The Joint Action Of CD59mAb And LPS On Activating Complement To Lyze Tumor Cells

Objectives:It was reported that the over expression of CD59 had been found in a variety of solid tumors cells such as intestinal,ovarian and prostate in recent years.It was related with the uncontrolled growth of tumors and malignant transformation.To recombine mutant and wide CD59 genes into eukaryotic expression vectors,establish eukaryotic expression systems and detect the expression on transfected A2780 cells and investigate their anti-complement ability,to explore biological function of the site W40.This study focused on the anti-complement ability of mutant CD59,and the joint inhibitory effect with LPS on ovarian cancer cell line A2780,and to observe the joint effect of CD59mAb and LPS on CD59's anti-complement function.Methods:The plasmids containing wide-type CD59 gene and containing mutant CD59 gene were transfected into A2780 cells by Lipfectine.Stable expression clones were selected by the addition of G418.RT-PCR,enzyme Immunohistochemistry,immunofluorescence,flow cytometry and Western-blot were used to determine the target gene and protein expression.MTT method and LDH method were used to observe the anti-complement ability of wide-type CD59 and mutant CD59,and to observe the joint effect of CD59mAb and LPS on CD59's anti-complement function.Results:The expressing of CD59 protein was analyzed successfully by enzyme Immunohistochemistry,immunofluorescence,flow cytometry,Western-blot and RT-PCR.The MTT results showed mutant CD59 lost its ability of protection role of being against human complement compared with wide-type CD59,and there was no significant difference compared with control group. After incubated for a half hour with 5 mg/L LPS,MTT results showed the inhibition ratios of A2780 cells with wide-type CD59 gene,A2780 cells with mutant CD59 gene and A2780 cells were(26.9±2.95)%,(36.3±4.87)%,(29.6±3.16)%respectively,a statistically significant difference. The LDH results suggested that the rate of destruction to the A2780 cells with mutant CD59 gene 65.13±9.77 was significantly higher than that in wild-type CD59 cells transfected A2780 cells,the difference was statistically significant(p＜0.05);compared with CD59mAb group and LPS group,At 5mg/L and 10mg/L,the combine group enhanced the complements' lysine effect,The differences were significant(p＜0.05).Conclusions:The W40 site of human CD59 is important to its activity,which prohibition of this site may be a potential way to raise complement activity and help LPS inhibiting tumor cells.The anti-complement ability of CD59 can be weakened when CD59mAb and LPS combined at small dose. It offers a new method to cure the cancer with highly expressing of CD59 on cancer cells.