Inhibition of mda-7/IL-24 recombinant adenovirus on proliferation of small cell lung cancer NCI-H446 cells
- VernacularTitle:mda-7/IL-24腺病毒对小细胞肺癌NCI-H446细胞增殖的抑制作用研究
- Author:
Qunfeng MA
;
Hong JIANG
;
Kun LIU
- Publication Type:Journal Article
- Keywords:
interleukin-24;
melanoma differentiation-associated gene-7;
recombinant adenovirus;
caicinaoma, small cell;
cell proliferation
- From:
Medical Journal of Chinese People's Liberation Army
2001;0(07):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare recombinant mda-7/IL-24 adenovirus to study its function on the proliferation of small cell lung cancer NCI-H446 cells. Methods According to the manufacturer's instructions of AdEasy vector system, mda-7/IL-24 cDNA was subcloned into the adenoviral shuttle vector pAdTrack-CMV. The efficient recombination of adenoviral backbone vector pAdEasy-1 and pAdTrack-CMV-mda-7/IL-24 was achieved in bacteria E. coli BJ 5183. The recombinant adenoviral vector pAd-mda-7/IL-24 linearized by Pac Ⅰ was transfected into HEK293 cells with lipofectamine 2000. To generate higher titer viral stocks, the amplification of recombinant adenovirus was accomplished in packing cells. Viral titers were measured by tissue culture infectious dose 50 (TCID_ 50 ) method. Ad.mda-7/IL-24 was identified by PCR. The expression of pAd-mda-7/IL-24 in NCI-H446 cells was detected by Western-blot analysis. The function of Ad.mda-7/IL-24 on the proliferation of NCI-H446 cells was assayed by MTT after cells were infected by 50 pfu/ml adenovirus. Results The recombinant adenoviral shutter vector pAdTrack-CMV-mda-7/IL-24 and recombinant adenoviral vector pAd-mda-7/IL-24 were constructed successfully as identified by sequence analysis. PCR assay showed that adenovirus Ad.mda-7/IL-24 contained mda-7/IL-24 cDNA. After amplification in packing cell HEK293, the titer of virus was 2?10~ 10 pfu/ml measured by TCID_ 50 assay. Western-blot results identified that MDA-7/IL-24 could be expressed in NCI-H446 cells. After infected by 50 pfu/ml adenovirus, the proliferation of NCI-H446 cells was inhibited by 21.37% with MTT method. Conclusion Ad.mda-7/IL-24 can inhibit the growth of NCI-H446 cell obviously. This result lays foundation to study its function mechanism and to apply it in gene therapy of the small cell lung cancer.
