阻断TGF-β信号通路增强NK细胞过继性治疗乳腺癌的体外效应

Objective: Construction of pTAR-GET-DNTβRⅡ eukaryotic expression vector and transfection it into NK cells. Investigate the antitumor effeet of blocking TGF-β insensive NK cells adoptive immunotherapy of breast cancer in vitro. Methods: The expression of TβRⅠand TβRⅡin parental NK cells were detected by fluorescence activated cell sorter. Mean levels of TGF-β1 secreted by 3 human breast cancer cell lines (MCF-7, MDA-MB231 and T47D) were determined by ELISA. Transfect pTAR-GET-DNTβRⅡ eukaryotic expression vector to NK cells using Amaxa Nucleofector technology. RT-PCR and inverted fluorescent microscope were used to identify the expression of DNTβRⅡ, then western blot was used to detect the phosphorylated satus of Smad2 and Smad3. The eytotoxieity of NK cells against MCF-7 cells was detected and analyzed by CCK-8 kit. Results: The highest level of TGF-β1 was secreted by MCF-7 cell line. High expression of TβRⅠand TβRⅡwas identified by FACS. The expression of DNTβRⅡin NK cells was confirmed by inverted fluorescent microscope and RT-PCR. By the irritation of TGF-β1, Western blot detected inactive Smad2 and Smad3 status in NK cells transfected with pTAR-GET-DNTβRⅡ. Parental NK cells displayed lower cytotoxity against MCF-7 incubated with TGF-β1 than that without TGF-β1, but DNTβRⅡ plasmid transfection unchanged NK cells activity in vitro. Conclusion: Eukaryotic expression plasmid pTAR-GET-DNTβR Ⅱ transfection blocking the NK TGF-β cell signaling pathways at receptor level. Blocking TGF-βsignaling pathway can enhance the NK adoptive treatment effects of breast cancer cell lines in vitro.