SHARP-2 gene silence by lentiviral-based short hairpin RNA interference prolongs survival time of rat kidney transplant recipients
10.3760/cma.j.issn.1001-7097.2009.06.015
- VernacularTitle:基于慢病毒的SHARP-2特异的RNA干扰延长肾移植受者大鼠存活时间
- Author:
Zhangfei SHOU
;
Huaying XIAO
;
Yifang XU
;
Yuanzhi WANG
;
Yi YANG
;
Hong JIANG
;
Huiping WANG
;
Ying CHEN
;
Yiwen LI
;
Jianghua CHEN
- Publication Type:Journal Article
- Keywords:
Lentivirus;
Lentivirus infections;
RNA interference;
Kidney transplantation;
Interferon type Ⅱ;
Interleukin-2;
SHARP-2
- From:
Chinese Journal of Nephrology
2009;25(6):458-464
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate if rat enhancer of split- and hairy-related protein-2 (SHARP-2) short hairpin RNA interference (shRNAi) prolongs the survival time of rat kidney transplant recipients. Methods Gene recombinant procedures, transfection and co-transfection were carried out to introduce short hairpin RNA interference sequences target for SHARP-2 into 3rd generation self-inactivated lentiviral-ViraPower packaging mix. Limiting dilution method was used for viral titration. Real-time PCR was employed for quantification of gene expression. Rat kidney transplantation was utilized to investigate the effect of SHARP-2 gene silence on the recipient survival. Results A lentiviral-based shRNAi construct LV-SHARP-2iC showed 84% SHARP-2 gene silence efficiency in normal rat kidney cells. At multiplicity of infection 20, 57% T cells could be transfected by lentivirus with spinoculation method. In activated T cells, SHARP-2 g ene silence resulted in 61.3% and 68.7% reduction of intedeukin 2 (IL-2) and interferon γ (IFN-γ) gene expression. When donor kidney was perfused with 5×107 TU LV-SHARP-2iC, the median survival time prolonged for 4-5 days as compared to blank and scramble control groups. Conclusions A recombinant lentivirus LV-SHARP-2iC that effectively silence SHARP-2 gene expression is constructed successfully, leading to the inhibition of IL-2 and IFN-γ. LV-SHARP-2iC treatment can prolong the survival time of rat kidney transplant recipients.
