小RNA干扰联合电针治疗对脊髓损伤后结缔组织生长因子的沉默作用

Effect of small interferon RNA combined with electro-acupuncture on the expression of connective tissue growth factor after spinal cord injury in rats

目的探讨结缔组织生长因子(CTGF)siRNA联合电针治疗对脊髓损伤(SCI)后CTGF阳性表达的沉默作用。方法将SD大鼠随机分为对照(control)组、脊髓损伤(SCI)组、电针(EA)组、CTGF干扰(CTGF)组、CTGF干扰联合电针(EA+CTGF)组。制备SCI模型,将含有CTGF siRNA/invivofectamine的复合物植入CTGF、EA+CTGF组大鼠的脊髓(T10)损伤断端,SCI、EA组用invivofectamine转染试剂代替。EA、EA+CTGF组在模型制备后每天给予电针治疗。各组在3 d、7 d、14 d取损伤脊髓,应用Western blotting、RT-PCR测定CTGF、转化生长因子β-1(TGF-β1)及GFAP蛋白和mRNA表达变化,应用免疫荧光做形态学观察。结果与SCI组相比,CTGF组、EA+CTGF组的CTGF、TGF-β1、GFAP蛋白表达下调,联合治疗效果更明显(P<0.05);EA+CTGF组CTGF、TGF-β1、GFAP mRNA表达明显低于SCI组、EA组和CTGF组(P<0.01)。免疫荧光发现,SCI组CTGF阳性细胞反应性增生;CTGF组CTGF表达减弱,阳性细胞数减少;EA+CTGF组CTGF阳性细胞数明显减少。结论 CTGF siRNA联合电针治疗使损伤脊髓CTGF表达下调,胶质反应减轻,抑制瘢痕形成。

Objective To investigate the effect of treatment with connective tissue growth factor（ CTGF） siRNA combined with electric acupuncture therapy on spinal CTGF expression in rats after spinal cord injury（ SCI）. Methods Seventy-eight Sprague-Dawley rats were randomly divided into five groups： Normal group,SCI group,electric acupuncture group（ EA）,CTGF siRNA treatment group（ CTGF）,CTGF siRNA treatment combined with electric acupuncture group（ EA ＋ CTGF）. The SCI model was made via completely transecting spinal cord at the T10 level,and gelatin sponges soaked with CTGF siRNA and invivofectamine were transplanted into spinal cord in the CTGF group and the EA ＋ CTGF group,while in vivo fectamine was injected into the rats of the SCI group and EA group instead. After transplantation,the rats in the EA ＋ CTGF and EA group were electro-acupunctured at a fixed time point every day. The injured spinal cords in each group were obtained at day 3,7 and 14,respectively. The morphology of tissue,spinal CTGF、transforming growth factor-β1（ TGF-β1） 、GFAP protein and CTGF、TGF-β1、GFAP mRNA were examined through immunofluorescent method,Western blotting and reverse transcription polymerase chain reaction（ RT-PCR） techniques. Results Western blotting showed that compared with the SCI group,spinal CTGF、TGF-β1、GFAP expression in both the CTGF group and the EA ＋CTGF group reduced significantly（ P〈 0. 05）,and compared with the CTGF group,proteins in EA ＋ CTGF group decreased significantly（ P〈 0. 05）. RT-PCR results revealed that CTGF、TGF-β1、GFAP mRNA levels in the EA ＋ CTGFgroup were significantly lower than that in the SCI、EA、CTGF group,demonstrating significant difference and consistent with the case of CTGF、TGF-β1、GFAP protein expression as revealed by Western blotting（ P〈 0. 01）.Immunofluorescence showed that CTGF-positive cells had hyperplasia and hypertrophy due to injury in the SCI group; CTGF increased expression significantly in the cells. Compared with SCI group,CTGF expression and the number of CTGFpositive cells showed decreasing tendency post treatment with CTGF siRNA. CTGF expression near the damaged area in the EA ＋ CTGF group was the weakest showing similar expression level to the intact spinal part. Conclusion CTGF siRNA combined with electric acupuncture can effectively prevent the expression of CTGF mRNA and CTGF and the formation colloid scar in spinal cord of rats after SCI,hereby conducive to the recovery of neurological function.