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Objective To observe the effect of a new cell delivery tool(MSC exo)on the proliferation of pancreatic cancer by transferring targeted genes.Methods Transmission Electron Microscope(TEM)and Nanoparticle Tracking Analysis(NTA)were used to identify human mesenchymal stem cell exosomes(MSC-exo)and transport miR-450a-5p into CFPAC-1,to explore the effect of miR-450a-5p targeting BZW2 on inhibiting the proliferation of pancreatic cancer cells.Results The expression of miR-450a-5p was low in pancreatic cancer tissue(P<0.05),and the expression of CD63 and TSG101 of MSC-exo-miR-450a-5p in CFPAC-1 cells was higher than that of MSC-exo by Western blot(P<0.05).CCK-8 and EdU results showed that MSC-exo-miR-450a-5p significantly inhibited the proliferation of CFPAC-1 cells(P<0.05).Cell scratch and Transwell experiments showed that MSC-exo-miR-450a-5p can inhibit the migration and invasion of CFPAC-1 cells(P<0.05).Through dual luciferase assay,it was confirmed that miR-450a-5p targets BZW2,and RT-qPCR and Western blotting showed a negative correlation(P<0.05)between miR-450a-5p and BZW2 expression.Overexpression of BZW2,CCK-8,EdU,cell scratch,and Transwell experiments confirmed that pc-BZW2 reversed the anti-cancer function of MSC-exo-miR-450a-5p on CFPAC-1.Western blot detected PCNA,Ki-67,MMP2,MMP9,and the results were consistent with the above experiments(P<0.05).Conclusion hMSC exo is a new delivery system,targeting BZW2 to transport miR-450a-5p to inhibit the biological malignancy of pancreatic cancer cells,which provides an important clue for the research of targeted treatment of pancreatic cancer.
RESUMO
According 1o the genome sequences of α.β,e,ι toxins of Clostridium perfringens in GenBank,four pairs of primers targeting α,β,ε,ι toxin genes were designed.After the multiplex PCR reaction condition was optimized,the multiplex PCR for identification and toxintyping of C.perfringens strains was developed.The specificity test showed that the expected fragments of C.perfringens reference strains including A.B,C,D,E five toxin types were amplified successfully from genomic DNA of C.perfringens,respectively.However,a band could not be amplified from Clostidrium novyi and Clostridium septicum as negative control groups.The sensitivity test showed that the limit detection of multiplex PCR was 9.0,17.8,12.2,13.8,18.5 pg DNA of A,B,C,D,E five toxin types C.perfringens,respectively.Repetitive testing showed that the established method had a good repeatability.Nine type A strains of and 1 type C strains of C.Perfringens from 21 clinical samples of dead goat were detected by the multiplex PCR developed in this study.This study establishes the multiple PCR method which not only can detect C.perfringens rapidly but also can identify five toxin types of C.perfringens.