part of UPR.When the ER function is severely impaired,the organelle elicits apoptosis signal activated by growth arrest and DNA damage inducible gene （GADD153） and caspase-12. The apoptotic crosstalk between ER and mitochondria is also controlled by Bcl-2 family.To evaluate the impact of bFGF-mediated activation on the anti-apoptosis and the relationship between mitochondria and ER in ovarian cancer, by means of free-serum starvation to induce ER stress and apoptosis in vitro,we investigate the effects of bFGF on survival, activity of ERK,PKB,Bad and CREB,mRNA or protein expression of Bcl-2,Bcl-xl,Bax, Grp78 and GADD153 in ovarian cancer CAOV3 cells, and explore the relationship between these effects and MEK/ERK, PI3K/PKB signal pathway.Methods1. CAOV3 cells were cultured in serum-free DMEM with or without bFGF which blocked by PD98059 （MEK1 inhibitor） or Wortmarmin （PI3K inhibitor）.2. The effects of bFGF, PD98059 and Wortmannin on the apoptosis in starvated CAOV3 cells were estimated by DNA ladder gelelectrophoresis, FCM analysis and staining with Hematoxylin-Eosin, Coomassie brilliant blue and Annexin-EGFP/PI, respectively.3. The changes of the activity of ERK, PKB and CREB, and the protein expression of Bcl-2、Bax、Bad、Grp78、GADD153 induced by starvation, bFGF,PD98059 and Wortmannin were accessed by western blotting.4. The mRNA expression of Bcl-2、Bcl-x1 and Grp78 induced by starvetion, bFGF, PD98059 and Wortmannin were determined by reverse transcription PCR（RT-PCR）.Results1. Starvated CAOV3 cells displayed typical signs of apoptosis such as nuclear condensation or DNA fragments.As compared to starvation group, the cells after bFGF treatment were still viable and had increased activation of ERK and PKB, which was prevented by PD98059 and Wortmannin effectively（P＜0.01）.Total ERK and PKB did not change under the same experimental conditions （P＞0.05）.2. bFGF could activate CREB^ser133 and upregulate mRNA and protein expression of Bcl-2 time-dependently as compared to starvation group, which peaked at 45 min, 4 h and 8 h after initiation of bFGF treatment, respectively（P＜0.01）.PD98059 could inhibit the effects of bFGF on Bcl-2 and CREB rather than Wortmannin. Total CREB remained unchanged under the same experimental conditions （P＞0.05）.3. Both bFGF and starvation were ineffective on Bcl-x1 mRNA expression.4.Starvation induced Bax and GADD153 protein expression time-dependently, but bFGF restraind their expression which were blocked both by PD98059 and Wortmannin （P＜0.01）.5.After bFGF exposure, the phosphorylated Badserf36 increased time-dependently as compared to starvation group （P＜0.01） and its peak was at 30 min which was prevented by Wortmannin but not PD98059. Total Bad protein levels did not change under the same experimental conditions （P＞0.05）.6. bFGF could upregulate Grp78 mRNA and protein expression more effectively and persistently than starvation group.Grp78 mRNA and protein expression achieved their peaks at 4 h and 8 h after bFGF treatment,respectively （P＜0.01）.Wortmannin could inhibit this process but PD98059 was ineffective on it.Conclusion1.Starvation could upregulate Bax and GADD153 that induce apoptosis in CAOV3 cells.bFGF could inhibit serum-free starvation-induced apoptosis through MEK/ERK, PI3K/PKB signal transduction pathway partly.3.bFGF activated MEK/ERK and PI3K/PKB by phosphorylating the kinases immediately rather than increasing their protein synthesis.4.bFGF upregulated Bcl-2 mRNA and protein expression through MEK/ERK/CREB^ser133 and increased Grp78 mRNA and protein expression through PI3K/PKB signal transduction pathway.5. bFGF induced Bad ^ser136 by PI3K/PKB signal transduction pathway and was ineffective on Bcl-x1 mRNA expression.6. bFGF blocked Bax protein expression through MEK/ERK and PI3K/PKB signal transduction pathway as well as GADD 153.更多还原