Wang Xiao-xia, Yao Xiao-bao, Ji Xian-sheng, Li Sheng-li, Zhu Hong-liang, Fan Dai-ming
Department of Otolaryngology, 451 Hospital of PLA, Xi'an 710054, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2007 Dec;27(12):1888-91.
To construct a recombinant adenovirus vector carrying antisense heat shock protein 70 (HSP70) cDNA and observe its effect on inhibiting the growth of laryngeal carcinoma Hep-2 cells.
The HSP70 gene fragment encoding the 5' region was cloned reversely into the shuttle plasmid PAdTrack-CMV, and the resultant plasmid was recombined with the backbone plasmid PadEasy-1 in the E.coli Bj5183 cells to generate the recombinant adenovirus vector. The adenovirus were then packaged and amplified in 293 cells, and the viral titer was determined using GFP.
The recombinant adenovirus vector carrying antisense HSP70 cDNA was constructed successfully with a viral titer of 8 x 10(9). HSP70 expression of Hep-2 cells was obviously blocked by antisense HSP70 RNA, and Western blotting and immuohistochemistry demonstrated that cells transfected with antisense HSP70 did not express or express HSP70 at low levels. Flow cytometry presented apoptotic peak in the antisense HSP70-transfected cells, but not in the control cells.
The recombinant adenovirus vector containing antisense HSP70 cDNA can effectively deliver antisense HSP70 gene into Hep-2 cells, suggesting the great potential of this gene therapy strategy in management of human laryngeal carcinoma.
