DEAE-葡聚糖在SHIV病毒TCID50滴定及扩增中的作用

Role of DEAE-dextran in SHIV TCID_(50) titration and propagation

目的确定DEAE-葡聚糖对CEMx174细胞的半数抑制浓度,明确其在SHIV病毒TCID50滴定及病毒扩增中的促进作用。方法分别使用含DEAE和无DEAE的DMEM完全培养基测定SHIVchn19p7的TCID50。用无血清DMEM培养基系列稀释DEAE,加入CEMx174细胞,使用cck-8测定细胞破坏率。分别选取DEAE浓度为28.125μg/mL和14.0625μg/mL的无血清DMEM培养基对CEMx174细胞预处理3 h。再加入SHIV-KB9病毒液,定期测定培养上清中的P24水平,同时做正常病毒对照和DEAE-1640对照,比对不同处理下的病毒扩增情况。结果使用了DEAE后,SHIVchn19p7的TCID50达到了3.16×104 TCID50/mL,不使用DEAE,病毒的TCID50测定为阴性。DEAE对CEMx174细胞的IC50为44.85μg/mL。经浓度为28.125μg/mL和14.0625μg/mL的DEAE预处理后,SHIV-KB9病毒扩增在13 d～17 d达到高峰。而用不含DEAE的1640生长液培养的实验孔在19 d才开始出现阳性反应。结论高浓度的DEAE对细胞有较强的杀伤作用,低浓度的DEAE对细胞的破坏率较低,并且能显著促进病毒扩增。DEAE在病毒进入细胞的过程中确实起了重要的作用。

Objective The aim of this study was to determine the IC50 of DEAE-dextran in cell line CEMx174 and its role in the SHIV TCID50 titration and propagation.Methods The TCID50 of SHIVchn19p7-transfected TZM-bl cells in DMEM containing DEAE-dextran（15ug/mL） and DMEM without DEAE-dextran were determined,respectively.2-fold series dilute the DEAE-dextran in DMEM without fetal bovine serum,dispense 100 μL of CEMx174 cells to all wells to make sure that the final concentrations of DEAE-dextran were from 450 μg/mL,225 μg/mL,112.5 μg/mL,56.25 μg/mL,28.125 μg/mL to 14.0625 μg/mL,to determine the survival rate with cck-8 kit and calculate the IC50.To culture the CEMx174 cells with DEAE-DMEM（28.125 μg/Ml and 14.0625 μg/mL） for 3 h before the infection of SHIV-KB9,then culture the cells in 1640 complete growth medium containing DEAE-dextran（14.0625 μg/mL） and 1640 GM without DEAE-dextran,respectively.The level of P24 antigen in the supernatant was tested continuously.Virus propagation control with 1640 GM and 1640 GM containing DEAE-dextran（14.0625 μg/mL） without preculture with DEAE in DMEM were done at the same times.Results The TCID50 of SHIVchn19p7 transfected TZM-bl cells in DMEM containing DEAE-dextran（15 μg/mL） was 3.16×104 TCID50/mL,negative results in the DMEM without DEAE-dextran.The IC50 of DEAE was 44.85 μg/mL for CEMx174 cells.The propagation of SHIV-KB9 reached a peak value of P24 antigen in supernatant at 13 d-17 d after the infection while positive conversion at 19 d,when SHIV-KB9 propagation with 1640 GM without DEAE-dextran（14.0625 μg/mL） and preculture with DEAE in DMEM.Conclusions High concentration of DEAE-dextran is toxic to normal cells.This effect can be reduced at a lower concentration.Definitely,it can enhance the SHIV propagation in cells and play an important role in transfection of mammalian cells.