Flt3 Ligand Gene Therapy Of Murine Liver Cancer Mediated By Recombinant Adenovirus Vector

F1t3 Ligand Gene Therapy of Murine Liver Cancer Mediated by Recombinant Adenovirus Vector F1t3 ligand, a recently cloned primitive hematopoietic growth factor, can stimulate hematopoisis in vivo and enhance immulogical function. To investigate the antitumor effect of in situ mFL transduction, adenovirus vector was seclected. First, recombinant adenovirus vector carrying mFL was constructed by using a new simplified bacterial homologous recombinant system. After cotransformation BJ5 183 E.coli. bacteria by lineared recombinant shuttle plasmid and adenovirus backbone plasmid pAdEasy- 1, recombinant adenovirus vector plasmid was generated by homologous recombination, which causes antibiotic change and makes selection easier and quicker. Following transfection 293 cells by lipofectamine, recombinant adenovirus was generated and amplified. After three times of nfection-collection-freeze/thaw? the lyses supematant of 293 was purified by CsCl gradient ultraspeed centrifuge. Titer was determined by GFP expression in infected 293 cells. AdmFL was 8.4x 10ftilml, and Ad-empty (no foreign gene control vector) was 6.2x 10fulml. In vitro infection of murine liver cancer cell line 1-lepal-6 with AdmFL at MOJ 1O~ 100 and 500, can lead to 5 % 60% and 95 % infection rate respectively, measured by GFP expression. There was no direct growth inhibition on Hepal-6 infected by AdimFL at MOI 100. The mRNA of mFL was detected in the infected Hepal-6 by RT-PCR, and the mFL protein was 80.5 ?7.3ng/ml in the infected Hepal-6 cell culture supematant. All data suggested AdmFL can infect Hepal-6 efficiently and mediate the expression of mFL in vitro. Intratumor injection of 1 x 1 O9efu AdmFL had significant therapeutic effect. Establish murine liver cancer model by subcutaneous inoculation 4 2x 106 Hepal-6 on right back flank of C57BL/6 mice. After single intratumor injection of lx 109efuAdmFL~ Ad control and PBS, the tumor mass and survival rate were observed. In contrast with control group, AdmFL can significantly inhibit tumor growth, lead to complete tumor regression in 89% of mice 37 days after treatment and prolong the survival rate. At the same time, AdmFL can confer specific protection against rechallenge of parental Hepal -6, but not EL4, in the other flank of tumor- free mice. 20 days after treatment with AdmFL, the in vitro splenetic CTL activity was greater than the control group and can confer adoptive protection against Hepal-6 . On day 7, pathological staining showed that there were significantly more intratumor inflammatory infiltration and CD8~ T lymphocytes than that of control group. The soluble human F1t3 ligand homologue was cloned from human peripheral mononuclear cells by RT-PCR. Besides the full-length soluble cDNA, two new splicing variant were cloned. Using bacterial homological recombinant system, recombinant adenovirus vector carrying the 540bp soluble hFL gene was constructed, whose titer was 7.6 x l0æ”…fulml. It paved a way for further gene therapy research base on human FL.