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目的:克隆小鼠的p27Kipl基因,并以此为探针分析p27Kipl在外周血B细胞中的表达,以及其在抗原受体和CD40介导的抑制和促进增殖信号中的作用。方法:从培养的B细胞中提取总RNA,用RT-PCR扩增并克隆p27Kipl基因。用流式细胞仪检测B细胞凋亡。用Northern blot观察p27Kipl mRNA的表达水平。结果:成功地克隆了小鼠的p27Kipl基因。发现抗原受体交联在引起外周血B细胞凋亡之前,出现p27Kipl表达上调,若同时激活CD40则使p27Kipl表达降低,B细胞也免于凋亡。用针对p27Kipl的反义寡核苷酸处理,可阻断抗原受体交联引 起的外周血B细胞凋亡。结论:p27Kipl可能在抗原受体信号 介导的B细胞凋亡和CD40信号介导的B细胞生存中发挥一 定的作用。
OBJECTIVE: To clone the p27Kipl gene from mice and use it as a probe to analyze the expression of p27Kip1 in peripheral blood B cells and its role in antigen receptor and CD40-mediated suppression and proliferation signaling. Methods: Total RNA was extracted from cultured B cells and the p27Kip1 gene was amplified and cloned by RT-PCR. B cell apoptosis was detected by flow cytometry. Northern blot was used to observe the expression of p27Kipl mRNA. Results: The mouse p27Kipl gene was successfully cloned. Found that antigen receptor cross-linked in the peripheral blood of B cells before apoptosis, the emergence of p27Kipl expression increased, if simultaneous activation of CD40 make p27Kipl expression decreased, B cells are also protected from apoptosis. Antisense oligonucleotides targeting p27Kipl blocked peripheral blood B cell apoptosis induced by antigen receptor cross-linking. Conclusion: p27Kipl may play a role in antigen receptor signaling-mediated B cell apoptosis and CD40 signaling-mediated B cell survival.