| AAV（Adeno-associated virus）and LV（Lentivirus）are commonly used viral vectors in gene therapy.At present,gene drugs based on AAV and LV have been successfully put on the market.LHPP is a new tumor suppressor protein discovered in recent years.Recent studies have shown that LHPP has inhibitory effects on rectal cancer,cervical cancer,pancreatic cancer and so on,and most of them are related to PI3K/AKT/m TOR pathway.Although the role of LHPP protein in gastric cancer and lung cancer has not been reported,many literatures have reported that PI3K/AKT/m TOR is closely related to the occurrence and development of gastric cancer and lung cancer.Therefore,in this study,the constructed LHPP gene overexpression plasmid was packaged and recombined to obtain virus particles,and then virus particles were used to infect lung cancer cells and gastric cancer cells to study the effect of LHPP gene overexpression on lung cancer cells and gastric cancer cells.Methods:（1）p AAV2.1-CMV-Lac Znls and p UC57-m LHPP plasmids were digested with enzyme,and the target bands were recovered after running electrophoresis.The target bands were recovered,ligated,transformed and extracted（D7-LHPP）with T4 ligase,and the correctness of each plasmid was identified.（2）p EMBL-AAV-D（+）-CMVGFP4,p AAV2 and p Helper plasmids were added into 293 T cells by three-plasmid cotransfection method,and the transfection rate was determined by flow cytometry to determine the optimal dose of transfection reagent.（3）After the experimental conditions were determined,D7-LHPP,p Helper and p AAV2/2 were packaged in 293 T cells by the same method,and the toxin was collected 72 hours later.（4）The protein was extracted from lung and gastric cancer cells 72 hours after virus infection,and the expression of LHPP was up-regulated by WB.（5）The proliferation of LHPP overexpression cells was detected by CCK8.（6）The effect of LHPP overexpression on tumor cell migration was detected by cell scratch test.（7）Determine the MOI value of lentivirus infected cells,the type of enhancement medium and the working concentration of puromycin.（8）Stable cell lines were selected according to the above conditions;（9）WB was used to detect whether LHPP gene mediated by lentivirus vector was inserted into cells,and whether the expression of LHPP in cells was up-regulated;（10）CCK8 was used to detect the proliferation of cells infected by lentivirus;（11）Scratch test was used to detect whether the migration function of cells was affected after lentivirus infection.Results:（1）The results of restriction endonuclease digestion and sequencing showed that the structure of the recombinant plasmid D7-LHPP was correct,and all the helper plasmids were correct.（2）Flow cytometry showed that the transfection rate was the highest when the transfection reagent dosage was 1.5μL/ well（24-well plate）.（3）The expression of LHPP in tumor cells was up-regulated by WB after tumor cells were infected with adeno-associated virus particles.（4）The up-regulation of LHPP expression mediated by adeno-associated virus vector did not inhibit the proliferation and migration of tumor cells.（5）Integration of foreign LHPP gene into tumor cells was detected by WB after lentivirus infection.（6）The up-regulation of LHPP expression mediated by lentivirus vector did not inhibit the proliferation and migration of tumor cells.Conclusion: The overexpression of LHPP mediated by AAV vectors and LV vectors had no effect on the proliferation and migration of lung cancer cells（H3122,A549,H2228）and gastric cancer cells（AGS,HGC-27,MGC-823）. |
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