阳离子脂质体介导的重组pEGFP-N1-IGF-1质粒体内转染在损伤后脊髓组织内的表达

目的探讨重组质粒pEGFP-N1-IGF-1在大鼠损伤脊髓内表达及mRNA定量分析,为基因治疗SCI提供依据。方法建立改良Nystrom’s大鼠脊髓不完全损伤动物模型,应用基因直接注射法,将阳离子脂质体与重组质粒pEGFP-N1-IGF-1混合后注入大鼠脊髓损伤部位,以空白质粒及生理盐水对照,转染1周后取材,行冰冻切片并于荧光显微镜下观察绿色荧光蛋白表达情况;行RealTimePCR脊髓内IGF-1的mRNA定量表达,并进行统计学分析。结果通过阳离子脂质体Transfectamreagent成功介导重组质粒pEGFP-N1-IGF-1在大鼠脊髓内转染其神经细胞,并表达EGFP和IGF-1的融合蛋白,通过RealtimePCR方法进行mRNA定量分析,进一步证实了重组质粒pEGFP-N1-IGF-1能在脊髓内明确地表达IGF-1,pEGFP-N1-IGF-1转基因组与IGF-1组和对照组mRNA表达相对数比较均有明显显著性差异(P<0.01)。结论重组质粒pEGFP-N1-IGF-1转染后能在脊髓内明确表达IGF-1。证明阳离子脂质体介导的IGF-1体内转基因方法的可行性,为今后应用IGF-1基因治疗脊髓损伤提供了理论依据。

The experimental study on the expression of recombinant plasmid pEGFP-N1-IGF-1 in the injured spinal cord of rats

Objective To study the expression plasmid of pEGFP-N1-IGF-1 and its quantitative analysis in injured spinal cord of rats in vivo, and to provide proof for gene therapy of spinal cord injury (SCI). MethodsThe trauma model of SCI with midrange paraplegia in rats was made according to the modified Nystrom's compression method. The recombinant plasmid pEGFP-N1-IGF-1 was injected into thoracic spinal cord of adult rats respectively mediated by Transfectam reagent in order to investigate the expression of IGF-1 on injured spinal cord following gene transfer in vivo. The expression of IGF-1 mRNA was measured by Real time PCR (Q-PCR) in the spinal cord at 1st week after gene transfer in vivo, and the expression of EGFP around the injection locus was observed by fluorescence microscope. Results The expression of EGFP and IGF-1 fusion protein was found in the nerve cell of the injection locus by fluorescence microscope at 1st week after gene transfer in pEGFP-N1-IGF-1 group, but not in saline(SAL) control group. In pEGFP-N1 group it can be found too. Quantitative analysis of IGF-1 mRNA showed that the expression of IGF-1 mRNA in pEGFP-N1-IGF-1 group was significantly higher than that in the other two groups by Real time PCR. There were significant differences between pEGFP-N1-IGF-1 group and pEGFP-N1 group and between pEGFP-N1-IGF-1 group and SAL control group (P<0.01). Conclusion The expression of IGF-1 mRNA in pEGFP-N1-IGF-1 group was significantly higher than that in the other two groups by Real time PCR. It proves that the method of IGF-1 gene transfer medicated by cationic liposome may be used for the gene therapy of SCI.