本文關(guān)鍵詞：導(dǎo)入β-半乳糖苷酶基因的重組腺病毒的純化　出處：《河北醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 腺病毒 LacZ基因 轉(zhuǎn)染 人腎胚293細(xì)胞 純化

【摘要】：目的：伴隨工業(yè)、農(nóng)業(yè)和交通事業(yè)的發(fā)展，高能量創(chuàng)傷日益增多，周圍神經(jīng)損傷已成為臨床上比較常見的致殘性損傷，而針對(duì)周圍神經(jīng)損傷的治療一直是臨床上比較棘手的問題，盡管顯微外科修復(fù)技術(shù)已經(jīng)十分精湛，但長期以來難獲得滿意效果。隨著分子生物及基因工程的發(fā)展，當(dāng)今針對(duì)基因治療研究已成為周圍神經(jīng)損傷研究領(lǐng)域的熱點(diǎn)課題。通過研究發(fā)現(xiàn)在中樞和周圍神經(jīng)系統(tǒng)中把攜帶目的基因的腺病毒作為載體(Adenovirus,AdV)導(dǎo)入后，目的基因就會(huì)適時(shí)地表達(dá)產(chǎn)物，從而達(dá)到治療或者研究的目的。 其中在介導(dǎo)基因轉(zhuǎn)染方面腺病毒載體具有諸多優(yōu)點(diǎn)，因此在神經(jīng)示蹤研究、神經(jīng)再生和神經(jīng)運(yùn)動(dòng)系統(tǒng)疾病的診治中成為應(yīng)用最廣泛的載體。在神經(jīng)系統(tǒng)內(nèi)導(dǎo)入以腺病毒作為載體的目的基因，通過目的基因在神經(jīng)系統(tǒng)內(nèi)的表達(dá)促進(jìn)周圍神經(jīng)的再生，同時(shí)也對(duì)神經(jīng)示蹤的研究的具有重要意義。 當(dāng)下，神經(jīng)修復(fù)研究領(lǐng)域的熱點(diǎn)是將各種神經(jīng)營養(yǎng)因子基因融合到復(fù)制缺陷型重組腺病毒（Adenovirus,AdV）中，再將攜帶目的基因的腺病毒轉(zhuǎn)入到脊髓或周圍神經(jīng)中來對(duì)受損的神經(jīng)元進(jìn)行保護(hù)或者在周圍神經(jīng)系統(tǒng)中誘導(dǎo)周圍神經(jīng)的再生過程。腺病毒載體具有較高的感染效率、廣泛的宿主范圍、可以將分裂期或靜息期細(xì)胞感染、巨大的基因裝載量、容易提取高濃度病毒成分、毒性低水平表達(dá)等優(yōu)點(diǎn)。利用分子生物技術(shù)克隆乳糖操縱子基因片段LacZ基因構(gòu)建腺病毒載體，制備病毒顆粒從而為進(jìn)一步研究腺病毒載體介導(dǎo)的LacZ基因在周圍神經(jīng)損傷中的應(yīng)用。 LacZ基因廣泛用于基因表達(dá)調(diào)控研究中的一種基因。LacZ基因編碼的beta一半乳糖普酶（簡稱beta-gal)是由4個(gè)亞基組成的四聚休，，可催化乳糖的水解．Beta-gal比較穩(wěn)定，用X-Gal為底物進(jìn)行染色時(shí)，呈藍(lán)色，便于檢測和觀察．LacZ基因的諸多優(yōu)點(diǎn)使它成為基因工程實(shí)驗(yàn)中的一個(gè)常用標(biāo)記基因，因此它被用于基因的時(shí)空表達(dá)、探測已知調(diào)控序列有效區(qū)段的位置和作用、尋找未知序列等領(lǐng)域。 因此，利用腺病毒安全、穩(wěn)定、高效以及LacZ基因特異性表達(dá)等特點(diǎn)當(dāng)下利用含LacZ基因的腺病毒對(duì)周圍神經(jīng)損傷修復(fù)進(jìn)行研究已成為基因治療的前沿。但腺病毒可以被中和抗體迅速地清除，這是由于腺病毒載體的免疫原性比較強(qiáng)，在日常工作生活中腺病毒侵入人體后會(huì)誘發(fā)機(jī)體產(chǎn)生抗腺病毒中和抗體。如果接受腺病毒載體介導(dǎo)的基因治療后因?yàn)橹泻涂贵w會(huì)消滅腺病毒載體，從而導(dǎo)致腺病毒載體在體內(nèi)的數(shù)量減少而使基因治療失敗。為了達(dá)到持續(xù)時(shí)間長、表達(dá)效果確切等效果，本實(shí)驗(yàn)將已有腺病毒通過轉(zhuǎn)染至293細(xì)胞，通過在293細(xì)胞中的繁殖，從而提取出具有侵染力的濃縮的高純度病毒。為下一步和今后研究將LacZ基因?qū)階dV后在中樞和周圍神經(jīng)系統(tǒng)內(nèi)的表達(dá)來促進(jìn)周圍神經(jīng)的再生奠定基礎(chǔ)。 方法： 1HEK293細(xì)胞的培養(yǎng)把凍存的細(xì)胞由-196℃的液氮中快速放入37℃水浴中融化，放入后緩慢搖晃凍存管，使細(xì)胞外冰晶融化。3000r/min離心3min融化好的細(xì)胞懸液。將上層的上清液吸去，并放入10ml培養(yǎng)液到離心管中，輕輕吹打成懸液，放入細(xì)胞培養(yǎng)瓶中。將盛有細(xì)胞和培養(yǎng)液培養(yǎng)瓶在培養(yǎng)箱中培養(yǎng)。培養(yǎng)箱的環(huán)境接近人體環(huán)境，為37℃和5％CO2。當(dāng)細(xì)胞生長均勻時(shí)，便可計(jì)數(shù)一定體積細(xì)胞懸液中的細(xì)胞數(shù)。然后根據(jù)公式換算出細(xì)胞在每毫升細(xì)胞懸液中含有的量。細(xì)胞生長到占瓶底面積的90％還需要2-3次傳代培養(yǎng)。顯微鏡下觀察細(xì)胞，挑選形狀飽滿、生長能力強(qiáng)的細(xì)胞接種病毒。 2病毒轉(zhuǎn)染把293細(xì)胞放入75cm2培養(yǎng)瓶中后加入10mlDMEM5％進(jìn)行培養(yǎng)。待細(xì)胞生長至60%-70%時(shí)將培養(yǎng)液倒去，小心加入病毒混合液。十字形輕輕晃動(dòng)培養(yǎng)瓶使培養(yǎng)液蓋住細(xì)胞層。放入培養(yǎng)箱中培養(yǎng)90分鐘后加入9mlDMEM5％，再經(jīng)過72小時(shí)孵育，便可以通過MOI測定來判斷病毒顆粒。 3腺病毒擴(kuò)增將細(xì)胞在-20℃到37℃分別放置半小時(shí)，反復(fù)三次。用離心管離心后收集上清液冰凍存于-20℃或-80℃。取三瓶培養(yǎng)理想的293細(xì)胞，倒掉里面的細(xì)胞培養(yǎng)液，將5ml混合液放到每個(gè)培養(yǎng)瓶中，輕輕晃動(dòng)混勻。放到培養(yǎng)箱中孵育，時(shí)間大約為90分鐘。向培養(yǎng)瓶中加入DMEM，繼續(xù)培養(yǎng)，時(shí)間大約48-72小時(shí)。同樣的方法從第一次被轉(zhuǎn)染的細(xì)胞中取上清后再次轉(zhuǎn)染細(xì)胞。便可得到再次擴(kuò)增的病毒。 4病毒的純化離心細(xì)胞，待細(xì)胞碎片沉淀后取上清液。5mlPEG8000放入每10ml上清液中，將上清放到冰上1小時(shí)使其沉淀。以12000rpm的速度離心上述混合物，時(shí)間大約20min。將上清液倒去后，在CsCl溶液中加入沉淀物。經(jīng)過5分鐘離心，收集病毒懸浮液。 550%組織培養(yǎng)感染劑量法測定病毒滴度取293細(xì)胞一瓶，用2%FCS/DMEM將細(xì)胞濃度調(diào)整為為7.5×105/ml；將100μl細(xì)胞懸液分別放到2塊96孔板中。10孔為一個(gè)稀釋度，其中2孔作為陰性對(duì)照；經(jīng)過10天培養(yǎng)后鏡下觀察，數(shù)出每一排出現(xiàn)細(xì)胞病變現(xiàn)象（CPE）的孔的數(shù)目。并利用Karber方程計(jì)算滴度T=101+d(s-0.5)，測出AdLacZ病毒液的TCID50：1010TCID50/ml。 結(jié)果： 1正常具有活力的HEK293細(xì)胞呈多角形，貼壁較疏松，細(xì)胞之間相互連成網(wǎng)狀，并有聚集成團(tuán)的傾向。 2含lacz基因的腺病毒轉(zhuǎn)染后的293細(xì)胞在培養(yǎng)24小時(shí)后即可在倒置顯微鏡下觀察到細(xì)胞變性即CPE現(xiàn)象而且通過Ｘ－ｇａｌ染色后可觀察到細(xì)胞出現(xiàn)藍(lán)染，即檢測到報(bào)告基因lacz的表達(dá)，細(xì)胞表達(dá)出攜帶目的基因的腺病毒顆粒，對(duì)照組未發(fā)現(xiàn)藍(lán)染表達(dá)。表明轉(zhuǎn)染成功。 3在96孔板上培養(yǎng)293細(xì)胞，用獲得病毒感染293細(xì)胞后計(jì)數(shù)出現(xiàn)CPE孔個(gè)數(shù)，測定病毒滴度。最后獲得病毒滴度為1010TCID50/ml（1×109pfu/ml）。 結(jié)論：成功的純化了AdLacZ腺病毒，并通過提高了病毒液滴度，為周圍神經(jīng)再生機(jī)制的研究及神經(jīng)損傷的基因治療奠定基礎(chǔ)。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1010TCID50/ml（1×109pfu/ml）的病毒液，為進(jìn)一步實(shí)驗(yàn)開辟了道路。
[Abstract]:Objective: with the development of industry, agriculture and transportation, high energy trauma is increasing, peripheral nerve injury has become a common clinical disabling injury, and for the treatment of peripheral nerve injury has been a clinical difficult problem, although microsurgical repair technique is superb, but for a long time is difficult to obtain satisfactory results. With the rapid development of molecular biological and genetic engineering, the study of gene therapy has become a hot topic in the research field of peripheral nerve injury. The study found that the target gene with adenovirus as a vector in the central and peripheral nervous system (Adenovirus, AdV) after the introduction, the purpose of gene expression product in a timelymanner, so to achieve the purpose of treatment or research.
In the aspect of gene transfection mediated by adenovirus vector has many advantages, so in the study of neural tracer, becomes the carrier of the most widely used in diagnosis and treatment of nerve regeneration and nerve system diseases. In the nervous system to import adenovirus as a gene vector, the gene expression in the nervous system to promote the regeneration of peripheral nerve at the same time, research on neural tracing is of great significance.
At present, the hot research in the field of nerve repair is the fusion of various neurotrophic factor gene by recombinant adenovirus (Adenovirus, AdV), then the adenovirus carrying purpose gene transferred to the spinal cord or peripheral nerve to protect or damaged neurons induced by peripheral nerve regeneration process in the peripheral nervous system. The infection efficiency of adenovirus vector with high, wide host range, can be split or resting cell infection, a huge amount of genetic load, easy to extract high concentration of viral components, has the advantages of low toxicity level expression. Construction of recombinant adenovirus vector using molecular biotechnology cloning lactose operon fragment of LacZ gene, preparation the virus particles so as to further study the application of LacZ gene mediated by adenovirus in peripheral nerve injury.
LacZ gene is widely used in gene expression of a gene encoding.LacZ gene regulation of beta in half lactose Cape enzyme (beta-gal) is composed of 4 subunits of four poly Hugh,.Beta-gal can catalyze the hydrolysis of lactose is relatively stable, the substrate X-Gal staining, blue, the advantages of convenient detection and observation the.LacZ gene to make it become a commonly used marker gene engineering experiments, so it is used for gene expression time, position and function of detecting effective segments of known regulatory sequences, for unknown sequences.
Therefore, the use of adenovirus is safe, stable, efficient and LacZ gene specific expression characteristics of the current use of adenovirus with LacZ gene on the repair of peripheral nerve injury has become the forefront of gene therapy. But adenovirus neutralizing antibodies can be removed quickly, this is the immunogenicity in adenovirus vector is relatively strong human adenovirus, invasion in daily life can induce neutralizing anti adenovirus antibody. If gene therapy mediated by adenovirus vector because the neutralizing antibody after elimination of adenovirus vector, resulting in adenovirus vector reduced gene therapy in vivo. The number of failed to achieve lasting for a long time, the expression of the exact effect and other effects, this experiment will have been transfected to 293 cells by adenovirus, through in 293 cell reproduction, in order to extract high purity concentrated with the infection of the disease Poison. It lays the foundation for the next and future research to promote the regeneration of the peripheral nerve after introducing the LacZ gene into AdV in the central and peripheral nervous system.
Method錛

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