CTLA-4 Ig腺病毒基因治疗联合供体细胞输注诱导混合嵌合体和大鼠心脏移植耐受

目的　在心脏移植手术中实施CTLA 4Ig腺病毒基因治疗并联合术后输注供体骨髓细胞 ,诱导异基因大鼠心脏移植耐受 ,并对相关机制进行研究。方法　将异基因DA大鼠的心脏移植给受体LEW大鼠 ,同时经门静脉输注供体DA的脾细胞 (SC ,3× 10 8)、CTLA 4Ig腺病毒 [( 1～ 5 )× 10 9PFU ml],第 4天由舌静脉输注DA的骨髓细胞 (BMC ,3× 10 8)。观察、记录心脏移植物的存活时间。并对皮肤移植的受体作同样处理 ,观察皮肤移植存活情况。通过MLR、IL 2逆转实验及嵌合体的测定 ,探讨耐受机理 ,并检测了CTLA 4Ig的体内表达、TH1 TH2型细胞因子的表达。结果　单用CTLA 4Ig腺病毒基因治疗 ,或CTLA 4Ig腺病毒基因治疗联合单独的供体脾细胞或骨髓细胞能不同程度地延长异基因心脏移植物的存活 ,但不能延长皮肤移植物的存活。脾细胞、CTLA 4Ig腺病毒和骨髓细胞(SC Ad BMC)处理组的心脏移植物存活时间明显超过其它各耐受诱导组 ,并且能够诱导皮肤耐受。RT PCR实验证明 ,在受体内不同的组织CTLA 4Ig基因的表达量有所不同 ,并且随着时间的推移表达下降。TH1和TH2型细胞因子的检测显示 ,耐受大鼠体内未发现这两类细胞因子的偏移现象。MLR证明耐受大鼠的免疫应答表现为供体特异性降低 ,IL 2逆转实验、嵌合体检测表明 ,该耐受可能与     

成年大鼠胰岛的体外基因转染

目的 探讨携带外源基因的慢病毒在体外有效感染胰岛及外源基因在胰岛中的表达，为通过移植前向胰岛细胞转入特定的免疫调节分子基因诱导胰岛移植物耐受奠定基础。方法 将目的基因CTLA4Ig导入慢病毒载体pWPTS，构建成pWPTS-CTLA4Ig载体。用磷酸钙沉淀法将pWPTS-EGFP、pWPTS-CTLA4Ig分别和其辅助载体pMD2．G、pCMVΔ8．92共转染293T细胞，收获病毒上清液，测定病毒滴度后感染新分离的胰岛。通过Western Blot测定胰岛培养上清液中CTLA4Ig蛋白的表达。结果 ①成功构建了携带CTLA4Ig基因的慢病毒载体pWPTS-CTLA4Ig；②包装产生的慢病毒Lenti-EGFP、Lenti-CTLA4Ig在体外可以感染胰岛，其中在Lenti-EGFP慢病毒感染的胰岛观察到了绿色荧光，及在Lenti-CTLA4Ig慢病毒感染的胰岛培养上清液中检测到了CTLA4Ig蛋白的表达。结论 慢病毒在体外可以有效感染大鼠胰岛，且携带的外源基因可以在胰岛细胞中稳定表达，其中Lenti-CTLA4Ig慢病毒感染的胰岛为进行胰岛移植并诱导体内特异的胰岛移植物耐受奠定了基础。     

CTLA4Ig重组腺病毒载体的构建与体外表达

目的:构建含有CTLA4Ig基因的重组腺病毒载体,为下一步在动物模型体内表达和基因治疗提供基础。方法:自行设计一对分别含有BglⅡ及HindⅢ酶切位点的CTLA4Ig基因上下游引物,以质粒PCDNA3.0/CTLA4Ig为模板,通过PCR扩增获得CTLA4Ig基因全部序列。片段回收以后经BglⅡ及HindⅢ双酶切,定向插入到腺病毒穿梭质粒pAdTrack-CMV的启动子下游BglⅡ及HindⅢ位点之间,获得重组质粒pAdTrack-CTLA4Ig。通过BglⅡ/HindⅢ双酶切、PCR及插入片段测序鉴定后,将正确重组体pAdTrack-CTLA4Ig与腺病毒骨架质粒pAdEasy-1共转化BJ5183细菌。同源重组后用选择性培养基筛选阳性克隆,提取质粒用脂质体介导转染293细胞,通过观察绿色荧光蛋白(GFP)的表达及PCR扩增目的基因等方法鉴定重组的腺病毒。结果:成功构建了含有CTLA4Ig基因的重组腺病毒,病毒滴度为1.65×10¹²PFU/L。结论:该重组腺病毒的构建为下一步研究其在哺乳动物细胞内表达、研究CTLA4Ig的生物学活性及移植排斥、自身免疫病的基因治疗提供了一定的基础。     

腺病毒介导的CD40Ig基因治疗诱导异基因大鼠心脏移植物的长期存活

目的:通过腺病毒介导的CD40Ig基因治疗,阻断受体体内CD40/CD154共刺激途径,诱导异基因大鼠之间的心脏移植耐受,并探讨相关机制。在将异基因DA大鼠的心脏移植给受体LEW大鼠后,即经门静脉输注携带CD40Ig基因的腺病毒(AdCD40Ig,5×109 pfu)。观察、记录心脏移植物的存活时间。ELISA检测受体大鼠体内CD40Ig蛋白的表达。通过MLR,IL-2逆转实验及Th1/Th2型细胞因子表达的检测,研究耐受的机制。结果:与未处理对照组相比,携带绿色荧光蛋白基因的腺病毒(AdEGFP)给予组,移植物的存活时间未见明显延长;AdCD40Ig腺病毒给予组,异基因大鼠心脏移植物平均存活时间延长至(142.8±26.8)天。ELISA检测表明,CD40Ig蛋白在受体体内表达时间较长,但随时间的推移表达水平逐渐下降。Th1和Th2型细胞因子的检测结果显示,耐受大鼠体内未发现这两类细胞因子偏移的现象。MLR证明耐受大鼠对供体抗原表现为特异性低应答。IL-2逆转实验表明,该耐受形成的早期与克隆无能有关。结论:经.AdCD40Ig腺病毒基因治疗的受体大鼠可以产生特异性的移植耐受,使异基因大鼠心脏移植物长期存活。     

腺病毒介导CTLA4Ig和Iκ-Bα双基因在ECV304细胞中的表达

目的: 通过制备细胞毒性T淋巴细胞相关抗原 4融合蛋白(CTLA4Ig)和核因子的抑制蛋白 (Iκ- Bα)双基因共表达腺病毒载体, 检测重组腺病毒在ECV304细胞中的表达。方法: 将Iκ- Bα和CTLA4Ig通过内部核糖体进入部位(IRES2 )连接, 并以基因重组的形式插入腺病毒表达载体, 构建重组腺病毒, 检测Iκ- Bα和CTLA4Ig在ECV304细胞中的蛋白表达情况及其对炎症介质TNF的调控效应。结果: ( 1 )构建pAdTrack -CMV- CTLA4Ig IRES2- Iκ-Bα, 与pAdEasy在BJ5183重组后转染 293, 获得重组腺病毒。(2)PCR鉴定重组腺病毒正确。(3)用重组腺病毒感染体外培养的ECV304后,该腺病毒可表达Iκ- Bα蛋白及CTLA4Ig蛋白, 且可以下调LPS攻击后TNF α的产生。结论: 构建人CTLA4Ig, Iκ- Bα双基因共表达腺病毒载体, 可以有效的抑制炎症因子的表达,为进一步的CTLA4Ig免疫耐受基因治疗中的抑制, 因NF- κB的过度激活而产生的炎性反应提供研究基础。     

腺病毒介导CTLA4Ig基因修饰骨髓间质干细胞体外抑制免疫应答的研究

目的:通过腺病毒载体介导CTLA4Ig在大鼠骨髓间质干细胞(MSC)中的表达,探讨MSC作为基因转移靶细胞的可行性和转染效率,研究其在体外对免疫应答的抑制作用。方法:构建含有CTLA4Ig基因的重组腺病毒载体pAd-CTLA4Ig,按照不同的感染倍率(MOI)转染大鼠MSC,用荧光显微镜和流式细胞仪分析转染效率,流式细胞术、Westernblotting等方法检测目的蛋白CTLA4Ig在MSC中的表达。将转基因MSC加入混合淋巴细胞反应(MLR)体系,观察其抑制淋巴细胞增殖的生物学效应。结果:重组腺病毒载体pAd-CTLA4Ig对MSC的最大转染率为80.7%±4.7%,转基因MSC可检测到目的蛋白的表达。基因修饰的MSC能有效抑制MLR体系中淋巴细胞的增殖,4d达到最大抑制效率51.46％;再次MLR证实这种抑制作用是供者特异性的。结论:MSC是一种较理想的基因转移靶细胞,其表达的转基因产物CTLA4Ig可在体外特异性地抑制免疫应答。     

重组大鼠脑源性神经营养因子腺病毒的构建及体外表达分析

目的构建含有大鼠脑源性神经营养因子(BDNF)基因的重组腺病毒(AD)载体,并分析其体外表达情况。方法将采用RT-PCR技术获取的大鼠BDNF的cDNA基因定向克隆入穿梭质粒pAdTrack-CMV中。通过与腺病毒骨架载体pAdeasy-1在细菌内同源重组形成重组腺病毒质粒pAd-BDNF,转染人胚肾293细胞后包装成有感染能力的重组腺病毒颗粒(Ad-BDNF)。重组病毒感染体外培养的Hela细胞后,用RT-PCR、Western blot及免疫细胞化学检测细胞BDNF基因及蛋白表达情况。结果 pAd-BDNF测序结果和预期一致,同源重组并经293细胞包装后形成重组腺病毒Ad-BDNF,重组病毒感染Hela细胞后能高水平表达BDNF。结论成功制备了重组腺病毒Ad-BDNF,感染细胞证实其呈分泌表达,为将其进一步应用于神经系统损伤性疾病治疗的研究打下了基础。     

携带人生长抑素2型受体的腺病毒载体的构建和表达

目的： 构建表达SSTR2的重组腺病毒载体，用于胰腺癌的基因治疗。 方法: 构建重组腺病毒穿梭质粒pDC316-SSTR2，通过脂质体与腺病毒骨架质粒pBHGlox(delta)E1,3Cre共转染293细胞，经位点特异性重组获得重组腺病毒Ad-SSTR2。通过PCR鉴定Ad-SSTR2。经293细胞扩增，纯化制备高滴度病毒感染液。以病毒感染液感染人胰腺癌细胞capan-2，应用免疫印迹检测SSTR2蛋白的表达。 结果: 成功构建腺病毒穿梭质粒pDC316-SSTR2，与pBHGlox(delta)E1,3Cre共转染293细胞获得了重组腺病毒Ad-SSTR2，以Ad-SSTR2基因组DNA为模板同时扩增出了人SSTR2 cDNA基因片段和腺病毒骨架基因片段。Ad-SSTR2感染capan-2 48 h后，免疫印迹检测到SSTR2蛋白的表达。 结论: 表达人SSTR2的重组腺病毒载体构建成功，并在胰腺癌细胞中得到正确表达，为SSTR2基因治疗胰腺癌奠定了基础。     

CTLA4-Ig重组腺相关病毒载体的构建及转染表达效果

重组腺相关病毒 (recombinantadeno associatedvirus,rAAV)载体是目前基因治疗领域中倍受瞩目的载体之一。本研究通过构建CTLA4 Ig基因重组腺相关病毒载体 (rAAV CTLA4 Ig) ,研究其对大鼠转染的表达效果及规律。大肠杆菌Sure 2和 2 93细胞均由武汉同济医院心血管内科研究室提供 ;质粒pUF1 CTLA4 Ig、pXX2和pXX6由美国匹兹堡大学分子生物实验室XiaoX教授提供 ;近交系Lewis大鼠 ,2 0 0～ 2 5 0g,雄性 ,购自中国医学科学院实验动物研究所。仓鼠抗小鼠的CTLA4 Ig购自Pharm ingen公司 ,辣根过氧化物酶标记的羊抗人IgG购自Vec…     

大鼠CTLA4-FasL融合基因重组腺相关病毒载体的构建、制备及表达

目的检测大鼠CTLA4-FasL重组腺相关病毒载体感染大鼠关节炎性成纤维样滑膜细胞后目的基因的表达。方法构建大鼠CTLA4-FasL融合基因重组腺相关病毒载体,制备重组病毒,体外感染从佐剂诱导关节炎的大鼠关节中分离的炎性成纤维样滑膜细胞,利用Flag标签纯化目的蛋白,ELISA和Western blot等方法检测目的蛋白的表达。结果获得含有大鼠CTLA4-FasL融合基因的重组腺相关病毒,感染炎性成纤维样滑膜细胞后能够分泌性表达目的蛋白。结论构建并制备的CTLA4-FasL重组腺相关病毒能够有效感染炎性成纤维样滑膜细胞并促使期表达CTLA4-FasL融合蛋白,为今后关节炎治疗的体内实验研究奠定了基础。     

A rapid and easy method for production and selection of recombinant adenovirus genomes

Adenoviruses are used widely as vectors for gene therapy. Due to the large size of their genome there is a low frequency of unique restriction sites and many techniques have been described to construct recombinant viruses. Whatever the considered technique, the Escherichia coli strain BJ5183 is used to obtain recombinant adenovirus genomes in a plasmid, or to construct defective viral backbones which will be used to produce infectious viral particles by homologous recombination in HEK293 cells. Unfortunately BJ5183 bacteria do not produce a sufficient amount of plasmid DNA to allow for restriction analysis. Plasmids have to be transferred into another strain to detect the expected construction. It is reported now that the common E. coli strain, Top10F′ can be used for the construction of recombinant adenovirus genomes. A plasmid carrying a kanamycin resistance gene and containing the two ends of the adenovirus genome was used. It permits modification by classical molecular biology techniques or homologous recombination at both ends of the genome. The remainder of the genome is introduced by homologous recombination in Top10F′. Several homologous recombination steps were successfully performed without the steps of extraction and introduction of plasmid DNA in another strain to check the plasmids obtained.     

前凋亡因子bimS基因的重组腺病毒载体构建

为探讨前凋亡因子bimS用于肿瘤基因治疗奠定基础,拟构建bimS的重组腺病毒载体。采用RT-PCR方法从人HL-60细胞扩增目的基因bimS;克隆至T载体测序正确。亚克隆bimS基因到腺病毒穿梭质粒pAdtrack-CMV上,形成含目的基因和绿色荧光蛋白(GFP)基因的穿梭载体。穿梭载体pAdtrack-CMV-bimS和病毒骨架载体pAdEasy-1经电穿孔法转入BJ5183大肠杆菌中行同源重组,获得重组pAdEasy-CMV-bimS,线性化后脂质体法转染人胚肾(HEK)293细胞进行病毒的包装、扩增、病毒滴度测定以及瞬时表达的观察;将病毒以MOI=100感染HEK293细胞,western blot检测bimS蛋白表达。结果显示病毒感染293细胞48~72h观察到GFP表达,7d出现典型细胞病变症状(CPE);提取培养上清中病毒DNA,以PCR法可检测到bimS的扩增产物;western blot检测病毒感染的293细胞总蛋白,与未感染的阴性对照细胞相比,bimS蛋白表达明显高于阴性对照细胞。表明重组bimS腺病毒构建成功;为进一步进行肿瘤基因治疗的体内外试验奠定基础。     

Local adenovirus-mediated CTLA4-immunoglobulin expression suppresses the immune responses to adenovirus vectors in the brain

The effect of local administration of two adenovirus vectors, one of which expressed CTLA4-immunoglobulin (AdCTLA), which blocks the B7-CD28 co-stimulatory pathway of T cell activation in the inflammatory response to adenovirus vectors was investigated. Mice injected with AdCTLA and an E1-deleted adenovirus vector that encodes the lacZ gene (AdRL) into the brain showed inflammatory cell infiltration from the early phase until day 6 after injection that was not different from that seen in control mice injected with an E1-deleted adenovirus vector containing no transgene (Ad0) and AdRL. After day 6 the inflammation in the control mice increased, peaked by day 15 and then decreased gradually but persisted until day 60. By contrast, in mice treated with AdCTLA and AdRL the inflammation, especially T cell infiltration, was suppressed after day 15. The anti-adenovirus antibody titer increased gradually until day 60 in the Ad0-AdRL control group, and whereas the mice injected with AdCTLA and AdRL showed lower anti-adenovirus antibody titers than the control group mice after day 15. Neutralizing antibody was not detected in either group. Expression of beta-galactosidase, the gene product of AdRL, at the injection site in the striatum and corpus callosum peaked on day 6 and remained until day 60 although it was very low in both groups; beta-galactosidase expression was similar in the two groups in spite of the difference in the degree and extent of the local immune response in the brain. This study demonstrated that the injection of an adenovirus vector expressing CTLA4-immunoglobulin into the brain suppressed not only local cell infiltration in the brain but also reduced the humoral immune response to adenovirus vectors.     

Local adenovirus-mediated CTLA4-immunoglobulin expression suppresses the immune responses to adenovirus vectors in the brain

The effect of local administration of two adenovirus vectors, one of which expressed CTLA4-immunoglobulin (AdCTLA), which blocks the B7-CD28 co-stimulatory pathway of T cell activation in the inflammatory response to adenovirus vectors was investigated. Mice injected with AdCTLA and an E1-deleted adenovirus vector that encodes the lacZ gene (AdRL) into the brain showed inflammatory cell infiltration from the early phase until day 6 after injection that was not different from that seen in control mice injected with an E1-deleted adenovirus vector containing no transgene (Ad0) and AdRL. After day 6 the inflammation in the control mice increased, peaked by day 15 and then decreased gradually but persisted until day 60. By contrast, in mice treated with AdCTLA and AdRL the inflammation, especially T cell infiltration, was suppressed after day 15. The anti-adenovirus antibody titer increased gradually until day 60 in the Ad0-AdRL control group, and whereas the mice injected with AdCTLA and AdRL showed lower anti-adenovirus antibody titers than the control group mice after day 15. Neutralizing antibody was not detected in either group. Expression of β-galactosidase, the gene product of AdRL, at the injection site in the striatum and corpus callosum peaked on day 6 and remained until day 60 although it was very low in both groups; β-galactosidase expression was similar in the two groups in spite of the difference in the degree and extent of the local immune response in the brain.This study demonstrated that the injection of an adenovirus vector expressing CTLA4-immunoglobulin into the brain suppressed not only local cell infiltration in the brain but also reduced the humoral immune response to adenovirus vectors.     

共表达人B7—1和HGF／NK4基因的重组腺病毒的制备及初步鉴定

目的构建能同时表达B7—1和HGF／NK4基因的重组腺病毒载体，同时制备相应的复制缺陷型重组腺病毒，检测其在喉癌细胞中的表达。方法构建以串联方式携带人B7-1和HGF／NK4基因的重组穿梭载体pAdtrack-NK4-B7-1。Pme I线性化后与腺病毒载体pAdEasy-1共转化大肠杆菌BJ5183，通过同源重组得到重组腺病毒载体pAd—NK4-B7—1。将重组腺病毒载体转染HEK293包装细胞制备重组腺病毒，应用病毒悬液感染人喉癌细胞株。用RT-PCR检测感染细胞中B7-1和NK4基因mRNA表达。结果酶切鉴定证实阳性pAdTrack—NK4-B7—1重组穿梭载体含有目的基因B7-1和HGF／NK4，同源重组后制备的病毒载体DNA经酶切鉴定获得了阳性重组腺病毒载体，该载体能有效转染HEK293细胞并在细胞内成功包装，转染3d后可以观察到绿色荧光蛋白（GFP）表达并逐渐增多、增强。制备的Ad—NK4-B7在体外能有效感染Hep-2细胞，感染后可维持6d高水平的B7—1和NK4基因的表达，整体表达可持续2周。结论成功构建了同时表达B7．1和NK4基因的重组腺病毒载体并制备出重组腺病毒颗粒，该病毒能有效地感染喉癌细胞并表达高水平的NK4和B7-1基因，为进一步研究B7—1和NK4基因的功能及应用B7—1和NK4进行喉癌的联合基因治疗提供了依据和素材。     

人骨形态发生蛋白2基因重组腺病毒的构建与鉴定

BACKGROUND:Bone morphogenetic protein 2 plays a key role in inducing osteogenesis. It involves in a series of bioprocess, including cell proliferation, determining the differentiation direction of germ line and cell death.  OBJECTIVE: To construct and identify the recombinant adenovirus vectors encoding human bone morphogenetic protein 2 gene by using AdMax system. METHODS:First, human bone morphogenetic protein 2 gene sequencing was amplified by PCR from human cDNA template and then cloned. Second, the recombinant shuttle plasmid was constructed and transformed into Escherichia coli competent cells DH5α. After the positive colonies were identified by colonies PCR and sequencing, the expression vectors were amplified and extracted. Next, the adenovirus expression vectors with target gene and the helper packaging plasmid carrying a majority of adenovirus genes were co-transfeced into 293 cells for virus packaging and amplification. Finally, target genes were detected by PCR, and target protein was detected by Western blot method, as well as infectious titer of the recombinant adenovirus was detected by end point dilution method. RESULTS AND CONCLUSION:Gene fragment of a length of 1 223 bp human bone morphogenetic protein 2 was obtained by PCR. The expression vectors constructed by homologous recombination techniques were identified by PCR cloning and sequencing; the results were correct. After virus packaging and amplification in 293 cells were identified by Western blot and PCR methods, the virus titer of recombinant adenovirus was 5×1013 pfu/L. These results suggest that the recombinant adenovirus vectors carrying human bone morphogenetic protein 2 gene have been constructed successfully.     

Simultaneous administration of a low-dose mixture of donor bone marrow cells and splenocytes plus adenovirus containing the CTLA4Ig gene result in stable mixed chimerism and long-term survival of cardiac allograft in rats

T-cell costimulatory blockade combined with donor bone marrow transfusion may induce mixed chimerism, rendering robust tolerance in transplanted organs and cells. However, most protocols entail high doses of donor bone marrow cells (BMCs) or repeated administration of costly agents that block costimulatory pathways, thus delaying clinical development. To circumvent these shortcomings, we developed a strategy in which the dosage of donor BMCs was reduced but compensated by donor splenocytes (SPLCs). Furthermore, repeated administration of costly agents was replaced with a single injection of adenovirus expressing a gene of interest. In rat cardiac transplantation models, cardiac allografts from DA (RT-1a) rats were transplanted heterotopically into the abdomen of LEW (RT-11) recipient rats. Immediately after cardiac transplantation, an adenovirus vector (AdCTLA4Ig; 5 x 10(9) plaque-forming units) containing the gene for CTLA4Ig was administered to recipients (n = 6) simultaneously with a low dose of donor BMCs (1 x 10(8)/rat) and SPLCs (5 x 10(7)/rat) via the portal vein. The treated LEW recipient rats developed long-lasting mixed chimerism (>10% at >100 days) and exhibited long-term cardiac allografts (mean survival time of > 200 days) compared with control recipients. Moreover, recipients displaying long-lasting mixed chimerism accepted subsequent donor skin allografts while promptly rejecting third-party skin allografts. These results suggest that blockade of the CD28-B7 pathway, using adenovirus-mediated CTLA4Ig gene transfer, in concert with a low dosage of donor BMCs and SPLCs, may represent a feasible strategy to induce stable mixed chimerism and permit long-term survival of cardiac allografts.     

重组人肝细胞生长因子-NK4腺病毒的制备及初步鉴定

目的制备表达人肝细胞生长因子（HGF）-NK4蛋白的复制缺陷型重组腺病毒。方法酶切pcDNA3-hNK4质粒获得NK4基因编码区序列并克隆至穿梭载体构建重组pAdTrack—CMV—NK4载体，线性化后与pAdEasy-1共转化BJ5183，通过同源重组得到重组pAd—NK4病毒载体。将重组腺病毒载体转染HEK293包装细胞制备重组腺病毒，用病毒悬液感染人肝癌细胞株HepG2。RT—PCR法检测感染肿瘤细胞中NK4mRNA表达。结果酶切鉴定得到阳性pAd—NK4重组腺病毒载体，该载体能有效转染HEK293细胞并在细胞内成功包装。在转染2d后能观察到绿色荧光蛋白（GFP）表达。制备的Ad—NK4在体外能有效感染HepG2细胞并获得NK4基因高水平表达。结论成功构建了NK4基因的重组腺病毒载体并制备重组腺病毒颗粒，为进一步研究NK4基因的功能及应用NK4进行基因治疗提供实验依据。