Study On The Effect Of Anti-cd5 Mcab On The Apoptosis Of CD5~+B1 Cells

Object: To observ the effect of Anti-CD5 McAb inducing CD5+B1 cells apoptosis and analysis the Mcl-1, Bcl-2 and Bax protein expresse levels. To investigat molecular mechanisms on apoptosis in CD5+B1 cells by Anti-CD5 McAb,which has laying foundations for using it to treat the tumors and immune diseases assoiated with CD5+B1 cells. Methods :1. To culture and freeze jeko-1 cell lines was cultured in RPMI-1640 with heat-inactiveated 20% FBS.Cultured cells were maintained in a conventional environment.The jeko-1 cell lines was resuspended with corresponding complete culture medium 10% DMSO and frozen in liquid nitrogen chamber.2.The effect of Anti-CD5 McAb inducing jeko-1 cell lines to apoptosis was tested with flow cytometry.At the same time,compared with TNF-αinducing jeko-1 cell lines to apoptosis and cells spontaneous apoptosis.Cells were divided into 3 groups and the cell concentration was adjusted to 1×106/ml respectively.Then fixed with anti-CD5 McAb(100μg/ml),TNF-α(600 ng/ml)and culture medium . The cells were cultured for 24 h, 48 h, 72 h.After being washed twice, the cells were stained with reagent according to directions.The effect of apoptosis was analyzed on a flow cytomer.This experiment was repeated three times.3.To assay the Mcl-1, Bcl-2, Bax mRNA level of the apoptosis in jeko-1 cell lines with Real-time quantitative (Real time Quantitative PCR RTQ-PCR):Set up the experimental group of CD5 McAb -inducing apoptosis in jeko-1 cell line and the control groups of TNF-α-inducing and spontaneous.Each group was divided into test and control group,that had 12 groups totally.Harvesting the cells after 48h,which were fixed with agents.RNA was extracted from jeko-1 cell line and reversely transcribed to cDNA.According to SYBR qPCR Mix of kit insructions for Mcl-1,Bcl-2,Bax and GAPDH amplifid by real-time floursent quantitative PCR.Each specimen Mcl-1,Bcl-2,Bax and GAPDH was detected at the same time, seting up a control group of distilled water and standard group. The relative expression of Mcl-1,Bcl-2,Bax mRNA was analyzed using the 2-△△ct method.This experiment was repeated three times.4.Satatistical analysis was completed with SPSS 11.5 statistical software packages.The significant different was significant when P < 0.05 or P < 0.01.Results:1.Cells detected by flow cytometry showed that the rate of Anti-CD5 McAb inducing apoptosis was significantly higher than TNF-αinducing apoptosis and the sponatantous apoptosis. There were Statistical significance between them (P < 0.05).Apoptosis rate was increased with time.2.To assay the Mcl-1, Bcl-2, Bax mRNA level of the apoptosis cell in different groups after 48 h..It was found that increased level of Bcl-2 in the group of Anti-CD5 McAb-inducing compared with TNF-α-inducing and the sponatantous apoptosis.There were statistical significance between them (P < 0.05).Bax,Mcl-1 had no statistically significant differences between them(P > 0.05);Bcl-2 and Bax ratio of anti-CD5 McAb inducing apoptosis cell was decreased relative to TNF-αinducing apoptosis and sponatantous apoptosis. Conclusion:1.This study found that the effect of Anti-CD5 McAb inducing CD5+B1 cell apoptosis was obvious.It be used for the treatment of clinical disease.2. The mechanisms of Anti-CD5 McAb inducing CD5+B1 cells apoptosis has the relationship with Bcl-2 family:when CD5+B1 cell occur to apoptosis, Bcl-2 gene expression is decreased, Bax and Mcl-1 gene had no change.