【作者】 陈刚；

【导师】 任华；

【作者基本信息】 中国协和医科大学 ， 胸外科学， 2010， 博士

【摘要】 目的考察外源性血管内皮生长因子对体外培养的A549肺癌细胞增殖,细胞凋亡,化疗药物敏感性的影响,并观察VEGF-VEGFR2-PI3K/Akt信号通路在其中的作用。从而初步探讨VEGF对肺癌细胞生物学影响及机制。方法分别在含有10%、0.5%、0%血清浓度的培养基中,加入不同浓度梯度外源性VEGF培养A549细胞,MTT实验检测VEGF对于细胞生长及生存的影响；确定顺铂、吉西他滨、多烯紫杉醇的适合作用浓度,MTT法比较加入与否外源性VEGF时A549细胞对上述化疗药物的敏感性；MTT法比较加入外源性VEGF时抑制VEGF-VEGFR2-PI3K/Akt信号通路与否对于A549细胞的药物敏感性的影响；Western-blot实验检测加入VEGF时细胞VEGF-VEGFR2-PI3K/Akt信号通路中Akt蛋白磷酸化的变化；流式细胞仪检测VEGF+化疗药物组与单纯化疗组相比,细胞周期及凋亡的变化情况。结果1、向含10%及0.5%血清的培养基中加入不同浓度梯度外源性VEGF,细胞的增殖及凋亡变化不显著。应用不含血清的培养基培养A549细胞,MTT示分别加入50ng/ml,100ng/ml,500ng/ml外源性VEGF均能使促进其增殖及生存,流式细胞分析示加入100ng/m1外源性VEGF细胞S期及G2期比例增加,G1期比例下降。2、MTT法示在0.5%血清的培养基中应用适宜浓度的顺铂、吉西他滨、多烯紫杉醇杀伤细胞的同时加入外源性VEGF能够显著提高细胞生存,应用VEGF-VEGFR2-PI3k/Akt信号通路的抑制剂能够阻断这种效应,流式细胞分析示加入VEGF组与对照组相比化疗诱导的细胞凋亡明显减少。3、Western-blot实验示外源性VEGF对细胞中Akt磷酸化的激活作用于30分钟至2小时达到最强,随时间发展逐渐减弱,加入VEGF信号通路抑制剂能够减弱VEGF对Akt磷酸化的激活作用。结论1、无血清培养基培养细胞时,VEGF可延长A549细胞的存活时间。2、向细胞培养基中加入外源性VEGF可拮抗顺铂、吉西他滨、多烯紫杉醇对于A549细胞诱导的凋亡作用。3、VEGF拮抗A549细胞的凋亡作用与VEGF-VEGFR-PI3K/Akt通路相关。目的探讨由质粒载体介导的针对VEGF的RNAi对A549肺癌细胞中VEGF的表达、A549细胞增殖的影响及RNAi联合化疗药物对肿瘤的杀伤作用,为RNA干扰技术在肺癌治疗领域中的应用提供实验依据。方法将携带有针对VEGF的RNAi表达框架的真核表达载体pRS-VEGF-shRNA质粒于DH5a大肠杆菌中扩增并进行测序鉴定,通过脂质体转染技术将该质粒转染入A549细胞,RT-PCR法检测VEGF mRNA表达被抑制情况,ELISA法检测培养细胞上清液中VEGF蛋白表达,MTT法检测转染该质粒对于A549细胞的增殖与凋亡的影响,MTT法检测RNAi与顺铂、吉西他滨或多烯紫杉醇共同作用时细胞的增殖与凋亡的变化。结果1、质粒pRS-VEGF-shRNA进行扩增后,测序结果显示其具有能够抑制VEGF表达的shRNA序列。2、RT-PCR法示与对照组比较,48小时后质粒pRS-VEGF-shRNA转染组VEGF mRNA表达量显著降低。3、ELISA法示转染组细胞上清液中VEGF蛋白含量显著降低,与RT-PCR结果相一致。4、转染后48h,MTT法示质粒转染组细胞的与对照组相比细胞增殖及存活受到抑制。5、RNAi质粒与化疗药物共作用组较单纯化疗药物组细胞存活数减少。结论1.质粒pRS-VEGF-shRNA能够成功转染入A549细胞,VEGF mRNA表达及VEGF蛋白分泌受到抑制。2.应用针对VEGF的RNAi阻断内源性VEGF表达后,A549细胞的增殖受到影响。3. RNAi能够通过抑制肿瘤自分泌的VEGF与化疗药物协同杀伤肺癌细胞。更多还原

【Abstract】 Objective To investigate the effect by exogenous vascular endothelial growth factor on proliferation, apoptosis, chemosensitivity in cultured A549 lung cancer cells and the involvement of VEGF-VEGFR2-PI3K/Akt pathway, hoping to provide the experimental data of biological effects of VEGF on lung cancer cells.Methods Culture A549 lung cancer cell with different concentrations of exogenous VEGF in the medium containing different concentrations of serum, then the cell growth was monitored by MTT assay. The cells were treated with anticancer drugs alone or in combination of exogenous VEGF. The proliferation and apoptosis was measured by MTT assay. The distribution of cell cycle or the apoptosis rate was analysed by flow cytometry. After inhibition VEGF-VEGFR2-PI3K/Akt signaling pathway, the chemosensitivity was evaluated by MTT assay. The phosphorylation of Akt was detected by Western blot.Results Culturing the cells with medium containing 10% or 0.5% serum, the exogenous VEGF did not change the growth rates significantly. When A549 cells in the serum free culture, applying exogenous VEGF, proliferation and survival could be stimulated, and the cell population in G1 phase was decreased while the cell population in S and G2 phase was increased. Compared to chemotherapy alone, the survival of the cells was improved when it treated with anticancer drugs together with exogenous VEGF, which was suppressed by VEGF-VEGFR-PI3K/Akt inhibitors. Flow cytometry analysis indicated the proportion of apoptotic cells was lesser in the VEGF plus chemotherapy group. Western blot revealed the level of phosphorylated Akt reached the highest after the cells were incubated with exogenous VEGF for 30 minutes or 2 hours.Conclusion 1. Exogenous VEGF could prolong survival of A549 human lung cancer cells cultured cells in serum-free medium.2. Exogenous VEGF could reduce chemotherapy-induced apoptosis of A549 human lung cancer cells.3. VEGF-VEGFR-PI3K/Akt signaling pathway was involved in the protection against chemotherapy toxicity by exogenous VEGF in A549 human lung cancer cells.Objective To study the effect of vector-mediated RNA interference targeting VEGF combined with chemotherapy on cell proliferation and survival in A549 human lung cancer cell line, providing some data of RNA interference therapy for lung cancer.Methods The VEGF-specific shRNA-expressing plasmid pRS-VEGF-shRNA was amplified in E. coli DH5α. The shRNA sequence targeting human VEGF was confirmed by DNA sequencing analysis. After the plasmid was transfected into A549 cells by liposome, VEGF mRNA expression and VEGF protein concentration was detected by RT-PCR and ELISA separately. The cytotoxity of RNAi plasmid alone or in combination with anticancer drugs was evaluated by MTT assay.Results The sequencing proved the targeting sequence was identical to part of VEGF cDNA. RT-PCR showed the down-regulation of VEGF mRNA expression. ELISA showed the concentration of VEGF protein in the supernatant of the medium was decreased. MTT assay indicated the growth of the plasmid transfected cells was inhibited. After the cells were treated with plasmid and anticancer drugs, compared to the control group, the number of apoptotic cells increased.Conclusion 1. Plasmid pRS-VEGF-shRNA could be successfully transfected into A549 cells, resulting in the inhibition of VEGF mRNA expression and VEGF protein secretion.2. After the expression of endogenous VEGF was suppressed, the growth of A549 cells was affected.3. The cytotoxicity of anticancer drugs was enhanced when the secretion of endogenous VEGF was down-regulated by plasmid-mediated RNAi in A549 lung cancer cells.更多还原