The Experimental Study Of Adenovirus-mediated CNTF Gene Expression In Spinal Cord

Objective :CNTF (Ciliary Neurotrophic Factor, CNTF) is a nervous system activity protein that can promote a variety of neuronal differentiation and survival, also can prevent neuron degeneration and protect neurons from damage. It was extracted from chick embryo ciliary body, choroid, iris gain by Barbin, etc 1984, and named after the effect of maintaining motor neurons biological activity in central and peripheral nervous system and nutritional role of ciliary ganglion neurons . The most prominent effect of CNTF is to maintain the biological activity of central and peripheral motor neurons, promote a variety of neuronal survival and differentiation, prevent neuronal degeneration after injury. The spinal cord of normal rats can express low level of CNTF, the CNTF expression in central and peripheral nervous can transient up-regulate after injury. But its hard to reach the level of resisting the nerve injury. With the rapid development of molecular biology and gene technology, Using recombinant adenovirus(Adenovirus, AdV)encoding neurotrophic factor gene transfer into target cells and tissues, in order to obtain a stable and lasting expression of neurotrophic factor has become a new research direction in the field of nerve repair. In this study, recombinant replication-defective adenovirus vectors carrying CNTF gene(AdCNTF)or adenovirus vectors without carrying any exogenous gene(Ad0) was transferred into lumbar enlargement of spinal cord, in order to observe adenovirus-mediated exogenous CNTF gene expression in rat spinal cord, to explore this new way of gene therapy.Methods: 126 wistar rats aged 7 weeks, weighing 200-250g was randomly divided into three groups. No treatment was given to 42 rats of normal control group (A).Recombinant adenovirus vectors without carrying any exogenous gene(Ad0)was transferred into 42 rats of experimental control group(B). Recombinant adenovirus vector carrying CNTF gene (AdCNTF) was transferred into 42 rats of experimental group (C).Wistar rats of group B and C were successfully anesthetized by intraperitoneal injection of 10% chloral hydrate, then fixed on the stereotaxic apparatus. 2 cm length of posterior median longitudinal incision was made, T13 vertebral plate was removed and the spinal cord was carefully exposed by separation of paraspinal muscles. Then , using gelatin sponge to stop bleeding. 1μL adenovirus solution were extracted quickly by micro-injection. Adenovirus solution was injected at the site of 0.8mm right to the posterior median artery of spinal cord, The needle tip is 45°head ward and downward at the depth of 2.5mm. The speed is 1μL/min. Ad0 was injected into experimental control group(B), AdCNTF was injected into experimental group(C). Needles were stuck 2 min and withdrew slowly after injection. The wound was rinsed by normal saline, stopped bleeding thoroughly,then closed the incision. At 7 different time points(2d, 7d, 2w, 4w, 6w, 8w, 10w)post injection, Lumbar enlargement of spinal cord was taken out. Each group was divided into two parts by experimental indicators. Part of the wistar rats was perfused by 4% paraformaldehyde solution, then spinal cord specimens were cut into 40μm successive Vibration slicing for the CNTF immunohistochemistry test. Another part of wistar rats were sacrificed without perfused , spinal cord specimens were taken out and frozen at -70℃for RT-PCR test in order to detect CNTF relative expression. The measured data expressed by"x±s"were analyzed by the SPSS 17.0 statistical software .First of all,three sets of data need homogeneity of variance test, if the homogeneity of variance is applied ,LSD-t test was used for Pairwise comparison at the same time between groups. Otherwise, Kruskal -Wills H test would be apllied, P <0.05 was considered statistically significant.Results:1 CNTF gene expression in the rat spinal cordCNTF immunohistochemistry test shows that means in group A was compared in pairwise comparison have no statistically significant difference (p>0.05)at different point. It is suggest that the expression of A group of CNTF have no change with time;In experimental control group(B), CNTF expression increased at 2d post Ad0 injection, reached the peak at 7d, and reduced to normal levels at 2w, Compared to nomal control group(A) at 2w ,it was no statistically significant difference (p>0.05);In experimental group (c) CNTF expression increased at 2d post AdCNTF injection ,reached the peak at 7d which is similar to group B and decreased gradually until 8w, then it was reduced to normal levels. Compared to nomal control group(A) at 8w , it was no statistically significant difference(p>0.05); CNTF expression of group C was higher than that of Group B at 2d,7d,2w,4w,6w; Statistical analysis: Means in group A,B,C was compared in pairwise comparison have statistically significant difference at 2d,7d (p<0.05). there was no statistically significant difference compared in pairwise comparison at 8w,10w(p>0.05).2 Adenovirus in spinal cord PCR detection:By using adenovirus-specific primers to amplify adenovirus DNA in 10×gradient dilution,the adenovirus with the minimum dilution is 10~4. Adenovirus DNA were detected at different time points on the B group (experimental control group)and C group (experimental group) spinal cord By PCR method. It was undetectable at 10 weeks post the adenovirus, adenovirus DNA decreased with time.3 CNTF mRNA expressionRT-PCR(Reverse transcription PCR)method had been used to detect CNTF mRNA expression in normal control grou(A), experimental control group(B), experimental group(C),the spinal cord of normal control group (A)can express low level of CNTFmRNA, but the expression does not change with time; In experimental control group(B), compared to nomal control group(A)at the same point ,CNTF expression increased at 2d post Ad0 injection, reached the peak at 7d, and reduced to normal levels at 2w. In experimental group(C)CNTFmRNA expression increased at 2d post AdCNTF injection , reached the peak at 7d which is similar to group B and decreased gradually until 8w, then it was reduced to normal levels.CNTF expression of group C was higher than that of Group B at 2d, 7d, 2w, 4w ,6w post injection; Statistical analysis: Means in group A,B,C was compared in pairwise comparison have statistically significant difference at 2d,7d(p<0.05).There was no statistically significant difference compared in pairwise comparison at 8w,10w.(p>0.05).Conclusion: The spinal cord of normal rats can express low level of endogenous CNTF. After injection of adenovirus vectors without carrying any exogenous gene(Ad0), we found that expression of endogenous CNTF increased transitorily. The up-regulation of CNTF was caused by inflammatory stimulation and immune responses or the process of removing vertebral and injection. While, Exogenous CNTF gene obtained a stable and lasting expression in the rat spinal cord tissue after injection of AdCNTF.