Objective To explore the effects of overexpression of human tissue inhibitors of metalloproteinase-1 (hTIMP-1) on proliferation of human liver cancer cell line HepG2 in vitro. Methods A recombinant adenoviral vector containing full-length cDNA of hTIMP-1 was generated and transfected into HepG2. The viral titer was checked by measuring GFP, and the expression of hTIMP-1 in vitro was detected by the techniques of Western blot and semi-quantitative RT-PCR. The ultrastructure was observed by transmission electron microscope and the effects of overexpression of hTIMP-1 on proliferation of HepG2 in vitro was analyzed by MTT assay and growth curve. Results The resultant AdhTIMP-1 was successfully constructed and the expression of hTIMP-1 was detected by Western blot and RT-PCR. The growth and proliferation of HepG2, which had been transfected with AdhTIMP-1, was significantly inhibited. Conclusion The proliferation of HepG2 was markedly inhibited by recombinant adenovirus-mediated overexpression of hTIMP-1, which may pave the way for further application in liver gene therapy.
【Abstract】ObjectiveTo construct a mrp1 expression vector and investigate its biological characteristics in HepG2 cells in vitro. MethodsThe 6.5 kb multidrug resistanceassociated protein （MRP) cDNA obtained from plasmid pGEM-mrp1 was cloned into the pCI-neo mammalian expression vector, which was later transferred into human hepatocarcinoma cell line HepG2 by liposome. Then the HepG2 cells resisting G418 were clustered and proliferated, and the mrp1 mRNA and MRP in these HepG2 cells were detected by means of RT-PCR and FCM respectively. ResultsThe mrp1 expression vector was established successfully, and the stable MDR hepatocarcinoma cell line (HepG2/mrp1) was developed as well. The content of the specific fragment of mrp1 mRNA was (56.8±6.37)% and MRP was 7.89 in the HepG2/mrp1 cells, the corresponding value in HepG2 cells was (9.67±3.26)% and 0.79 respectively. The difference was statistically significant (P＜0.05). ConclusionIt is practicable to establish MDR hepatocarcinoma cell line by transferring mrp1 cDNA into HepG2 cells, which is useful in the research of MDR mechanism.
Objectives To investigate contents and clinical significances of CD19+IL-10+ B cell and its subsets in peripheral blood monouclear cell (PBMC) and cancer tissue of patient with colorectal cancer (CRC). Methods Thirty-eight patients with CRC underwent surgery from November 2017 to November 2018 in this hospital were enrolled as a trail group. The proportions of CD19+IL-10+ B cell, CD19+IL-10+CD24hiCD38hi B cell, CD19+IL-10+CD24intCD38int B cell, and CD19+IL-10+CD24hiCD38– B cell in the CD19+ B cells in the PBMC, cancer tissue and paracancer tissue of these patients were detected by the flow cytometry. Thirty-seven healthy volunteers were selected as a control group and the same cell types same as the trail group were detected by the same method in the PBMC only. Results ① The contents of CD19+IL-10+ B cell and CD19+IL-10+CD24hiCD38hi B cell of the PBMC in the trail group were significantly higher than those in the control group (t=9.09, P<0.01; t=9.36, P<0.01, respectively), which of the cancer tissues in the trail group were significantly higher than those in the corresponding paracancer tissues (t=11.67, P<0.01; t=19.64, P<0.01, respectively), while the content of CD19+IL-10+CD24hiCD38– B cell of the PBMC in the trail group was significantly lower than that in the control group (t=6.15, P<0.01). But the CD19+IL-10+CD24intCD38int B cell had no significant difference of the PBMC between these two groups (t=1.78, P=0.08). ② The contents of CD19+IL-10+ B cell and CD19+IL-10+CD24hiCD38hi B cell of the PBMC in the CRC patients with stage Ⅲ+Ⅳ were significantly higher than those in the CRC patients with stage Ⅰ+Ⅱ (t=5.39, P<0.01; t=3.13, P<0.01, respectively). The others all had no significant differences (P>0.05). Conclusion CD19+IL-10+ B cells are significantly increased in PBMC and cancer tissue of patient with CRC and with advanced CRC (TNM stage Ⅲ+Ⅳ), further more, main increasing subgroup of CD19+IL-10+ B cell is CD19+IL-10+CD24hiCD38hi B cell, which indicates that CD19+IL-10+ B cell and CD19+IL-10+CD24hiCD38hi B cell might participate in tumorigenesis and tumor progress of CRC.