| PART 1 THE LOADING CAPACITY OF PCNS FOR PENTR-SHBMP9 AND THE CYTOTOXICITY OF PCNS IN HEPG2 CELLSObjective: To investigate the loading capacity of PCNs for pENTR-shBMP9 and the cytotoxicity of PCNs in Hep G2 cells.Methods: The loading capacity of PCNs for pENTR-shBMP9 was tested by agarose gel electrophoresis,and PCNs was transfected into HepG2 cells,the cytotoxicity of PCNs in HepG2 cells was detected by CCK-8 assay.Results: When the mass ratio of PCNs / pENTR-shBMP9 reached 60: 1,the absorbency reached 100%,and no excess plasmids were observed.After 48 hours of treatment,PCNs had the highest cytotoxic effect in Hep G2 cells and then returned to baseline after 72 hours of treatment.The survival rate of Hep G2 cells was 97.7% at the concentration of 2?g / ?l.The survival rate of HepG2 cells was still> 85%(85.0 ± 0.23%)after 48 hours,even though the concentration of PCNs was 4?g / ?l,indicating that PCNs were less cytotoxic to HepG2 cells.Conclusions: The optimal mass ratio of PCNs carrying pENTR-shBMP9 was 60: 1,and the cytotoxicity of PCNs in HepG2 cells is low.PART 2 BIOLOGICAL EFFICIENCY OF PCNS/ PENTR-SHBMP9 TRANSFECTION IN HEPG2 CELLSObjective: To investigate the biological efficiency of PCNs / p ENTR-sh BMP9 transfection in Hep G2 cells.Methods: PCNs / p ENTR-sh BMP9 were transfected into Hep G2 cells at a concentration gradient of 75: 1.25,150: 2.5 and 300: 5 ?g / ?g and cultured for 12 h,24 h,48 h and 72 h respectively.The optimal inhibitory concentration and time of PCNs / p ENTR-sh BMP9 transfection in Hep G2 cells were determined by PCR and WB.The biological efficiency of PCNs / p ENTR-sh BMP9 transfection into Hep G2 cells was tested by PCR and WB when compared with Lipofectamine 2000 transfected sh BMP9.Results: At the mass ratio of 150?g: 2.5?g and 48 hours after transfection,the expression of m RNA and protein of BMP-9 showed significant inhibitory effect(p?0.01).Compared with lipofectamine 2000 / sh BMP9 transfection,PCNs/ p ENTR-sh BMP9 induced BMP-9 inhibition in m RNA and protein levels was more obvious than that of lipofectamine 2000/sh BMP9 in Hep G2 cells(p?0.01),indicating that the transfection efficiency of PCNs / p ENTR-sh BMP9 was higher than that of lipofectamine 2000 / sh BMP9 in Hep G2 cells in vitro.Ccnclusion: When PCNs / p ENTR-sh BMP9 mass ratio was 150?g: 2.5?g and 48 hours of transfection,the expression of BMP-9 m RNA and protein were significantly inhibited.The transfection efficiency of PCNs / p ENTR-sh BMP9 was higher than that of lipofectamine 2000 / sh BMP9 in vitro.PART 3 BIOLOGICAL EFFICIENCY OF PCNS/ PENTR-SHBMP9 TRANSFECTION IN INSULIN RESISTANCE ANIMAL MODELObjective: To investigate the biological efficiency of PCNs / p ENTR-sh BMP9 transfection in insulin resistance animal model.Methods: The C57BL/6J mice were randomly divided into 4 groups(5 mice per group).After fasting for 10-12 hours,PCNs,PCNs/ p ENTRsh BMP9,or p ENTR-sh BMP9 was given by tail vein at week 11 of receiving the HFD.The optimal inhibitory concentration and time of PCNs / p ENTR-sh BMP9 transfection in C57 mice were determined by PCR and WB.The biological efficiency of PCNs / p ENTR-sh BMP9 transfection into C57 mice was tested by PCR and WB when compared with p ENTR-shBMP9 transfection.Results: when the mass ratio is 150:2.5?g/?g(PCNs: p ENTR-sh BMP9),BMP-9 expression in m RNA and protein levels in the liver was lower than other mass ratio(p?0.01).In addition,the most significant inhibition of hepatic BMP-9 expression was found on day 5(p?0.01).PCNs/ p ENTR-sh BMP9 treatment led to a significant decrease of hepatic BMP-9 expression in both m RNA and protein levels compare with p ENTR-sh BMP9 treatment(p?0.01).Ccnclusion: when the mass ratio(PCNs: p ENTR-sh BMP9)is 150:2.5 ?g/?g,PCNs/p ENTR-sh BMP9 resulted in the highest inhibition rate of BMP-9 m RNA and protein expression on day 5 of tail vein injection in mice,and the transfection efficiency of PCNs/ p ENTR-sh BMP9 is higher than that of p ENTR-sh BMP9 in vivo.PART4 THE EFFECTS OF PCNS/ PENTR-SHBMP9 TRANSFER ON INSULIN SIGNALING PATHWAY IN VIVOObjective: To investigate the effects of PCNs/ p ENTR-sh BMP9 transfer on insulin signaling pathway in vivoMethods: The C57BL/6J mice were randomly divided into 4 groups(5 mice per group).After fasting for 10-12 hours,PCNs,PCNs/ p ENTRsh BMP9,or p ENTR-sh BMP9 was given by tail vein at week 11 of receiving the HFD.The expression of PEPCK,p-IR,p-AKT,IR and AKT were detected by WB.Results: PEPCK protein levels in PCNs/ p ENTR-sh BMP9-transfected mice are significantly higher than in p ENTR-sh BMP9-treated mice(p?0.01).The phosphorylation of Ins R and Akt was also significantly decreased in PCNs/p ENTR-sh BMP9-mice compare with p ENTR-sh BMP9-mice(p?0.01).Conclusions: The biological effects of PCNs/p ENTR-sh BMP9 on the insulin signaling pathway are stronger than that of p ENTR-sh BMP9 in vivo. |
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