本文選題：腺病毒載體 + CNTF基因　； 參考：《河北醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:睫狀神經(jīng)營養(yǎng)因子(Ciliary Neurotrophic Factor ,CNTF )是一種可以促進(jìn)多種神經(jīng)元分化和存活,阻止神經(jīng)元退變并保護(hù)神經(jīng)元免受損傷的神經(jīng)活性蛋白,1984年由Barbin等從雞胚睫狀體、脈絡(luò)膜、虹膜中提取獲得,因其能維持副交感神經(jīng)節(jié)的存活并對(duì)睫狀節(jié)神經(jīng)元具有營養(yǎng)作用而得名。CNTF最突出的作用是維持中樞和周圍運(yùn)動(dòng)神經(jīng)元的生物活性,可以促進(jìn)多種神經(jīng)元的存活和分化,抑制神經(jīng)元受損后的退變。正常大鼠的脊髓可以表達(dá)少量的CNTF,且損傷后在中樞及周圍神經(jīng)損傷局部會(huì)發(fā)生暫時(shí)性的表達(dá)上調(diào),但其難以達(dá)到對(duì)抗神經(jīng)損傷的水平。隨著分子生物學(xué)和基因工程技術(shù)的快速發(fā)展,利用復(fù)制缺陷型重組腺病毒(Adenovirus,AdV)將編碼神經(jīng)營養(yǎng)因子的基因?qū)氲桨屑?xì)胞和組織中,以期得到穩(wěn)定持久的表達(dá)已成為神經(jīng)損傷修復(fù)領(lǐng)域新的研究方向。本實(shí)驗(yàn)將攜帶有CNTF的復(fù)制缺陷型腺病毒(AdCNTF)和不攜帶任何外源基因的復(fù)制缺陷型腺病毒(Ad0)分別導(dǎo)入大鼠脊髓腰膨大中,觀察腺病毒介導(dǎo)的外源性CNTF基因在大鼠脊髓的表達(dá)水平,以探討這一基因治療新途徑。 方法:將7周齡、體重200-250g的健康Wistar大鼠126只隨機(jī)分至三組當(dāng)中。其中正常對(duì)照組(A組)42只,正常飼養(yǎng)不予處理;實(shí)驗(yàn)對(duì)照組(B組)42只,導(dǎo)入不攜帶外源基因的重組腺病毒載體Ad0;實(shí)驗(yàn)組(C組)42只導(dǎo)入攜帶外源性CNTF基因的重組腺病毒載體AdCNTF。腹腔注射10%水合氯醛麻醉,麻醉成功后將大鼠固定于立體定位儀上,切取長約2cm后正中縱切口,逐層切開并分離椎旁肌肉顯露T13椎板及棘突,仔細(xì)咬除T13椎板后明膠海綿輕微壓迫止血。微量注射器迅速吸取1μL病毒液,于T13脊髓后正中動(dòng)脈稍偏右0.8mm處,斜向頭端45°刺入,進(jìn)針長度約2.5mm,緩慢推送1μL/min。實(shí)驗(yàn)對(duì)照組(B組)注入Ad0,實(shí)驗(yàn)組(C組)注入AdCNTF,完畢滯針2min后緩慢拔針。充分止血,生理鹽水沖洗后局部應(yīng)用抗生素,關(guān)閉傷口。三組大鼠在B、C組注射腺病毒后2天、7天、2周、4周、6周、8周、10周時(shí)取材。將同一時(shí)間點(diǎn)不同組別的wistar大鼠(6只)其中3只經(jīng)多聚甲醛灌注后取出脊髓腰膨大節(jié)段。另外3只大鼠在麻醉后不進(jìn)行灌注直接取出脊髓腰膨大節(jié)段,凍存于-70℃冰箱中留備RT-PCR實(shí)驗(yàn)之用。分別對(duì)三組大鼠不同時(shí)間點(diǎn)的脊髓灌注標(biāo)本進(jìn)行40μm連續(xù)振動(dòng)切片,進(jìn)行CNTF免疫組化實(shí)驗(yàn),計(jì)數(shù)脊髓前角CNTF陽性細(xì)胞數(shù)目。對(duì)凍存標(biāo)本進(jìn)行RT-PCR實(shí)驗(yàn),逆轉(zhuǎn)錄脊髓組織中的CNTFmRNA,并以β-actin為內(nèi)參照,計(jì)算CNTFmRNA的相對(duì)表達(dá)量。所測數(shù)據(jù)使用SPSS 17.0統(tǒng)計(jì)軟件處理,以x±s表示,首先對(duì)三組數(shù)據(jù)進(jìn)行方差齊性檢驗(yàn),若方差齊同則應(yīng)用LSD-t檢驗(yàn)進(jìn)行同一時(shí)間點(diǎn)組間兩兩比較,方差不齊采用Kruskal-Wills H檢驗(yàn), P0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果: 1 CNTF基因在大鼠脊髓中的表達(dá)趨勢 脊髓切片CNTF免疫組化檢測顯示A組(正常對(duì)照組)組內(nèi)不同時(shí)間點(diǎn)陽性細(xì)胞計(jì)數(shù)比較均無顯著差異(P0.05),認(rèn)為A組CNTF表達(dá)量不具有隨時(shí)間變化的趨勢;B組(實(shí)驗(yàn)對(duì)照組)手術(shù)導(dǎo)入脊髓空白腺病毒Ad0后第2天CNTF表達(dá)量增加,第7天達(dá)高峰,2周時(shí)CNTF降至正常水平,在第2周及以后時(shí)間點(diǎn)與A組(正常對(duì)照組)比較無顯著差異(P0.05);C組(實(shí)驗(yàn)組)在手術(shù)導(dǎo)入脊髓攜帶有CNTF基因的AdCNTF后第2天CNTF表達(dá)量增加,表達(dá)高峰同為第7天,此后逐漸下降,至第8周時(shí)降至正常水平,且C組的CNTF表達(dá)量在第2天、7天、2周、4周、6周時(shí)均高于同時(shí)間點(diǎn)B組和A組。統(tǒng)計(jì)分析顯示:在手術(shù)導(dǎo)入腺病毒后第2天、7天,三組兩兩比較均有顯著差異(P0.05);在第2周、4周、6周,B、C組與A組(正常對(duì)照組)比較,C組與其有顯著差異(p0.05);在第8周、10周,三組兩兩比較均無顯著差異(p0.05)。 2脊髓標(biāo)本腺病毒PCR檢測 利用腺病毒特異性引物對(duì)腺病毒10×梯度稀釋液中的DNA進(jìn)行擴(kuò)增,測出AdCNTF與Ad0具有共同的最低稀釋度10~4。在不同時(shí)間點(diǎn)對(duì)B組(實(shí)驗(yàn)對(duì)照組)和C組(實(shí)驗(yàn)組)脊髓標(biāo)本中腺病毒DNA進(jìn)行檢測,發(fā)現(xiàn)腺病毒DNA含量隨時(shí)間下降,導(dǎo)入后第10周已測不到腺病毒DNA。 3 CNTF mRNA表達(dá)趨勢 用逆轉(zhuǎn)錄PCR技術(shù)(RT-PCR)檢測CNTF mRNA在正常對(duì)照組(A組)、實(shí)驗(yàn)對(duì)照組(B組)、實(shí)驗(yàn)組(C組)中的變化趨勢:正常對(duì)照組(A組)存在低水平的CNTF mRNA表達(dá),表達(dá)量不隨時(shí)間變化;實(shí)驗(yàn)對(duì)照組(B組)在手術(shù)導(dǎo)入無外源基因的腺病毒Ad0 2天后,與同時(shí)間點(diǎn)正常對(duì)照組(A組)比較CNTF mRNA表達(dá)量增多,第7天達(dá)到高峰,2周時(shí)降至正常水平;實(shí)驗(yàn)組(C組)手術(shù)導(dǎo)入攜帶有CNTF基因的腺病毒AdCNTF后2天可檢測到CNTF mRNA升高,表達(dá)高峰同為第7天,此后呈下降趨勢,至第8周時(shí)降至正常水平。在第2天、7天、2周、4周、6周時(shí)C組CNTF mRNA表達(dá)量均高于同時(shí)間點(diǎn)實(shí)驗(yàn)對(duì)照組(B組)和正常對(duì)照組(A組)。統(tǒng)計(jì)分析顯示:手術(shù)導(dǎo)入病毒后第2天、7天時(shí)三組兩兩比較均有顯著差異(P0.05),第2周、4周、6周時(shí)另兩組與A組(正常對(duì)照組)比較,C組與其有顯著差異(p0.05),8周、10周時(shí)三組兩兩比較均無顯著差異(p0.05)。 結(jié)論:正常大鼠脊髓組織中即存在低水平的內(nèi)源性CNTF表達(dá)。大鼠脊髓在導(dǎo)入不攜帶外源基因的腺病毒Ad0后,內(nèi)源性CNTF表達(dá)水平較正常大鼠短暫增高,這是由于腺病毒引起的炎性刺激、免疫反應(yīng)及手術(shù)造模過程對(duì)脊髓造成了損傷。而在導(dǎo)入攜帶CNTF基因的AdCNTF后,外源性CNTF基因在大鼠脊髓組織內(nèi)得到了高強(qiáng)度持久的表達(dá)。
[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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346

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本文編號(hào)：1885091