CTGF siRNA联合电针治疗对大鼠损伤脊髓星形胶质细胞GFAP表达的影响

Effect of treatment with connective tissue growth factor small interferon RNA combined with electric acupuncture on expression of glial fibrillary acidic protein of astrocytes after spinal cord injury in rats

目的:探讨抗CTGF小干扰RNA(small interferon RNA,siRNA)局部注射联合电针治疗对脊髓损伤(spinal cord injury,SCI)后胶质纤维酸性蛋白(GFAP)表达的影响。方法:将SD大鼠随机分为SCI组、CTGF siR-NA干扰(CTGF)组、CTGF siRNA干扰联合电针(EA+CTGF)组。制作大鼠SCI模型,将含有CTGF siRNA/Invivo-fectamine复合物溶液的明胶海绵移植入CTGF组和EA+CTGF组大鼠的脊髓(T10)损伤断端,SCI组用Invivo-fectamine转染试剂代替。EA+CTGF组在模型制备后每天的固定时间给予电针治疗。各组在3 d、7 d、14 d后分别取材,应用免疫荧光组织化学、Western Blot和逆转录聚合酶链反应(RT-PCR)观察星形胶质细胞的形态、数量和GFAP、GFAP mRNA表达变化。结果:SCI组星形胶质细胞反应性增生、胞体肥大,损伤边缘区形成GFAP强表达的胶质界膜;CTGF siRNA干预后,GFAP表达减弱,胞体减小,GFAP阳性细胞数减少;EA+CTGF组GFAP免疫反应表达明显降低,损伤边缘区GFAP荧光强度与脊髓其它区域基本相同。Western Blot结果显示:与SCI组相比,CTGF组与EA+CTGF组的GFAP均减少,有统计学意义(P<0.05),与CTGF组相比,EA+CTGF组的GFAP减少,有统计学意义(P<0.05);RT-PCR电泳结果表明损伤早期EA+CTGF组GFAP mRNA表达明显低于损伤组并有显著性差异(P<0.01),变化与Western Blot结果是一致的。结论:SCI后,CTGF siRNA联合电针治疗能有效阻止GFAP表达,使胶质反应减轻,有助于神经功能恢复。

Objective： To investigate the effect of treatment with connective tissue growth factor（CTGF） siRNA combined with electric acupuncture therapy on spinal glial fibrillary acidic protein（GFAP） expression in rats after spinal cord injury（SCI）.Methods： Fifty four female Sprague-Dawley rats were randomly divided into three groups： SCI group,CTGF siRNA treatment group（CTGF）,CTGF siRNA treatment combined with electric acupuncture group（EA＋CTGF）.The SCI model was made via transecting spinal cordat T10 of rats completely,and gelatin sponges soaked with CTGF siRNA and Invivofectamine were transplanted into spinal cord in the CTGF group and the EA＋CTGF group,while Invivofectamine was injected into the rats of the SCI group instead.After transplantation,rats in the EA＋CTGF group were electro-acupunctured at a fixed time points every day.The injured spinal cords in each group were obtained at 3 d,7 d and 14 d,respectively.Number and morphology of astrocytes,spinal GFAP protein and GFAP mRNA were examined through immunofluorescence histochemical method,Western Blot and reverse transcription-polymerase chain reaction（RT-PCR） techniques.Result： Astrocytes had hyperplasia and hypertrophy due to injury in the SCI group.Glial limiting membrane with strong expression of GFAP formed around damaged spinal area.GFAP expression and the number of GFAP-positive cells showed decreasing tendency post treatment with CTGF siRNA.GFAP expression near damaged area in the EA＋CTGF group was the weakest,showing similar expression level to the intact spinal part.Western Blot showed that compared with the SCI group,spinal GFAP expression in rats of both the CTGF group and the EA＋CTGF group reduced significantly（P〈0.05）;and compared with the CTGF group,GFAP protein in EA＋CTGF group decreased significantly（P〈0.05）.RT-PCR results revealed that GFAP mRNA level in the EA＋ CTGF group was significantly lower than that in the SCI group,demonstrating significant difference（P〈0.01） and consistent with the case of GFAP protein expression as was revealed by Western Blot.Conclusion： CTGF siRNA combined with electric acupuncture can effectively prevent the expression of GFAP mRNA and GFAP and the formation colloid scar in spinal cord of rats after SCI,hereby conducive to the recovery of neurological function.