Objective： To construct recombinant Apoptin gene （vp3） retrovirus pLVP3 and to study its apoptosis inducing effect on human breast cancer cells 435 as well as to discuss its mechanism in vitro and in vivo. Methods...Objective： To construct recombinant Apoptin gene （vp3） retrovirus pLVP3 and to study its apoptosis inducing effect on human breast cancer cells 435 as well as to discuss its mechanism in vitro and in vivo. Methods： vp3 gene was cloned and recombinated into retrovirus vector pLP-LNCX-VP3 （pLVP3） at loxP site, which was transformed into package cell line PT67 and then into NIH3T3 cells for titer assay. The human breast cancer cell line 435 was infected with retrovirus pLVP3, and then MTT assay and Western blotting were adopted to detect cellular proliferation and Apoptin protein expression. Forty-eight hours after infection flow cytometry （FCM） was used for apoptosis detection and Surface Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometry （SELDI-TOF-MS） was used for protein profile assay. Nude mice model of human breast cancer cells 435 was set up to observe the tumor inhibiting rates of pLVP3, and TUNEL assay was used to detect tumor apoptosis as well as real-time PCR for vp3 gene expression. Results： Recombinant plasmid pLVP3 was successfully constructed. Virus titer reached to 5×10^8 pfu/ml in the PT67 culture supernatant. Forty-eight hours after infection, cellular inhibition rate was 65.1% in MTT assay, higher than that in blank control （P〈0.05） and Apoptin protein expressed more in test group in Western blotting. FCM assay showed apoptotic peaks with a percentage of 15.42%. SELDI-TOF-MS findings suggested that two protein peaks, M_2544.1＋H and M_3712.4＋H, were statistically different between infection group and control group （P〈0.05）. The tumor inhibition rates in pLVP3 group were 65.52% and 68.23%, much higher than that of control group （t=4.06, P〈0.01）. TUNEL assay findings showed that positive yellow stains were seen in pLVP3 retrovirus group and 5-FU positive control group without difference （t1=1.05, t2=0.84, P〉0.05）. Conclusion： The experiment demonstrated that vp3 could induce apoptosis in tumor cells in vivo and in vitro, which laid a basis for further study on molecular mechanism of tumor cell apoptosis induced by Apoptin and provided valuable reference for tumor gene therapy.展开更多
Apoptin is a chicken anemia virus-derived,p53-independent,bcl-2-insensitive apoptotic protein with the ability to specifically induce apoptosis of various human tumor cells,but not of normal diploid cells.To explore t...Apoptin is a chicken anemia virus-derived,p53-independent,bcl-2-insensitive apoptotic protein with the ability to specifically induce apoptosis of various human tumor cells,but not of normal diploid cells.To explore the application of apoptin in tumor gene therapy,we used a recombinant fowlpox virus expressing apoptin protein （vFV-Apoptin） to investigate the anti-tumor effectes of vFV-Apoptin on human cervical carcinoma（HeLa） cells in vivo and in vitro through 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide（MTT） assay,acridine orage/ethidium bromide（AO/EB） and annexin V staining test,respectively.The results show that vFV-Apoptin inhibites the proliferation of HeLa cells in vitro through inducing the apoptosis of HeLa cells,and the inhibition effect of vFV-Apoptin has a dose-effect and time-effect relationship.The results of animal models show that vFV-Apoptin significantly inhibits tumor growth,extends the lifespan of animals and improves the mean survival.Experimental results indicate that vFV-Apoptin has a potential application in the tumor gene therapy.展开更多
文摘Objective： To construct recombinant Apoptin gene （vp3） retrovirus pLVP3 and to study its apoptosis inducing effect on human breast cancer cells 435 as well as to discuss its mechanism in vitro and in vivo. Methods： vp3 gene was cloned and recombinated into retrovirus vector pLP-LNCX-VP3 （pLVP3） at loxP site, which was transformed into package cell line PT67 and then into NIH3T3 cells for titer assay. The human breast cancer cell line 435 was infected with retrovirus pLVP3, and then MTT assay and Western blotting were adopted to detect cellular proliferation and Apoptin protein expression. Forty-eight hours after infection flow cytometry （FCM） was used for apoptosis detection and Surface Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometry （SELDI-TOF-MS） was used for protein profile assay. Nude mice model of human breast cancer cells 435 was set up to observe the tumor inhibiting rates of pLVP3, and TUNEL assay was used to detect tumor apoptosis as well as real-time PCR for vp3 gene expression. Results： Recombinant plasmid pLVP3 was successfully constructed. Virus titer reached to 5×10^8 pfu/ml in the PT67 culture supernatant. Forty-eight hours after infection, cellular inhibition rate was 65.1% in MTT assay, higher than that in blank control （P〈0.05） and Apoptin protein expressed more in test group in Western blotting. FCM assay showed apoptotic peaks with a percentage of 15.42%. SELDI-TOF-MS findings suggested that two protein peaks, M_2544.1＋H and M_3712.4＋H, were statistically different between infection group and control group （P〈0.05）. The tumor inhibition rates in pLVP3 group were 65.52% and 68.23%, much higher than that of control group （t=4.06, P〈0.01）. TUNEL assay findings showed that positive yellow stains were seen in pLVP3 retrovirus group and 5-FU positive control group without difference （t1=1.05, t2=0.84, P〉0.05）. Conclusion： The experiment demonstrated that vp3 could induce apoptosis in tumor cells in vivo and in vitro, which laid a basis for further study on molecular mechanism of tumor cell apoptosis induced by Apoptin and provided valuable reference for tumor gene therapy.
基金Supported by the National High-Tech Research and Development Program of China(No.2007AA021004)the National Basic Research Program of China(No.2005CB523005)+1 种基金the National Science and Technology Major Project of China (Nos.2008ZX10004-015,2009ZX08006-002B)the National Natural Science Foundation of China(No.30771609)
文摘Apoptin is a chicken anemia virus-derived,p53-independent,bcl-2-insensitive apoptotic protein with the ability to specifically induce apoptosis of various human tumor cells,but not of normal diploid cells.To explore the application of apoptin in tumor gene therapy,we used a recombinant fowlpox virus expressing apoptin protein （vFV-Apoptin） to investigate the anti-tumor effectes of vFV-Apoptin on human cervical carcinoma（HeLa） cells in vivo and in vitro through 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide（MTT） assay,acridine orage/ethidium bromide（AO/EB） and annexin V staining test,respectively.The results show that vFV-Apoptin inhibites the proliferation of HeLa cells in vitro through inducing the apoptosis of HeLa cells,and the inhibition effect of vFV-Apoptin has a dose-effect and time-effect relationship.The results of animal models show that vFV-Apoptin significantly inhibits tumor growth,extends the lifespan of animals and improves the mean survival.Experimental results indicate that vFV-Apoptin has a potential application in the tumor gene therapy.
