摘要
目的 探索淋巴细胞增殖时RNA编辑酶ADAR1的变化。方法 ①ADAR1底物的合成 ,利用含有α -tropomyosin基因的质粒pBluescriptSK(+/- ) ,体外转录的方法 ,合成双链RNA底物 ,掺入α - 3 2 P -ATP标记 ;②分离小鼠脾脏和淋巴结内的淋巴细胞 ,加入IL - 2 /ConA刺激淋巴细胞增殖 ,于不同的时间点收取细胞 ,裂解后用合成好的底物测定ADAR1的活性 ;③应用薄层色谱 ,观察细胞裂解物作用后的RNA中有多少腺嘌呤核苷变成了次黄嘌呤核苷 ;④用MTT法测定细胞的增殖率。结果 加入IL - 2 /ConA的淋巴细胞与未加入的比较 ,前者于 30h起ADAR1的活性升高 ,72h达高峰 ,其变化与细胞增殖曲线相一致。结论 首次发现淋巴细胞增殖过程中ADAR1的活性升高 。
Aim To explore the changes of ADAR1 activity during mouse primary lymphocytic proliferation. Methods ① Synthetic dsRNA substrate labeled with 32 p-ATP was prepared by in vitro transcription using pBluescriptSK (+/-) vector containing a gene of α-tropomyosin; ② Lymphocytes were isolated from mouse spleen and lymph nodes, incubated in RPMI1640 with or without IL-2/ConA. The cells were harvested at different time points, and whole-cell extract was prepared so that the ADAR1 activities could be detected; ③The production of inosine in the synthetic dsRNA incubated with whole-cell extract was measured by TLC and radioautogram; ④Lymphocytic proliferation rate was detected by MTT colorimetry. Results ADAR1 activity was increased from 30 hours of incubation and reached higher level around 72 hours, which was consistent with the cell's proliferation curve. Conclusion Increased ADAR1 acitivity is first found during lymphocytic proliferation. ADAR1 might play an important role in the lymphocytic function.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2002年第3期208-210,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助课题 No .3 0 170 92 3
