Construction Of Recombinant Adenovirus Vector Expressing The Human Endostatin And The Research Of Its Related Function

BackgroundEndometriosis (EMs for short) is one kind of estrogen dependence disease,whose real cause of disease and morbidity mechanism are fairly ambiguous. The aimof clinical therapy at present clinically is focused on alleviating pain, improvingpregnancy rate and delaying recurrence to the greatest extent as far as possible, stillthe medicine treating specifically for cause of disease do not exist, and the diseasealso has no way to get fundamental settlement. Though peritoneoscope operationcures EMS, which is one of the method that surgery cures at present, the operationvalue is difficult to embody in not only low and medium grade EMS, dysgenesia andrelapse patient, but also the existence dispute. In machine-made EMs morbidityresearch, the recipient at present common viewpoint is that Sampson prefers in 1927propose that the "cultivating theory", pointing out ectopia has to fall in newbornblood vessel support, then it is possible maintain the development and maintenanceof endometriosis. The blood vessel generates mechanism already becoming one ofuniversally accepted important EMs morbidity mechanism.Endostatin, the most potent endogenous angiogenic inhibitor at present, iswidely investigated and applied in cancer therapy, while its application in thetreatment of endometriosis is seldom reported. Nowadays, more and more studyresults showed that it can restrain vascular endothelial celI(VEC) distinctively, thisforebodes the enormous potential treatment in EMs.Now the research on the antiangiogenesis with tumor blood vessel generates is paid close attention to, whose proteinum treats and already having entered to clinicalexperimental stage of scheduled timeⅡin abroad. But Endostatin proteide structure isunsteady usually, half-life period is short, and it require many times repetition to giveadministration, whose productive technology has been complicated ,which haverestricted its application greatly. If being able to adopt the gene therapy strategies,making organism become a "factory" producing Endostatin protein, may resolve theabove-mentioned problems completely. Adenovirus vector is usually used to genetherapy having provided a very good approach by broad field, applying to theextracorporeal gene introduction, the vivo vaccination and gene therapy.ObjectiveIn order to go deep into study the therapeutic effect in studying Endostatin inEndometriosis, we intent to construct the recombinant adenovirus vector expressingthe humam Endostatin, and prepared adenovirus particle which have the infectingability using corresponding package cell line. After that, we infected vascularendothelial cell with recombination hEndostatin adenovirus, and observed thepossible influence on VEC. According to the results, we could deeply investigate thefunction and mechanism of Endostatin, and would lay down basis for the research onendostatin's therapeutical effect on EMs using animal model in future.Methods and Results1. Using genetic engineering method to obtain hunman endostatin gene, the genefragment was inserted into the same restrictive enzyme cutting points of a defectadenovirus plasmid vector. Through sequencing analysis, the endostatin gene ofrecombinant plasmid has a point mutation from A to G in the 471 st amino acid base.To compare the endostatin gene data of GenBank, this mutation belongs to a silentmutation type completely and the coded amino acid is still arginine, which can notaffect the protein expression of endostatin.2. The integrated adenovirus particle was obtained by infection package cellHEK293T with linearfizated defect type recombination hEndostatin adenovirusplasmid using Lipofectamine 2,000 liposome. After purification with cesium chloridedensity gradient centrifugation, the virus titer was adjusted to 5.2×10~9pFU/mLdetection with OD260nm assay. 3. ECV-304 cell line was infected with the integrated hEndostatin adenovirus(Ad-hEndo). Western blot was performed to detect the expression of hEndostatin andflow cytometry was to assess the level of apoptosis after Ad-hEndo infection forECV-304 and then different groups' growth curves were drawn, respectively. Tocompared with control groups, amount of hEndostatin were expressed after Ad-hEndoinfection for 48h. And apoptosis were fotmd at day 3 after infection. Cell growthcurve assay also showed that growth velocity was markedly impaired in theAd-hEndo treatment group.ConclusionBy structuring the defect type recombination hEndostatin adenovirus plasmid,we get the integrated hEndostatin adenovirus (Ad-hEndo) with package cell line,which can infect target cell ECV-304 efficiently. After infection, ECV-304 expressedhEndostatin significantly, and the expressive hEndostatin can lead to obviousapoptosis and suppression phenomenon in this cell. These results will establish abasis for researching the therapeutic effect of Ad-hEndo on EMs animal models infuture.