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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?
OBJECTIVE: To establish the CR2 and HOS CD4CR2 cell lines stably expressing CR2 and to study the changes of MAPKs activation and cell proliferation in CR2 cells and to clarify the effect of CR2 signaling on the CD4 + cells induced by HIV gp160 and so on. Methods HOS CR2 and HOS CD4CR2 cell lines stably expressing CR2 were established by stable transfection of mammalian cells. After the cells were positively purified by magnetic beads, the expression of CR2 was identified by FACS and Western blot. The cells were treated with PMA, 10% NHS, HIV gp160 and the like, and then the activation level of MAPKs was detected by Western blotting. Differences in the proliferation of the cells were detected by CellTiter 96AqueousOneSolutionReagent. Results The results of FACS analysis and statistical analysis showed that the positive rate of CR2 expression was over 96%. Western blot analysis showed that CR2 expression in HOS CR2 and HOS CD4CR2 cells was similar to that in Raji cells. PMA and pre-activated complement 10% NHS treatment of cells and found that the activation of ERK, JNK, P38 reached the peak at 10min; PD98059, Wortmanin and anti-CR2 can block the activation of ERK, JNK, P38. HIV gp160, pre-activated complement 10% NHS corresponding activation of HOS CR2, HOS CD4 and HOS CD4CR2 cells ERK, JNK, P38, these activation can be blocked by the corresponding antibody; cell proliferation experiments showed that HIV gp160 Inhibition of cell proliferation (P <0. 01), CR2 expression can increase HIV gp160-induced cell proliferation inhibition (P <0.01). Conclusion The positive purification method of magnetic beads can enrich the cells stably expressing the target gene,