Construction Of A Human Adiponectin Expression Vector And Its Expression In Vitro

Background: With the discovery of adipose factors, it is suggestedthat adipose tissue serves not only as an energy reservoir but also anendocrine organ. Adiponectin is exclusively expressed in differentiatedadipocytes. It is proved to have the function of enhancing fatty acidoxidation, reverse insulin resistance and resist atherosclerosis in vitro andin vivo. Decreased expression and lower level of adiponectin correlateswith obesity, type 2 diabetes and cardiovascular diseases. Heterozygousadiponectin-deficient (adipo+/-) mice showed mild insulin resistance, whilehomozygous adiponectin-deficient (adipo-/-) mice showed moderateinsulin resistance with glucose intolerance. Moreover, adipo(-/-) miceshowed 2-fold more neointimal formation in response to external vascularcuff injury than wild-type mice . This study provides the first directevidence that adiponectin plays a protective role against insulin resistanceand atherosclerosis in vivo. Many research indicate that the replenishmentof adiponectin might provide a novel treatment modality for obesity, type 2diabetes and atherosclerosis. Type 2 diabetes usually coexists with insulinresistance, hypertension, dyslipideamia, and chronic low level ofinflammation. The constellation of these metabolic components is generallyreferred to metabolic syndrome and these are the atherosclerosis riskfactors, and they might share a common mechanism. So it is of relevance toconstruct the adiponectin gene expression vector for further study of itsfunction, the control of gene expression and for its possible application ingene theraphy. Perspectives: To construct an human adiponectin gene expressionplasmid and detect if it can express in cell line after being transfected toeukaryote. To discuss whether the plasmid is being successfully constructedand can be used for further research. Methods: 1.Construction of plasmid pMD18-hAPN. Total RNA were extractedfrom human adipose tissue. Primers were designed according to thepublished sequence. The fragement from Xhoâ… and Hind III digestion ofthe RT-PCR production was cloned to the plasmid pMD18-T simple Vector.The resultant plasmid was designed as pMD18-hAPN. Direct sequencingwas undertaken to check the nucleotide sequence of the insert DNAfragement. 2.Construction of plasmid pLNCX-hAPN. The hAPN fragment fromXhoâ… and Hind III digestion of plasmid pMD18- hAPN was cloned to theXhoâ… and Hind III digested plasmid pLNCX2. The final plasmidpLNCX2-hAPN was thus constructed. Direct sequencing was undertakento check the nucleotide sequence of the insert hAPN fragement. 3 . Cell transfection and examination of expression. PlasmidpLNCX2-hAPN and pLNCX2 were transfected into cells of HFL by the aidof lipofectant, respectively. After selection with Geneticin(G418) in culturemedium, the cells were used to extract total RNA. Human adiponectinmRNA was detected with reverse transcription-polymerase chainreaction(RT-PCR). Results: 1.Direct sequencing of the final plasmid showed that the direction ofinsertion and nucleotide sequence of the inserted human adiponectin cDNAwere correct. 2.Human adiponectin mRNA could be transcribed from the plasmidas proved with the results of RT-PCR of the RNA from the transfectedcells. Conlusions: An adiponectin gene plasmid vector for expression ineukaryote was constructed.