bb基因診斷、治療

1、基因診斷與基因治療Gene Diagnosis and Gene TherapyLaboratory of Molecular Virology,Institute of Virology, Wuhan University School of Medicine 7/25/20221第一節(jié) 基因診斷 （gene diagnosis）一、定義： Definition of gene diagnosis 基因診斷：用分子生物學(xué)技術(shù)對導(dǎo)致疾病的原因進行病原學(xué)和細胞遺傳基因的檢測分析,從而對相應(yīng)疾病進行診斷。 Genetic diagnosis is a test using molecular bi

2、ology technologies to seek the causes of diseases through analysis of pathogens and genes.7/25/20222 基因診斷的靶分子是基因。 The targets of gene diagnosis are genes7/25/20223傳統(tǒng)的疾病診斷：Conventional diagnostic approaches 細胞形態(tài)、數(shù)量 celluar morphology& celluar quantity生化反應(yīng)、酶活性 biochemical reaction, activities of enzym

3、es 免疫學(xué)檢驗 Immunologic tests 7/25/20224 檢測表型或形態(tài)學(xué)改變 detection of phenotypic or morphologic features 檢測對象是基因的產(chǎn)物-蛋白質(zhì)、多肽。 Detection objects are products of genes , such as protein, polypeptide 7/25/20225二、基因診斷常用的技術(shù)： Technologies in common use 分子雜交技術(shù) (Nucleic acid molecular hybridization )PCR技術(shù) (Polymerase

4、-Chain-Reaction)3. 以上二者的衍生技術(shù)（包括DNA芯片 ） derivative technologies from above two technologies, DNA-Chips4. DNA序列測定 DNA sequencing 7/25/20226基因診斷獨特的優(yōu)點：Advantages of gene therapy 從分子水平上找到特異的致病基因 To find pathogenic genes at the molecular levels 具有很高的靈敏度 High sensitivity 7/25/20227獲得穩(wěn)定的結(jié)果 Stable Results 核酸

5、比蛋白質(zhì)穩(wěn)定 Nucleic acid is more stable than that of protein.快速、早期診斷技術(shù) Speedy & early 甚至在未出現(xiàn)癥狀的情況下早期診斷應(yīng)用廣、適用性強 Wide applicability7/25/20228三、基因診斷策略 Strategies for gene therapy（一）通過致病基因的檢測診斷疾病 Diagnosis of diseases via pathogenic genes 7/25/20229 基因的結(jié)構(gòu)清楚、突變點明確 、致病機制基本清楚。 Structure, mutation and pathogenic

6、 mechanism of pathogenic genes are clearly understood .7/25/2022107/25/2022111)單基因遺傳病eg. monogenetic or single-gene disease 鐮狀細胞貧血Sickle cell anemia 珠蛋白基因 globin gene Codon 6 GAGGTG A-T突變(mutation)谷氨酸(glutamic) 纈氨酸(valine) 7/25/2022122)、Gene 缺失遺傳病的診斷1)地貧的Gene診斷基因不同程度的缺失引起不同類型的地貧，基因缺失14個。正常Gene組用BamH

7、I切割，可得到14kb的片段，而缺失一個 Gene時可得到10kb片段。BamHIBamHI212 10 kb14 kbprobe7/25/202213以Gene為探針，Southern blot 方法檢測/-/- - /- - - - / - - 正常 缺1 缺2 缺3 缺414kb10kb7/25/2022143)DMD/BMD的缺失診斷（進行性肌營養(yǎng)不良或假肥大型）DMD是一種嚴重致死性疾?。╔R），臨床癥狀表現(xiàn)肌肉進行性萎縮和乏力。BMD臨床癥狀與DMD相似，但癥狀較輕。Gene 定位Xp21 ， Gene 2300kb,79 個Exon ，cDNA全長13974bp 編碼3685 A

8、a，分子量427kD。DMD/BMD 70%左右為缺失型，基因很大，缺失可能發(fā)生在不同的部位。應(yīng)用多重PCR擴增檢測，判斷缺失狀態(tài)。7/25/202215（二）通過連鎖遺傳標(biāo)志檢測診斷疾病 Diagnosis via linkage genetic marker 基因結(jié)構(gòu)、突變情況不清楚，但已知定位在染色體的特定位置上。 Chromosome location of pathologic genes is clear, but the structure and mutation of the genes are unclear. 7/25/202216同一染色體上位置十分靠近的基因或其他遺傳

9、標(biāo)志,常常一起遺傳,形成連鎖（linkage）. The genes or other genetic markers located near at the same chromosome can be inherited together, and to form linkage.7/25/202217例如：具體基因不明的遺傳病 Hereditary disease caused by unidentified genes 用內(nèi)切酶切正常母親DNA，雜交后出現(xiàn)7.6kb帶；同酶同方法檢查有遺傳病的父親，發(fā)現(xiàn)6.0和7.6kb兩種片斷，表明6.0kb與基因病有連鎖關(guān)系。這樣當(dāng)胎兒羊水或胚胎絨

10、毛細胞同樣處理后，僅出現(xiàn)7.6kb片段，表明胎兒正常，如果同時有6.0和7.6時，表明胎兒攜帶有父親的遺傳病基因。 7/25/2022181、Gene異常不明確的遺傳病的診斷例：成年型多囊腎病APKD，AD ，臨床表現(xiàn)為腰痛，蛋白尿，血尿，高血壓，腎盂性腎炎，腎結(jié)石。最終導(dǎo)致腎功能衰竭和尿毒癥。APKDGene定位16p13，但致病基因尚未克隆，基因產(chǎn)物的生化性質(zhì)和疾病發(fā)病機理也尚未闡明，但已證實APKD Gene與珠蛋白基因3端附近的一段小衛(wèi)星DNA序列（3HVR）緊密連鎖，因此，可以通過RFLP連鎖分析進行診斷。7/25/202219 以3HVR為probe與PvuII酶切后的家系有關(guān)成員

11、的Gene組DNA雜交Southern Blot雜交 Gene組DNA probe（3HVR）PvuII酶切 DNA片段 雜交 結(jié)果 7/25/202220 英國女王維多利亞。她帶有血友病的基因，并將其傳給了她的兒女。血友病是一種遺傳性凝血障礙疾病，患者可能因很小的傷口而出血不止導(dǎo)致死亡。血友病在女性一般表現(xiàn)為隱性遺傳，較少發(fā)病，但會傳給后代；在男性則表現(xiàn)為顯性遺傳，顯示出病癥。維多利亞女王在科堡主持的一次歐洲王室成員集會，與會者有17人是她的后裔，其中有她的孫女亞歷山德拉（21），她同俄國沙皇尼古拉二世結(jié)婚，導(dǎo)致他們的兒子患有血友病。7/25/202221PCRRFLP診斷甲型血友病的基因診

12、斷，已知甲型血友病XR，獲得家系成員基因組DNA 。 PCR 142 bp BcLI 142bp 99 bp 43bp 電泳 結(jié)果分析？p1p2142bp99bp43bpBcl IBcl I -Bcl I +7/25/202222問:1/ 2、1診斷；2/ 1是男或是女發(fā)??？ 142bp99bp1 2123III 17/25/202223結(jié)果分析1）先癥者II2具有99bp片段而發(fā)病，該片段來自母親。2）II2有142bp/99bp雜合子。142bp來自父親，為正常片段，99bp片段是來自母親而成為攜帶者。3）1為99bp片段 如果是 男性 為患者（99bp片段來自母親）；女性為攜帶者（來自父

13、母雙方）7/25/202224連鎖分析(genetic linkage analysis )就是在家系成員中尋找患病成員共同具有的比正常成員出現(xiàn)頻率更高的染色體片段. The search for certain chromosome fragments that are found in people with a disorder are more frequently than you would expect in people without the disorder. 7/25/202225在這個區(qū)域內(nèi)具有疾病傾向的基因 The gene with susceptibility t

14、o disease is located in the region.7/25/202226 遺傳標(biāo)記選擇 Genetic marker selection ：多標(biāo)記原則 Mult-marker principle靠近原則 Convergence principle7/25/202227不是尋找DNA缺陷(DNA deficiency),而是通過分析DNA遺傳標(biāo)記多態(tài)性來判斷被檢者是否具有帶致病基因的染色體,判斷被檢者患病的可能性 It is an analysis of polymorphism of genetic marker to decide whether the chromoso

15、me is carrying the pathogenic gene, but not a search for DNA deficiency.7/25/202228（三）通過基因表達的定量分析診斷疾病 Diagnosis of disorders via quantitative analysis of gene expression 基因表達水平的變化,可引起相應(yīng)蛋白質(zhì)的含量變化。 variation in gene expression levels is related to variation in its protein levels 7/25/202229 定量PCR技術(shù)可用于基

16、因診斷，也可用于治療效果監(jiān)控。 Application of real-time polymerase chain reaction to gene diagnosis and monitoring outcome of therapy7/25/2022307/25/202231四、基因診斷存在的問題及前景 Problems and prospects in the gene diagnosis 1、理論問題 theory problems.7/25/202232 已找到的致病基因不多，弄清機制的就更少?；蛟\斷失去了基礎(chǔ)。 Little is known about pathogenic g

17、enes, and less is known about the mechanism. Gene diagnosis loses its foundation7/25/2022332、技術(shù)問題 technical problems 對設(shè)備及條件要求高、所需試劑價格昂貴。 high requirements for equipments and conditions & high expense for essential regents 7/25/202234技術(shù)復(fù)雜、精細，對操作人員的要求高。The variety and complexity of technology has also

18、 grown and we need a profession that deals with this situation7/25/202235技術(shù)靈敏度高，很容易受到污染而出現(xiàn)假陽性。False positive results may be obtained by contamination due to the high sensitivity of techniques . 7/25/2022363、倫理問題 ethical concerns 可能引起基因歧視（It may cause a genetic discrimination by gene diagnosis ） 7/25

19、/2022377/25/202238 基因診斷的發(fā)展前景 Future prospects for gene diagnosis 呈現(xiàn)十分廣闊的應(yīng)用前景，發(fā)展?jié)摿薮蟆?gene diagnosis has a wide application and a great potential in future.7/25/202239五、已進行基因診斷的遺傳病 Inherited disorders detected by gene diagnosis（一）血紅蛋白?。╤ypochromia）鐮狀細胞貧血（sickle cell anemia）珠蛋白基因 globin gene Codon 6GA

20、GGTG A-T突變谷氨酸(Ghe) 纈氨酸（vol） 7/25/202240 （）地中海貧血 （） Mediterranean Sea anemia （）珠蛋白基因 缺失，珠蛋白mRNA含量減少，珠蛋白（）鏈合成不平衡。Defect in （）globin , and reduction of globin mRNA production , which leads to an imbalance in : chain ratio.7/25/202241診斷 Diagnosis ：PCR-ASO（allele specific oligonucleotide,ASO )7/25/202242

21、定性分析 (qualitative analysis )：PCR定量分析 (quantitative analysis) ： 實時定量 RT-PCR Real-time quantitative PCR assay 7/25/202243杜氏肌營養(yǎng)不良（Duchenne muscular dystrophy, DMD)-性連鎖隱性遺傳病 （Sex-Linked Recessive Inheritance），抗肌萎縮蛋白（dystrophin）基因突變所致 Duchenne Muscular Dystrophy is caused by the gene mutation of dystroph

22、in.7/25/202244苯丙酮尿癥（phenylketonuria，）常染色體隱性遺傳氨基酸代謝病 autosomal recessive genetic disorders of aromatic amino acid metabolism 苯丙氨酸羥化酶基因突變 phenylalanine hydroxylase gene mutation 丙氨酸羥化酶缺乏 phenylalanine hydroxylase deficiency7/25/202245 脆性綜合癥 fragile X syndromeFMR-1基因的微衛(wèi)星序列（CGG）n改變，導(dǎo)致功能變異。The mutation i

23、n CGG repeat of FMR1 gene leads to altered function.7/25/202246可通過檢測串聯(lián)重復(fù)序列的長度來進行。 We can examine the disease by detecting the length of tandem repeat sequences.7/25/202247 基因診斷技術(shù)檢測腫瘤 The roles of gene diagnosis technology in detection of cancer）腫瘤的早期診斷和預(yù)警診斷Early diagnosis & early warning of tumours7

24、/25/202248）高危（易感）人群篩查及危險性評估 Screen for high risk or susceptible group & Risk Assessment 7/25/202249）腫瘤鑒別診斷、分期、預(yù)后判斷Distinctively diagnosis , staging and prognosis）微小轉(zhuǎn)移灶識別Detection of micrometastases 7/25/202250）治療效果監(jiān)測及評價 Assessment & surveillance of therapy effects7/25/202251對不同腫瘤、不同目的，采取不同的基因診斷策略 Di

25、fferent strategies for different tumor cells, different purposes7/25/202252 腫瘤基因診斷主要策略有 Main strategies for identifying genes involved in tumours檢查腫瘤染色體易位及融合基因Detection of chromosome translocation & fusion genes 7/25/202253檢查癌基因與抗癌基因 Detection of oncogenes & tumor suppressor genes 7/25/202254檢測腫瘤相關(guān)基

26、因 Detection of tumor-related gene 檢測腫瘤標(biāo)志物或m等 Detection of markers and mRNA of tumor 7/25/202255基因診斷技術(shù)檢測感染性疾病 Gene therapy technologies in the diagnosis of infectious diseases 細菌 、病毒、寄生蟲 感染 (infection of bacteria, virus and parasite ）7/25/2022567/25/202257第二節(jié) 基因治療（genetherapy）是現(xiàn)代分子生物學(xué)技術(shù)與醫(yī)學(xué)學(xué)科交叉滲透而形成的一

27、個全新治療領(lǐng)域。 Gene therapy is a new cross-discipline, which covers technologies in modern molecular biology and medical science.7/25/2022587/25/202259基因治療早期是將目的基因?qū)氚屑毎麅?nèi)，成為宿主細胞遺傳物質(zhì)的一部分， Early purpose of gene therapy is to make target genes become a permanent part of the host cells genetic material by inse

28、rting their genetic material into the chromosomes of host cells. 7/25/202260目的基因的表達產(chǎn)物對疾病起治療作用。 Therapeutic effect of expression products of genes of interest on diseases.7/25/202261 現(xiàn)在:將正常基因?qū)氩∽兗毎?，替代或與缺陷基因共存，產(chǎn)生正常表達產(chǎn)物補充缺陷蛋白質(zhì) Deliver normal copy of a gene into pathological cells to supplement the pro

29、tein that are not expressing enough.7/25/202262抑制細胞內(nèi)過度表達的基因； Deliver nucleic acid that will inactivate an over-expressed deleterious protein.7/25/202263將特定基因?qū)敕遣∽兗毎?，在體內(nèi)表達特定產(chǎn)物； and transfer given gene into normal cells to let it express given gene products.7/25/202264向功能異常細胞中導(dǎo)入該細胞中本來不存在的基因，利用表達產(chǎn)物達到治療

30、疾病的目的。 add a functional copy of the therapeutic gene to cells with dysfunction that originally do not express it. 利用基因產(chǎn)生治療效應(yīng)。 and make use of genes to produce the treatment effect7/25/202265第一節(jié)基因治療策略 Strategies for gene therapy基因置換（genereplacement ）或基因矯正（genecorrection）將特定目的基因?qū)胩囟毎脫Q原有的缺陷基因。 A norm

31、al copy of a gene may be introduced into cells to replace a defective copy.7/25/202266必要條件是：Essential conditions對導(dǎo)入的基因及其產(chǎn)物有詳盡的了解. Full understanding about the transferred gene and its gene product is essential . 能有效的導(dǎo)入靶細胞 The ability of being transferred efficiently into target cells 在靶細胞中長期穩(wěn)定存在 Lon

32、g-term and stable gene expression in target cells is desired. 7/25/202267導(dǎo)入基因有適度水平的表達 Keeping transferred gene expressing at a moderate level. 導(dǎo)入方法及載體對細胞無害 The methods of gene transfer and vectors must be harmless to the target cells.7/25/202268一般采用同源重組技術(shù)（homologousrecombination）又稱基因打靶（genetargeting

33、） Generally adopt this technique of homologousrecombination, gene targeting. 7/25/2022697/25/2022707/25/202271.基因替代(gene replacement)、基因添加(gene addition)、基因補充(gene supplement)、 .7/25/2022721) 針對特定的缺陷基因,導(dǎo)入相應(yīng)的正?；?缺陷基因并未除去) Transfer a functional copy of the gene into target cells by aiming at a partic

34、ular defect gene (and the defected gene can be kept ).2)導(dǎo)入靶細胞本來不表達的基因(將細胞因子導(dǎo)入腫瘤細胞) Transfer the interest gene into target cells that are originally not expressing the gene product (by getting cytokine into tumor cells)7/25/2022733.基因干預(yù)(gene interference) 采用某些方式抑制某個基因的表達或通過破壞某個基因的結(jié)構(gòu)使之不能表達,達到治療目的. To

35、attain the treatment purpose, gene interference is either to extinguish expression from the mutation allele or break the structure of a certain gene.7/25/202274 三種策略是以恢復(fù)細胞正常功能或干預(yù)細胞功能為目的. The purpose of above three strategies is to resume the normal function or intervene the function of cells.7/25/20

36、2275 4.細胞自殺效應(yīng) Suicide gene therapy 通過導(dǎo)入自殺基因,誘發(fā)細胞自殺效應(yīng)也是重要的治療策略. Induction of suicide effect via delivery of “suicide” genes into target cells, such as tumor cells is also an important treatment strategy 7/25/202276自殺基因是病毒、細菌等原核生物中具有特殊功能的酶類基因，此類基因轉(zhuǎn)入哺乳動物細胞后產(chǎn)生的酶能將無毒或毒性極低的藥物前體轉(zhuǎn)化成細胞毒性代謝產(chǎn)物，導(dǎo)致腫瘤細胞自殺，因此又稱為病毒

37、導(dǎo)向酶解藥物前體療法(VDEPT). 7/25/2022777/25/2022785、基因修飾(gene modification)也屬于基因添加，但目的不是修復(fù)、改善細胞功能，而是改變細胞功能。7/25/2022791）基因修飾腫瘤細胞的“疫苗”療法：將IL-2、TNF-導(dǎo)入腫瘤細胞，誘發(fā)抗腫瘤細胞毒性淋巴細胞反應(yīng)；另分泌的細胞因子增強CTL和效應(yīng)細胞反應(yīng)。7/25/202280 2）基因修飾TIL 的過繼療法：將細胞因子導(dǎo)入腫瘤侵潤淋巴細胞中，使TIL具有更強的抗腫瘤作用7/25/2022813）免疫增強基因療法：將MHC-I 類基因?qū)肽[瘤細胞，增加其免疫原性。7/25/202282 第

38、二節(jié) 基因轉(zhuǎn)移技術(shù) Gene transfer technologies 基因治療方式有兩種：Manners of gene therapy1）體內(nèi)法（in vivo）2）回體法（ex vivo）7/25/202283Cells removed from bodyTransgene deliveredCells culturedCells returned to the bodyEx VivoIn VivoTransgene delivereddirectly into hostStrategies for Transgene Delivery7/25/202284in vivo 即將外源性功

39、能基因以逆轉(zhuǎn)錄病毒、質(zhì)粒載體或脂質(zhì)體類包囊，或以裸露DNA的形式直接注入體內(nèi)，讓基因在體內(nèi)表達。 A method of letting a foreign functional gene express in vivo by transfer of retrovirus vector, plasmid vector, liposome or the direct injection of nude DNA.7/25/202285ex viro 即將受體細胞（例如淋巴細胞、骨髓干細胞、皮膚或纖維細胞等）在體外培養(yǎng)導(dǎo)入外源性功能基因，然后把這種經(jīng)遺傳修飾的受體細胞輸回或移植入患者體內(nèi)，讓外源基因

40、表達。 A method of letting a foreign functional gene express by transferring the gene into target cells (eg. Lymphocytes, hematopoietic stem cells, skin or fiber cells, etc.) cultured ex viro, and then transfusing or transplanting the genetic modified cells into the patient body.7/25/2022867/25/2022877

41、/25/202288基因治療必須通過適當(dāng)?shù)幕蜣D(zhuǎn)移(gene transfer)技術(shù),將外源基因轉(zhuǎn)移至特定細胞內(nèi). Gene therapy relies in the concept that “foreign” genes may be transfer to target cells using appropriate gene transfer technologies7/25/202289 基因轉(zhuǎn)移技術(shù)有兩大類 Methods of gene delivery :生物學(xué)方法 Biological methods (病毒介導(dǎo)的基因轉(zhuǎn)移系統(tǒng)Viral methods)非生物學(xué)方法 Non

42、-Biological methods (物理或化學(xué)方法 Physical or Chemical methods)7/25/202290逆轉(zhuǎn)錄病毒載體(retrovirus vector)是基因治療中最常用的載體,轉(zhuǎn)移效率較高. The most common virus vectors that are used in gene therapy are retrovirus vectors 一、基因轉(zhuǎn)移生物學(xué)方法Biologic methods of gene transfer7/25/2022917/25/202292基因組是二倍體ss+RNA The genome is diploid

43、.類似真核mRNA， similar in structure to eucaryotic mRNA 5端帽狀結(jié)構(gòu)， 5 capping structure 3端多聚A尾, 3 poly-A tail 7/25/202293 前病毒基因組結(jié)構(gòu)特點： Characters of provirus genome 1)兩端各有一長末端重復(fù)序列 LTR（由U3、R、和U5組成），LTR中含有啟動子、增強子、加尾信號。 A long terminal repeat (LTR) of endogenous retrovirus is located at either end of genome (com

44、pose of U3,R and U5). LTR contains the elements including promoter, enhancer and tailing signal. 7/25/202294 是病毒整合進細胞基因組的過程的關(guān)鍵結(jié)構(gòu)。 It is the key structure for the integration of viral genome into the chromosome of host cells, forming the provirus. 7/25/2022952）有三個結(jié)構(gòu)基因: gag、plo和env It contains 3 struc

45、ture genes, gag, plo and env 分別編碼核心蛋白、逆轉(zhuǎn)錄酶和外殼（包膜）糖蛋白 ,encoding core protein, reverse transcriptase and envelope glycoprotein, respectively.7/25/202296 7/25/2022973）5端LTR下游有病毒包裝所必需的序列、引物結(jié)合位點等。 There are a sequence required for efficient packaging of virus and a region of primer binding site at the do

46、wnstream of 5 LTR.7/25/202298retrovirus vector7/25/202299 用于基因治療的逆轉(zhuǎn)錄病毒載體系統(tǒng)由兩部分組成：Retroviral vector system that can be used for gene therapy is constituted by two parts. 將前病毒DNA中編碼蛋白 的基因切除，Cut off the gene genes encoding viral proteins, 保留順式元件、包裝信號, and keep cis-acting elements and packaging signals

47、插入質(zhì)粒 to insert a plasmid -逆轉(zhuǎn)錄病毒載體 forming a retroviral vector. 7/25/2022100包裝細胞（packaging cell）是將缺失了包裝信號及相關(guān)序列的缺陷型逆轉(zhuǎn)錄病毒（輔助病毒）導(dǎo)入哺乳動物細胞而制備成的特殊細胞。 Packaging cells are a kind of special cell lines that can enable defective retrovirus (helper virus ) that lack packaging signals and correlation sequence to

48、 transfer into mammal cells. 7/25/2022101將上兩部分結(jié)合使用即可產(chǎn)生攜帶治療基因，而只有一次感染能力的病毒顆粒-假病毒顆粒（pseudoviral particles） Combine above two part to form pseudoviral particles that carry therapy gene and can infect (or transduce) target cells only once.7/25/2022102假病毒感染效率非常高 High efficiency of transfection. 用于體內(nèi)基因治療時

49、，只能感染增殖期細胞，有一定局限性。 The limited ability of only infecting dividing cells when used in gene therapy.7/25/2022103逆轉(zhuǎn)錄病毒 載體 (retrovirus vectors) 腺病毒載體 （adenovirus, Ad vectors ）腺相關(guān)病毒（adenoassociated virus，AAv vectors ）載體單純皰疹病毒（herpes simpex virus vectors ）載體Types Of Viruses7/25/2022104Naked DNATarget Cell

50、Therapeutic ProteinAAVRetrovirus/LentivirusAdenovirusNucleusGene Therapy Principles7/25/20221057/25/20221067/25/2022107二、基因轉(zhuǎn)移非生物學(xué)方法脂質(zhì)體（liposome）介導(dǎo)基因轉(zhuǎn)移 利用陽離子脂質(zhì)單體與DNA混合，可自動形成包埋外源DNA的脂質(zhì)體，再通過細胞的內(nèi)吞作用（endocytosis）將外源DNA轉(zhuǎn)移至細胞內(nèi)。 Liposome, positively charged lipid droplets, can interact with negatively charg

51、ed foreign DNA to wrap it up and deliver to cells via endocytosis. 7/25/20221082.細胞表面受體介導(dǎo)的基因轉(zhuǎn)移 Gene transfer via cell surface receptors 受體介導(dǎo)的細胞內(nèi)吞作用是轉(zhuǎn)移特異性外源物質(zhì)進入細胞的一種方式。Receptor-mediated endocytosis is one of manners of transferring foreign matter into cells.7/25/2022109細胞膜上存在一些專一性受體，與相應(yīng)的配體結(jié)合后，通過細胞的內(nèi)吞

52、作用，實現(xiàn)配體向細胞內(nèi)轉(zhuǎn)移。 The specific receptors of cell membrane can combine with corresponding ligands ,and transfer the ligands into cells via endocytosis.7/25/20221107/25/2022111將外源DNA與已證明有細胞或組織親和性的配體偶聯(lián)，可使DNA具有靶向性。 The target-specific action of gene delivery can be increased by coupling ligands with high c

53、ell or tissue affinity with foreign DNA. . 7/25/2022112偶聯(lián)通常用多聚陽離子 (condensed cation )，如多聚賴氨酸 (polylysine) 來實現(xiàn)。 Coupling can be realized by condensed cation, such as polylysine7/25/2022113如：僅在肝細胞表面的去唾液酸糖蛋白（ASGP）受體，它的天然配體是ASGP。帶有這種配體的DNA復(fù)和物可被定向送入肝細胞。 Asialoglycoprotein receptor(ASGPR) is only express

54、on the surface of hepatocytes, and its natural ligand is asialoglycoprotein. DNA complex carrying the ligand can be transferred directionally into hepatocytes.7/25/20221143.直接注射進行基因轉(zhuǎn)移 (DNA injection) 直接將裸DNA注入動物或器官組織內(nèi)。 Nude DNA is directly injected into animals or tissues. 7/25/2022115外源基因在肌肉中的表達量與注

55、入的DNA含量成正比； The amount of foreign gene expressions in muscle is in direct ratio to that of DNA delivered by intramuscular injection. 重復(fù)注射比一次注射效果好。 The effect of repeated injection is more effective than that of single injection.7/25/20221167/25/2022117優(yōu)點： Advantages制備簡單 easy to handle排除病毒致癌的潛在危險 wit

56、hout carcinogenic risk of virus7/25/2022118不需整合即可表達 expression without needing integration可反復(fù)使用 and can be administered again and again.7/25/2022119三、基因轉(zhuǎn)移的靶細胞Target cells used in gene transfer目前條件下，基因治療僅限于體細胞（嚴禁進行生殖細胞的基因治療操作）All current human gene therapy approaches are targeted to somatic cells not

57、 germ-line cells.7/25/20221207/25/2022121 Somatic gene therapy 7/25/2022122 選擇轉(zhuǎn)移基因的靶細胞，要考慮幾點： How to select target cells for gene transfer? 1）最好是組織 特異性 細胞 Tissue specific cells are better.2）細胞較易獲得， It is easy to obtain. 3) 生命周期較長 Lifecycle is long. 7/25/20221234）易受外源物質(zhì)轉(zhuǎn)化 It is easy to be transform b

58、y foreign factors5）轉(zhuǎn)染后細胞仍易成活 It is easy to survive after transfection. 7/25/2022124 目前已開展的研究的靶細胞有： Target cells in present studies 造血干細胞 (hematopoietic stem cells )、淋巴細胞 (lymphocytes)、皮膚成纖維細胞(skin fibroblasts)、肝細胞 (hepatocytes)、血管內(nèi)皮細胞 (vascular endothelial cells,)、肌細胞 (muscular cells, )、神經(jīng)元和神經(jīng)膠質(zhì)細胞 (

59、neurons & glial cells )、腫瘤細胞 (tumour cells ) 等 and so on.7/25/2022125ex vivoretroviral vectors(oncoretroviral or lentiviral)in vivoadenoviral vectorsAAV vectorslentiviral vectors7/25/2022126第三節(jié) 基因治療的應(yīng)用applications of gene therapy1990年 基因治療的第一個病例是先天性腺苷脫氨酶(ADA)缺乏癥 The first disease approved for treatm

60、ent with gene therapy was adenosine deaminase (ADA) deficiency in 1990. 7/25/2022127 美國醫(yī)學(xué)家WF安德森等人對腺甘脫氨酶缺乏癥（ADA缺乏癥）的基因治療，是世界上第一個基因治療成功的范例。 1990年9月14日，安德森對一例患ADA缺乏癥的4歲女孩謝德爾進行基因治療。這個4歲女孩由于遺傳基因有缺陷，自身不能生產(chǎn)ADA，先天性免疫功能不全，只能生活在無菌的隔離帳里。他們將含有這個女孩自己的白血球的溶液輸入她左臂的一條靜脈血管中，這種白血球都已經(jīng)過改造，有缺陷的基因已經(jīng)被健康的基因所替代。在以后的10個月內(nèi)她又接