【作者】 崔斐；

【导师】 罗荣城；

【作者基本信息】 南方医科大学 ， 肿瘤学， 2008， 博士

【摘要】 研究背景和目的:原发性肝癌（hepatic cell carcinoma,HCC,以下简称肝癌）是世界上常见的恶性肿瘤之一,肝癌死亡率占我国恶性肿瘤死亡率的第二位。包括肝癌在内的多数恶性实体肿瘤的生长和转移依赖于血管生成,血管生成可为肿瘤细胞的增殖提供营养和代谢的通路,促进肿瘤的生长和转移,加快病情的恶化,故肿瘤血管生成现已成为热门的研究领域。贝伐单抗（Bevacizumab,Avastin,rhuMAb-VEGF）是人工合成的一种针对血管内皮生长因子（vascular endothelial growth factor,VEGF）的重组人源化IgG1型单克隆抗体。Bevacizumab特异性结合于VEGF后能阻碍后者与内皮细胞表面受体Flt-1及KDR结合,阻断VEGF介导的下游信号通路,使VEGF不能发挥促进血管内皮细胞增殖以及肿瘤内血管新生的作用,从而阻断对肿瘤生长至关重要的血液、氧气和其他生长必需的营养供应,使肿瘤无法在体内生长和散播转移,令化疗能够发挥有效的抗肿瘤作用,进而延缓肿瘤的生长和转移。Bevacizumab与放、化疗联合具有协同作用:①可使肿瘤区血管生成正常化,组织间高压减轻,利于化疗药物向肿瘤区输送。②放、化疗所致局部肿瘤区缺氧可促VEGF表达,帮助肿瘤细胞抵抗放、化疗的凋亡诱导机制;而联用Bevacizumab将预防此继发保护效应,使治疗反应增敏。临床观察到联合VEGF单抗和化疗所建立的生物化疗模式取得的有效率不仅远高于单用化疗或VEGF单抗,而且超过二者代数和,特别是对一部份复发/转移病人,已经无效的化疗药物在使用Bevacizumab等分子靶向药物后重新恢复敏感性。似乎Bevacizumab的抗肿瘤作用除了抑制肿瘤血管形成以外,还包括增加肿瘤细胞对化疗药物的敏感性。国内外目前均无对这种现象的合理解释。本课题研究的主要目的是探讨靶向药物与化疗药物单独及联合应用对肝癌的抑制作用,靶向药物与化疗的协同作用,为肝癌的综合治疗、提高疗效提供实验资料。第一章贝伐单抗对肝癌细胞增殖的抑制作用一、方法1、采用RT-PCR、免疫细胞化学的方法分析肝癌细胞株中VEGF、Flt-1、KDR基因和蛋白水平的表达:选用肝癌细胞株HepG2、SMMC-7721为实验细胞并选择人脐静脉内皮细胞系ECV-304作为VEGF受体表达的阳性对照,采用独立样本t检验从中筛选VEGF mRNA表达量高,Flt-1、KDR阳性表达且mRNA表达量高的肝癌细胞株,用于本课题的研究;2、MTT法检测Bevacizumab对肝癌细胞的增殖抑制作用:无血清条件下,0.1μg/ml、1μg/ml、10μg/ml、20μg/ml Bevacizumab作用于HepG2细胞48h,采用单因素方差分析（LSD法）比较不同浓度间及与对照组的增殖情况;3、Annexin V-FITC/PI双染检测细胞凋亡:无血清条件下,10μg/mlBevacizumab作用于HepG2细胞48h后,经Annexin V-FITC/PI双染,流式细胞仪检测凋亡情况。采用独立样本t检验分析比较Bevacizumab组和对照组;4、RT-PCR分析肝癌细胞株VEGF、Flt-1、KDR mRNA表达量的变化:无血清条件下,10μg/ml Bevacizumab作用于HepG2细胞48h后,行RT-PCR检测。采用独立样本t检验分析。二、结果1、RT-PCR结果示:肝癌细胞株HepG2中均有VEGF、Flt-1、KDR mRNA的表达,而SMMC-7721中只有VEGF、Flt-1 mRNA的表达;且HepG2中VEGF、Flt-1 mRNA的表达量均高于SMMC-7721（VEGF mRNA:P=0.001,t=9.056;Flt-1mRNA:P=0.000,F=55.975）;免疫细胞化学结果示:HepG2见VEGF、Flt-1及KDR的表达,SMMC-7721见VEGF、Flt-1的表达,无KDR的表达。因此,我们选用VEGF、Flt-1和KDR均表达的HepG2细胞作为实验用细胞;2、不同浓度Bevacizumab对肝癌细胞株HepG2的增殖有抑制作用。0.1μg/ml、1μg/ml、10μg/ml、20μg/ml组细胞增殖抑制率分别为8.8%、27%、32%、26%。其中1μg/ml、10μg/ml、20μg/ml组与对照组比较差异均有统计学意义（P值分别为0.001,0.000,0.001）,但此三组间差异无统计学意义（P＞0.05）;3、Bevacizumab可诱导肝癌细胞株HepG2的凋亡。Bevacizumab作用于HepG2肝癌细胞48h后,10μg/ml Bevacizumab组的细胞凋亡率为（17.04±0.14）%,明显高于对照组（2.85±0.08）%（t=198.533,P=0.000）;4、Bevacizumab作用于肝癌细胞株HepG2后,其VEGF、KDR mRNA表达量均减少,与空白对照组比较差异具有统计学意义（t值分别为:12.476和5.071,P值分别为:0.000和0.007）;而Flt-1 mRNA表达量与空白对照组比较无显著差异（t=2.449,P=0.07）。三、结论1、人肝癌细胞株中存在VEGF及其受体的共同表达;2、Bevacizumab作用于肝癌细胞株HepG2后,其增殖受抑;细胞凋亡率增高;3、Bevacizumab对HepG2的增殖抑制作用可能是通过KDR介导的,同时随着肿瘤细胞的增殖抑制,其VEGF表达减少;4、Bevacizumab可能通过阻滞VEGF的自分泌作用而发挥对肝癌细胞的增殖抑制作用。第二章贝伐单抗联合吉西他滨对肝癌细胞增殖的抑制作用一、方法1、MTT法检测Bevacizumab联合吉西他滨对肝癌细胞的增殖抑制作用:实验分组为:10μg/ml Bevacizumab组,0.01μg/ml、0.1μg/ml、1μg/ml、10μg/ml吉西他滨组和Bevacizumab+不同浓度吉西他滨联合组,对照组加入无血清RPMI-1640培养基。培养72h后行MTT检测。采用析因设计资料的方差分析;2、Western Blot检测Bevacizumab联合吉西他滨对HepG2中ERK1/2、Bcl-2、Bcl-xl表达的影响:分别将Bevacizumab、吉西他滨、Bevacizumab联合吉西他滨及安慰剂作用于HepG2细胞,提取细胞蛋白后,行Western Blot检测。二、结果1、不同浓度吉西他滨对肝癌细胞株HepG2增殖的影响差异有显著性意义（F=1318.002,P=0.000）,吉西他滨单用与Bevacizumab联用对HepG2增殖的影响差异有显著性意义（F=1663.503,P=0.000）。且两者之间交互效应显著（F=41.832,P=0.000）。0.01μg/ml、0.1μg/ml、1μg/ml、10μg/ml吉西他滨组的抑制率分别为2.72%、15.31%、36.42%、74.87%,联合10μg/ml Bevacizumab后其抑制率分别为31.2%、50.48%、63.43%、83.66%;2、实验四组均在分子量为28 KD（Bcl-2、Bcl-xl）、44 KD和42 KD（ERK1/2）处出现特异性反应条带,其中空白组的Bcl-2及Bcl-xl的表达明显强于其余三组的表达。而Bcl-xl的表达在联合组最弱。空白组和单药Bevacizumab组的ERK表达与联合用药组及单药吉西他滨组比较明显减弱。三、结论1、Bevacizumab联合吉西他滨可增强吉西他滨单药对肝癌细胞的增殖抑制作用;2、应用了化疗药物后,ERK基因的表达被明显抑制,进而促进细胞凋亡。无论化疗药物还是分子靶向药物,均可以下调Bcl-2及Bcl-xl的表达,而当两药联合时,Bcl-2及Bcl-xl的表达下降更为明显。提示两者协同显著增强对靶向药物及化疗敏感性的机制可能与促进细胞凋亡及调控Bcl-2及Bcl-xl的表达有关。第三章贝伐单抗联合吉西他滨对肝癌裸鼠皮下移植瘤治疗作用的研究一、方法1、Bevacizumab联合吉西他滨对肝癌移植瘤体积和MVD的影响:建立人肝癌细胞HepG2裸鼠移植瘤模型,每隔3天观察一次,用游标卡尺测量肿瘤的大小。实验分A组:空白对照组,予以无菌生理盐水,0.2ml/只;B组:Bevacizumab组,5mg/kg:C组:吉西他滨组,100mg/kg:D组:Bevacizumab 5mg/kg+吉西他滨100mg/kg联合组。各组给药均为2次/周,共4周,腹腔注射。自接种后第19天起,每周检测肿瘤体积。连续给药4周后停药一周,处死裸鼠,取移植瘤组织进行CD34染色检测各组微血管密度（MVD）。对照组与各治疗组间移植瘤体积比较采用重复测量数据的方差分析;对照组与各治疗组间MVD的比较采用单因素方差分析;2、Bevacizumab联合吉西他滨对荷瘤裸鼠皮下移植瘤VEGF、Flt-1、KDR、ERK、Bcl-2、Bcl-xl的影响:分别将Bevacizumab、吉西他滨、Bevacizumab联合吉西他滨及安慰剂作用于荷人肝癌HepG2裸鼠皮下移植瘤,各组留取新鲜活组织标本10%中性多聚甲醛固定,常规脱水,石蜡包埋,4μm厚连续切片,进行苏木素-伊红（HE）染色后制备成免疫组化切片,采用标准EnVision^TM法进行免疫组化流程染色;3、Western Blot检测Bevacizumab联合吉西他滨对荷人肝癌HepG2裸鼠皮下移植瘤中ERK1/2、Bcl-2、Bcl-xl表达的影响:分别将Bevacizumab、吉西他滨、Bevacizumab联合吉西他滨及安慰剂作用于荷人肝癌HepG2裸鼠皮下移植瘤,提取组织蛋白后,行Western Blot检测。二、结果1、各治疗组给药后体积与对照组相比增长均较缓慢,其中Bevacizumab联合吉西他滨组最为明显。Bevacizumab联合吉西他滨组与Bevacizumab、吉西他滨组之间均有显著差异（P值分别为0.004,0.010）,其抑瘤率分别为40.35%、45.61%、84.21%;2、与对照组比较,单药吉西他滨组MVD无显著差异（P=0.267）;单药Bevacizumab组及联合吉西他滨组的MVD均减少,与对照组比较差异均有统计学意义（P值均为0.000）;Bevacizumab联合吉西他滨组的MVD减少较Bevacizumab组更为显著,差异有统计学意义（P=0.000）;3、Flt-1在四组中均有较高的表达,其中在空白对照组和联合用药组中的表达均为强阳性;单药吉西他滨组和单药Bevacizumab组中Flt-1的表达均为中度阳性;各组间的差异并不明显。KDR在空白对照组中的表达为强阳性;单药吉西他滨组中KDR的表达为弱阳性;联合用药组和单药Bevacizumab组中KDR为阴性;4、实验四组均在分子量为28 KD（Bcl-2、Bcl-xl）、44 KD和42 KD（ERK1/2）处出现特异性反应条带,其中空白组的Bcl-2及Bcl-xl的表达明显强于其余三组的表达。而Bcl-2及Bcl-xl的表达均在联合组最弱。空白组和单药Bevacizumab组的ERK表达与联合用药组及单药吉西他滨组比较明显减弱。三、结论1、Bevacizumab与吉西他滨均可延缓肿瘤的生长,且Bevacizumab联合吉西他滨可显著增加二者单药的疗效;2、Bevacizumab可抑制移植瘤新生血管的形成,联合吉西他滨治疗后的移植瘤组织中新生血管形成减少更为显著;3、Bevacizumab极有可能直接封闭VEGF,阻断VEGF经由VEGFR-2进行的信号转导,阻止肿瘤细胞中新生血管的生成;4、应用化疗药物后,ERK基因的表达被明显抑制,进而促进细胞凋亡。无论化疗药物还是分子靶向药物,均可以下调Bcl-2及Bcl-xl的表达,而当两药联合时,Bcl-2及Bcl-xl的表达下降更为明显。提示两者协同显著增强对靶向药物及化疗敏感性的机制可能与促进细胞凋亡及调控Bcl-2及Bcl-xl的表达有关。更多还原

【Abstract】 Background and Objective:Primary hepatic carcinoma（PHC）is one of the common malignant tumors in the world,of which the mortality rate ranked second in our country.Growth and metastasis of most malignant solid tumors including PHC depend on angiogenesis which could provide the proliferation of tumor cell with nutrition and metabolism pathway,promote the growth and metastasis of tumor and aggravate the patients’ condition.Consequently tumor angiogenesis now has became a popular area for studying.Bevacizumab（Avastin,rhuMAb-VEGF）is a synthetic recombinant humanized monoclonal IgG1 antibody aiming directly at VEGF.Bevacizumab specifically binds with VEGF,results in interfering with the combination of VEGF and Flt-1,KDR which are the endotheliocyte surface receptors,blocking the downstream signal pathway mediated by VEGF,preventing VEGF from promoting the proliferation of vascular endothelial cell and angiogenesis in tumor,thus block blood,oxygen and other nutrition supply necessary for the tumor growth,all of which make the tumor unable to grow,perfuse and metastasize,the chemotherapy function effectively to retard the growth and metastasis of the tumor.Bevacizumab combined with radiotherapy and chemotherapy has synergistic effects:①enable the normalization of angiogenesis in tumor regions,relive high pressure between tissues,help the transport of chemotherapeutics toward tumor regions.②regional tumor hypoxia led by radiotherapy and chemotherapy could promote the expression of VEGF,which help tumor cell to resist apoptosis-induced mechanism result from radiotherapy and chemotherapy;But the combination of radiotherapy and chemotherapy with bevacizumab can obviate this secondary protective effect and sensitize the therapeutic reaction.Clinical observation shows that the effective power of biologic chemotherapy mode constructed by the combination of VEGF monoclonal antibody and chemotherapy is far stronger than either single use of chemotherapy or VEGF monoclonal antibody,even surpass the sum of both,expecially for some patients with recurrent or metastatic tumors.These patients can regain saitivity for chemotherapeutics which are already invalid after applying molecule targeted drugs like bevacizumab.Besides inhibition of tumor angiogenesis,the anti-tumor effect of bevacizumab seems include increasing the sensitivities of tumor cells for chemotherapeutics.There’s no rational explanation for this phenomenon at home and abroad which make it necessary for us to do researches to perfect the theoryetical basis of biologic chemotherapy mode and then give an impulse to the combined therapy of tumor.The main purpose of this research is to investigate the depressant effects on PHC which were caused by using targeted drugs or chemotherapeutics alone and the combination of two drugs,the synergistic action of targeted drugs plus chemotherapy, thus found a theoretic basis for the combined therapy of PHC and improving its effectiveness.Chapter 1 Inhibition of bevacizumab on the proliferation of PHC cellsMethods:1.Analyzed the expression of gene and protein of VEGF,Flt-1 and KDR in PHC cell lines using RT-PCR and immuocytochemistry:choose two PHC cell lines HepG2 and SMMC-7721 as experiment cells,while human allantoic vein endothelial cell line ECV-304 as positive controls of VEGF receptor expression,sieved out PHC cell lines of high VEGFmRNA expressing,positive Flt-1 and KDR expressing as well as high mRNA expressing with independent sample t test for this research.2.Evaluated by MTT assay the inhibition of PHC cell resulted from bevacizumab: with no blood serum,0.1μg/ml、1μg/ml、10μg/ml、20μg/ml bevacizumab separately acted on HepG2 cells for 48 hours,then compared the proliferation of control group with other groups of different concentration using one-way ANOVA（LSD test）.3.Detected the apoptosis by Annexin V-FITC/PI staining:with no blood serum, 10μg/ml bevacizumab acted on HepG2 cells for 48 hours,then staining by Annexin V-FITC/PI,detected the apoptosis with flow cytometry.Then,differences between group of bevacizumab and control group were tested with Independent T Test.4.Analyzed expression changes of VEGF,Flt-1 and KDR mRNA in PHC cell lines by RT-PCR:with no blood serum,10μg/ml bevacizumab acted on HepG2 cells for 48 hours,then tested by RT-PCR.Analyzed the results by independent sample t test.Results:1.Result of RT-PCR:all PHC cell lines HepG2 expressed VEGF,Flt-1 and KDR mRNA,while SMMC-7721 only expressed VEGF and Flt-1 mRNA.The expression of VEGF and Flt-1 mRNA in HepG2 are higher than that in SMMC-7721（VEGF mRNA:P=0.001,t=9.056;Flt-1mRNA:P=0.000,F=55.975）;Results of immunocytochemistry:VEGF and Flt-1 expressed in both cell lines,but KDR expressed only in HepG2.Therefore,we adopted HepG2 cells for experiments because of their high VEGF expression as well as Flt-1 and KDR expression.2.Bevacizumab of different concentration could inhibit the proliferation of PHC cell lines HepG2,cytostasis rate of groups with 0.1μg/ml、1μg/ml、10μg/ml、20μg/ml bevacizumab were 8.8%、27%、32%、26%,respectively.Compared with control group, cytostasis rate in groups with 1μg/ml、10μg/ml、20μg/ml bevacizumab showed statistical significance.（P valued 0.001,0.000,0.001）. 3.Bevacizumab may induce the apoptosis of HCC cell HepG2.The apoptosis rates of HepG2 cells in bevacizumab groups were（17.04±0.14）%,higher than that of control group（2.85±0.08）%（t=198.533,P=0.000）.4.Expression of VEGF and KDR mRNA in PHC cell lines HepG2 declined after effecting by bevacizumab.Compared with that of blank control group,the differences showed no statistical significance（t valued 12.476 vs 5.071,P valued 0.000 vs 0.007）. Expression of Flt-1 mRNA was not significant different with that of blank control group（t=2.449,P=0.07）.Conclusions:1.Expression of VEGF and its receptors both existed in all human PHC cell lines.2.Effecting by bevacizumab,the proliferation of PHC cell lines HepG2 was inhibited with the apoptosis rate increased.3.Depressant effects on the proliferation of HepG2 by bevacizumab could be mediated by KDR.Expression of VEGF decreased with the inhibition of proliferation in tumor cells.4.Bevacizumab may inhibit the proliferation of PHC cells by blocking the autocrine of VEGF.Chapter 2 Effects on PHC cell lines HepG2 by bevacizumab combined with gemcitabineMethods:1.Evaluated the inhibition of proliferation in PHC cells with bevacizumab plus gemcitabine by MTT assay:Experiment groups were dealt with 10μg/ml bevacizumab or 0.01μg/ml,0.1μg/ml,1μg/ml,10μg/ml gemcitabine or bevacizumab combined with gemcitabine of different concentration,while control group with RPMI-1640 medium of no blood serum.Detected by MTT assay after 72 hours cultivation and analyzed with factorial analysis. 2.Evaluated by Western Blot the effects on expression of ERK1/2,Bcl-2 and Bcl-xl in HepG2 cells after using bevacizumab plus gemcitabine:using bevacizumab, gemcitabine,combination of both and placebo separately in HepG2 cells,then extracted cell proteins and evaluated by Western Blot.Results:1.Effects on the proliferation of PHC cell lines HepG2 by gemcitabine of different concentration showed significant differences（F=1318.002,P=0.000）.Effects on proliferation of HepG2 with gemcitabine alone and that plus bevacizumab showed significant differences（F=1663.503,P=0.000）.The reciprocation between gemcitabine and bevacizumab is significant（F=41.832,P=0.000）.Inhibition ratios of groups with 0.01μg/ml、0.1μg/ml、1μg/ml、10μg/ml gemcitabine were respectively 2.72%、15.31%、36.42%、74.87%,while combining with 10μg/ml bevacizumab,the inhibition ratios were respectively 31.2%、50.48%、63.43%、83.66%.2.Specific reaction strap appeared at 28KD Mol wt（Bcl-2,Bcl-xl）,44KD and 42KD（ERK1/2） in all four groups.Expression of Bcl-2 and Bcl-xl in blank control group was significantly stronger than that of the other three groups.Bcl-xl expressed the weakest in the group with combination of bevacizumab and gemcitabine.ERK expressed significantly weaker in blank group and bevacizumab group than combination group and gemcitabine group.Conclusions:1.The combination with bevacizumab could amplify depressant effect of gemcitabine on PHC cells.2.Expression of ERK gene was evidently depressed with chemotherapeutics, which led to the promotion of apoptosis.Either chemotherapeutics or molecule targeted drugs can down regulate the expression of Bcl-2 and Bcl-xl.Furthermore, this action was far clearer with the combination of two drugs.These results suggested that the synergic mechanism of increasing patients’ sensitivities for targeted drugs and chemotherapeutics by combining two drugs may be related with the promotion of apoptosis as well as regulation of Bcl-2 and Bcl-xl expression. Chapter 3 Treatment effect of bevacizumab combined with gemcitabine on subcutaneous transplantation tumor of nude mice bearing PHC cells HepG2Methods:1.Effects on the volumes and MVD of PHC transplantation tumors Treated with bevacizumab plus gemcitabine:constructed nude mice model of transplantation tumor with human PHC cells HepG2,obsered every three days and measured the sizes of tumors with vernier caliper.Groups were assigned as follows:group A,as blank control group,gave stroke-physiological saline solution,0.2ml each.Group B, as bevacizumab group,gave bevacizumab 5mg/kg.Group C,as gemcitabine group, gave gemcitabine 100mg/kg.Group D,combination group,gave bevacizumab 5mg/kg combined with gemcitabine 100mg/kg.All groups were intraperitoneal injected twice a week for four weeks.Measured the volumes of tumors every week since the 19th day after inoculation.Withdrawalled drugs for one week after four weeks’ successive administration.Then put the nude mice to death,got transplantation tumor tissues and measured the microvessel density（MVD） of each groups by CD34 dyeing.Compared the volumes of transplantation tumors in control group and other groups with repeated measure ANOVA,the MVD with one-way ANOVA.2.Effects on VEGE Flt-1,KDR,Bcl-2 and Bcl-xl of subcutaneous transplatation tumor in nude mice beating cancer with bevacizumab plus gemcitabine:use bevacizumab,gemcitabine,combination of both and normal saline in the subcutaneous transplantation tumors in nude mice bearing human PHC cells HepG2, separately took fresh living tissues in all groups,then fixed with 10%neutral paraformaldehyde,dehydrated conventionally,paraffin imbedded,serial sectioned in 4μm and dyeing by HE,finally made into immunohistochemical slices and dyeing immunohistochemically by standard En Vision^TM method.3.Evaluated effects on expression of ERK1/2,Bcl-2 and Bcl-xl in subcutaneous transplatation tumor in nude mice bearing human PHC cells HepG2 with bevacizumab plus gemcitabine by Western Blot:separately gave bevacizumab, gemcitabine,combination of both and normal saline to the subcutaneous transplantation tumors in nude mice bearing human PHC cells HepG2,then evaluated by Western Blot after extracting proteins in tissues.Results:1.Volumes in all experiment groups growed slower after administration compared with that in the control group.The effect was most significant in group with bevacizumab plus gemcitabine.There were significant differences among groups with bevacizumab,gemcitabine and combination of both（P valued 0.004 vs 0.010）.The inhibition ratio of experiment groups were 40.35%、45.61%、84.21% respectively.2.MVD of group with gemcitabine alone showed no significant difference compared with control group（P=0.267）,while that of groups with bevacizumab and combination of both decreased and showed statistical significance compared with control group（P all valued 0.000）.Decrease of MVD in group with gemcitabine plus bevacizumab was more significant than that in bevacizumab group and the difference between two groups showed statistical significance（P=0.000）.3.Flt-1 expressed relatively high in all four groups.Expression of Flt-1 in blank control and combination group showed strongly positive,while that in gemcitabine and bevacizumab group showed midrange positive.The differences among groups were not significant.Expression of KDR in blank control group showed strongly positive,while that in gemcitabine group showed weakly positive,bevacizumab and combination group negative.4.Specific reaction strap appeared at 28KD Mol wt（Bcl-2,Bcl-xl）,44KD and 42KD（ERK1/2） in all four groups.Expression of Bcl-2 and Bcl-xl in blank control group was significantly stronger than that of the other three groups.Bcl-xl expressed the weakest in the group with combination of bevacizumab and gemcitabine.ERK expressed significantly weaker in blank group and bevacizumab group than combination group and gemcitabine group.Conclusions: 1.Both bevacizumab and gemcitabine could depress the growth of tumor,the combination of two drugs can amply the effects.2.Bevacizumab inhibited the formation of new vessels in transplantation tumors. The effects became more evident with the combination of gemcitabine.3.There was a strong likelihood that bevacizumab block the signal transduction of VEGF which was conducted by VEGFR-2 and prevent the formation of new vessels in tumors.4.Expression of ERK gene had been obviously depressed after using chemotherapeutics,thus promoted the apoptosis.Both chemotherapeutics and molecule targeted drugs can down regulated the expression of Bcl-2 and Bcl-xl.This down regulation became more evident with the combination of two drugs,which indicated that the synergic mechanism of increasing patients’ sensitivities for targeted drugs and chemotherapeutics with the combination of two drugs may be related with the promotion of apoptosis as well as regulation of Bcl-2 and Bcl-xl expression.更多还原