Xu Kai-lin, Zhang Ying, Pan Xiu-ying, Lu Qun-xian
Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
Chin Med J (Engl). 2005 Mar 20;118(6):480-6.
The B7/CD28 pathway provides critical costimulatory signals for complete T cell activation, and members of this pathway have served as useful targets for immunotherapeutic strategies. In this study, we investigated the RNA interference (RNAi) effect induced by small interfering RNA (siRNA) targeting CD28 mRNA on human lymphocytes and its specificity.
According to CD28 gene sequence, we designed and synthysized three different siRNAs (siRNA-1, siRNA-2, siRNA-3) containing 21 bases using Silencertrade mark siRNA construction kit. These siRNAs were transfected into freshly isolated human lymphocytes with Lipofectamine 2000 reagent. At 24-hour, 48-hour and 72-hour post transfection, these cells were collected and analyzed. The changes of surface expression of CD28 gene were detected by flow cytometry, and the changes of CD28 mRNA levels were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The cell viability of transfected lymphocytes was determined by methyl thiazolyl tetrazolium (MTT) assay and trypan blue dye exclusion assay.
Three siRNAs (siRNA-1, siRNA-2, siRNA-3) specifically targeting CD28 mRNA were successfully designed and constructed. Flow cytometry analysis showed that a decrease in CD28 expression was detectable at 24-hour post transfection. Different siRNA showed different inhibition effects on CD28 expression. At 48-hour post transfection, the degrees of reduction with siRNA-1, siRNA-2 and siRNA-3 were 22.10% +/- 1.63%, 73.50% +/- 1.02% and 42.90% +/- 0.89% respectively compared with the control (P < 0.001). Neither of the groups transfected only with siRNA or lipo showed marked reduction in CD28 expression (3.15% +/- 0.75% and 4.55% +/- 0.80%) (P > 0.05). Moreover, lymphocytes treated with siRNA-co showed no marked reduction in CD28 expression (5.07% +/- 0.96%) (P > 0.05). The results of semi-quantitative RT-PCR assay indicated CD28 mRNA level was inhibited after transfection of specific siRNAs. At least 4-fold of reduction in siRNA-2 group occurred at 48-hour post transfection compared with the control (P < 0.001). MTT assay and trypan blue dye exclusion assay demonstrated that the viable cell rations of transfected lymphocytes were significantly reduced in siRNA-1, siRNA-2 and siRNA-3 groups at 48-hour post transfection (P < 0.01). The control groups showed no marked reduction in cell viability (P > 0.05).
Three different siRNAs were synthesized and transfected into lymphocytes. They could reduce the expression of CD28 and the CD28 mRNA level. siRNA-2 was the most efficient. The cell viability reduced correspondingly. Therefore, the silencing effect on CD28 mRNA induced by siRNA may contribute to costimulatory blockade. This result show that siRNA may be useful for further study on graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT).
B7/CD28通路为T细胞的完全活化提供关键的共刺激信号，该通路的成员已成为免疫治疗策略的有用靶点。在本研究中，我们研究了靶向CD28 mRNA的小干扰RNA（siRNA）对人淋巴细胞诱导的RNA干扰（RNAi）效应及其特异性。
根据CD28基因序列，使用Silencer™ siRNA构建试剂盒设计并合成了三种不同的含21个碱基的siRNA（siRNA-1、siRNA-2、siRNA-3）。这些siRNA用Lipofectamine 2000试剂转染到新鲜分离的人淋巴细胞中。在转染后24小时、48小时和72小时收集这些细胞并进行分析。通过流式细胞术检测CD28基因表面表达的变化，通过半定量逆转录聚合酶链反应（RT-PCR）测定CD28 mRNA水平的变化。用噻唑蓝（MTT）法和台盼蓝拒染法测定转染淋巴细胞的细胞活力。
成功设计并构建了三种特异性靶向CD28 mRNA的siRNA（siRNA-1、siRNA-2、siRNA-3）。流式细胞术分析显示，转染后24小时可检测到CD28表达降低。不同的siRNA对CD28表达显示出不同的抑制作用。转染后48小时，与对照组相比，siRNA-1、siRNA-2和siRNA-3的降低程度分别为22.10%±1.63%、73.50%±1.02%和42.90%±0.89%（P<0.001）。仅用siRNA或脂质体转染的组均未显示CD28表达有明显降低（3.15%±0.75%和4.55%±0.80%）（P>0.05）。此外，用siRNA-对照处理的淋巴细胞在CD28表达上也没有明显降低（5.07%±0.96%）（P>0.05）。半定量RT-PCR分析结果表明，转染特异性siRNA后CD28 mRNA水平受到抑制。与对照组相比，siRNA-2组在转染后48小时至少降低了4倍（P<0.001）。MTT法和台盼蓝拒染法表明，转染后48小时，siRNA-1、siRNA-2和siRNA-3组转染淋巴细胞的活细胞比例显著降低（P<0.01）。对照组细胞活力无明显降低（P>0.05）。
合成了三种不同的siRNA并转染到淋巴细胞中。它们可以降低CD28的表达和CD28 mRNA水平。siRNA-2最有效。细胞活力相应降低。因此，siRNA对CD28 mRNA的沉默作用可能有助于共刺激阻断。这一结果表明，siRNA可能有助于异体骨髓移植（allo-BMT）后移植物抗宿主病（GVHD）的进一步研究。
