ABSTRACT
Objective To construct and identify replication deficient recombinant adenovirus expressing human c-Jun N-terminal kinase(JNK)by homologous recombination adenovirus dominant-negative type JNK(Ad-DN-JNK).Methods The linearized recombinant shuttle vector pAdTrack-CMV-DN-JNK Was co-transformed with backbone vector pAdEasy-l into bacteria BJ5183 for recombinant adenoviral vector.The recombinant adenoviral vector was transfected into HEK293 packing cells tO construct replication deficient recombinant adenovirus,and then the recombinant edenovirns WaS detected by PCR and DNA sequencing.Western blot analysis was utilized to detect the Cxpression of Ad-DN-JNK and the level of insulin receptor substrate l Serine307 phosphorylation.Results JNK recombinant adenoviral vectorcould be effectively transfeeted into HEK 293 cell and successfully packed by intracellular enzyme.The expression of green fluorescent protein(GFP)Was observed on the 5th day after transfection.The fragment of JNK gene waS amplified by PCR and identified by sequencing.The titer of the prepared Ad-DN-JNK is 2.5×1010 pfu/ml.The animal experiment confirmed that constructed Ad-DN-JNK could be effectively expressed in liver tissue.Conclusion The research successfully constructed recombinant adenoviral vector and recombinant adenoviral particle.Animal experiment demonstrated the Ad-DN-JNK could effectively mediated the expression of DN-JNK gene and down-regulated the level of IRSlscfine307 phosphorylation.The achievement laid a foundation for further investigation of the function and application of JNK.
ABSTRACT
Objective To construct a rat model of nonalcoholic fatty liver disease (NAFLD) by feeding rats fat-rich diet and analyze the effect of insulin resistance (IR)in the development of NAFLD. Methods Male SD rats were randomly divided into normal diet group (NG, n =24) and fat-rich diet group (FG, n =24). At the end of feeding for2 weeks, 4 weeks, 6 weeks or 8weeks, 6 rats in NG and FG were randomly took out. Their weight were recorded, then the serum fasting blood sugar, fasting insulin, triglyceride, total cholesterol, ala-nine aminotransferase and aspartate aminotransferase were measured, and the fasting insulin resistance index and liver index (liver weight (g)/body weight(g) × 100%)was calculated. Then liver tissues were homogenized, and muleic dialdehyde and superoxide dismutase were determined. The hepatic steatosis in all rats was assessed according to the results under light microscope. Results The body weight of rats in NG increased faster than those in FG after six weeks. The liver index of rats in FG was markedly higher than that in NG since the second weekend. The rats in FG began to have hepatocyte steatosis from the second weekend, had insulin resistance, hyperlipidemia, dysfunction of liver and lipid peroxide of liver from the fourth weekend, suffered mild fatty liver from the sixth weekend, and developed to moderate fatty liv-er from the eighth weekend. Conclusions NAFLD with IR model was successfully developed by feeding SD rats with an improved rich-fat diet for 6 weeks. IR may play an important role in the development of NAFLD.
ABSTRACT
Objective To construct replication deficient recombinant adenovirus expressing human c-Jun N-terminal kinase by homologous recombination.Methods The linearized recombinant shuttle vector pAdTrack-CMV-WT-JNK was co-transformed with backbone vector pAdEasy-1 into bacteria BJ5183 for recombinant adenoviral vector.The recombinant adenoviral vector was transfected into HEK293 packing cells to construct replication deficient recombinant adenovirus,and then the recombinant adenovirus was detected by PCR and DNA sequencing.Results JNK recombinant adenoviral vector was effectively transfected into HEK 293 cells and was successfully packed by intracellular enzyme.The expression of green fluorescent protein(GFP)was observed on the 5th day after transfection.The fragment of JNK gene was amplified by PCR and identified by sequencing.The animal experiment confirmed that Ad-WT-JNK was effectivety expressed in liver tissue. Conclusion The research successfully constructed recombinant adenoviral vector and recombinant adenoviral particle.And the achievement laid a foundation for further investigation of the function and application of JNK.