【作者】 杨闯；

【导师】 傅仲学；

【作者基本信息】 重庆医科大学 ， 外科学， 2011， 博士

【摘要】 第一部分抗VEGFR2-奥沙利铂免疫脂质体的制备目的探讨生物素―链霉亲和素桥接作用制备抗VEGFR2-奥沙利铂免疫脂质体。方法采用逆向旋转蒸发法制备生物素化的聚乙二醇脂质体(PEG-脂质体),通过生物素―链霉亲和素桥接作用将生物素化VEGFR2单克隆抗体与生物素化的PEG-脂质体连接,构建抗VEGFR2-免疫脂质体。采用激光粒度仪、扫描电镜分析脂质体的粒径和电位大小;激光共聚焦显微镜和流式细胞仪检测抗VEGFR2-免疫脂质体的特征及抗体活性;高效液相色谱仪分析抗VEGFR2-奥沙利铂免疫脂质体的包封率及体外药物释放率。结果免疫脂质体的平均粒径为185.34±28.08 nm,电位为-19.98±2.96 mv。脂质体呈球型,分散均匀,无聚集粘附成团现象;激光共聚焦显示罗丹明标记的二抗可在抗VEGFR2-免疫脂质体表面有效结合,流式细胞仪检测结合率达75%以上;高效液相色谱仪分析免疫脂质体的包封率为(58.07±6.94)%,体外累积释放率在120 h可达89%以上。结论通过链霉亲和素与生物素桥接作用将生物素化VEGFR2构建在生物化PEG-脂质体表面是一种可行的方法,制备的抗VEGFR2-奥沙利铂免疫脂质体具有一定的包封率和体外释放率。第二部分抗VEGFR2-奥沙利铂免疫脂质体对人结直肠癌THC8307/L-OHP细胞的靶向效应目的探讨抗VEGFR2-奥沙利铂免疫脂质体对人结直肠癌THC8307/L-OHP细胞的凋亡诱导作用及可能机制。方法采用免疫荧光、Western blot和RT-PCR分析VEGFR2在THC8307/L-OHP细胞的表达;流式细胞仪、免疫荧光、扫描电镜分析抗VEGFR2-免疫脂质体对THC8307/L-OHP细胞的靶向结合。MTT筛选给药浓度,分析抗VEGFR2-奥沙利铂免疫脂质体对THC8307/L-OHP细胞的毒性。THC8307/L-OHP细胞经抗VEGFR2-奥沙利铂免疫脂质体处理,流式细胞仪及TUNEL分析细胞的凋亡与增殖;RT-PCR分析Bcl-2、Bax、Caspase-3、Ki-67 mRNA的表达;Western blot检测Bcl-2、Bax、活化Caspase-3/P17蛋白的表达;免疫组化分析Ki-67阳性细胞数;检测P38、ERK、JNK的mRNA和蛋白的表达变化。THC8307/L-OHP细胞经SP600125预处理后,分析抗VEGFR2-奥沙利铂免疫脂质体对细胞凋亡调节。结果与HUVEC比较,VEGFR2在THC8307/L-OHP细胞有高表达。抗VEGFR2-免疫脂质体与THC8307/L-OHP细胞的结合能力高于非免疫脂质体(P<0.05);游离奥沙利铂(50μg/ml),奥沙利铂PEG-脂质体(含奥沙利铂50μg/ml)和抗VEGFR2-奥沙利铂免疫脂质体(含奥沙利铂50μg/ml)分别处理THC8307/L-OHP细胞12 h,细胞凋亡率分别为(10.02±0.69)%,(15.29±1.70)%和(55.89±8.90)%,正常组为(6.69±0.35)%。各组间比较,差异有统计学意义(P<0.01,P<0.05)。TUNEL检测THC8307/L-OHP细胞凋亡量的变化趋势与流式结果类似;CFSE平均荧光强度的相对比值分别为1.83±0.21,1.56±0.16和1.29±0.10。各组间比较,P<0.05。Bcl-2、Ki-67的mRNA和蛋白的表达显著下调,Bax、Caspase-3的mRNA和蛋白的表达上则调(P< 0.01,P<0.05);在MAPK信号通路中,P38的mRNA和蛋白表达各组间比较无显著变化(P>0.05),ERK表达随细胞凋亡的增加下调,JNK表达上调(P<0.05)。SP600125预处理THC8307/L-OHP细胞后,抗VEGFR2-奥沙利铂免疫脂质体对细胞的凋亡作用不被SP600125抑制剂所抑制,Bcl-2的mRNA和蛋白的表达显著下调(P<0.01),Bax、Caspase-3则上调(P<0.01)。结论抗VEGFR2-奥沙利铂免疫脂质体具有增强与THC8307/L-OHP细胞相结合的能力,并对细胞有显著凋亡诱导作用,可能与ERK、JNK信号通路参与凋亡调节有关。第三部分抗VEGFR2-奥沙利铂免疫脂质体对裸鼠异种移植瘤的靶向作用目的探讨抗VEGFR2-奥沙利铂免疫脂质体在裸鼠异种移植瘤体内的靶向效应。方法建立裸鼠异种移植瘤模型,经尾静脉注射荧光标记(Dio标记)的抗VEGFR2-免疫脂质体,运用活体荧光显像和免疫荧光技术检测免疫脂质体在瘤体组织内的聚集能力;分析游离奥沙利铂(5μg/g),奥沙利铂PEG-脂质体(含奥沙利铂5μg/g),抗VEGFR2-奥沙利铂免疫脂质体(含奥沙利铂5μg/g)对荷瘤鼠的瘤体抑制率和生存期的影响;高效液相色谱法检测瘤体组织内药物的含量;免疫组化和Western blot分析瘤体细胞凋亡与增殖的变化。结果活体荧光显像观察到Dio标记的抗VEGFR2-免疫脂质体在瘤体的聚集能力高于非免疫脂质体,免疫双标显示其在瘤体细胞内和血管周围大量分布;分别用游离奥沙利铂,PEG-奥沙利铂及抗VEGFR2-奥沙利铂免疫脂质体处理裸鼠,瘤体的抑制率为(39.55±9.38)%,(50.34±8.86)%和(61.20±9.77)%,组间比较差异有统计学意义(P<0.05)。抗VEGFR2-奥沙利铂免疫脂质体处理组裸鼠的生存率高于其它各组;给药2 h后,游离奥沙利铂处理组瘤体组织内药物的含量明显降低。给药24 h后,奥沙利铂PEG-脂质体处理组和抗VEGFR2-奥沙利铂免疫脂质体处理组在瘤体组织内药物的含量均达高值,但抗VEGFR2-奥沙利铂免疫脂质体组的含量高于奥沙利铂PEG-脂质体组(P<0.05),并且在72 h后仍可维持较高值;瘤体细胞的凋亡指数在抗VEGFR2-奥沙利铂免疫脂质体处理组显著高于其它各组(P<0.001,P<0.01,P<0.05),而增殖指数显著低于其它各组(P<0.001,P<0.01,P<0.05);Bcl-2蛋白表达下调,Bax、P17蛋白表达上调(P<0.01,P<0.05)。结论抗VEGFR2-奥沙利铂免疫脂质体能有效抑制荷瘤鼠瘤体的生长,并影响瘤体细胞的凋亡与增殖。第四部分抗VEGFR2-奥沙利铂免疫脂质体对人结直肠癌THC8307/L-OHP细胞多药耐药的影响目的探讨抗VEGFR2-奥沙利铂免疫脂质体对人结直肠癌THC8307/L-OHP细胞的耐药逆转作用。方法MTT方法分析抗VEGFR2-奥沙利铂免疫脂质体对THC8307/L-OHP细胞的耐药逆转倍数。免疫组化、RT-PCR、Western blot分析游离奥沙利铂和不同剂量的抗VEGFR2-奥沙利铂免疫脂质体处理细胞12 h,P-gp/MDR1、ABCG2的表达变化。分析相同剂量的抗VEGFR2-奥沙利铂免疫脂质体处理细胞不同时间,P-gp/MDR1、ABCG2的表达变化。采用Western blot分析游离奥沙利铂和不同剂量的抗VEGFR2-奥沙利铂免疫脂质体作用细胞48 h,ABCG2、P-gp/MDR1、GST3、Bcl-2和P17蛋白的表达变化。采用免疫组化技术分析瘤体组织ABCG2和P-gp/MDR1的阳性表达。结果抗VEGFR2-奥沙利铂免疫脂质体对THC8307/L-OHP细胞的耐药逆转倍数为1.21。25μg/ml的游离奥沙利铂,抗VEGFR2-奥沙利铂免疫脂质体组Ⅰ(含25μg/ml奥沙利铂)和抗VEGFR2-奥沙利铂免疫脂质体组Ⅱ(含50μg/ml奥沙利铂)作用细胞12 h,P-gp/MDR1的mRNA表达分别为2.22±0.33,2.67±0.40,3.26±0.4(7组间比较,P<0.05);ABCG2的mRNA表达分别为2.85±0.43,7.74±1.16,8.68±1.30(组间比较,P<0.05与P<0.01);P-gp/MDR1的蛋白表达分别为5.68±0.85,10.04±1.51,17.62±2.64。组间比较差异有显著性(P<0.01);ABCG2蛋白表达分别为2.06±0.31,3.74±0.56,4.56±0.68。组间比较差异有显著性,P<0.05和P<0.01;抗VEGFR2-奥沙利铂免疫脂质体(含25μg/ml奥沙利铂)处理细胞12 h,24 h,48 h和72 h,P-gp/MDR1蛋白表达分别为8.57±1.28,16.36±2.45,3.42±0.51,0.75±0.11。各组间比较,差异有统计学意义(P<0.01);ABCG2蛋白含量分别为1.27±0.19,2.13±0.32,1.63±0.24,0.55±0.08。各组间比较,差异有统计学意义(P<0.05);P-gp/MDR1、ABCG2耐药基因的mRNA表达在24 h升高明显,72 h显著降低。与正常对照组比较,差异有统计学意义(P<0.01,P<0.05)。游离奥沙利铂和抗VEGFR2-奥沙利铂免疫脂质体(组Ⅰ、Ⅱ)作用细胞48 h,游离奥沙利铂处理组的P-gp/MDR1、ABCG2、GST3、Bcl-2、P17蛋白的表达无显著变化(P>0.05,与正常对照组比较);抗VEGFR2-奥沙利铂免疫脂质体处理组(组Ⅰ,组Ⅱ)P-gp/MDR1、ABCG2、GST3、Bcl-2蛋白的表达呈下调趋势,P17则上调。各组间比较,差异有统计学意义(P<0.01,P<0.05);瘤体组织中存在P-gp/MDR1和ABCG2阳性细胞,但随处理因素增加其表达减弱。结论抗VEGFR2-奥沙利铂免疫脂质体逆转THC8307/L-OHP细胞的多药耐药可能与P-gp/MDR1、ABCG2、GST3的下调有关。抗凋亡蛋白Bcl-2,Caspase-3可能参与抗VEGFR2-奥沙利铂免疫脂质体对THC8307/L-OHP细胞耐药性的调节。更多还原

【Abstract】 PART ONE PREPARATION OF ANTI-VEGFR2 IMMUNOLIPOSOMAL OXALIPLATINObjective We aimed to investigate a novel method for the preparation of anti-VEGFR2 immunoliposomal oxaliplatin to elucidate the bridging role of Streptavidin-biotin.Methods Biotinylated PEG-liposomes were prepared using reverse rotary evaporation. The biotinylated PEG-liposomes were linked to streptavidin molecules that act as a bridge between the biotinylated VEGFR2 antibody and the biotinylated PEG-liposomes. The size and zeta-potential of the liposomes were determined by a Laser particle size analyzer and by scanning electron microscopy; Laser confocal microscopy and flow cytometry detection were used to determine the characteristics and antibody activity of anti-VEGFR2 immunoliposomes; and the encapsulation efficiency and in-vitro drug release rate of anti-VEGFR2 immunoliposomal oxaliplatin were detected by HPLC.Results We obtained immunoliposomes with a particle size of 185.34±8.08 nm and zeta-potential of 19.98±2.96 mv. The evenly shaped liposomes had smooth surfacesand were evenly dispersed without aggregation or adhesion into groups. Confocal microscopy revealed a strong interaction between the rhodamine-labeled secondary antibodies and the anti-VEGFR2 primary antibodies. Flow cytometry analysis revealed binding of the secondary antibody to over 75% of the liposomes. The entrapment efficiency of the liposomes was (58.07±6.94)%, and after 120 h we observed an increase in cumulative percentage release of over 89%, as determined by HPLC.Conclusion The bridging role of Streptavidin-biotin is a feasible method for biotinylated VEGFR2 antibody binding to biotinylated PEG-liposomes. The preparation of anti-VEGFR2 immunoliposomal oxaliplatin is important as it determines the entrapment efficiency and in-vitro release rate of the therapy.PART TWO ANTI-VEGFR2 IMMUNOLIPOSOMAL OXALIPLATIN TARGETING IN HUMAN COLORECTAL CARCINOMA THC8307/L-OHP CELLS Objective We aimed to investigate anti-VEGFR2 immunoliposomal oxaliplatin induction of THC8307/L-OHP cell apoptosis and to elucidate the underlying mechanism.Methods We used immunofluorescence, Western blot, and RT-PCR to analyse the effect of VEGFR2 expression on THC8307/L-OHP cells, and the binding of anti-VEGFR2 immunoliposomes to cells was analyzed by flow cytometry, immunofluorescence, and scanning electron microscopy. MTT analysis was used to determine the administration drug dose and the cytotoxicity of anti-VEGFR2 immunoliposomal oxaliplatin. Apoptosis or proliferation was detected by flow cytometry and TUNEL analyses; RT-PCR analysis was performed to measure the mRNA expression of Bcl-2, Bax, Caspase-3, Ki-67, P38, ERK, and JNK. We performed Western blot analysis to determine protein expression of Bcl-2, Bax and activation of Caspase-3/P17, P38, ERK, and JNK. Whereas Ki-67 positive cells were visualized using immunohisto- chemisty. SP600125 was used as the pretreatment for THC8307/L-OHP cells in order to detect the induction of apoptosis by anti-VEGFR2 immunoliposomal oxalipolatin.Results We found that VEGFR2 was highly expressed in THC8307/L-OHP cells, compared with HUVE cells. The anti-VEGFR2 immunoliposomes were proactively combined with cells than non-immunoliposomes (P<0.05). THC8307/L-OHP cells were treated with free oxaliplatin (50μg/ml), PEG-liposomal oxaliplatin (containing oxaliplatin 50μg/ml), or anti-VEGFR2 immunoliposomal oxaliplatin (containing oxaliplatin 50μg/ml) for 12 h and the apoptosis rate was (10.02±0.69)%, (15.29±1.70)% and (55.89±8.90)%, respectively. There was statistical significance, compared with each group (P <0.01, P <0.05). The results of apoptosis analyzed by TUNEL were similar to the results obtained through flow cytometry. The mean CFSE relative fluorescence intensity ratio was 1.83±0.21, 1.56±0.16, and 1.29±0.10, respectively. It was statistical significance (P <0.05). The mRNA and protein expression of Bcl-2 and Ki-67 were significantly down-regulated, however, Bax and Caspase-3/P17 were significantly up-regulated (P <0.01, P<0.05). In the MAPK signaling pathway, the mRNA and protein expression of P38 were not changed significantly (P >0.05) and expression of ERK was down-regulated, while apoptosis was enhanced and JNK was up-regulated (P <0.05). After SP600125 pre-treatment, THC8307/L-OHP cells, apoptosis induced by anti-VEGFR2 immunoliposmal oxaliplatin was not inhibited by SP600125. Additionally, the mRNA and protein expression of Bcl-2 were down-regulated significantly (P <0.01), whereas Bax and Caspase-3/P17 were up-regulated (P <0.01).Conclusion The anti-VEGFR2 immunoliposomes were fortified combined with THC8307/L-OHP cells, and anti-VEGFR2 immunoliposomal oxaliplatin significantly induced apoptosis. These findings indicated that the ERK and JNK signaling pathways may be involved in apoptotic regulation.PART THREE ANTI-VEGFR2 IMMUNOLIPOSOMAL OXALIPLATIN TARGETING IN A NUDE MOUSE TUMOR-XENOGRAFT MODEL OF COLORECTAL CARCINOMAObjective We investigated the antitumor efficiency of anti-VEGFR2 immunoliposomal oxaliplatin potentialization in a nude mouse tumor-xenograft model of colorectal carcinoma.Methods In a tumor-bearing nude mouse model, we observed intravenous tail vein injections of (Dio)-labeled anti-VEGFR2 immunolipsomes using fluorescence imaging and in-vivo imaging systems. Mice were treated with free oxaliplatin (5μg/g), PEG-liposomal oxaliplatin (containing oxaliplatin 5μg/g), and anti-VEGFR2 immunoliposomal oxaliplatin (containing oxaliplatin 5μg/g) via the tail vein intravenous injections, followed by analysis of the accumulation of oxaliplatin in tumor tissues by HPLC, observation of the tumor volume, and the survival rate. Cell apoptosis and the proliferation of tumors was detected by the TUNEL assay, immunohistochemistry, and Western blot. Results The Dio-labeling anti-VEGFR2 immunoliposomal aggregation was greater than that of the non-immunoliposomal aggregation in the tumor tissues as visualized by the in-vivo imaging system: the majority of anti-VEGFR2 liposomes were found in the tumor cells and the surrounding capillary vessels in the tumor. The tumor volume suppression rate was determined to be (39.55±9.38)%, (50.34±8.86)%, and (61.20±9.77)%, respectively. There was statistical significance, compared with each group (P <0.01, P <0.05). Longer survival was observed in the nude mice that were treated with anti-VEGFR2 immunoliposomal oxaliplatin. After intravenous injection of free oxaliplatin, tumor tissue accumulation of oxaliplatin quickly decreased at 2 h. Upon administration of PEG-liposomal oxaliplatin and anti-VEGFR2 immunoliposomal oxaliplatin, the maximum value was detected at 24 h. Moreover, treatment with anti-VEGFR2 immunoliposomal oxaliplatin led to greater values than that treatment with PEG-liposomal oxaliplatin, with values maintaining a high level 72 h. The apoptotic index in tumor tissue was higher compared to the other groups (P <0.001, P <0.01 vs. P <0.05, respectively), but the proliferation index predominance was reduced (P <0.001, P <0.01vs. P <0.05, respectively). Additionally, protein expression of Bcl-2 was down-regulated, whereas Bax and P17 expression was up-regulated (P <0.01, P <0.05).Conclusion In this study, we demonstrated that anti-VEGFR2 immunoliposomes exhibit a strong aggregation ability. Moreover, treatment with anti-VEGFR2 immunoliposomal oxaliplatin is able to suppress growth of tumor tissue, contribute to tumor cellular apoptosis, and suppress tumor cell proliferation in a nude mouse tumor-xenograft model of colorectal carcinoma.PART FOUR ANTI-VEGFR2 IMMUNOLIPOSOMAL OXALIPLATIN INFLUENCES MULTIDRUG RESISTANCE OF HUMAN COLORECTAL CARCINOMA THC8307/L-OHP CELLSObjective We next analysed the effect of anti-VEGFR2 immunoliposomal oxaliplatin on the multidrug resistance of human colcrectal carcinoma THC8307/L-OHP cells.Methods A significant level of drug resistance reversal was observed in THC8307/L-OHP cells treated with anti-VEGFR2 immunoliposomal oxaliplatin as analyzed by MTT. Immunohistochemistry, RT-PCR, Western blot were performed to determine the expression of P-gp/MDR1, and ABCG2 when THC8307/L-OHP cells were treated with free oxaliplatin, various doses of Anti-VEGFR2 immunoliposomal oxaliplatin at 12 h, or one dose of Anti-VEGFR2 immunoliposomal oxaliplatin at distinct time points. The protein expression of ABCG2, P-gp/MDR1, GST3, Bcl-2, and Caspase-3/P17 were obtained by Western blot after THC8307/L-OHP cells were treated with either free oxaliplatin or various doses of anti-VEGFR2 immunoliposomal oxaliplatin for 48 h. P-gp/MDR1 and ABCG2 positive cells were analyzed by immunohistochemistry.Results A 1.21-fold reversal of drug resistance was observed. After THC8307/L-OHP cells were treated with free oxaliplatin (25μg/ml) and anti-VEGFR2 immunoliposomal oxaliplatin (containing oxaliplatin 25μg/ml vs. 50μg/ml) at 12 h, the observed mRNA expression of P-gp/MDR1 and ABCG2 was 2.22±0.33 and 2.67±0.40 vs. 3.26±0.47; and 2.85±0.43 and 7.74±1.16 vs. 8.68±1.30, respectively. Group comparison, P<0.05 vs. P<0.01. Furthermore, the protein expression of P-gp/MDR1 and ABCG2 was 5.68±0.85 and 10.04±1.51 vs. 17.62±2.64 (P <0.01); and 2.06±0.31 and 3.74±0.56 vs. 4.56±0.68 (P <0.05 vs. P <0.01), respectively. THC8307/L-OHP cells treated with anti-VEGFR2 immunoliposomal oxaliplatin (containing oxaliplatin 25μg/ml) at 12 h, 24 h, 48 h, and 72 h exhibited P-gp/MDR1 protein expression levels of 8.57±1.28, 16.36±2.45, 3.42±0.51, and 0.75±0.11, respectively. There was statistical significance, compared with each group (P <0.01, P <0.05). Protein expression levels of ABCG2 were found to be 1.27±0.19, 2.13±0.32, 1.63±0.24, and 0.55±0.08 (P <0.05), respectively. The P-gp/MDR1 and ABCG2 mRNA expression of resistance genes was significantly increased at 24 h, followed by a significant reduction in expression at 72 h (P <0.01 and P <0.05, compared with the control group). After treatment with free oxaliplatin (25μg/ml) at 48 h, the protein expression levels of P-gp/MDR1, ABCG2, GST3, Bcl-2, and Caspase-3/P17 were not changed significantly (P >0.05, vs. control). After treatment with anti-VEGFR2 immunoliposomal oxaliplatin (containing oxaliplatin 25μg/ml vs. 50μg/ml), the protein expression levels of P-gp/MDR1, ABCG2, GST3, and Bcl-2 were reduced, whereas Caspase-3/P17 was up-regulated. Compared with each group, P <0.01 vs. P <0.05. There were positive cells of P-gp/MDR1 and ABCG2 in tumor tissue. Nevertheless, the protein expression was to weaken following administration agent augmentation.Conclusion We found that the anti-VEGFR2 immunoliposomal oxaliplatin reversed multidrug resistance of human colorectal carcinoma THC8307/L-OHP cells, this may be attributed to the down-regulation of P-gp/MDR1, ABCG2, and GST3. Moreover, anti-apoptotic proteins Bcl-2 and Caspase-3 may be involved in the anti-VEGFR2 immunoliposomal oxaliplatin regulation leading to the multidrug resistance of THC8307/L-OHP cells.更多还原