| Part I The construction and identification of new oncolytic adenovirus M5 and M6[Objective] To construction and identification of two oncolytic adenovirus which express HSV-TK gene[Methods] HSV-TK gene was acquired by PCR and linked to PcDNA3.1+/E3⊿6.7k/gp19k and PcDNA3.1+/E3⊿ADP, M5 and M6 were constructed by homologous recombination. HirtDNA of M5 and M6 was acquired from the infected 293 cells, then PCR and enzyme cut were used to identification M5 and M6.[Results] The sequence of PCR product was identified to be right by sequencing company,PCR was done using the template of virus DNA,and the product was separated on a 1.0% agarose gel,a bond was seen in around 1.2kb, virus DNA was enzyme cut by BanⅢand the product was also separated on a 0.8% agarose gel,and the purpuse bands were seen in around 1.2kb and above 30kb.[Conclusion] oncolytic adenovirus which express HSV-TK gene were constructed successfully.Part II The in vitro effect comparation of oncolytic adenovirus M5, M6 and replication-deficient adenovirus Adv-TK[Objective] To compare the in vitro effect of oncolytic adenovirus M5, M6 and replication-deficient adenovirus Adv-TK[Methods] After tumor cells and normal cells were infected with M5, M6 and Adv-TK, the expression of TK protein was detected by Western blot, the CPE effect was detected by crystallization violet stain, the replication ablity in tumor cells and normal cells was detected by realtime PCR, the lethal effect to normal cells and tumor cells was detected by MTT and flow cytometry.[Results] In tumor cells, the TK protein expression of M5 and M6 was higher than Adv-TK, and the expression of M6 was the highest, however, in normal cells, the TK protein expression of M5 and M6 was much lower than Adv-TK, and there was almost no TK protein detected in M6 infected normal cells; crystallization violet stain showed Adv-TK had no lysis activity to normal and tumor cells, three days after infection, the lysis activity of M5 was higher than M6, but five days after infection, the lysis activity of M6 was higher then M5, even infected with very high MOI, normal cells could not be lysised by M5 and M6; the replication ability of M6 was lower than M5 before 36h after infection, but after 36h, the replication ability of M6 was much higher than M5, and M5 and M6 both could not replicate in normal cells; when tumor cells were infected with high MOI, the lethal effect of M5 and M6 was similar with Adv-TK, but when infected with low MOI, the lethal effect of M5 and M6 was much higher than Adv-TK, moreover the lethal effect of M6 was higher than M5; even infected with very high MOI, there was almost no lethal effect of M6 to normal cells, and the the lethal effect of M5 was much lower than Adv-TK.[Conclusion] When infected with high MOI, the lethal effect of M5, M6 and Adv-TK were similar, when infected with low MOI, the ethal effect of M5 and M6 were higher than Adv-TK, and the ethal effect of M6 was higher than M5, however, in normal cells, the ethal effect of M5 and M6 were much lower than Adv-TK. PartⅢThe anti-tumoral effect comparation of oncolytic adenovirus M5, M6 and replication-deficient adenovirus Adv-TK in nude mice[Objective] To compare the in vivo effect of oncolytic adenovirus M5, M6 and replication-deficient adenovirus Adv-TK[Methods]The orthotopic gastric carcinoma xenograft model was constructed, Ad5/dE1A, M5, M6 and Adv-TK were injected into mice,60mg/kg GCV was injected for fourteen consecutive days, the effect of M5, M6 and Adv-TK to orthotopic gastric carcinoma xenograft and tumor metastasis was compared, and the survival rate was observed, HE stain was used to detect histopathological changes, in situ hybridization was carried out to detect adenovirus replication, IHC was used to detect the expression of adenovirus in tumor, and Western blot was used to detect the expression of TK protein.[Results] M5, M6 and Adv-TK all could inhibit the growth of orthotopic gastric carcinoma, the inhibition rate of M5 and M6 was higher than Adv-TK, and the inhibition rate of M6 was also higher than M5; the inhibit tumor metastasis ability of M5 and M6 was stronger than Adv-TK; compared with other adenovirus, M6 showed the most adenovirus expression and adenovirus replication in tumor, the expression of M5 was higher than that of Adv-TK in tumor; the TK protein of M5 and M6 was higher than Adv-TK; the survival rate of M5 and M6 was higher than Adv-TK, and the survival rate of M6 was also higher than M5.[Conclusion] The inhibition of M5 and M6 to orthotopic gastric carcinoma growth was higher than Adv-TK, and the survival rate of M5 and M6 was higher than Adv-TK too. Part IV Compare the therapy effect and kinetics of oncolytic adenovirus M5, M6 and replication-deficient adenovirus Adv-TK in tumor model of C57BL/6 mice[Objective] To compare the therapy effect and kinetics of M5, M6 and Adv-TK in tumor model of C57BL/6.[Methods] Colon subcutaneous tumor was constructed, and mice were divided into five group randomly, PBS, Ad5/dElA, M5, M6 and Adv-TK was injected into mice,60mg/kg GCV was injected for fourteen consecutive days, the inhibition effect of M5, M6 and Adv-TK to subcutaneous tumor growth was compared, mice were sacrificed at the indicated time points, real-time PCR was used to detect the expression of adenoviruse and TK.[Results] The growth of subcutaneous tumor was inhibited by M5, M6 and Adv-TK, and the inhibition rate of M5 and M6 was higher than Adv-TK, moreover, the inhibition rate of M6 was higher than M6; the time of M5 and M6 stayed in subcutaneous tumor was longer than Adv-TK; and the levels of M5 and M6 reached the second peak seven days after injection, the TK expression of M5 and M6 was higher than Adv-TK,and the TK expression duration of M5 and M6 was also longer than Adv-TK.[Conclusion] The inhibition of M5 and M6 to colon subcutaneous tumor growth was higher than Adv-TK, which was associated with TK expression and adenovirus levels.Part V Assessment of safety of oncolytic adenovirus M5 and M6 in vivo[Objective] To investigate the safety of oncolytic adenovirus M5 and M6 in vivo [Methods] PBS, Ad5/dE1A, M5, M6 and Adv-TK was injected into mice, blood was harvested for hematological indices, hematoxylin and eosin (H&E) stain was carried out to detect the pathological response of organs, real-time PCR was used to detect the adenovirus levels, CD4+ and CD8+ lymphocytes in peripheral blood and spleen was detected by flow cytometry, anti-adenovirus neutralization antibody was detected by TCID50 methods.[Results] Compared with PBS group, a transient slight increase in Cr was detected in M5, M6 and Adv-TK group, there was on obvious pathological change in liver, spleen and kidney; M5, M6 and Adv-TK was all detected in liver, spleen and kidney, the time of M5, M6 and Adv-TK stayed in liver was longer, and the expression level of the three adenovirus was similar, CD4+ and CD8+ lymphocytes in peripheral blood were detected no change after M5, M6 and Adv-TK injected, but CD4+ and CD8+ lymphocytes in spleen were detected a transient slight decrease; the quantity of anti-adenovirus neutralization antibody in M5 and M6 group was similar with Adv-TK.[Conclusion] oncolytic adenovirus M5 and M6 are safe in vivo... |
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