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

Inhibition of mda-7/IL-24 recombinant adenovirus on proliferation of small cell lung cancer NCI-H446 cells

目的构建mda-7/IL-24腺病毒,检测其对小细胞肺癌NCI-H446细胞增殖的影响。方法将mda-7/IL-24cDNA亚克隆至腺病毒穿梭质粒载体pAdTrack-CMV中,在大肠杆菌BJ5183中将重组病毒穿梭质粒和骨架质粒重组。在HEK293细胞中进行病毒包装和扩增,用PCR方法鉴定后,通过TCID50法测定病毒滴度。用滴度为50pfu/细胞的重组腺病毒Ad·mda-7/IL-24感染NCI-H446细胞72h后,经MTT实验检测其对细胞增殖的影响,并用Westernblot方法检测mda-7/IL-24在NCI-446中的表达。结果核酸序列测定和PCR鉴定表明成功构建Ad·mda-7/IL-24;TCID50法测定扩增的腺病毒滴度为2×1010pfu/ml。Ad·mda-7/IL-24在NCI-H446细胞中表达MDA-7/IL-24,MTT检测表明Ad·mda-7/IL-24明显抑制NCI-H446细胞生长,抑制率为21·37%。结论重组腺病毒Ad·mda-7/IL-24能显著抑制NCI-H446细胞增殖,为研究mda-7/IL-24的作用机制及将其用于小细胞肺癌的基因治疗提供了条件。

Objective To prepare recombinant mda-7/IL-24 adenovirus to study its function on the proliferation of small cell hmg 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 （TCIDs0） 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 adenoviru~ Rmdts 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 2×10^30pfu/ml measured by TCIDs0 assay. Western-blot resuits identified that MDA-7/IL-24 could be expressed in NCI-H446 cells. After infected by 50 pfu/ml adenovirus, the proliferation of NCIH446 cells was inhibited by 21. 37% with MTT method. C_amelusion 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.