慢病毒簡介醫(yī)學(xué)宣教培訓(xùn)課件

1、慢病毒簡介醫(yī)學(xué)宣教慢病毒簡介醫(yī)學(xué)宣教慢病毒載體概況HIV-1的基因結(jié)構(gòu)慢病毒載體歷史與構(gòu)建重組慢病毒的產(chǎn)生慢病毒在中樞神經(jīng)系統(tǒng)中的應(yīng)用慢病毒在造血干細胞系統(tǒng)中的應(yīng)用慢病毒在眼科疾病治療中的應(yīng)用2慢病毒簡介醫(yī)學(xué)宣教慢病毒載體概況2慢病毒簡介醫(yī)學(xué)宣教慢病毒是逆轉(zhuǎn)錄病毒科亞科之一，分為靈長類和非靈長類慢病毒。靈長類慢病毒包括HIV-1，HIV-2，猴免疫缺陷病毒（SIV），非靈長類慢病毒包括貓免疫缺陷病毒（FIV），牛免疫缺陷病毒（BIV），馬免疫缺陷病毒（EIAV）等，其中HIV研究最為透徹。研究表明慢病毒核蛋白質(zhì)前整合復(fù)合物具有噬核特性，病毒基因組運輸至細胞核，從而使慢病毒可以感染和在非有絲分裂

2、細胞中復(fù)制。這一特性是慢病毒成為基因治療的轉(zhuǎn)移載體。31.慢病毒載體概況慢病毒簡介醫(yī)學(xué)宣教慢病毒是逆轉(zhuǎn)錄病毒科亞科之一，分為靈長類和非靈長類慢病毒。靈 HIV-1為雙鏈RNA病毒，共有9個基因。 gag基因編碼病毒的核心蛋白，包括基質(zhì)蛋白、衣殼蛋白、核衣殼蛋白。pol基因編碼病毒復(fù)制所需的酶，如反轉(zhuǎn)錄酶、整合酶、蛋白酶。 env基因編碼病毒的包膜糖蛋白，決定病毒感染宿主的靶向性。rev編碼的蛋白調(diào)節(jié)gag、pol、env的表達水平。tat編碼的蛋白參與RNA轉(zhuǎn)錄的控制。4個輔助基因vif、vpr、vpu、nef編碼的蛋白則作為毒力因子參與宿主細胞的識別和感染。兩端為長末端重復(fù)序列(LTR)，內(nèi)

3、含復(fù)制所需的順式作用元件。42.HIV-1的基因結(jié)構(gòu)編碼病毒的基本結(jié)構(gòu)調(diào)節(jié)基因慢病毒簡介醫(yī)學(xué)宣教 HIV-1為雙鏈RNA病毒，共有9個基因。4 3.1第一代HIV-1來源的慢病毒載體 以Naldini及Kafri構(gòu)建的三質(zhì)粒系統(tǒng)為代表，該系統(tǒng)由包裝質(zhì)粒、包膜質(zhì)粒及載體質(zhì)粒3種質(zhì)粒組成。包裝質(zhì)粒是HIV-1前病毒基因組5端LTR由巨細胞病毒早期啟動子取代，3LTR由SV40 polyA序列取代。包裝成分分別構(gòu)建在兩個質(zhì)粒上，一個表達gag和pol，另一個表達env。載體質(zhì)粒攜帶了5端LTR，和全部5端非翻譯區(qū)域，另外還帶有rev應(yīng)答元件（RRE）。包膜表達質(zhì)粒使用水皰性口炎病毒糖蛋白G基因(VS

4、V-G)用來代替了原病毒的env基因。 53.慢病毒載體歷史與構(gòu)建慢病毒簡介醫(yī)學(xué)宣教 3.1第一代HIV-1來源的慢病毒載體53.慢 3.2第二代HIV-1來源的慢病毒載體 1997年,Zufferey等將包裝質(zhì)粒上的vif、vpr、vpu和nef基因(即輔助基因)敲除,從而得到的,其他方面與第一代載體系統(tǒng)一致。 6慢病毒簡介醫(yī)學(xué)宣教 3.2第二代HIV-1來源的慢病毒載體6慢病毒 3.3第三代HIV-1來源的慢病毒載體 為減少復(fù)制型病毒的產(chǎn)生，可通過減少輔助質(zhì)粒與載體質(zhì)粒的同源性，或者將gag/ pol和rev編碼序列隔離,分散在不同的質(zhì)粒上。這樣的包裝系統(tǒng)由四質(zhì)粒代替原有的三質(zhì)粒包裝系統(tǒng)。

5、 質(zhì)粒一攜帶了gag/ pol編碼序列及RRE ; 質(zhì)粒二包含了編碼rev的序列; 質(zhì)粒三是載體質(zhì)粒; 質(zhì)粒四表達env。7慢病毒簡介醫(yī)學(xué)宣教 3.3第三代HIV-1來源的慢病毒載體7慢病毒 3.4自身失活型（SIN）慢病毒載體 SIN載體的構(gòu)建是在原病毒載體基礎(chǔ)上刪除了病毒3端LTR的U3區(qū)增強子和啟動子序列的片段。該區(qū)域出現(xiàn)突變則在HIV-1載體轉(zhuǎn)錄后，其5LTR會因為缺失HIV-1所需要的啟動子和增強子序列而無法復(fù)制出完整長度的病毒基因組。8慢病毒簡介醫(yī)學(xué)宣教 3.4自身失活型（SIN）慢病毒載體8慢病毒簡介 常用瞬時轉(zhuǎn)染法，即將包膜質(zhì)粒，包裝質(zhì)粒和載體質(zhì)粒共轉(zhuǎn)染人胚腎293T細胞直接產(chǎn)

6、生生產(chǎn)細胞。最后慢病毒分泌到培養(yǎng)基中進行培養(yǎng)而得到大量載體慢病毒。94.重組慢病毒的產(chǎn)生慢病毒簡介醫(yī)學(xué)宣教 常用瞬時轉(zhuǎn)染法，即將包膜質(zhì)粒，包裝質(zhì)粒和載體質(zhì)慢病毒在中樞神經(jīng)系統(tǒng)中的應(yīng)用10慢病毒簡介醫(yī)學(xué)宣教慢病毒在中樞神經(jīng)系統(tǒng)中的應(yīng)用10慢病毒簡介醫(yī)學(xué)宣教 Recombinant viral vectors have been used to study a variety of fundamental issues in developmental neurobiology, as well as pathogenesis and treatments for various neurodeg

7、enerative diseases. Lentiviral vectors are valuable tools for neurobiology research owing to their ability to transduce nondividing cells, such as neurons, and to introduce therapeutic or reporter genes into central nervous system (CNS) cells in vivo and in vitro. 重組病毒載體已被用于研究各種發(fā)育神經(jīng)生物學(xué)中的基礎(chǔ)問題，以及各種神經(jīng)退

8、行性疾病的發(fā)病機理和治療。慢病毒載體能轉(zhuǎn)導(dǎo)分裂的細胞，如神經(jīng)元，并介導(dǎo)中樞神經(jīng)系統(tǒng)（CNS）細胞在體內(nèi)和體外基因治療或報道基因而作為神經(jīng)生物學(xué)研究的有價值的工具。 111. Introduction慢病毒簡介醫(yī)學(xué)宣教 Recombinant viral vect Lentivirus preintegration complexes interact with the nuclear pore and undergo active transport into the nucleus of nondividing cells, where the proviral DNA is integra

9、ted into the genomic DNA of the host cell. This feature is the primary reason why lentiviruses are being devel-oped as gene-transfer vectors for postmitotic cells in the CNS. 慢病毒整合前復(fù)合物與核孔相互作用進入細胞核分裂的細胞，其中的前病毒DNA整合到宿主細胞的基因組DNA并進行主動運輸。此功能是為什么慢病毒在有絲分裂后的細胞在中樞神經(jīng)系統(tǒng)的基因轉(zhuǎn)移載體的主要原因。 121.1. Lentiviral Gene Deli

10、very to CNS Cells慢病毒簡介醫(yī)學(xué)宣教 Lentivirus preintegrat Self-inactivating (SIN) vectors reduce the probability of oncogenesis by pro-moter insertion. In SIN vectors, viral promoter activity is deleted from the inte-grated provirus by deletions in the U3 region of the 3 long terminal repeat (LTR) that are

11、copied during reverse transcription to the 5LTR. 自身失活型（SIN）慢病毒載體3端LTR的U3區(qū)啟動子發(fā)生失活突變后，在逆轉(zhuǎn)錄過程中轉(zhuǎn)移至5LTR。這樣的載體整合入靶細胞，將不會產(chǎn)生完整長度的載體RNA，因此命名為“自身失活型載體”。13慢病毒簡介醫(yī)學(xué)宣教 Self-inactivating (SIN To increase gene delivery in brain, newer generations of lentiviral vectors incorporate the central poly-purine tract (cPPT

12、), an approx 180 bp region derived from the gag region, which increases nuclear import of the proviral DNA and transduction efficiency in the brain. 為了增加基因在腦中的傳遞，新一代的慢病毒載體包含cPPT，一個來自gag區(qū)約180bp的區(qū)域，從而增加了原病毒DNA進入核，也提高了在大腦中的轉(zhuǎn)導(dǎo)效率。 14慢病毒簡介醫(yī)學(xué)宣教 To increase gene deliv慢病毒簡介醫(yī)學(xué)宣教培訓(xùn)課件 The development of stable

13、packaging cell lines that produce high titers of lentiviral vectors has been hindered by the toxicity of constitutive VSV-G expression. For this reason, many groups continue to use transient triple transfection to generate their vector stocks. 產(chǎn)生高滴度的慢病毒載體的穩(wěn)定的包裝細胞系的發(fā)展受到VSV-G表達的毒性的阻礙。因此，大多數(shù)人使用三質(zhì)粒系統(tǒng)。16

14、1.3. Lentiviral Vector Production慢病毒簡介醫(yī)學(xué)宣教 The development of stab Three plasmids are required: encoding the envelope glycoprotein (most commonly VSV-G), encoding the packaging proteins (minimally including gag and pol, often including tat and rev on the same plasmid, and in some cases the accessory

15、 genes vif , vpr, vpu, and nef),encoding the genome (including the intact or self-inactivating LTRs, the packaging signal , the promoter and cDNA of interest and, in some cases, posttranslational regulatory elements and the central poly-purine tract (cPPT), which increase titer and expression.17慢病毒簡

16、介醫(yī)學(xué)宣教 Three plasmids are required1. A highly transfectable cell line such as human embryonic kidney 293T.2. Growth media for 293T cells.3. Poly-D-lysine.4. Borate buffer.5. Reagents for calcium phosphate transfection.182. Materials2.1. Transfection慢病毒簡介醫(yī)學(xué)宣教1. A highly transfectable cel6. High-qualit

17、y plasmid DNA: transfer plasmid, packaging plasmid, envelope plasmid purified through cesium chloride/ethidium bromide equilibrium centrifugation or an anion-exchange matrix.7. Polybrene, 800 g/mL stock solution in PBS.8. Large polyallomer centrifuge tubes for concentrating the vector in a Beckman S

18、W28 ul-tracentrifuge rotor.19慢病毒簡介醫(yī)學(xué)宣教6. High-quality plasmid DNA: Anaesthesia mice.Surgical Preparation.Drilling and injection.Postoperative care.Perfusion.A manual for stereotaxic surgery such as Stereotaxic Surgery in the Rat.A mouse brain atlas such as The Mouse Brain in Stereotaxic Coordinates.

19、202.2. Stereotactic Surgery慢病毒簡介醫(yī)學(xué)宣教Anaesthesia mice.202.2. StereoTransfection轉(zhuǎn)染Collection and Concentration of the Viral Supernatant 收集和集中的病毒上清Titering Lentiviral Vectors 滴定慢病毒載體213. Methods慢病毒簡介醫(yī)學(xué)宣教Transfection轉(zhuǎn)染213. Methods慢病慢病毒在造血干細胞系統(tǒng)中的應(yīng)用22慢病毒簡介醫(yī)學(xué)宣教慢病毒在造血干細胞系統(tǒng)中的應(yīng)用22慢病毒簡介醫(yī)學(xué)宣教造血干細胞（HSC）具有自我更新和分化為

20、血液及免疫系統(tǒng)中各種成熟細胞的能力，許多HSC疾病如遺傳性、代謝性和感染性疾病或惡性腫瘤等有望通過基因治療的方法得到糾正。目的基因轉(zhuǎn)移HSC后，可隨著HSC的自我更新和分化在體內(nèi)長期表達，因此HSC是較理想的基因轉(zhuǎn)移靶細胞。23慢病毒簡介醫(yī)學(xué)宣教造血干細胞（HSC）具有自我更新和分化為血液及免疫系統(tǒng)中各種研究表明，VSV-G假構(gòu)型HIV-I載體可不經(jīng)過對HSC的預(yù)刺激就能有效地將基因轉(zhuǎn)移到人早期干細胞并能在重度聯(lián)合免疫缺陷(SCID)鼠骨髓中穩(wěn)定地長期表達， Miyoshi等構(gòu)建VSV-G假構(gòu)型HIV-I載體，以綠色熒光蛋白（GFP）作為標記基因，將新鮮分離的人臍血CD34 +細胞在無血清及細

21、胞因子培養(yǎng)基中轉(zhuǎn)染5小時，然后植入經(jīng)亞致死量照射的非肥胖型糖尿病/重度聯(lián)合免疫缺陷(NOD/SCID)鼠，可在受鼠脾臟、骨髓及外周血GFP的長期表達。24慢病毒簡介醫(yī)學(xué)宣教研究表明，VSV-G假構(gòu)型HIV-I載體可不經(jīng)過對HSC的預(yù)Isolation of Human CD34+ Hematopoietic Progenitor Cells 人CD34+造血祖細胞的分離Preparation of Lentiviral Vectors 慢病毒載體的制備Transduction of CD34+ Cells by Lentiviral Vectors 慢病毒載體轉(zhuǎn)導(dǎo)CD34+細胞Analysis

22、 of Transduced Human CD34+ Cells in NOD/SCID Mice分析轉(zhuǎn)人CD34+細胞的NOD/ SCID小鼠25Methods慢病毒簡介醫(yī)學(xué)宣教Isolation of Human CD34+ Hemat慢病毒在眼科疾病治療中的應(yīng)用26慢病毒簡介醫(yī)學(xué)宣教慢病毒在眼科疾病治療中的應(yīng)用26慢病毒簡介醫(yī)學(xué)宣教 The primary aim of gene transfer into the retinal cells has been to investigate the developmental mechanisms of the retinal cells

23、 or to reverse retinal diseases. Currently, lentivirus and adenoassociated virus vectors are being used for studying and correcting gene therapy of retinal degenerative diseases. 視網(wǎng)膜細胞基因轉(zhuǎn)導(dǎo)的主要目的是研究視網(wǎng)膜細胞的發(fā)病機制或逆轉(zhuǎn)視網(wǎng)膜疾病。目前，慢病毒和腺病毒載體在被用于研究和糾正的視網(wǎng)膜變性性疾病的基因治療。 271. Introduction慢病毒簡介醫(yī)學(xué)宣教 The primary aim of gene Using an HIV vector carrying the green fluorescent protein (GFP) gene expressed from the cytomegalovirus (CMV) promoter, we showed that efficient and long-lasting gene expression could be obtained in the r