腺病毒介导的CNTF基因在大鼠脊髓表达的实验研究

发布时间：2018-05-13 22:24

本文选题：腺病毒载体 + CNTF基因　；参考：《河北医科大学》2011年硕士论文

【摘要】：目的:睫状神经营养因子(Ciliary Neurotrophic Factor ,CNTF )是一种可以促进多种神经元分化和存活,阻止神经元退变并保护神经元免受损伤的神经活性蛋白,1984年由Barbin等从鸡胚睫状体、脉络膜、虹膜中提取获得,因其能维持副交感神经节的存活并对睫状节神经元具有营养作用而得名。CNTF最突出的作用是维持中枢和周围运动神经元的生物活性,可以促进多种神经元的存活和分化,抑制神经元受损后的退变。正常大鼠的脊髓可以表达少量的CNTF,且损伤后在中枢及周围神经损伤局部会发生暂时性的表达上调,但其难以达到对抗神经损伤的水平。随着分子生物学和基因工程技术的快速发展,利用复制缺陷型重组腺病毒(Adenovirus,AdV)将编码神经营养因子的基因导入到靶细胞和组织中,以期得到稳定持久的表达已成为神经损伤修复领域新的研究方向。本实验将携带有CNTF的复制缺陷型腺病毒(AdCNTF)和不携带任何外源基因的复制缺陷型腺病毒(Ad0)分别导入大鼠脊髓腰膨大中,观察腺病毒介导的外源性CNTF基因在大鼠脊髓的表达水平,以探讨这一基因治疗新途径。方法:将7周龄、体重200-250g的健康Wistar大鼠126只随机分至三组当中。其中正常对照组(A组)42只,正常饲养不予处理;实验对照组(B组)42只,导入不携带外源基因的重组腺病毒载体Ad0;实验组(C组)42只导入携带外源性CNTF基因的重组腺病毒载体AdCNTF。腹腔注射10%水合氯醛麻醉,麻醉成功后将大鼠固定于立体定位仪上,切取长约2cm后正中纵切口,逐层切开并分离椎旁肌肉显露T13椎板及棘突,仔细咬除T13椎板后明胶海绵轻微压迫止血。微量注射器迅速吸取1μL病毒液,于T13脊髓后正中动脉稍偏右0.8mm处,斜向头端45°刺入,进针长度约2.5mm,缓慢推送1μL/min。实验对照组(B组)注入Ad0,实验组(C组)注入AdCNTF,完毕滞针2min后缓慢拔针。充分止血,生理盐水冲洗后局部应用抗生素,关闭伤口。三组大鼠在B、C组注射腺病毒后2天、7天、2周、4周、6周、8周、10周时取材。将同一时间点不同组别的wistar大鼠(6只)其中3只经多聚甲醛灌注后取出脊髓腰膨大节段。另外3只大鼠在麻醉后不进行灌注直接取出脊髓腰膨大节段,冻存于-70℃冰箱中留备RT-PCR实验之用。分别对三组大鼠不同时间点的脊髓灌注标本进行40μm连续振动切片,进行CNTF免疫组化实验,计数脊髓前角CNTF阳性细胞数目。对冻存标本进行RT-PCR实验,逆转录脊髓组织中的CNTFmRNA,并以β-actin为内参照,计算CNTFmRNA的相对表达量。所测数据使用SPSS 17.0统计软件处理,以x±s表示,首先对三组数据进行方差齐性检验,若方差齐同则应用LSD-t检验进行同一时间点组间两两比较,方差不齐采用Kruskal-Wills H检验, P0.05为差异有统计学意义。结果: 1 CNTF基因在大鼠脊髓中的表达趋势脊髓切片CNTF免疫组化检测显示A组(正常对照组)组内不同时间点阳性细胞计数比较均无显著差异(P0.05),认为A组CNTF表达量不具有随时间变化的趋势;B组(实验对照组)手术导入脊髓空白腺病毒Ad0后第2天CNTF表达量增加,第7天达高峰,2周时CNTF降至正常水平,在第2周及以后时间点与A组(正常对照组)比较无显著差异(P0.05);C组(实验组)在手术导入脊髓携带有CNTF基因的AdCNTF后第2天CNTF表达量增加,表达高峰同为第7天,此后逐渐下降,至第8周时降至正常水平,且C组的CNTF表达量在第2天、7天、2周、4周、6周时均高于同时间点B组和A组。统计分析显示:在手术导入腺病毒后第2天、7天,三组两两比较均有显著差异(P0.05);在第2周、4周、6周,B、C组与A组(正常对照组)比较,C组与其有显著差异(p0.05);在第8周、10周,三组两两比较均无显著差异(p0.05)。 2脊髓标本腺病毒PCR检测利用腺病毒特异性引物对腺病毒10×梯度稀释液中的DNA进行扩增,测出AdCNTF与Ad0具有共同的最低稀释度10~4。在不同时间点对B组(实验对照组)和C组(实验组)脊髓标本中腺病毒DNA进行检测,发现腺病毒DNA含量随时间下降,导入后第10周已测不到腺病毒DNA。 3 CNTF mRNA表达趋势用逆转录PCR技术(RT-PCR)检测CNTF mRNA在正常对照组(A组)、实验对照组(B组)、实验组(C组)中的变化趋势:正常对照组(A组)存在低水平的CNTF mRNA表达,表达量不随时间变化;实验对照组(B组)在手术导入无外源基因的腺病毒Ad0 2天后,与同时间点正常对照组(A组)比较CNTF mRNA表达量增多,第7天达到高峰,2周时降至正常水平;实验组(C组)手术导入携带有CNTF基因的腺病毒AdCNTF后2天可检测到CNTF mRNA升高,表达高峰同为第7天,此后呈下降趋势,至第8周时降至正常水平。在第2天、7天、2周、4周、6周时C组CNTF mRNA表达量均高于同时间点实验对照组(B组)和正常对照组(A组)。统计分析显示:手术导入病毒后第2天、7天时三组两两比较均有显著差异(P0.05),第2周、4周、6周时另两组与A组(正常对照组)比较,C组与其有显著差异(p0.05),8周、10周时三组两两比较均无显著差异(p0.05)。结论:正常大鼠脊髓组织中即存在低水平的内源性CNTF表达。大鼠脊髓在导入不携带外源基因的腺病毒Ad0后,内源性CNTF表达水平较正常大鼠短暂增高,这是由于腺病毒引起的炎性刺激、免疫反应及手术造模过程对脊髓造成了损伤。而在导入携带CNTF基因的AdCNTF后,外源性CNTF基因在大鼠脊髓组织内得到了高强度持久的表达。
[Abstract]:Objective: Ciliary Neurotrophic Factor (CNTF) is a neuroactive protein that can promote the differentiation and survival of a variety of neurons, prevent neuron degeneration and protect neurons from injury. In 1984, Barbin was extracted from the ciliary body, choroid, and iris of the chicken embryo, because it can maintain the parasympathetic ganglion. The most prominent role of the named.CNTF is to survive and have a nutritional role in the ciliary ganglion neurons. It maintains the biological activity of the central and peripheral motor neurons, promotes the survival and differentiation of a variety of neurons and inhibits the degeneration of neurons after damage. The spinal cord of normal rats can express a small amount of CNTF, and it is injured in the central and peripheral nerves after injury. With the rapid development of molecular biology and genetic engineering, the genes encoding neurotrophic factors are introduced into target cells and tissues with the rapid development of molecular biology and gene engineering technology, with the rapid development of molecular biology and genetic engineering technology, in order to achieve a stable and lasting table. It has become a new research direction in the field of neural injury repair. This experiment will introduce the replication deficient adenovirus (AdCNTF) with CNTF and the replication deficient adenovirus (Ad0) without any foreign gene into the spinal lumbar enlargement of the rat, and observe the expression level of the exogenous CNTF gene mediated by adenovirus in the spinal cord of rats. This gene therapy is a new way.
Methods: 126 healthy rats of 7 weeks old and body weight 200-250g were randomly divided into three groups, of which 42 were in the normal control group (group A), and the normal control group (group B) had 42 recombinant adenovirus vector Ad0 without exogenous gene, and 42 of the experimental group (group C) imported the recombinant adenovirus vector carrying the exogenous CNTF gene. AdCNTF. intraperitoneal injection of 10% chloral hydrate anaesthesia, after the success of the anesthesia, the rats were fixed on the stereotaxis and were cut into the median longitudinal incision after a long period of 2cm. The T13 vertebral plate and spinous process were cut out and separated by the paravertebral muscles, and the T13 vertebral plate was carefully bitten by the gelatin sponge and the hemostasis was slightly oppressed. The micro syringe quickly absorbed the 1 mu L virus, and was in the T13 spinal cord. The middle artery was slightly deviated from the right 0.8mm, the oblique head end was 45 degrees, the needle length was about 2.5mm, the experimental control group (group B) was slowly pushed into the Ad0, the experimental group (group C) was injected AdCNTF, and after the stagnation needle 2min, the needle was slowly drawn. The blood was fully stopped, the saline was washed and the wound was closed. The three groups of rats were in B, and the C group injected adenovirus 2 days after the C group, 7 In days, 2 weeks, 4 weeks, 6 weeks, 8 weeks, and 10 weeks, the Wistar rats (6 rats) from different groups of the same time (6) were injected with paraformaldehyde to take out the spinal cord swelling segment. The other 3 rats were not directly taken out of the spinal cord after anaesthesia, and stored in the RT-PCR experiment in the refrigerator at -70 centigrade. To three groups of rats respectively. The specimens of spinal cord perfusion at different time points were sliced 40 m continuous vibration, and the number of CNTF positive cells in the anterior horn of the spinal cord was counted by CNTF immunohistochemical test. The RT-PCR experiment of the frozen specimens was carried out, CNTFmRNA in the spinal cord tissue was reverse transcribed, and the relative expression of CNTFmRNA was calculated with the internal reference of beta -actin. The measured data were calculated using SPSS 17 statistics. The software was treated with x + s. First, three groups of data were tested for homogeneity of variance, and Kamo Sai was compared with 22 in the same time point group with LSD-t test. The variance was not homogeneous by Kruskal-Wills H test, and P0.05 was statistically significant.
Result:
The expression trend of 1 CNTF gene in the spinal cord of rats
CNTF immunohistochemical detection showed that there was no significant difference in the positive cell count at different time points in the A group (P0.05), and that the CNTF expression in the A group did not change with time, and the CNTF expression in the B group (experimental control group) was increased second days after the operation was introduced to the blank adenovirus Ad0, seventh Tianda peak, 2 weeks. When CNTF was reduced to normal level, there was no significant difference between the second weeks and the A group (P0.05). The expression of CNTF in the C group (experimental group) increased in the second day after the operation of the spinal cord carrying CNTF gene AdCNTF, and the expression peak was seventh days, and then decreased gradually to the normal level and the CNTF expression in C group. Second days, 7 days, 2 weeks, 4 weeks and 6 weeks were higher than the same time point B group and A group. Statistical analysis showed that there were significant differences in three groups 22 (P0.05) at second days after the operation, 7 days, 7 days, and second weeks, 6 weeks, B, C group and A group (P0.05) compared with group C (P0.05). There was no significant difference (P0.05).
Detection of adenovirus PCR in 2 spinal cord specimens
The adenovirus specific primers were used to amplify the DNA in the adenovirus 10 x gradient diluent, and the common minimum dilution degree of AdCNTF and Ad0 was measured. The adenovirus DNA was detected in the B group (experimental control group) and the C group (experimental group) at different time points. It was found that the DNA content of the adenovirus decreased with time and was measured at tenth weeks after the introduction. No adenovirus DNA.
3 CNTF mRNA expression trend
The change trend of CNTF mRNA in normal control group (group A), experimental control group (group B) and experimental group (group C) was detected by reverse transcriptase PCR technique (RT-PCR). There was a low level CNTF mRNA expression in normal control group (A group), and the expression amount did not change with time; the experimental control group (B group) was introduced to the same time point at the same time point at the same time after introducing the adenovirus Ad0 without exogenous gene for 2 days after operation. In the normal control group (group A), the expression of CNTF mRNA increased, reached the peak at seventh days, and dropped to the normal level at 2 weeks. The experimental group (group C) was introduced into the adenovirus AdCNTF with CNTF gene in 2 days to detect the elevation of CNTF mRNA, the peak expression was seventh days, and then descended down to the normal level at eighth weeks, at second days, 7 days, 2 weeks, 4. The expression of CNTF mRNA in group C was higher than that at the same time point (group B) and normal control group (group A) at 6 weeks. The statistical analysis showed that there were significant differences between the three groups (P0.05), second weeks, 4 weeks at 7 days after the operation and 7 days, and the other two groups were compared with the A group (normal control group) at the 6 weeks (P0.05), 8 weeks, 10. There was no significant difference between the 22 groups in the weeks of the week (P0.05).
Conclusion: there is a low level of endogenous CNTF expression in the spinal cord of normal rats. The endogenous CNTF expression level of the spinal cord in the rat spinal cord is significantly higher than that of the normal rats after the introduction of the adenovirus Ad0 without exogenous gene. This is due to the inflammatory stimulation of the adenovirus, the immune response and the operation process have caused damage to the spinal cord. After carrying AdCNTF of CNTF gene, the exogenous CNTF gene was expressed in high intensity and persistent expression in rat spinal cord tissue.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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