ABSTRACT
To construct recombinant eukaryotic expression plasmid vector of human IL-34 gene, and to study the effects of IL-34 expressed by human bone marrow-derived mesenchymal stem cells (hBM-MSCs) on THP-1 cells. Full-length IL-34 encoding sequence was amplified by PCR. And this fragment was cloned into the plasmid pIRES2-EGFP. Western blotting and ELISA were used to analyze the expression of IL-34 in hBM-MSCs. THP-1 cells were cultured with hBM-MSCs medium containing IL-34 protein. Real-time PCR detected the effects of IL-34 on the expression of IL-10 and TNFα in THP-1 cells. Restrictive enzyme analysis and sequencing demonstrated that IL-34 eukaryotic expression vector was successfully constructed. IL-34 protein expressed by hBM-MSCs could promote IL-10 and TNFα expression in THP-1 cells. Those results show that IL-34 expressed by hBM-MSCs has regulating effect on THP-1 cells.
ABSTRACT
Objective To investigate the antitumor effects and the possible mechanisms of dendrit-ic cells co-cultured with cytokine-induced killer cells(DC-CIK)in combination with sorafenib on two lung adenocarcinoma cell lines,A549 cells(harboring KRAS gene mutation)and PC-9 cells(harboring EGFR gene mutation). Methods DC and CIK cells were routinely generated in vitro by stimulating PBMCs isola-ted from lung cancer patients with different cytokines and then co-cultured after a week of culturing. Flow cy-tometry analysis(FCM)was used to analyze the phenotype of DC-CIK cells after 7 days of co-culturing. The 50% inhibitory concentration(IC50 )of sorafenib against tumor cells was detected by MTT assay. The tumor cells were treated with DC-CIK cells alone or in combination with sorafenib. The proliferation of tumor cells was tested by CCK-8 kit and dynamically monitored by real-time cellular analysis(RTCA). Annexin-V/ PI staining was used to examine the apoptosis rates in each group. Real-time fluorescent quantitative PCR and FCM were respectively performed to detect the expression of natural killer group 2 member D ligands (NKG2DLs)at mRNA and protein levels after the treatment with sorafenib for 24 h. Results There was no significant difference between the IC50 of sorafenib against A549 and PC-9 cells after a 24-hour exposure(P﹥0. 05). Compared with the DC-CIK biotherapy,treating the tumor cells with DC-CIK cells in combination with sorafenib significantly inhibited the cell proliferation and increased the total apoptosis rates of tumor cells(P﹤0. 05). Moreover,the inhibition rates to tumor cell proliferation were enhanced along with the in-crease of effect-to-target ratio(E/ T). Compared with the single-factor treatment groups,the normalized cell index(NCI)in the combined treatment group was significantly decreased. Blocking NKG2D could abate the inhibitory effect of DC-CIK cells on tumor cell proliferation(P﹤0. 05). The expression of NKG2DLs(inclu-ding ULBP1,UBLP2 and ULBP3)on tumor cells at mRNA and protein levels were increased to different ex-tent after treating with 5 μmol/ L of sorafenib for 24 h. Conclusion There was no significant different be-tween the inhibitory effects of sorafenib on the proliferation of lung adenocarcinoma cancer cells harboring KRAS or EGFR gene mutation. The antitumor effects of DC-CIK cells combined with sorafenib on lung ade-nocarcinoma cells might be induced by regulating the NKG2D-NKG2DLs pathway and enhancing apoptosis. Moreover,the antitumor effects of the combined treatment were better than those of single-factor treatments.
ABSTRACT
Objective:To predict the secondary structure and B-cell epitope of human IL-37.Methods:Based on IL-37b ami-no acid sequence, the secondary structure was predicted by SOPMA; hydrophilicity, flexibility, accessibility index were predicted by software of ProScale, Bcepred, respectively.Combined the results according to these methods , the B cell epitopes of IL-37b were pre-dicted.Results: The second structure of IL-37b contained extended strand (31.65%), random coil (52.75%), alpha helix (8.26%), beta turn (7.34%) and the most possible epitopes of IL-37b were located in or adjacent to amino acid 21-27, 34-75, 175-192 , 213-215 .Conclusion:These results will be helpful for the estimate of the epitopes and provide a theory basis for developing mon -oclonal antibodies against human IL-37.