携带凋亡素基因的重组腺相关病毒抑制膀胱癌作用的研究
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摘要
研究目的
1.制备携带凋亡素基因的重组腺相关病毒rAAV-VP3,并鉴定其在体外能否发挥生物学活性。
2.探讨制备与纯化凋亡素蛋白多克隆抗体的方法,并对纯化后抗体的有效性和特异性进行免疫学评价。
3.探讨并分析重组腺相关病毒rAAV-VP3对人膀胱癌细胞的感染特点、体外抑制膀胱癌效应及其可能的作用机理。
4.评价携带凋亡素基因的重组腺相关病毒rAAV-VP3对荷人膀胱癌Balb/c裸鼠的基因治疗作用与效果,并对其作用机理进行探讨。
研究方法
1.携带凋亡素基因的重组腺相关病毒的包装、纯化、鉴定及体外生物学活性分析：将VP3基因通过基因重组的方法克隆至pAAV-MCS载体中,经EcoRI/Sal Ⅰ双酶切鉴定和基因测序无误后将其与包装质粒pAAV-RC、辅助质粒pAAV-Helper三质粒共转染AAV-293细胞,包装重组腺相关病毒rAAV-VP3。收获病毒后采用氯仿处理-聚乙二醇/氯化钠沉淀-氯仿抽提3个步骤进行病毒的分离、浓缩和纯化；PCR扩增病毒液中的目的基因鉴定重组病毒的包装是否成功。病毒液负染后透射电镜观察病毒颗粒,并用地高辛标记的CMV探针点杂交方法检测病毒液中rAAV-VP3的物理滴度；利用RT-PCR检测感染重组病毒后VP3基因在Vero细胞中的转录,免疫荧光检测VP3蛋白在Vero细胞中的表达。
2.VP3蛋白多克隆抗体的制备及免疫学评价：用真核表达载体pcDNA3.0-VP3为模板构建原核表达载体pET8a-VP3,经Nde I/BamH I双酶切鉴定和基因测序无误后,在IPGT诱导下表达VP3蛋白并对其进行纯化,将纯化后的VP3蛋白与弗氏完全佐剂或不完全佐剂乳化后,分别对两只新西兰大耳白兔进行皮下多点注射,间接ELISA检测免疫后血清效价,效价达到指标后第2天以心脏穿刺的方法采全血后分离抗血清。抗血清效价高的兔子进一步采用Protein A纯化总IgG,用重组腺相关病毒rAAV-VP3感染EJ、T24以及Vero细胞72h后行免疫荧光.Western blot检测,对抗体的特异性进行免疫学评价。
3.携带凋亡素基因的重组腺相关病毒对膀胱癌的体外抑制作用：利用MTT法检测凋亡素重组腺相关病毒对T24、EJ细胞的MOI值及其对膀胱癌细胞增殖的影响；RT-PCR检测凋亡素在T24、EJ细胞中的转录,Western Blot检测凋亡素蛋白在膀胱癌细胞中的表达；透射电镜、DNA Ladder法观察和判定膀胱癌细胞凋亡；流式细胞术PI法检测重组病毒对膀胱癌细胞周期的影响,Annexin V-FITC/PI双染法检测感染重组病毒后T24、EJ细胞的凋亡；Western Blot检测凋亡素对膀胱癌细胞中的Survivin表达的影响；Elisa检测凋亡素对膀胱癌细胞凋亡通路中caspase-3的激活作用。
4.凋亡素重组腺相关病毒对荷人膀胱癌Balb/c裸鼠的基因治疗评价：建立荷人膀胱癌Balb/c裸鼠皮下移植瘤模型,将动物随机分为未治疗组、对照病毒rAAV-EGFP组、丝裂霉素C(MMC)治疗组、rAAV-VP3治疗组共4组,病毒组注射剂量为1×1010v.g./50μl/只,MMC用量1mg/kg,均采用瘤周及瘤内多点注射法。通过绘制皮下移植瘤生长曲线、计算平均瘤重和抑制率、大体观察和病理组织学检查观察凋亡素重组腺相关病毒对Balb/c裸鼠皮下移植瘤的抑制作用,通过观察各组裸鼠治疗过程中一般情况、各组裸鼠存活率的比较评价治疗效果。免疫组化染色观察注射重组病毒后VP3蛋白在瘤内的表达时效性,免疫组化法计数阳性细胞所占百分比观察rAAV-VP3治疗组与对照病毒rAAV-EGFP组Ki.67、C-erbB-2、Rb、nm23的表达差异。实验结果
1.携带凋亡素基因的重组腺相关病毒的包装、纯化、鉴定及体外生物学活性分析：重组质粒pAAV-VP3经酶切鉴定和基因测序正确,三质粒共转染AAV-293细胞72小时后重组腺相关病毒rAAV-VP3包装成功,收获的重组病毒纯化后滴度为5.1×1011v.g./ml,电镜下可见病毒颗粒。感染Vero细胞后,可检测到VP3基因的转录和蛋白表达。
2.VP3蛋白多克隆抗体的制备及免疫学评价：原核表达载体pET8a-VP3的构建成功并经PCR鉴定和基因测序正确；原核表达菌株pET8a-VP3/BL21经终浓度为0.4mM的IPTG诱导4小时后在14.4kDa与20kDa之间出现目的条带,其大小与预期结果相符合,外源蛋白成功表达；收集洗脱峰,SDS-PAGE检测VP3蛋白纯度良好,无明显杂带。用纯化后的VP3蛋白免疫新西兰大耳白兔后抗血清效价可达1：243000,采用Protein A纯化总IgG, SDS-PAGE电泳检测纯化后抗体纯度较高,无明显杂带,间接ELISA法检测抗体灵敏度为5.4ng/ml;将rAAV-VP3感染EJ、T24以及Vero细胞72小时后行免疫荧光检测,可见特异性内源性表达的VP3蛋白以及核定位效应。在EJ和T24细胞中凋亡素主要定位于细胞核,而在Vero细胞中则定位于细胞质,行Western blot检测,于接近14kDa处可见特异性的目的条带。
3.携带凋亡素基因的重组腺相关病毒对膀胱癌的体外抑制作用：MTT法显示以MOI值2.0×104v.g./cell、1.0×105v.g./cell、2.0×105v.g./cell、5.0×105v.g./cell、1.0×106v.g./cell分别感染T24细胞和EJ细胞后72h, MOI=5.0×105v.g./cell时rAAV-VP3对T24细胞和EJ细胞的杀伤效应最为明显,增加感染复数不能进一步增强rAAV-VP3对膀胱癌细胞的杀伤效应；感染重组腺相关病毒rAAV-VP3的T24、EJ细胞增殖活性均明显下降,且随时间的推移效果更明显,而细胞对照组和rAAV-eGFP的OD490值都逐渐小幅上升,60h以后则逐渐下降(P<0.01)。透射电镜观察膀胱癌细胞出现核固缩、凋亡小体等典型的凋亡形态学改变,DNA Ladder法显示在100bp左右形成断裂的DNA片段；通过RT-PCR和Western Blot可检测到重组腺相关病毒rAAV-VP3感染膀胱癌细胞后能在T24、EJ细胞中转录与表达；流式细胞术PI法检测发现重组腺相关病毒rAAV-VP3感染膀胱癌细胞后与对照组比较S期细胞比例降低和G2M期细胞比例增多(P<0.01)。Annexin V-FITC/PI双染法检测到感染rAAV-VP3后T24、EJ细胞发生凋亡的比例明显升高。Western Blot检测腺相关病毒介导的凋亡素蛋白表达可抑制内源性Survivin的表达,而对照病毒rAAV-LacZ作用的T24、EJ细胞内源性Survivin为高表达状态。Elisa法检测感染第3d、4d、5d后T24和EJ细胞中激活型caspase-3的含量明显增高,与对照病毒组和细胞对照组比较差异有统计学意义(P<0.01)。
4.重组腺相关病毒rAAV-VP3能明显抑制裸鼠皮下移植瘤生长,有较高的肿瘤抑制率并能提高裸鼠的存活率,生存中位数时间为55天,瘤内注射重组腺相关病毒rAAV-VP3后,可以观察到VP3蛋白表达量呈逐渐升高的趋势,于21天左右达到顶峰,以后一直维持强阳性表达；免疫组化检测发现重组腺相关病毒rAAV-VP3可抑制Ki-67、C-erbB-2的表达,并能上调肿瘤转移抑制基因Rb、nm23的表达。结论
1.包装的重组腺相关病毒rAAV-VP3在体外能够发挥生物学活性。
2.制备的凋亡素蛋白多克隆抗体具有较强的灵敏度和特异性,证实了凋亡素在正常细胞中定位于细胞质,而在肿瘤细胞中则定位于细胞核。
3.重组腺相关病毒rAAV-VP3对T24、EJ细胞的最适感染复数为5.0×105v.g./cell,感染rAAV-VP3后膀胱癌细胞的增殖受到抑制,主要作用于肿瘤细胞的S期和G2/M期,发生G2/M期阻滞,引起细胞DNA合成受阻和降解。腺相关病毒介导的凋亡素蛋白表达可抑制内源性Survivin的表达,提示VP3与Survivin的存在拮抗性,可能是凋亡素诱导肿瘤细胞凋亡的作用机制之一；caspase-3的激活是凋亡素诱导的细胞凋亡通路中的关键分子信号之一
4.重组腺相关病毒rAAV-VP3能明显抑制裸鼠皮下移植瘤生长,并能减少肿瘤对周围组织的侵犯与瘤栓形成。基因治疗后能提高裸鼠的存活率,与MMC治疗组相比具有一定的优越性。其介导的VP3蛋白表达量呈逐渐升高的趋势,凋亡素体内抑制膀胱癌的作用机制可能与其抑制Ki-67、C-erbB-2的表达,并上调肿瘤转移抑制基因Rb、nm23的表达有关。
5.利用腺相关病毒介导凋亡素基因的表达治疗膀胱癌的策略可行,为下一步运用该重组腺相关病毒进行临床前研究奠定了基础。
Objective
1. To construct and prepare the recombinant adeno-associated virus (AAV) expressing apoptin and evaluate the biological activities in vivo and in vitro.
2. To investigate a method of the preparation and purification of the apoptotic protein polyclonal antibody and to make an immunological evaluation of the effectiveness and specificity of the purified antibody.
3. To analyze the characterization of rAAV-VP3to human bladder cancer cell lines and the potential mechanism of its anti-tumor effect.
4. To assess and explore the mechanism and outcomes of gene therapy to Balb/c nude mice of human bladder cancer model by recombinant adeno-associated virus delivering VP3(rAAV-VP3). Methods
1. In this study the AAV Helper-Free System was used to generate the recombinant adeno-associated virus expressing VP3gene. The VP3gene was cloned into the expression vector pAAV-MCS and the recombinant plasmid pAAV-VP3is confirmed by double enzymatic digestion using EcoR I and Sal I. The recombinant expression plasmid is co-transfected into the AAV-293cells with pHelper and pAAV-RC to produce recombinant AAV viral particles. The morphology of the virus particles were examined by electron microscopy. The physical titer of recombinant AAV was measured through Digoxigenin labeled CMV probe dot blot method, and the recombinant virus was verified by PCR of the exogenous interest genes of apoptin. RT-PCR and Western blot were performed to detect the transcription and expression of apoptin.
2. VP3gene was cloned into the prokaryotic expression vector pET8a. The prokaryotic expression vector pET8a-VP3was employed to express VP3protein in BL21which was induced by IPTG, and the protein was extracted from the cell and purified. The purified VP3protein was applied to immunize the New Zealand rabbits combined with the Freund's complete adjuvant or incomplete adjuvant by multi-points subcutaneous injection. ELISA test was performed to measure the titer of the antibody after immunization. Two days after the measurement the whole blood was harvested by cardiac puncture and the serum was separated from the whole blood. The immunglobin IgG was purified by Protein a method from the antiserum.
3. Concerning on the anti-tumor activity of rAAV-VP3in vitro, a variety of methods have been applied to the assays. Firstly, we observed the different MOI influence of rAAV-VP3to the growth of the bladder cell lines T-24and EJ, and determined the optimal infection MOI. Then, RT-PCR and Western Blot were performed to detect the expression of the Apoptin on mRNA level and protein level respectively. The anti-tumor effect of the virus was confirmed by employing transmission electron microscope and DNA ladder based method. To further investigate the apoptosis of the T-24and EJ cells after infection, we analyzed the cell cycle by using flow cytometry and apoptosis by Annexin V-FITC/PI double staining. Some bonus assays including the evaluation of the effect on expression of surviving and activation of caspase-3pathway have also been carried out by Western blot and Elisa.
4. Gene therapy evaluation of the recombinant adeno-associated virus expressing VP3(rAAV-VP3) of the Balb/c nude mice model of human bladder cancer:We have established the Balb/c nude mice model of human bladder cancer. The animal models were divided into4groups, including negative control group, rAAV-eGFP treated group as control, MMC treated group and VP3infusion group. The dosage of the injection was1×1010v.g./50μl/mouse, and positive control was lmg/kg. The virus and drugs were delivered by multiple injection of the tumor cells. The anti-tumor effect of rAAV-VP3to Balb/c nude mice model of human bladder cancer was analyzed by growth curve of the tumor, calculation of the average tumor weight and histochemistry of the tumor section. The comparisons of the outcomes were performed by observation of the general condition and the survival ratio in each group. The staining of the histochemistry were used to detect the expression efficiency of the VP3in tumor cells after rAAV-VP3infection. The counting of the immuno-positive cells was also applied to compare the differential expression pattern of Ki-67、C-erbB-2、Rb、nm23between rAAV-VP3delivered group and control group.
Results
1. The expression vector pAAV-VP3was successfully constructed and the sequence of the VP3was confirmed by DNA sequencing. The rAAV-VP3were obtained by three plasmids co-transfection into AAV-293cells after72hours. The recombinant adeno-associated virus has a high titer of5.1×1011v.g./ml, and virus particles can be observed by electron microscope. By infection the Vero cells with rAAV-VP3, the transcription and expression of VP3gene was detected by RT-PCR and Western blotting.
2. The immunglobin IgG was purified by Protein A method from the antiserum. The final purified antibody titers were up to1:243,000. The immunological evaluation of the polyclonal antibody specific binding was assayed by using recombinant adeno-associated virus rAAV-VP3infected different cell lines. First, it was used to detect the VP3gene expression by immunofluorescence in human bladder cancer cells T24and EJ, also in Vero cells. Apoptin was observed in T24, EJ cells, mainly located in the nucleus, whereas in Vero cells were localized in the cytoplasm. Secondly, the specific binding of the purified antibodies to VP3protein in different human bladder cancer cells were detected by Western blotting.
3. The anti-tumor effect of the rAAV-VP3has been observed by MTT assay. We infected the T-24and EJ cell lines with the MO1:2.0×104v.g./cell,1.0×105v.g./cell,2.0×105v.g./cell,5.0×105v.g./cell and1.0×106v.g./cell respectively for72hours. Interestingly, the evidences indicated that the most significant anti-tumor effect was observed with MOI=5.0×105v.g./cell, and no further better effect could be produced with high MOI. The proliferation of the bladder cells T-24and EJ have been blocked after infection, and obvious results could be obtained as time went on. The values of OD490have increased by small magnitude and gradually decreased after60hours(P<0.01). Some classical morphology of apoptosis including the shrunk of nucleus and appearance of apoptotic body have been observed by transmission electron microscope and100bp DNA ladders have also been visualized in cells infected with rAAV-VP3. The transcription of expression of VP3could be detected by RT-PCR and Western Blot. We have found that the cell ratio of S phase decreased and G2/M was blocked compared with control group after rAAV-VP3infection(P<0.01). The FACS analysis also shown that the percentage of the apoptotic cells have increased after the infection through double staining Annexin V-FITC/PI. Moreover, significant inhibition of Survivin has been observed by VP3, and high expression level of Survivin were in control groups. Active form of caspase-3has been detected in T-24and EJ cells on3days,4days and5days after infection by Elisa based methods, and the significance has been validated by statistical analysis with the comparison of control groups(P<0.01).
4. The adeno-associated virus rAAV-VP3could significantly inhibit the growth of the tumor in animal model. The inhibitory efficiency of the rAAV-VP3was coupled with high survival ratio, and the median survival time of55days. An increasing expression level trend of VP3could be detected after infection, and the maximum was attained at21days. The strong expression could be maintained for a long time. Importantly, the expression of VP3in vivo could dramatically downregulate the expression of Ki-67、C-erbB-2, and upregulate the expression of Rb、nm23in target cells.
Conclusion
1. The rAAV-VP3we have prepared have played good biological activities both in vitro and in vivo.
2. The polyclonal antibody we produced has a strong sensitivity and specificity. The results indicated that the location of VP3in normal cell was cytoplasm, and in tumor cell was nucleus.
3. The optimal MOI of rAAV-VP3to T24and EJ was5.0×105v.g./cell, and the growth of the bladder cancer cells were significantly inhibited, which mainly target at phase S and phase G2/M. The inhibition of phase G2/M could cause the inhibition of synthesis and degradation of DNA. The expression of VP3mediated by rAAV could downregulate the endogenous expression of Survivin. It has demonstrated that antagonism exists between Apoptin and Survivin, which may be one of the mechanisms that Apoptin could induce the apoptosis in tumor cells. Furthermore, the evidence we obtained indicated that caspase-3activation was one of the critical molecular signal in Apoptin-induced apoptosis pathway.
4. The delivery of VP3by using rAAV could significantly inhibit the growth of tumor, and reduce the invasion and formation of tumor embolus in the normal tissue nearby. The gene therapy by employing VP3could elevate the survival ratio of the nude mice, which was advantageous over anti-tumor drug MMC. The expression of VP3has indicated gradual increase trend in vivo. The mechanism of the anti-tumor effect may be related to the inhibition of the expression of Ki-67and C-erbB-2, and upregulation of the anti-tumor gene Rb, nm23.
5. AAV-mediated VP3gene administration may be a new therapeutic technique for the treatment of bladder neoplasms. It will supply a foundation for further studies of anti-tumor effect of bladder cancer.
1.制备携带凋亡素基因的重组腺相关病毒rAAV-VP3,并鉴定其在体外能否发挥生物学活性。
2.探讨制备与纯化凋亡素蛋白多克隆抗体的方法,并对纯化后抗体的有效性和特异性进行免疫学评价。
3.探讨并分析重组腺相关病毒rAAV-VP3对人膀胱癌细胞的感染特点、体外抑制膀胱癌效应及其可能的作用机理。
4.评价携带凋亡素基因的重组腺相关病毒rAAV-VP3对荷人膀胱癌Balb/c裸鼠的基因治疗作用与效果,并对其作用机理进行探讨。
研究方法
1.携带凋亡素基因的重组腺相关病毒的包装、纯化、鉴定及体外生物学活性分析：将VP3基因通过基因重组的方法克隆至pAAV-MCS载体中,经EcoRI/Sal Ⅰ双酶切鉴定和基因测序无误后将其与包装质粒pAAV-RC、辅助质粒pAAV-Helper三质粒共转染AAV-293细胞,包装重组腺相关病毒rAAV-VP3。收获病毒后采用氯仿处理-聚乙二醇/氯化钠沉淀-氯仿抽提3个步骤进行病毒的分离、浓缩和纯化；PCR扩增病毒液中的目的基因鉴定重组病毒的包装是否成功。病毒液负染后透射电镜观察病毒颗粒,并用地高辛标记的CMV探针点杂交方法检测病毒液中rAAV-VP3的物理滴度；利用RT-PCR检测感染重组病毒后VP3基因在Vero细胞中的转录,免疫荧光检测VP3蛋白在Vero细胞中的表达。
2.VP3蛋白多克隆抗体的制备及免疫学评价：用真核表达载体pcDNA3.0-VP3为模板构建原核表达载体pET8a-VP3,经Nde I/BamH I双酶切鉴定和基因测序无误后,在IPGT诱导下表达VP3蛋白并对其进行纯化,将纯化后的VP3蛋白与弗氏完全佐剂或不完全佐剂乳化后,分别对两只新西兰大耳白兔进行皮下多点注射,间接ELISA检测免疫后血清效价,效价达到指标后第2天以心脏穿刺的方法采全血后分离抗血清。抗血清效价高的兔子进一步采用Protein A纯化总IgG,用重组腺相关病毒rAAV-VP3感染EJ、T24以及Vero细胞72h后行免疫荧光.Western blot检测,对抗体的特异性进行免疫学评价。
3.携带凋亡素基因的重组腺相关病毒对膀胱癌的体外抑制作用：利用MTT法检测凋亡素重组腺相关病毒对T24、EJ细胞的MOI值及其对膀胱癌细胞增殖的影响；RT-PCR检测凋亡素在T24、EJ细胞中的转录,Western Blot检测凋亡素蛋白在膀胱癌细胞中的表达；透射电镜、DNA Ladder法观察和判定膀胱癌细胞凋亡；流式细胞术PI法检测重组病毒对膀胱癌细胞周期的影响,Annexin V-FITC/PI双染法检测感染重组病毒后T24、EJ细胞的凋亡；Western Blot检测凋亡素对膀胱癌细胞中的Survivin表达的影响；Elisa检测凋亡素对膀胱癌细胞凋亡通路中caspase-3的激活作用。
4.凋亡素重组腺相关病毒对荷人膀胱癌Balb/c裸鼠的基因治疗评价：建立荷人膀胱癌Balb/c裸鼠皮下移植瘤模型,将动物随机分为未治疗组、对照病毒rAAV-EGFP组、丝裂霉素C(MMC)治疗组、rAAV-VP3治疗组共4组,病毒组注射剂量为1×1010v.g./50μl/只,MMC用量1mg/kg,均采用瘤周及瘤内多点注射法。通过绘制皮下移植瘤生长曲线、计算平均瘤重和抑制率、大体观察和病理组织学检查观察凋亡素重组腺相关病毒对Balb/c裸鼠皮下移植瘤的抑制作用,通过观察各组裸鼠治疗过程中一般情况、各组裸鼠存活率的比较评价治疗效果。免疫组化染色观察注射重组病毒后VP3蛋白在瘤内的表达时效性,免疫组化法计数阳性细胞所占百分比观察rAAV-VP3治疗组与对照病毒rAAV-EGFP组Ki.67、C-erbB-2、Rb、nm23的表达差异。实验结果
1.携带凋亡素基因的重组腺相关病毒的包装、纯化、鉴定及体外生物学活性分析：重组质粒pAAV-VP3经酶切鉴定和基因测序正确,三质粒共转染AAV-293细胞72小时后重组腺相关病毒rAAV-VP3包装成功,收获的重组病毒纯化后滴度为5.1×1011v.g./ml,电镜下可见病毒颗粒。感染Vero细胞后,可检测到VP3基因的转录和蛋白表达。
2.VP3蛋白多克隆抗体的制备及免疫学评价：原核表达载体pET8a-VP3的构建成功并经PCR鉴定和基因测序正确；原核表达菌株pET8a-VP3/BL21经终浓度为0.4mM的IPTG诱导4小时后在14.4kDa与20kDa之间出现目的条带,其大小与预期结果相符合,外源蛋白成功表达；收集洗脱峰,SDS-PAGE检测VP3蛋白纯度良好,无明显杂带。用纯化后的VP3蛋白免疫新西兰大耳白兔后抗血清效价可达1：243000,采用Protein A纯化总IgG, SDS-PAGE电泳检测纯化后抗体纯度较高,无明显杂带,间接ELISA法检测抗体灵敏度为5.4ng/ml;将rAAV-VP3感染EJ、T24以及Vero细胞72小时后行免疫荧光检测,可见特异性内源性表达的VP3蛋白以及核定位效应。在EJ和T24细胞中凋亡素主要定位于细胞核,而在Vero细胞中则定位于细胞质,行Western blot检测,于接近14kDa处可见特异性的目的条带。
3.携带凋亡素基因的重组腺相关病毒对膀胱癌的体外抑制作用：MTT法显示以MOI值2.0×104v.g./cell、1.0×105v.g./cell、2.0×105v.g./cell、5.0×105v.g./cell、1.0×106v.g./cell分别感染T24细胞和EJ细胞后72h, MOI=5.0×105v.g./cell时rAAV-VP3对T24细胞和EJ细胞的杀伤效应最为明显,增加感染复数不能进一步增强rAAV-VP3对膀胱癌细胞的杀伤效应；感染重组腺相关病毒rAAV-VP3的T24、EJ细胞增殖活性均明显下降,且随时间的推移效果更明显,而细胞对照组和rAAV-eGFP的OD490值都逐渐小幅上升,60h以后则逐渐下降(P<0.01)。透射电镜观察膀胱癌细胞出现核固缩、凋亡小体等典型的凋亡形态学改变,DNA Ladder法显示在100bp左右形成断裂的DNA片段；通过RT-PCR和Western Blot可检测到重组腺相关病毒rAAV-VP3感染膀胱癌细胞后能在T24、EJ细胞中转录与表达；流式细胞术PI法检测发现重组腺相关病毒rAAV-VP3感染膀胱癌细胞后与对照组比较S期细胞比例降低和G2M期细胞比例增多(P<0.01)。Annexin V-FITC/PI双染法检测到感染rAAV-VP3后T24、EJ细胞发生凋亡的比例明显升高。Western Blot检测腺相关病毒介导的凋亡素蛋白表达可抑制内源性Survivin的表达,而对照病毒rAAV-LacZ作用的T24、EJ细胞内源性Survivin为高表达状态。Elisa法检测感染第3d、4d、5d后T24和EJ细胞中激活型caspase-3的含量明显增高,与对照病毒组和细胞对照组比较差异有统计学意义(P<0.01)。
4.重组腺相关病毒rAAV-VP3能明显抑制裸鼠皮下移植瘤生长,有较高的肿瘤抑制率并能提高裸鼠的存活率,生存中位数时间为55天,瘤内注射重组腺相关病毒rAAV-VP3后,可以观察到VP3蛋白表达量呈逐渐升高的趋势,于21天左右达到顶峰,以后一直维持强阳性表达；免疫组化检测发现重组腺相关病毒rAAV-VP3可抑制Ki-67、C-erbB-2的表达,并能上调肿瘤转移抑制基因Rb、nm23的表达。结论
1.包装的重组腺相关病毒rAAV-VP3在体外能够发挥生物学活性。
2.制备的凋亡素蛋白多克隆抗体具有较强的灵敏度和特异性,证实了凋亡素在正常细胞中定位于细胞质,而在肿瘤细胞中则定位于细胞核。
3.重组腺相关病毒rAAV-VP3对T24、EJ细胞的最适感染复数为5.0×105v.g./cell,感染rAAV-VP3后膀胱癌细胞的增殖受到抑制,主要作用于肿瘤细胞的S期和G2/M期,发生G2/M期阻滞,引起细胞DNA合成受阻和降解。腺相关病毒介导的凋亡素蛋白表达可抑制内源性Survivin的表达,提示VP3与Survivin的存在拮抗性,可能是凋亡素诱导肿瘤细胞凋亡的作用机制之一；caspase-3的激活是凋亡素诱导的细胞凋亡通路中的关键分子信号之一
4.重组腺相关病毒rAAV-VP3能明显抑制裸鼠皮下移植瘤生长,并能减少肿瘤对周围组织的侵犯与瘤栓形成。基因治疗后能提高裸鼠的存活率,与MMC治疗组相比具有一定的优越性。其介导的VP3蛋白表达量呈逐渐升高的趋势,凋亡素体内抑制膀胱癌的作用机制可能与其抑制Ki-67、C-erbB-2的表达,并上调肿瘤转移抑制基因Rb、nm23的表达有关。
5.利用腺相关病毒介导凋亡素基因的表达治疗膀胱癌的策略可行,为下一步运用该重组腺相关病毒进行临床前研究奠定了基础。
Objective
1. To construct and prepare the recombinant adeno-associated virus (AAV) expressing apoptin and evaluate the biological activities in vivo and in vitro.
2. To investigate a method of the preparation and purification of the apoptotic protein polyclonal antibody and to make an immunological evaluation of the effectiveness and specificity of the purified antibody.
3. To analyze the characterization of rAAV-VP3to human bladder cancer cell lines and the potential mechanism of its anti-tumor effect.
4. To assess and explore the mechanism and outcomes of gene therapy to Balb/c nude mice of human bladder cancer model by recombinant adeno-associated virus delivering VP3(rAAV-VP3). Methods
1. In this study the AAV Helper-Free System was used to generate the recombinant adeno-associated virus expressing VP3gene. The VP3gene was cloned into the expression vector pAAV-MCS and the recombinant plasmid pAAV-VP3is confirmed by double enzymatic digestion using EcoR I and Sal I. The recombinant expression plasmid is co-transfected into the AAV-293cells with pHelper and pAAV-RC to produce recombinant AAV viral particles. The morphology of the virus particles were examined by electron microscopy. The physical titer of recombinant AAV was measured through Digoxigenin labeled CMV probe dot blot method, and the recombinant virus was verified by PCR of the exogenous interest genes of apoptin. RT-PCR and Western blot were performed to detect the transcription and expression of apoptin.
2. VP3gene was cloned into the prokaryotic expression vector pET8a. The prokaryotic expression vector pET8a-VP3was employed to express VP3protein in BL21which was induced by IPTG, and the protein was extracted from the cell and purified. The purified VP3protein was applied to immunize the New Zealand rabbits combined with the Freund's complete adjuvant or incomplete adjuvant by multi-points subcutaneous injection. ELISA test was performed to measure the titer of the antibody after immunization. Two days after the measurement the whole blood was harvested by cardiac puncture and the serum was separated from the whole blood. The immunglobin IgG was purified by Protein a method from the antiserum.
3. Concerning on the anti-tumor activity of rAAV-VP3in vitro, a variety of methods have been applied to the assays. Firstly, we observed the different MOI influence of rAAV-VP3to the growth of the bladder cell lines T-24and EJ, and determined the optimal infection MOI. Then, RT-PCR and Western Blot were performed to detect the expression of the Apoptin on mRNA level and protein level respectively. The anti-tumor effect of the virus was confirmed by employing transmission electron microscope and DNA ladder based method. To further investigate the apoptosis of the T-24and EJ cells after infection, we analyzed the cell cycle by using flow cytometry and apoptosis by Annexin V-FITC/PI double staining. Some bonus assays including the evaluation of the effect on expression of surviving and activation of caspase-3pathway have also been carried out by Western blot and Elisa.
4. Gene therapy evaluation of the recombinant adeno-associated virus expressing VP3(rAAV-VP3) of the Balb/c nude mice model of human bladder cancer:We have established the Balb/c nude mice model of human bladder cancer. The animal models were divided into4groups, including negative control group, rAAV-eGFP treated group as control, MMC treated group and VP3infusion group. The dosage of the injection was1×1010v.g./50μl/mouse, and positive control was lmg/kg. The virus and drugs were delivered by multiple injection of the tumor cells. The anti-tumor effect of rAAV-VP3to Balb/c nude mice model of human bladder cancer was analyzed by growth curve of the tumor, calculation of the average tumor weight and histochemistry of the tumor section. The comparisons of the outcomes were performed by observation of the general condition and the survival ratio in each group. The staining of the histochemistry were used to detect the expression efficiency of the VP3in tumor cells after rAAV-VP3infection. The counting of the immuno-positive cells was also applied to compare the differential expression pattern of Ki-67、C-erbB-2、Rb、nm23between rAAV-VP3delivered group and control group.
Results
1. The expression vector pAAV-VP3was successfully constructed and the sequence of the VP3was confirmed by DNA sequencing. The rAAV-VP3were obtained by three plasmids co-transfection into AAV-293cells after72hours. The recombinant adeno-associated virus has a high titer of5.1×1011v.g./ml, and virus particles can be observed by electron microscope. By infection the Vero cells with rAAV-VP3, the transcription and expression of VP3gene was detected by RT-PCR and Western blotting.
2. The immunglobin IgG was purified by Protein A method from the antiserum. The final purified antibody titers were up to1:243,000. The immunological evaluation of the polyclonal antibody specific binding was assayed by using recombinant adeno-associated virus rAAV-VP3infected different cell lines. First, it was used to detect the VP3gene expression by immunofluorescence in human bladder cancer cells T24and EJ, also in Vero cells. Apoptin was observed in T24, EJ cells, mainly located in the nucleus, whereas in Vero cells were localized in the cytoplasm. Secondly, the specific binding of the purified antibodies to VP3protein in different human bladder cancer cells were detected by Western blotting.
3. The anti-tumor effect of the rAAV-VP3has been observed by MTT assay. We infected the T-24and EJ cell lines with the MO1:2.0×104v.g./cell,1.0×105v.g./cell,2.0×105v.g./cell,5.0×105v.g./cell and1.0×106v.g./cell respectively for72hours. Interestingly, the evidences indicated that the most significant anti-tumor effect was observed with MOI=5.0×105v.g./cell, and no further better effect could be produced with high MOI. The proliferation of the bladder cells T-24and EJ have been blocked after infection, and obvious results could be obtained as time went on. The values of OD490have increased by small magnitude and gradually decreased after60hours(P<0.01). Some classical morphology of apoptosis including the shrunk of nucleus and appearance of apoptotic body have been observed by transmission electron microscope and100bp DNA ladders have also been visualized in cells infected with rAAV-VP3. The transcription of expression of VP3could be detected by RT-PCR and Western Blot. We have found that the cell ratio of S phase decreased and G2/M was blocked compared with control group after rAAV-VP3infection(P<0.01). The FACS analysis also shown that the percentage of the apoptotic cells have increased after the infection through double staining Annexin V-FITC/PI. Moreover, significant inhibition of Survivin has been observed by VP3, and high expression level of Survivin were in control groups. Active form of caspase-3has been detected in T-24and EJ cells on3days,4days and5days after infection by Elisa based methods, and the significance has been validated by statistical analysis with the comparison of control groups(P<0.01).
4. The adeno-associated virus rAAV-VP3could significantly inhibit the growth of the tumor in animal model. The inhibitory efficiency of the rAAV-VP3was coupled with high survival ratio, and the median survival time of55days. An increasing expression level trend of VP3could be detected after infection, and the maximum was attained at21days. The strong expression could be maintained for a long time. Importantly, the expression of VP3in vivo could dramatically downregulate the expression of Ki-67、C-erbB-2, and upregulate the expression of Rb、nm23in target cells.
Conclusion
1. The rAAV-VP3we have prepared have played good biological activities both in vitro and in vivo.
2. The polyclonal antibody we produced has a strong sensitivity and specificity. The results indicated that the location of VP3in normal cell was cytoplasm, and in tumor cell was nucleus.
3. The optimal MOI of rAAV-VP3to T24and EJ was5.0×105v.g./cell, and the growth of the bladder cancer cells were significantly inhibited, which mainly target at phase S and phase G2/M. The inhibition of phase G2/M could cause the inhibition of synthesis and degradation of DNA. The expression of VP3mediated by rAAV could downregulate the endogenous expression of Survivin. It has demonstrated that antagonism exists between Apoptin and Survivin, which may be one of the mechanisms that Apoptin could induce the apoptosis in tumor cells. Furthermore, the evidence we obtained indicated that caspase-3activation was one of the critical molecular signal in Apoptin-induced apoptosis pathway.
4. The delivery of VP3by using rAAV could significantly inhibit the growth of tumor, and reduce the invasion and formation of tumor embolus in the normal tissue nearby. The gene therapy by employing VP3could elevate the survival ratio of the nude mice, which was advantageous over anti-tumor drug MMC. The expression of VP3has indicated gradual increase trend in vivo. The mechanism of the anti-tumor effect may be related to the inhibition of the expression of Ki-67and C-erbB-2, and upregulation of the anti-tumor gene Rb, nm23.
5. AAV-mediated VP3gene administration may be a new therapeutic technique for the treatment of bladder neoplasms. It will supply a foundation for further studies of anti-tumor effect of bladder cancer.
引文
1.重组质粒pAAV-VP3经酶切鉴定和基因测序正确,三质粒共转染AAV-293细胞72小时后重组腺相关病毒rAAV-VP3包装成功,收获的重组病毒纯化后滴度为5.1×1011 v.g./ml,电镜下可见病毒颗粒；
2.感染Vero细胞后,可检测到VP3基因的转录和蛋白表达；
3.构建的重组腺相关病毒rAAV-VP3在体外能够发挥生物学活性,其介导的凋亡素表达生物学效应为下一步实验奠定了基础。
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1.构建了原核表达载体pET8a-VP3,在IPGT诱导下表达VP3蛋白并对其进行纯化,获得高纯度的VP3蛋白；
2.将纯化后的VP3蛋白与弗氏完全佐剂或不完全佐剂乳化后,通过对新西兰大耳白兔进行皮下多点注射,分离抗血清进一步采用Protein A纯化总IgG,最终纯化后的抗体效价可以达到1：243,000；
3.利用免疫荧光技术检测VP3基因在人膀胱癌细胞株T24、EJ细胞以及Vero细胞中的表达情况,观察到凋亡素在T24、EJ细胞中主要定位于细胞核,而在Vero细胞中则定位于细胞质；
4.通过Western blotting检测纯化后的抗体能与细胞内腺相关病毒介导表达的凋亡素蛋白特异性结合；
5.实验研究证明了制备的凋亡素蛋白多克隆抗体的有效性和特异性,为进一步阐明凋亡素抗肿瘤效应的分子机制及生物学特性奠定了基础。
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11. Liu X, Zeidan YH, Elojeimy S, et al. Involvement of sphingolipids in apoptin-induced cell killing. Mol Ther.2006,14(5):627-636.
12. Wadia JS, Wagner MV, Ezhevsky SA, et al. Apoptin/VP3 contains a concentration-dependent nuclear localization signal (NLS), not a tumorigenic selective NLS. J Virol,2004,78(11):6077-6078.
13.王春晖,王剑松,詹辉,等.改良型TAT-凋亡素体外抗人膀胱肿瘤活性的研究.中国肿瘤临床,2011,38(5)：246-249.
14. Heilman DW, Teodoro JG, Green MR. Apoptin nucleocytoplasmic shuttling is required for cell type-specific localization, apoptosis, and recruitment of the anaphase-promoting complex/cyclosome to PML bodies. J Virol,2006,80(15): 7535-7545. 瘤细胞凋亡的作用机制之一；
6. Elisa法检测感染rAAV-VP3后T24和EJ细胞中激活型caspase-3的含量明显增高,caspase-3的激活是凋亡素诱导的细胞凋亡通路中的关键分子信号之一
1. van der Eb MM, Pietersen AM, Speetjens FM, et al. Gene therapy with apoptin induces regression of xenografted human hepatomas. Cancer Gene Ther,2002,9(1): 53-61.
2. Ferreira CG, Tolis C, Giaccone G. p53 and chemosensitivity. Ann Oncol,1999, 10(9):1011-1021.
3. Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med,1997,3(8):917-921.
4. Altieri DC, Marchisio PC. Survivin apoptosis:an interloper between cell death and cell proliferation in cancer. Lab Invest,1999,79(11):1327-1333.
5. Tamm I, Wang Y, Sausville E, et al. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res,1998,58(23):5315-5320.
6. Ku JH, Kwak C, Lee HS, et al. Expression of survivin, a novel inhibitor of apoptosis, in superficial transitional cell carcinoma of the bladder. J Urol,2004, 171(2 Pt 1):631-635.
7. Swana HS, Grossman D, Anthony JN, et al. Tumor content of the antiapoptosis molecule survivin and recurrence of bladder cancer. N Engl J Med,1999,341(6): 452-453.
8. Sharp JD, Hausladen DA, Maher MG, et al. Bladder cancer detection with urinary survivin, an inhibitor of apoptosis. Front Biosci,2002,7:e36-41.
9. Noteborn MH. Apoptin acts as a tumor-specific killer:potentials for an anti-tumor therapy. Cell Mol Biol,2005,51(1):49-60.
10. Danen-Van Oorschot AA, van Der Eb AJ, Noteborn MH. The chicken anemia virus-derived protein apoptin requires activation of caspases for induction of apoptosis in human tumor cells. J Virol,2000,74(15):7072-7078.
11 Maddika S, Booy EP, Johar D, et al. Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway. J Cell Sci,2005,118(Pt 19):4485-4493.
12. Burek M, Maddika S, Burek CJ, et al. Apoptin-induced cell death is modulated by Bcl-2 family members and is Apaf-1 dependent. Oncogene,2006,25(15): 2213-2222.
13. Noteborn MH. Chicken anemia virus induced apoptosis:underlying molecular mechanisms. Vet Microbiol,2004,98(2):89-94.
14. Wadia JS, Wagner MV, Ezhevsky SA, et al. Apoptin/VP3 contains a concentration-dependent nuclear localization signal (NLS), not a tumorigenic selective NLS. J Virol,2004,78(11):6077-6078.
1.重组腺相关病毒rAAV-VP3能明显抑制裸鼠皮下移植瘤生长,有较高的肿瘤抑制率,并能减少肿瘤对周围组织的侵犯与瘤栓形成；
2.重组腺相关病毒rAAV-VP3对荷瘤裸鼠基因治疗后,能提高裸鼠的存活率,生存中位数时间为55天,而MMC治疗组生存中位数时间为46天,治疗副作用方面与MMC治疗组相比具有一定的优越性；
3.瘤内注射重组腺相关病毒rAAV-VP3后,可以观察到VP3蛋白表达量呈逐渐升高的趋势,于21天左右达到顶峰,以后一直维持强阳性表达；
4.免疫组化检测发现重组腺相关病毒rAAV-VP3可抑制Ki-67、C-erbB-2的表达,并能上调肿瘤转移抑制基因Rb、nm23的表达,可能是凋亡素抑制膀胱癌的作用机制之一。
1. Noteborn MH. Chicken anemia virus induced apoptosis:underlying molecular mechanisms. Vet Microbiol,2004,98(2):89-94.
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8. Danen-Van Oorschot AA, Zhang YH, Leliveld SR, et al. Importance of nuclear localization of apoptin for tumor-specific induction of apoptosis. J Biol Chem,2003, 278(30):27729-27736.
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10. Heilman DW, Teodoro JG, Green MR. Apoptin nucleocytoplasmic shuttling is required for cell type-specific localization, apoptosis, and recruitment of the anaphase-promoting complex/cyclosome to PML bodies. J Virol,2006,80(15): 7535-7545.
11. Backendorf C, Visser AE, de Boer AG, et al. Apoptin:therapeutic potential of an early sensor of carcinogenic transformation. Annu Rev Pharmacol Toxicol,2008, 48:143-169.
12. Danen-Van Oorschot AA, Fischer DF, Grimbergen JM, et al. Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells. Proc Natl Acad Sci U S A,1997,94(11):5843-5847.
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14. Leliveld SR, Zhang YH, Rohn JL, et al. Apoptin induces tumor-specific apoptosis as a globular multimer. J Biol Chem,2003,278(11):9042-9051.
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16. Leliveld SR, Dame RT, Mommaas MA, et al. Apoptin protein multimers form distinct higher-order nucleoprotein complexes with DNA. Nucleic Acid Res,2003, 31(16):4805-4813.
17. Leliveld SR, Dame RT, Rohn JL, et al. Apoptin's functional N- and C- termini independently bind DNA. FEBS Lett,2004,557(1-3):155-158.
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21. Danen-Van Oorschot AA, van Der Eb AJ, Noteborn MH. The chicken anemia virus-derived protein apoptin requires activation of caspases for induction of apoptosis in human tumor cells. J Virol,2000,74(15):7072-7078.
22. Burek M, Maddika S, Burek CJ, et al. Apoptin-induced cell death is modulated by Bcl-2 family members and is Apaf-1 dependent. Oncogene,2006,25(15): 2213-2222.
23. Maddika S, Booy EP, Johar D, et al. Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway. J Cell Sci,2005,118(Pt 19):4485-4493.
24. Noteborn MH. Apoptin acts as a tumor-specific killer:potentials for an anti-tumor therapy. Cell Mol Biol,2005,51(1):49-60.
25. Zhuang SM, Shvarts A, Van Ormondt H, et al. Apoptin, a protein derived from chicken anemia virus, induces p53-independent apoptosis in human osteosarcoma cells. Cancer Res,1995,55(3):486-489.
26. Danen-Van Oorschot AA, van der Eb AJ, Noteborn MH. BCL-2 stimulates Apoptin-induced apoptosis. Adv Exp Med Biol,1999,457:245-249.
27. Danen-Van Oorschot AA, Zhang Y, Erkeland SJ, et al. The effect of Bcl-2 on Apoptin in'normal'vs transformed human cells. Leukemia,1999,13(Suppl 1): S75-S77.
28. Liu X, Elojeimy S, El-Zawahry AM, et al. Modulation of ceramide metabolism enhances viral protein apoptin's cytotoxicity in prostate cancer. Mol Ther,2006, 14(5):637-646.
29. Lin B, Kolluri SK, Lin F, et al. Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3. Cell,2004,116(4):527-540.
30. van der Eb MM, Pietersen AM, Speetjens FM, et al. Gene therapy with apoptin induces regression of xenografted human hepatomas. Cancer Gene Ther,2002, 9(1):53-61.
31. Ferreira CG, Tolis C, Giaccone G. p53 and chemosensitivity. Ann Oncol,1999, 10(9):1011-21.
32. Rohn JL, Zhang YH, Aalbers RLJ, et al. A tumor-specific kinase activity regulates the viral death protein Apoptin. J Biol Chem,2002,277(52):50820-50827.
33. Lee YH, Cheng CM, Chang YF, et al. Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization but partially affects its apoptotic activity. Biochem Biophys Res Commun,2007,354(2):391-395.
34. Maddika S, Panigrahi S, Wiechec E, et al. Unscheduled Akt-triggered activation of cyclin-dependent kinase 2 as a key effector mechanism of apoptin's anticancer toxicity. Mol Cell Biol,2009,29(5):1235-1248.
35. Maddika S, Wiechec E, Ande SR, et al. Interaction with PI3-kinase contributes to the cytotoxic activity of apoptin. Oncogene,2008,27(21):3060-3065.
36. Liu X, Zeidan YH, Elojeimy S, et al. Involvement of sphingolipids in apoptin-induced cell killing. Mol Ther.2006,14(5):627-636.
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41. Zheng L, Schickling O, Peter ME, et al. The death effector domain-associated factor plays distinct regulatory roles in the nucleus and cytoplasm. J Biol Chem, 2001,276(34):31945-31952.
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46. Olijslagers SJ, Zhang YH, Backendorf C, et al. Additive cytotoxic effect of apoptin and chemotherapeutic agents paclitaxel and etoposide on human tumour cells.Basic Clin Pharmacol Toxicol,2007,100(2):127-131.
47. Peng DJ, Sun J, Wang YZ, et al. Inhibition of hepatocarcinoma by systemic delivery of Apoptin gene via the hepatic asialoglycoprotein receptor. Cancer Gene Ther,2007,14(1):66-73.
48. Lian H, Jin N, Li X, et al. Induction of an effective anti-tumor immune response and tumor regression by combined administration of IL-18 and Apoptin. Cancer Immunol Immunother,2007,56(2):181-192.
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50. Schoop RA, Baatenburg de Jong RJ, Noteborn MH. et al. Apoptin induces apoptosis in an oral cancer mouse model. Cancer Biol Ther,2008,7(9): 1368-1373.
51. Sun J, Yan Y, Wang XT, et al. PTD4-apoptin protein therapy inhibits tumor growth in vivo. Int J Cancer,2009,124(12):2973-2981.
52. Tolboom TC, Zhang YH, Henriquez NV, et al. Fibroblast-like synoviocytes from patients with rheumatoid arthritis are more sensitive to apoptosis induced by the viral protein, apoptin, than fibroblast-like synoviocytes from trauma patients. Clin Exp Rheumatol,2006,24(2):142-147.
2.感染Vero细胞后,可检测到VP3基因的转录和蛋白表达；
3.构建的重组腺相关病毒rAAV-VP3在体外能够发挥生物学活性,其介导的凋亡素表达生物学效应为下一步实验奠定了基础。
1. Flotte TR. Gene therapy progress and prospects:recombinant adeno-associated virus (rAAV) vectors. Gene Ther,2004,11(10):805-810.
2.吴小兵,董小岩,伍志坚,等.一种快速高效分离和纯化重组腺病毒伴随病毒载体的方法.科学通报.2000,45(19)：2071-2075.
3. Noteborn MH. Chicken anemia virus induced apoptosis:underlying molecular mechanisms. Vet Microbiol,2004,98(2):89-94.
4. Noteborn MH. Apoptin acts as a tumor-specific killer:potentials for an anti-tumor therapy. Cell Mol Biol,2005,51(1):49-60.
5.王春晖,王剑松,詹辉,等.改良型TAT-凋亡素体外抗人膀胱肿瘤活性的研究.中国肿瘤临床,2011,38(5)：246-249.
6.潘建刚,周兴,李伟,等.基因重组腺相关病毒介导内皮抑素治疗裸鼠膀胱癌.中华实验外科杂志,2010,27(3)：285－286.
7. Pruthi RS, Baldwin N, Bhalani V, et al. Conservative management of low risk superficial bladder tumors. J Urol,2008,179(1):87-90.
1.构建了原核表达载体pET8a-VP3,在IPGT诱导下表达VP3蛋白并对其进行纯化,获得高纯度的VP3蛋白；
2.将纯化后的VP3蛋白与弗氏完全佐剂或不完全佐剂乳化后,通过对新西兰大耳白兔进行皮下多点注射,分离抗血清进一步采用Protein A纯化总IgG,最终纯化后的抗体效价可以达到1：243,000；
3.利用免疫荧光技术检测VP3基因在人膀胱癌细胞株T24、EJ细胞以及Vero细胞中的表达情况,观察到凋亡素在T24、EJ细胞中主要定位于细胞核,而在Vero细胞中则定位于细胞质；
4.通过Western blotting检测纯化后的抗体能与细胞内腺相关病毒介导表达的凋亡素蛋白特异性结合；
5.实验研究证明了制备的凋亡素蛋白多克隆抗体的有效性和特异性,为进一步阐明凋亡素抗肿瘤效应的分子机制及生物学特性奠定了基础。
1. Noteborn MH. Apoptin acts as a tumor-specific killer:potentials for an anti-tumor therapy. Cell Mol Biol,2005,51(1):49-60.
2. Backendorf C, Visser AE, de Boer AG, et al. Apoptin:therapeutic potential of an early sensor of carcinogenic transformation. Annu Rev Pharmacol Toxicol,2008, 48:143-169.
3. Rohn JL, Noteborn MM. The viral death effector Apoptin reveals tumor-specific process. Apoptosis,2004,9(3):315-322.
4.王文举,孙茂盛,严敏,等Neurturin嵌合神经营养因子重组腺病毒的构建及体外活性测定.中国生物工程杂志,2010,30(9)：7-12.
5. Schoop RA, Kooistra K, Baatenburg De Jong RJ, et al. Bcl-xL inhibits p53-but not apoptin-induced apoptosis in head and neck squamous cell carcinoma cell line. Int J Cancer.2004,109(1):38-42.
6. Noteborn MH. Chicken anemia virus induced apoptosis:underlying molecular mechanisms. Vet Microbiol,2004,98(2):89-94.
7. Danen-Van Oorschot AA, Zhang YH, Leliveld SR, et al. Importance of nuclear localization of apoptin for tumor-specific induction of apoptosis. J Biol Chem,2003, 278(30):27729-27736.
8. Rohn JL, Zhang YH, Aalbers RLJ, et al. A tumor-specific kinase activity regulates the viral death protein Apoptin. J Biol Chem,2002,277(52):50820-50827.
9. Leliveld SR, Dame RT, Mommaas MA, et al. Apoptin protein multimers form distinct higher-order nucleoprotein complexes with DNA. Nucleic Acid Res,2003, 31(16):4805-4813.
10. Leliveld SR, Dame RT, Rohn JL, et al. Apoptin's functional N-and C-termini independently bind DNA. FEBS Lett,2004,557(1-3):155-158.
11. Liu X, Zeidan YH, Elojeimy S, et al. Involvement of sphingolipids in apoptin-induced cell killing. Mol Ther.2006,14(5):627-636.
12. Wadia JS, Wagner MV, Ezhevsky SA, et al. Apoptin/VP3 contains a concentration-dependent nuclear localization signal (NLS), not a tumorigenic selective NLS. J Virol,2004,78(11):6077-6078.
13.王春晖,王剑松,詹辉,等.改良型TAT-凋亡素体外抗人膀胱肿瘤活性的研究.中国肿瘤临床,2011,38(5)：246-249.
14. Heilman DW, Teodoro JG, Green MR. Apoptin nucleocytoplasmic shuttling is required for cell type-specific localization, apoptosis, and recruitment of the anaphase-promoting complex/cyclosome to PML bodies. J Virol,2006,80(15): 7535-7545. 瘤细胞凋亡的作用机制之一；
6. Elisa法检测感染rAAV-VP3后T24和EJ细胞中激活型caspase-3的含量明显增高,caspase-3的激活是凋亡素诱导的细胞凋亡通路中的关键分子信号之一
1. van der Eb MM, Pietersen AM, Speetjens FM, et al. Gene therapy with apoptin induces regression of xenografted human hepatomas. Cancer Gene Ther,2002,9(1): 53-61.
2. Ferreira CG, Tolis C, Giaccone G. p53 and chemosensitivity. Ann Oncol,1999, 10(9):1011-1021.
3. Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med,1997,3(8):917-921.
4. Altieri DC, Marchisio PC. Survivin apoptosis:an interloper between cell death and cell proliferation in cancer. Lab Invest,1999,79(11):1327-1333.
5. Tamm I, Wang Y, Sausville E, et al. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res,1998,58(23):5315-5320.
6. Ku JH, Kwak C, Lee HS, et al. Expression of survivin, a novel inhibitor of apoptosis, in superficial transitional cell carcinoma of the bladder. J Urol,2004, 171(2 Pt 1):631-635.
7. Swana HS, Grossman D, Anthony JN, et al. Tumor content of the antiapoptosis molecule survivin and recurrence of bladder cancer. N Engl J Med,1999,341(6): 452-453.
8. Sharp JD, Hausladen DA, Maher MG, et al. Bladder cancer detection with urinary survivin, an inhibitor of apoptosis. Front Biosci,2002,7:e36-41.
9. Noteborn MH. Apoptin acts as a tumor-specific killer:potentials for an anti-tumor therapy. Cell Mol Biol,2005,51(1):49-60.
10. Danen-Van Oorschot AA, van Der Eb AJ, Noteborn MH. The chicken anemia virus-derived protein apoptin requires activation of caspases for induction of apoptosis in human tumor cells. J Virol,2000,74(15):7072-7078.
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1.重组腺相关病毒rAAV-VP3能明显抑制裸鼠皮下移植瘤生长,有较高的肿瘤抑制率,并能减少肿瘤对周围组织的侵犯与瘤栓形成；
2.重组腺相关病毒rAAV-VP3对荷瘤裸鼠基因治疗后,能提高裸鼠的存活率,生存中位数时间为55天,而MMC治疗组生存中位数时间为46天,治疗副作用方面与MMC治疗组相比具有一定的优越性；
3.瘤内注射重组腺相关病毒rAAV-VP3后,可以观察到VP3蛋白表达量呈逐渐升高的趋势,于21天左右达到顶峰,以后一直维持强阳性表达；
4.免疫组化检测发现重组腺相关病毒rAAV-VP3可抑制Ki-67、C-erbB-2的表达,并能上调肿瘤转移抑制基因Rb、nm23的表达,可能是凋亡素抑制膀胱癌的作用机制之一。
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