ABSTRACT
AIM: To explore the effect of Lycium barbarum polysaccharides (LBP) on cis-dichlorodiamineplatinum (II) (CDDP)-induced apoptosis in mouse testis sertoli cells TM4 and its possible mechanism. METHODS: TM4 cells were cultured in vitro, the effect of LBP on the survival rate of TM4 cells induced by CDDP was detected by MTT assay, the effect of LBP on the expression of apoptosis related genes Bcl-2, Bax and Caspase-3 in TM4 cells induced by CDDP was detected by Western blot, and the change of cell apoptosis rate was detected by flow cytometry. RESULTS: Compared with control group, TM4 cell apoptosis was significantly increased in CDDP group, the expression of anti-apoptotic gene Bcl-2 and pro-caspase-3 in proenzyme state were significantly decreased, the expression of pro-apoptotic gene Bax and caspase-3 were significantly increased. Compared with CDDP group, the apoptosis of TM4 cells in CDDP+LBP group was significantly decreased, the expression levels of anti-apoptotic genes Bcl-2 and Pro-Caspase3 were significantly increased, the expression levels of pro-apoptotic gene Bax and Caspase-3 were significantly decreased. CONCLUSION: LBP, by acting on CDDP induced TM4 cells, can inhibit CDDP induced TM4 cell apoptosis by enhancing the expression of Bcl-2 and inhibiting the expression of Bax and Caspase-3, thus alleviating the damage caused by CDDP to TM4 cells.
ABSTRACT
We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.