摘要
目的 评价RNA干扰(RNAi)是否能有效抵御补体介导的对猪内皮细胞的细胞毒作用。方法 将小分子干扰RNA(SiRNA)转染猪内皮细胞系PED,检测PED转染前后α1,3 半乳糖转移酶(α1,3 GT)mRNA及α- 半乳糖糖链(αGal)抗原表位的表达,并评价RNAi对补体介导的细胞毒的影响。结果 SiRNA 1/PED的α1,3 GT基因异构体(isoform)表达量与空转组(Mock)相比减少了70%(isoform1,69%;isoform2,72%)(P<0 05),但SiRNA 2/PED的α1,3 GT表达量与错配组及空转组相比差异无统计学意义(P>0. 05),流式细胞学检查显示,SiRNA 1/PED的αGal平均荧光强度(52 9)明显小于错配组(493 9,P<0 01)及空转组(505 7,P<0. 01)。标准4h51Cr释放实验中SiRNA 1/PED的细胞溶解率较空转组分别减少了70%(20%正常人血清组)和60%(40%正常人血清组)(P<0. 05)。结论 PED在转染SiRNA- 1后发生了基因沉默,猪内皮细胞可以成为RNAi作用的靶细胞。本研究为更深层次探讨RNAi在异种移植中的作用奠定了基础。
Objective To evaluate whether RNA interference can protect porcine endothelial cells from complement mediated cytotoxicity.Methods Immortalized porcine aortic endothelial cells of the line PED were cultured and transfected with α1, 3-galactosyltransferase (α1, 3-GT) specific siRNAs. Cells transfected with mismatch SiRNA was used as negative controls. Forty-eight hours later the cells were collected. The expression of α1, 3-GT mRNA was examined by RT-PCR. The expression of α-Gal was examined by flow cytometry. PED cells ere labeled with 51 Cr and mixed with normal human serum (NHS). The release of 51 Cr was measured by γ-ray counter. Heat inactivated NHS (HINHS) was used as control. Results Two isoforms (isoform 1 and isoform 2) were amplified from the PED cells. The expression of α1, 3-GT in the PED cells transfected with SiRNA-1 was lower by 70% in comparison with the mock group (69% for the isoform 1 and 72% for the isoform 2, both P <0.05). However, the expression of α1, 3-GT in the PED cells transfected with SiRNA21 was not different from those in the mock group and mismatch group (both P >0.05). Flow cytometry showed that the average fluorescence intensity of the PED cells transfected with SiRNA-1 was 52,9, significantly lower than that of the mismatch group and mock group (493.9 and 5-5.7 respectively, both P <0.0). Fluorescence microscopy observed the “silence effect” of αGal after SiRNA-1 transfection. Added with 20% and 40% NHS, the cell dissolution rate of the SiRNA transfection group was lower than that of the mock group by 70% and 60% respectively.Conclusions α1, 3GT gene silencing actually occurs following transfection of SiRNA-1. Porcine endothelial cells can be the targets of RNAi.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2005年第16期1133-1136,共4页
National Medical Journal of China
基金
国家 863高技术发展计划基金资助项目 ( 2003 AA 205092)
