Construction Of Long-acting Stable Expression Baculovirus Gene Delivery System And Its Application In Tumor Anti-angiogenesis Gene Therapy

With the development of molecular biology,gene therapy has become one of the most potential methods for treating tumors after surgery,radiotherapy,and chemotherapy.Because the development and metastasis of primary tumors depend on the generation of new blood vessels,anti-angiogenesis gene therapy has become a very attractive anti-cancer strategy.In order to successfully implement gene therapy,in addition to finding suitable genes,the selection or development of gene delivery vectors is also crucial.In recent years,baculovirus has attracted the attention of many researchers as a gene delivery vector,but baculovirus is easily inactivated and transiently expressed by the serum complement system in mammals,which limits its further application as a gene delivery vector.In order to overcome the shortcomings of the baculovirus gene delivery vector,this study intends to build a long-acting and stable baculovirus gene delivery vector system by using the SB transposition subsystem and surface display technology,using the green fluorescent protein gene eGFP as the target gene,and The decay acceleration factor?DAF?was displayed on the surface of the virus.Flow cytometry,Western blot,laser confocal and immunofluorescence were used to observe the expression efficiency of baculovirus-mediated green fluorescent protein in mammals.To further construct a recombinant baculovirus expressing a fusion protein?hEA?composed of human endostatin and angiostatin,which was breast-fed by ELISA,MTT,scratch,tube formation experiment and mouse liver cancer model The expression efficiency,anti-angiogenesis function and anti-tumor activity in animal cells were studied.In order to further improve the anti-tumor effect of hEA,the recombinant baculovirus expressing hEA was used in combination with the chemotherapy drug gemcitabine to investigate the synergistic effect of the combined drug.The research results obtained are as follows:1.Expression efficiency of baculovirus-mediated eGFP in vivo and in mammals?1?In vitro long-term expression study:Recombinant baculovirus was transduced into HUVEC cells,and long-term stable expression of baculovirus?BacSC-DAF-SB-T2CE?was observed through an inverted fluorescence microscope,which significantly prolonged the expression time of green fluorescent protein?>90 days?,while the number of fluorescent cells in the transient expression baculovirus?BacSC-DAF-CE?group gradually decreased to complete disappearance within 15 days;the total fluorescence intensity?TFI?of Human umbilical vein endothelial?HUVEC?cells was measured by flow cytometry,and the results showed that BacSC-The DAF-SB-T2CE group can continuously detect signals within 90days(from 6.7×10¹⁰ au on the 3rd day to 5×10⁹ au on the 90 th day),while the BacSC-DAF-CE group cannot detect fluorescence after the 15th day?Below the detection limit,the TFI is 10⁶¹0⁷ au?.?2?DAF protein expression:Western Blot results showed that the recombinant viruses BacSC-DAF-CE and BacSC-DAF-SB-T2CE infected Sf-9 cells and purified virus expressed fusion protein at about 70 kDa.The results of laser confocal microscopy showed that DAF protein was successfully expressed on the surface of virus-infected Sf-9 cells.?3?In vitro transduction efficiency study:The transfection efficiency of recombinant virus was verified by serum complement inactivation experiment.As the serum concentration increases,the percentage of eGFP positive cells gradually decreases.Whether it is human serum or mouse serum,BacSC-DAF-CE and BacSC-DAF-SB-T2CE are significantly different from BacSC-CE when the serum concentration is 20%,40%,60%and 80%?P<0.05?,but there was no significant difference between BacSC-DAF-CE and BacSC-DAF-SB-T2CE?P>0.05?.It shows that the recombinant virus displaying DAF protein on the surface can effectively resist the inactivation of serum complement.?4?In vivo transduction efficiency study:Immunofluorescence results showed that 1 day after intratumoral injection of recombinant virus,compared with the negative control BacSC-CE group,the green fluorescence of the BacSC-DAF-CE group and the BacSC-DAF-SB-T2CE group was significantly stronger.5 days after injection,the BacSC-DAF-CE group could not see green fluorescence,while the BacSC-DAF-SB-T2CE group continued to express for at least 15 days.This result indicates that the new recombinant baculovirus can increase the expression level in mammals and effectively extend the eGFP expression time.2.Antitumor study of baculovirus-mediated hEA protein?1?hEA expression efficiency study:ELISA results showed that with the increase of time,the expression level of the BacSC-DAF-ChEA group decreased rapidly,and its expression decreased close to 0 at 9 days,while the BacSC-DAF-SB-T2ChEA group mediated The expression level of hEA also decreased rapidly at the beginning,but the expression was stable from 9 days,and remained at about 100 pg/ml.?2?Anti-angiogenesis ability research:MTT,scratch and tube formation experiments showed that compared with the control group,long-term stable expression of baculovirus BacSC-DAF-SB-T2ChEA and transient expression of baculovirus BacSC-DAF-ChEA both Inhibition of angiogenic activity?P<0.05?;compared with BacSC-DAF-ChEA group,BacSC-DAF-SB-T2ChEA group had stronger anti-angiogenic ability?P<0.05?.?3?Antitumor activity:In the subcutaneous transplanted tumor model,compared with the control group?24 days?,both recombinant viruses can significantly prolong the survival time of mice?42 days?.At the end of the experiment,the survival rate in the BacSC-DAF-ChEA group was 40%,and the survival rate in the BacSC-DAF-SB-T2ChEA group was even higher,at 80%.3.Recombinant baculovirus-mediated combination chemotherapy with gemcitabine for synergistic antitumor studies?1?Compared with the control group,the use of BacSC-DAF-SB-T2ChEA or gemcitabine alone has a good therapeutic effect on subcutaneous liver cancer in mice?P<0.05?,and there is no difference between the two groups of single drugs?P>0.05?.The effect of the combined drug group was stronger than that of the single drug group?P<0.05?.?2?There were metastatic nodules visible to the naked eye in the control group,while there were no metastatic nodules in the other three groups.?3?In order to prove the effect of different groups on tumor proliferation,Ki67 antibody was used to mark the proliferation of tumor cells through immunohistochemistry.The results showed that the expression of Ki67 was highest in the control group,followed by the recombinant virus group and gemcitabine group,and the lowest in the combined drug group.?4?In order to prove the effect of different groups on tumor microvessel density,CD31antibody was used to mark tumor angiogenesis through immunohistochemistry experiments.The results showed that the expression of CD31 in the control group was the highest,followed by the gemcitabine group,and the recombinant virus group and the combination drug group had the lowest expression.In summary,the new recombinant baculovirus gene delivery system constructed in this study can resist complement inactivation and can be stably expressed in mammals in vitro and in vivo.Compared with the transiently expressed baculovirus?BacSC-DAF-ChEA?,the long-acting stable baculovirus expressing hEA?BacSC-DAF-SB-T2ChEA?has stronger anti-angiogenesis in the mouse subcutaneous liver cancer model Ability and anti-tumor ability in the body.When the long-acting stable baculovirus expressing hEA and gemcitabine commonly used in clinical application in the treatment of mouse subcutaneous liver cancer model,it showed a synergistic antitumor effect with gemcitabine.