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

SHARP-2 gene silence by lentiviral-based short hairpin RNA interference prolongs survival time of rat kidney transplant recipients

目的研究SHARP-2（rat enhancer of split-and hairy-related protein-2）特异性基因沉默后对白细胞介素2（IL-2）和1干扰素（IFN-γ）基因表达及肾移植受者大鼠存活时间的影响。方法采用基因重组、转染、共转染等分子生物学方法将针对SHARP-2的短发夹式RNA干扰序列导入到第3代自失活慢病毒载体Vira Power packaging mix，以构建重组慢病毒；有限稀释法确定病毒滴度；实时定量PCR法研究基因表达变化；原位大鼠肾移植模型研究SHARP-2基因沉默后受者存活时间变化。结果在正常大鼠肾脏细胞中，重组慢病毒LV-SHARP-2iC对SHARP-2的基因沉默效率达84％。在复感染指数（MOI）=20时，用spinoeulation法可使57％的T细胞受转染。在活化T细胞中，SHARP．2基因沉默后对IL-2和IFN-γ的基因表达抑制率分别达61．3％和68．7％。和空白对照以及随意序列对照组相比，5×10^7TUSHARP。2特异的RNA干扰重组慢病毒LV-SHARP-2iC灌注供肾后，肾移植受者大鼠的中位存活时间延长4～5d。移植肾局部组织分析发现SHARP-2基因沉默效果达61％，IL-2和IFN-γ的基因表达则分别下降了47．3％和58．2％。结论成功构建了以第3代自失活慢病毒为载体的、针对SHARP-2基因的短发夹式RNA干扰重组病毒LV—SHARP-2iC，该体系能使SHARP-2基因表达有效沉默，进而抑制活化T细胞中与排斥相关的IL-2和IFN-γ的基因表达，在肾移植模型中延长受者大鼠存活时间。

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 mnhiplicity 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 interleukin 2 （IL-2） and interferon γ （IFN-γ） gene expression. When donor kidney was perfused with 5×10^7 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.