發(fā)布時間：2018-04-24 05:12

  本文選題：腺病毒 + 睫狀神經(jīng)營養(yǎng)因子��； 參考：《河北醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 目的:周圍神經(jīng)損傷是臨床上常見疾病,周圍神經(jīng)損傷的顯微外科修復(fù)技術(shù)已經(jīng)十分先進,但長期以來難以獲得滿意得治療下效果。隨著基因技術(shù)的發(fā)展和分子生物學(xué)不斷進步,基因治療在神經(jīng)修復(fù)的研究中正逐步興起。 應(yīng)用復(fù)制缺陷型重組腺病毒(Adenovirus,AdV)為載體,將各種神經(jīng)營養(yǎng)因子基因轉(zhuǎn)入脊髓用來保護和促進受損的周圍神經(jīng)再生的研究,已成為周圍神經(jīng)修復(fù)方面的前沿課題。腺病毒的研究和開發(fā)進展非常迅猛,已成功應(yīng)用于多項基因治療臨床試驗中。腺病毒載體具有感染效率高、基因裝載容量大、能感染分裂期或靜息期細胞、宿主范圍廣、易制備高滴度病毒、遺傳毒性較低、不插入宿主基因組等優(yōu)點。CNTF是由200個氨基酸組成的酸性蛋白,與NTF家族沒有同源性,被稱為非靶源性營養(yǎng)因子。 CNTF在神經(jīng)再生過程中起著重要的營養(yǎng)支持作用,能支持感覺、交感及運動神經(jīng)元存活和分化,防止軸突切斷后運動神經(jīng)元的凋亡以及神經(jīng)細胞在發(fā)育過程中的程序性死亡。它還能促進在體和離體運動神經(jīng)細胞存活,防止神經(jīng)組織退變的關(guān)鍵因素。CNTF和其受體在神經(jīng)系統(tǒng)中分布很廣,具有廣泛的生物學(xué)活性,可以提高體內(nèi)外各種類型外周神經(jīng)細胞的存活。在周圍神經(jīng)系統(tǒng)、髓鞘和睫狀神經(jīng)節(jié)的雪旺氏細胞(SC),CNTF呈高水平表達,主要位于細胞體和突起,而核中無陽性反應(yīng)。CNTF主要在于星形膠質(zhì)細胞中存在;通過逆向傳遞機制作用機制,睫狀神經(jīng)營養(yǎng)因子(CNTF)的生物功能多樣,能促進運動神經(jīng)元群細胞存活,對中樞神經(jīng)系統(tǒng)及周圍神經(jīng)系統(tǒng)神經(jīng)細胞的生長、分化都具有明顯的營養(yǎng)作用。利用分子生物技術(shù)克隆大鼠睫狀神經(jīng)生長因子(CNTF)基因片段構(gòu)建腺病毒載體,制備病毒顆粒從而進一步為研究腺病毒載體介導(dǎo)的睫狀神經(jīng)營養(yǎng)因子在周圍神經(jīng)損傷中的應(yīng)用提供了基礎(chǔ)。 方法: 1利用實驗室保存的普通感受態(tài)制作超級感受態(tài)。從感受態(tài)大腸桿菌菌種中抽吸菌液培養(yǎng)過夜轉(zhuǎn)接于200mlLB培養(yǎng)基中,在OD600值在0.4左右時收獲細菌。加入100ul預(yù)冷0.1M CaCL2溶液,冷凍離心用移液槍輕輕上下吸動打勻,使細胞重新懸浮,動作輕柔。細胞懸浮液可立即用于轉(zhuǎn)化試驗或加入15%-30%的甘油后-80℃保存待用。(具體見實驗步驟) 2利用公司已經(jīng)包裝的含有CNTF的腺病毒載體的質(zhì)粒在超級感受態(tài)中轉(zhuǎn)化從而擴增得到能夠轉(zhuǎn)染計量的質(zhì)粒。在感受態(tài)中加入質(zhì)粒5ul冰上解凍,熱擊后冰上冷卻,加入培養(yǎng)基后震蕩培養(yǎng)1小時,在篩選平板上鋪板培養(yǎng)16-24小時,長出單克隆菌群后挑菌培養(yǎng),離心后按質(zhì)粒提取試劑盒步驟提取質(zhì)粒。 3擴增后得到的質(zhì)粒使用脂質(zhì)體2000轉(zhuǎn)染試劑盒轉(zhuǎn)染人腎胚293細胞,培養(yǎng)細胞,包裝帶有CNTF基因的重組腺病毒,在-70℃和37℃溫度下通過反復(fù)凍融制備高滴度腺病毒。 4病毒擴增后在96孔板中感染培養(yǎng)好的人腎胚293細胞,在顯微鏡下可觀察到293細胞有明顯的CPE現(xiàn)象,利用50%組織培養(yǎng)感染劑量法測定病毒滴度。 結(jié)果: 1將擴增質(zhì)粒按照實驗步驟的體系配好,制膠測定篩選擴增質(zhì)粒與公司合成質(zhì)粒酶切結(jié)果相同。 2重組質(zhì)粒轉(zhuǎn)染后的293細胞在培養(yǎng)24小時后即可在倒置熒光顯微鏡下觀察到細胞發(fā)出綠色熒光,即檢測到報告基因CNTF的表達,細胞表達出攜帶目的基因的腺病毒顆粒,對照組未發(fā)現(xiàn)熒光表達。轉(zhuǎn)染成功。 3在96孔板上培養(yǎng)293細胞,用獲得病毒感染293細胞后計數(shù)出現(xiàn)CPE孔個數(shù),測定病毒滴度。最后獲得病毒滴度為2.5×107TCID50/ml。 結(jié)論: 1成功的制備了用于質(zhì)粒轉(zhuǎn)化擴增的超級感受態(tài),并成功擴增了所需足夠計量的質(zhì)粒。 2成功的包裝了CNTF腺病毒,并通過反復(fù)凍融提高病毒液滴度,為進一步研究睫狀神經(jīng)營養(yǎng)因子在周圍神經(jīng)修復(fù)中的影響奠定了基礎(chǔ)。 3病毒擴增后感染人腎胚293細胞,培養(yǎng)細胞14至18天后在顯微鏡下可觀察到293細胞有明顯的CPE現(xiàn)象,利用50%組織培養(yǎng)感染劑量法測定病毒滴度,最終得到滴度為2.5×107TCID50/ml的病毒液,為進一步研究睫狀神經(jīng)營養(yǎng)因子在周圍神經(jīng)修復(fù)中的影響開辟了道路。
[Abstract]:Objective : Peripheral nerve injury is a common disease in clinic . The microsurgical repair technique of peripheral nerve injury is very advanced , but it is difficult to obtain satisfactory results for a long time . With the development of gene technology and the progress of molecular biology , gene therapy is gradually emerging in the study of nerve repair .



The research and development of adenovirus vector have been successfully applied in many kinds of gene therapy clinical trials . The research and development of adenovirus have been successfully applied in many clinical trials . The adenovirus vector has the advantages of high infection efficiency , large gene loading capacity , low genetic toxicity and no insertion into host genome . The CNTF is an acid protein composed of 200 amino acids , which has no homology with the NTF family , and is called non - target nutrient factor .



CNTF plays an important role in nerve regeneration . It can support sensory , sympathetic and motor neuron survival and differentiation , prevent apoptosis of motor neurons after axonal injury and procedural death of nerve cells during development . The CNTF has a wide distribution in the nervous system . It can promote the survival of motor neuron group cells .



Method :



1 . It was transferred to 200 ml of LB medium overnight by suction of bacterial liquid from competent Escherichia coli strain . 100 ul of pre - cooled 0.1 M CaCL2 solution was added , and the cells were resuspended and the action was gentle . The cell suspension could be immediately used in the conversion test or 15 % to 30 % glycerol was added to the cell suspension for later use . ( See experimental procedure for specific details ) .



2 . The plasmid containing CNTF - containing adenovirus vector which has been packaged by the company is transformed in a super - competent state so as to be amplified to obtain a plasmid capable of transfection and measurement .



3 . The plasmid obtained after amplification was transfected into 293 cells of human kidney embryo with liposome 2000 transfection reagent kit , the cells were cultured , the recombinant adenovirus with CNTF gene was packed , and high titer adenovirus was prepared by repeated freeze - thaw at -70 鈩，

本文編號：1795288