【摘要】 目的　建立CR2稳定表达的HOS CR2、HOS CD4CR2细胞系 ,研究细胞中MAPKs的活化和细胞增殖变化 ,阐明经CR2的信号传导、CR2的表达对HIV gp16 0等所致的CD4 +细胞的影响。方法　用哺乳动物细胞稳定转染法建立稳定表达CR2的HOS CR2、HOS CD4CR2细胞系 ,细胞经磁珠阳性纯化后 ,用FACS和Westernblot对CR2的表达进行鉴定。用PMA、10 %NHS、HIV gp16 0等处理细胞后 ,经Westernblot检测细胞的MAPKs的活化水平 ;用CellTiter 96AqueousOneSolutionReagent检测和分析处理后细胞的增殖差异。结果　FACS检测和统计学方法分析结果表明CR2表达的阳性率高达96 %以上 ;Westernblot结果显示所建HOS CR2、HOS CD4CR2细胞系的CR2的表达水平与Raji细胞相近 ;细胞经PMA、预先激活补体的 10 %NHS处理细胞 ,发现在处理 10min时ERK、JNK、P38的活化达高峰 ;PD980 5 9、Wortmanin和抗 CR2能阻断ERK、JNK、P38的活化。HIV gp16 0、预先激活补体的 10 %NHS能相应激活HOS CR2 ,HOS CD4和HOS CD4CR2细胞ERK、JNK、P38,这些激活均可被相应的抗体所阻断 ;细胞增殖实验结果显示HIV gp16 0能抑制细胞增殖 (P <0 .0 1) ,CR2的表达能加重HIV gp16 0所致的细胞增殖抑制 (P <0 .0 1)。结论　磁珠阳性纯化法可浓集稳定表达靶基因的细胞 ,提高阳?更多还原

【Abstract】 Objective To explore the signaling pathway mediated by complement receptor type 2 (CR2) and demonstrate the molecular mechanism of the deletion of CD4 + cell. Methods CR2 stably expressing HOS cell (HOS-CR2) and HOS-CD4 cell (HOS-CD4CR2) were established according to protocol for stable transfection of adherent cell; the cells were purified with Dynabeads M-280 Streptavidin by positive selection. Fluorescent-activated cell sorting (FACS) and Western blot were applied to determine CR2 expressions of HOS-CR2 and HOS-CD4CR2 respectively. Serum-starved HOS-CR2 cell line was treated with PMA and 10% complement pre-activated normal human serum (NHS) respectively for 5 min, 10 min, 20 min, 30 min, 1 h, 2 h and 3 h. Phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH 2-terminal protein kinase(JNK) and P38 mitogen-activated protein kinase(MAPK) were measured by Western blot using monoclonal antibodies (McAb) against phosphorylated-ERK, -JNK and -P38 MAPK respectively. Blocking test of the signal pathway was carried out by using PD98059, Wortmanin and anti-CR2 McAb. Serum-starved HOS-CD4 and HOS-CD4CR2 cell lines and pretreated (with anti-CD4 McAb, anti-CR2 McAb) HOS-CD4 and HOS-CD4CR2 cells were treated with HIV-gp160 and NHS, the signal transduction were detected by Western blot. The proliferation of HOS-CD4 and HOS-CD4CR2 cells treated with HIV-gp160 and NHS were detected by CellTiter 96  Non-radioactive One Solution cell proliferation assay. Results The FACS result shows the positive rate of CR2 expressing HOS-CR2 and HOS-CD4CR2 cells is higher than 96% and Western blot result shows that the CR2 expressions of the stable cell lines HOS-CR2 and HOS-CD4CR2 are as high as that of Raji cell. The activation of MAPKs in HOS-CR2 cell stimulated with PMA and NHS was similar. The activation of MAPKs increased at 5 min, reached peak at 10 min, decreased to basal line within 30 min, in a time-dependent manner. The activation of MAPKs blocked by PD98059 (inhibitor of ERK) and Wortmanin (inhibitor of PI-3K). ERK, JNK, P38 MAPK were activated by HIV-gp160 in HOS-CD4 cell; ERK, JNK, P38 MAPK were activated by HIV-gp160, NHS and HIV-gp160+NHS; compared with others, P38 MAPK was dramatically increased by HIV-gp160+NHS in HOS-CD4CR2 cells compared with other groups. The time curve of P38 MAPK activation by HIV-gp160+NHS showed the activation could last for 1 h. HIV-gp160 inhibited the proliferation of HOS-CD4 and HOS-CD4CR2 cells ( P <0.01), NHS enhanced the effect of HIV-gp160 ( P <0.01) and anti-CR2, anti-CD4, or anti-CR2+anti-CD4 partially blocked it ( P <0.05). Conclusion The MAPKs can be activated via CR2 and anti-CR2 McAb, PD98059 and Wortmanin can block the activation, these results indicate that CR2 medicates signal transduction independently. The signal transduction in HOS-CD4CR2 cells and cell proliferation show that CR2 plays a role in pathology of HIV, especially in CD4 + cell deletion, this introduces new insights into the mechanism of AIDS and the strategy of AIDS therapy.更多还原