Inhibitory Effect Of Adenovirus-mediated Mouse Endostatin On Lung Metastasis Of Osteosarcoma-bearing Nude Mice

Objective:Construct the Adenovirus-mediated Mouse Endostatin (Ad-mEndo) tostudy it's inhibitory Effect on angiogenesis and Lung Metastasis ofOsteosarcoma-bearing nude mice.Method: 1 The mEndostatin DNA extracted from vector containing mouse endostatingene was successfully amplified using PCR and cloned into adenovirus vectorPDC315 named PDC315-mEndo,Recombinant adenovirus plasmid PDC315-mEndowas cotransfected with pBGHE3 into 293 packaging cells by lipofectamine 2000 andthe replication-deficient recombinant adenovirus Ad-mEndo, recombinant adenoviruswas purified by CsC1 density purification. Osteosarcoma cells(MG-63)were infectedwith Ad—mEndo in vitro. We used western-blot analysis to measure the expressionof endostatin protein and analyzed their ability to inhibit angiogenesis by CAM. 2To establish translantable model of osteosarcoma-bearing nude mice: Osteosarcimacell line MG-63 (100million each) was inoculated in subcutaneous part of nudemice'right leg in logarithmic growth stage.After osteosarcoma appeared all in aweek,These mice were divided randomly into three groups: Ad—mEndo group (10mice),Ad—EGFP group (10mice),PBS group (10 mice). Besides the three groups,agroup (10 mice) which have not implanted tumor as the healthy controlgroup.According the groups, Ad—mEndo,Ad—EGFP,PBS were injected to themonce a week,200ul each time and for 5 consecutive weeks.Subsequently,the serumendostatin expression level was measured by ELISA analysis.At last, all the micewere killed to observe whether lung metastasis of implanted tumor had appeared.Results The mEndostatin DNA was confirmed by sequence meusurement. The virustiter was 2.5×10¹⁰ pfu/ml. Ad—mEndo can efficiently infected osteosarcomacells and the expressed mEndostatin can inhbit angiogenesis. The tumor volume ofAd—mEndo group was apparently less than that of the other groups,but endostatinexpression level apparently higher than them after treated (P＜0.05). Pasalogicalexamination of the tung tissue of the mice in Ad—mEndo group found no visiblesigns of tumor metastasis,which,in contrast, were widespread in,Ad—EGFP groupand PBS group.Conclusion: Adenovirus-mediated Mouse Endostatin gene had obtained high level ofexpression in vitro and in vivo, and significantly inhibited the angiogenesis and lungmetastasis of osteosarcoma-bearing nude mice, which had proved direct relationship between level of Endostatin and lung metastasis.