shRNA介导BTLA基因沉默对小鼠脾淋巴细胞增殖的影响

目的 探讨RNA干扰负性共刺激分子B和T淋巴细胞衰减因子(BTLA)表达后小鼠淋巴细胞增殖的变化.方法 构建小鼠BTLA基因小发夹RNA(shRNA)质粒载体pSiencer 3.1-BTLA/shRNA,与H1启动子共同亚克隆至慢病毒载体pLB,包装病毒后转导小鼠脾淋巴细胞,流式细胞术(FCM)检测感染效率及BTLA分子表达水平.以刀豆蛋白A(ConA)及抗CD3单抗刺激BTLA shRNA干扰组为实验组,以未行基因沉默组为对照组,CCK-8法测定各组淋巴细胞增殖水平.结果 成功构建3个pLB-BTLA shRNA慢病毒干扰载体和一个非特异性shRNA载体,并制备高滴度病毒.特异性shRNA干扰组BTLA表达水平较对照组明显降低(约40%).抗CD3单抗刺激BTLA shRNA转染的淋巴细胞后增殖水平(A450值)显著增高(3.80±0.58),与未转染shRNA对照组(2.42±0.72)相比差异有统计学意义(P＜0.05),而ConA刺激后细胞增殖水平shRNA转染组与未转染组相比差异无统计学意义(P＞0.05);组间相比,BTLA基因沉默的小鼠脾淋巴细胞经抗CD3单抗处理后细胞增殖水平较ConA处理组明显增高(P＜0.05).结论 慢病毒载体携带的特异性shRNA可有效干扰小鼠脾淋巴细胞BTLA基因的表达,BTLA基因沉默对淋巴细胞增殖具有促进作用.

Effects of shRNA mediated BTLA silence on proliferation of mouse splenic lymphocytes

Objective To investigate the proliferation of mouse splenic lymphocytes after shRNA mediated BTLA gene silence. Methods Three specific shRNAs and one nonspecific shRNA (scrambled)were ligated to pSilencer3.1 -H1 -neo plasmid and then subcloned into lentiviral vector pLB. Recombinant viral particles were harvested post-transduction to 293T cells. Mouse lymphocytes were infected with viral supernatant after 24 h incubation and continuously cultured till 4 days. Expression of eGFP was detected by fluorescence microscopy, efficiency of infection and expression of BTLA on lymphocyte cell by FCM. CCK-8 assay was used to detect the proliferation of lymphocytes. Results Lentiviral expression vectors pLB-shRNA/BTLA were successfully generated. The lentiviral particles were correctly packaged. Expression of BTLA protein in specific shRNA group was significantly decreased comparing to those in control group. O.D. value at A450 of lymphocytes stimulated by anti-CD3 antibody showed significant difference compared with normal BTLA group (P ＜ 0. 05 ), while there was no difference between ConA stimulated group and control (P ＞ 0.05 ). Conclusion Gene-specific shRNA can knockdown the expression of BTLA. The proliferation of lymphocytes stimulated by anti-CD3 antibody after RNAi demonstrates significant enhancement as compared to the unstimulated lymphocytes, while stimulated by ConA showed no difference compared to normal lymphocytes.