Gene Transfer Mediated Through Adeno-associated Virus Induces KiSS-1/p27～(Kip1) In Adult MG-63 Cells In Vitro

[Objective] The aim of this study is To construct vectors based on adeno-associated virus type2 (rAAV2) carrying KiSS-1/p27Kip1 and observe their expression in humen osteosarcoma cells, To investigate the proliferation and survival ability of osteosarcoma cell in vitro after gene transfection for gene therapy of osteosarcoma.[methods] 1. To construct vectors based on adeno-associated virus carrying KiSS-1/ p27Kip1. 2. rAAV-KiSS-1, rAAV-p27Kip1 were transferred into packaging cell line HEK-293,then harvested two different adeno-associated virus, examined the transfection efficiency by histochemical stain. 3. The medium containing virus particles was used for transducing MG-63 cell . 4. To examine the expression of KiSS-1/p27Kip1 by immuocytochemistry. 5.To examine the effect of the transfection in vitro by cell cell proliferation,cycle detection, and estimate the feasibility of the gene therapy. [results] 1. Vector construction:Gene KiSS-1/p27Kip1 was extract from the placenta and connected into the vector pAAV-MCS through T vector. The result was correct by sequencing. 2. Package,harvest,transduce: The CPE phenomenon can be examined by the microscope after transfection. Many blue-stain cells were found after X-gal histochemical stain, and the transfection efficiency is 50%. 3.Titre determination: The titre of virus can be determinateD by CPE phenomenon, and we can get the titre which was 1.8×108 PFU/ml. 4. Viral infection: The cell proliferation was observed to be slow down, and small amounts of cells died after infection. 5. Expression determination by immuocytochemistry: The expression of KiSS-1/ p27Kip1 protein in infected osteosarcoma cell lines increase compared with control group. 6. Effect of the transfection: The cell proliferation was slow down compared with control group. And the cell ratio of G2/M after rAAV-KiSS-1 infection increased compared with control group, and the dead cells were increased compared with control group; the cell ratio of S,G2 after rAAV-p27Kip1 infection decreased compared with control group.[conclusions]1.Construct vectors based on adeno-associated virus carrying KiSS-1/ p27Kip1, Collect two kinds of recombinated adeno-associated viral particle with high titer and infectivity by transduce. 2. After infection of the rAAV-KiSS-1/ rAAV-p27Kip1, KiSS-1/p27Kip1 can be observed high expression in MG-63 cell line by immunocytochemistry. 3. KiSS-1 inhibits MG-63 cell line proliferation by blockage G2/M cell cycle and induces cells dead. 4. p27Kip1 inhibits MG-63 cell line proliferation by blockage G1/S cell cycle.