利用腺病毒载体导入CTLA4Ig基因对大鼠同种心脏移植免？…

目的 探索ＣＴＬＡ４Ｉｇ基因在实验动物体内的表达以及对大鼠同种心脏移植后的免疫抑制作用。方法 利用腺病毒作载体,将ＣＴＬＡ４Ｉｇ基因导入同种心脏移植的本鼠体内,以编码β－半乳苷酶的有复制缺陷的腺病毒重组体（Ａｄｅｘ／ＬａｃＺ）为对照,观察基因在实验动物体内的表达以及对同种异体心脏移植后的免疫抑制作用。逆转录多聚酶链反应（ＲＴ０ＰＣＲ０法检测ＣＴＬＡ４Ｉｇ基因在大鼠体内的表达,并用流式细胞仪来测定血     

重组腺病毒载体局部介导CTLA4Ig延长小鼠异体移植皮肤的存活

目的 寻求一种简单可行的延长异体移植皮肤存活的方法。方法 将CTLA4Ig cDNA插入重组腺病毒载体柯氏质粒中,再通过同源重组构建CTLA如一重组腺病毒载体;观察该表达载体对体外培养的皮肤组织块的转染情况;并应用该载体转染皮肤移植物及局部创面,观察其对昆明小鼠→Balb c小鼠背部创面异体移植皮肤存活期的影响。结果 CTLA4Ig重组腺病毒载体能成功转染并表达于体外培养的皮肤组织块中;局部应用CTLA4Ig重组腺病毒载体使小鼠异体移植皮肤存活期延长至21d。结论用腺病毒载体局部转染CTLA4Ig能有效延长异体移植皮肤的存活。     

利用腺病毒载体导入CTLA4Ig基因对大鼠同种心脏移植免疫抑制作用的研究

目的　探索CTLA4Ig基因在实验动物体内的表达以及对大鼠同种心脏移植后的免疫抑制作用。方法　利用腺病毒作载体 ,将CTLA4Ig基因导入同种心脏移植的受体鼠体内 ,以编码 β 半乳糖苷酶的有复制缺陷的腺病毒重组体 (Adex/LacZ)为对照 ,观察该基因在实验动物体内的表达以及对同种异体心脏移植后的免疫抑制作用。逆转录多聚酶链反应 (RT PCR)法检测CTLA4Ig基因在大鼠体内的表达 ,并用流式细胞仪来测定血中CTLA4Ig蛋白质的浓度变化。 结果　导入的CT LA4Ig基因能够在大鼠肝脏中表达 ,血中CTLA4Ig的浓度明显高于对照组 (P <0 .0 5 ) ,而且移植后心脏的存活时间显著长于对照组 (P <0 .0 1)。结论　CTLA4Ig基因能在大鼠体内良好表达并明显拮抗同种移植排斥反应     

携带融合基因CTLA4-Ig重组腺病毒载体的构建及表达

目的　为进行基因转移阻断T细胞共刺激通路诱导移植免疫耐受的研究 ,构建携带融合基因CTLA4 Ig的重组腺病毒载体。方法　构建含人CTLA4 Ig的重组腺病毒载体质粒pShuttle Tracker CMV CTLA4 Ig ,与腺病毒骨架质粒 pAdEasy 1 △E1、△E3共转化大肠杆菌BJ5 183 ,经细菌内同源重组 ,获得重组腺病毒质粒pAd Tracker CTLA4 Ig ,转染 2 93细胞 ,制备携带CTLA4 Ig的重组腺病毒 ,体外感染L O2细胞 72h后ELISA检测其外分泌性表达。 结果　获得携带CTLA4 Ig的重组腺病毒 ,滴度为 6× 10 1 3 PFU L ;在其感染的L O2细胞培养上清中能检测到可溶性重组蛋白CTLA4 Ig的表达 (P N =4.6 ,阳性 )。结论　制备的重组腺病毒在体外能有效感染L O2细胞 ,受感染细胞能表达、分泌可溶性的融合蛋白CTLA4 Ig ;为进行体内移植免疫耐受的基因治疗研究奠定基础。     

示踪法进行臂丛神经显微解剖的实验研究

目的研究大鼠臂丛神经主要分支在神经干中的分布规律。方法用1，1’-双十八烷．3，3，3’，3’．四甲基吲哚羰花青一高氯酸盐（1，1’-diodadecyl-3，3，3’，3’tetramethylindocarbocyanine perchlofate，DiI）作臂丛神经远端主要分支的逆行神经示踪，观察其纤维在近端神经干不同平面内的分布范围。结果DiI在神经干内的扩散速度：2d组为：（8．819±0．32）mm／d、4d组为：（8．482±0．262）mm／d、6d组为：（8．062±0．108）mm／d。对于最低安全平面，桡神经中肱三头肌支位置最高，正中神经分支中桡侧屈腕肌、旋前圆肌支与主干融合的位置最高，而尺神经的分支最低安全平面均较低。结论DiI对神经束标记具有轴突特异性，可以用于活体神经束路的研究。DiI在活体臂丛神经中顺行、逆行扩散速度相同。正中神经、尺神经作为部分动力神经源移位于肌皮神经，在入肌点平面切取均是安全的。     

腺病毒载体介导蛋白激酶B基因转染大鼠胰岛的研究

目的 观察携带外源基因的腺病毒在体外感染胰岛及外源基因在胰岛中的表达.方法 构建表达Akt1的腺病毒载体(Ad5-Akt1).按病毒细胞比(MOI值)500感染新分离的胰岛.通过免疫印迹分析(Western blot)测定胰岛培养上清液中Akt1的表达.结果 (1)成功构建了携带Akt1基因的腺病毒载体Ad5-Akt1,所获得病毒滴度为5.3×109pfu/ml;(2)表达增强型绿色荧光蛋白的腺病毒(Ad5-EGFP)和Ad5-Akt1在体外可以感染胰岛,其中Ad5-EGFP转染率可达60%～70%.在Ad5-Akt1腺病毒感染的胰岛培养上清液中检测到了Akt1蛋白的表达.结论 腺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.     

腺病毒载体介导蛋白激酶B基因转染大鼠胰岛的研究

Objective To confirm in vitro gene delivery to rat islets by adenovirus vectors. Methods The adenovirus vector, AdS-Aktl, was constructed following the standard molecular methods. The purified islets were infected with either Ad5-EGFP or Ad5-Akt1 adenovirus the multiplicity of infection (MOI) was 500). The expression of Aktl in Ad5-Akt1 islet transfectants was confirmed by using Western blot. Results Adenovirus vector Ad5-Akt1 was constructed and the the titer of the virus was 5.3 × 109 pfu/ml. The transfection efficiency of Ad5-EGFP was 60% -70%, and Akt1 protein was detected in Ad5-Aktl islet transfectants. Conclusion Adenovirus is efficient to deliver exogenous gene into rat islets in vitro.     

腺病毒载体介导蛋白激酶B基因转染大鼠胰岛的研究

Objective To confirm in vitro gene delivery to rat islets by adenovirus vectors. Methods The adenovirus vector, AdS-Aktl, was constructed following the standard molecular methods. The purified islets were infected with either Ad5-EGFP or Ad5-Akt1 adenovirus the multiplicity of infection (MOI) was 500). The expression of Aktl in Ad5-Akt1 islet transfectants was confirmed by using Western blot. Results Adenovirus vector Ad5-Akt1 was constructed and the the titer of the virus was 5.3 × 109 pfu/ml. The transfection efficiency of Ad5-EGFP was 60% -70%, and Akt1 protein was detected in Ad5-Aktl islet transfectants. Conclusion Adenovirus is efficient to deliver exogenous gene into rat islets in vitro.     

腺病毒载体介导蛋白激酶B基因转染大鼠胰岛的研究

Objective To confirm in vitro gene delivery to rat islets by adenovirus vectors. Methods The adenovirus vector, AdS-Aktl, was constructed following the standard molecular methods. The purified islets were infected with either Ad5-EGFP or Ad5-Akt1 adenovirus the multiplicity of infection (MOI) was 500). The expression of Aktl in Ad5-Akt1 islet transfectants was confirmed by using Western blot. Results Adenovirus vector Ad5-Akt1 was constructed and the the titer of the virus was 5.3 × 109 pfu/ml. The transfection efficiency of Ad5-EGFP was 60% -70%, and Akt1 protein was detected in Ad5-Aktl islet transfectants. Conclusion Adenovirus is efficient to deliver exogenous gene into rat islets in vitro.     

腺病毒载体介导蛋白激酶B基因转染大鼠胰岛的研究

Objective To confirm in vitro gene delivery to rat islets by adenovirus vectors. Methods The adenovirus vector, AdS-Aktl, was constructed following the standard molecular methods. The purified islets were infected with either Ad5-EGFP or Ad5-Akt1 adenovirus the multiplicity of infection (MOI) was 500). The expression of Aktl in Ad5-Akt1 islet transfectants was confirmed by using Western blot. Results Adenovirus vector Ad5-Akt1 was constructed and the the titer of the virus was 5.3 × 109 pfu/ml. The transfection efficiency of Ad5-EGFP was 60% -70%, and Akt1 protein was detected in Ad5-Aktl islet transfectants. Conclusion Adenovirus is efficient to deliver exogenous gene into rat islets in vitro.     

Long-term survival of cardiac allografts induced by cyclophosphamide combined with CTLA4Ig-gene transfer mediated by adenoviral vector

There is a need to achieve donor-specific tolerance in clinical organ transplantation, where potential benefits remain overshadowed by chronic rejection and the side-effects of long-term immunosuppressive therapy. It is known that the mature immune system in mice can be reprogrammed to accept a foreign graft as if it was "self". The AdCTLA4Ig-mediated gene transfer (SC) + cyclophosphamide (CP) treatment alone prolongs allograft survival but does not induce tolerance. However, in our study, the AdCTLA4Ig-mediated gene transfer combined with SC + CP treatment yielded significantly prolonged mean survival times (149.7 +/- 18.0 days), while those in the untreated or AdLacZ treated mice were rejected in normal fashion (5.3 +/- 0.5 and 5.2 +/- 0.4 days, respectively), and survival in the AdCTLA4Ig or SC + CP treated groups were 45.7 +/- 9.6 or 50.2 +/- 5.3 days, respectively. In conclusion, a protocol of AdCTLA4Ig + SC + CP improved the survival of DA-->LEW cardiac allografts.     

Long-term acceptance of rat cardiac allografts on the basis of adenovirus mediated CD40Ig plus CTLA4Ig gene therapies

BACKGROUND: We have previously demonstrated that blockade of either CD80/86-CD28 or CD40-CD154 costimulatory pathways by using adenovirus vector coding CTLA4Ig (AdCTLA4Ig) or CD40Ig (AdCD40Ig) genes induced donor-specific tolerance in rat liver transplantation. In this study, we asked whether these gene-therapy-based costimulation blockade would induce tolerance in cardiac transplantation. METHODS: Heterotopic heart transplantation was performed in a full major histocompatibility complex (MHC) barrier combination of ACI (RT1avl) to Lewis (LEW, RT1l) rats. Vector (1 x 10(9) plaque forming unit [PFU]), AdLacZ, AdCTLA4Ig, or AdCD40Ig, was administered intravenously to recipient animals immediately after grafting, and graft survival, serum CTLA4Ig/CD40Ig levels, and graft histology were assessed. Tolerance was determined by secondary skin-graft challenging. RESULTS: Allografts of both untreated and AdLacZ controls were promptly rejected within 7 days, whereas a single treatment with AdCTLA4Ig or AdCD40Ig significantly prolonged median graft survival to 55.5 and 28.5 days, respectively. In contrast, the combined AdCTLA4Ig and AdCD40Ig gene therapy maintained high CTLA4Ig and CD40Ig levels through the posttransplant period and allowed long-term cardiac allograft survival for more than 270 days. However, both donor and third-party skin grafts were rejected in the animals who harbored cardiac grafts over 150 days. Also, typical features of chronic rejection were evident in the long-term surviving grafts. CONCLUSION: Simultaneous blockade of CD28 and CD154 pathways by AdCTLA4Ig plus AdCD40Ig induces a strong immunosuppression that allows long-term acceptance of full MHC mismatched cardiac graft in rats. This strategy, however, was not enough to induce tolerance to skin grafts and to avoid chronic rejection, as shown in the liver-transplantation model.     

负载Ad-CTLA4Ig的未成熟树突状细胞延长大鼠移植肾存活时间的探讨

目的 探讨负载细胞毒性淋巴细胞抗原4免疫球蛋白基因重组腺病毒(Ad-CTLA4Ig)的受者未成熟树突状细胞(DC)对大鼠移植肾存活时间的影响.方法 选择雄性SD大鼠为供者,雄性Wistar大鼠为受者,建立大鼠肾移植模型.将受者随机分为4组,每组12只.制备Ad-CTLA4Ig及受者未成熟DC悬液,37℃混合孵育6 h,于移植前7 d,实验组经腹腔内注射负载Ad-CTLA4Ig的DC悬液;Ad-CTLA4Ig对照组、重组腺病毒空载体(Ad-VG)对照组和生理盐水(NS)对照组分别经腹腔内注射1 ml Ad-CTLA4Ig、Ad-VG和NS.观察各组移植肾的存活时间、组织形态学改变、受者血液中腺病毒中和抗体滴度、血清CTLA4Ig水平及混合淋巴细胞反应(MLR)的变化.结果 实验组移植肾存活时间为(94.6±9.0)d,较各对照组显著延长:[Ad-CTLA4Ig对照组为(39.6±10.6)d,Ad-VG对照组为(8.6±2.8)d,NS对照组为(8.4±2.6)d],差异均有统计学意义(P＜0.01),实验组移植肾组织损伤程度较轻,血液中腺病毒中和抗体滴度及混合淋巴细胞反应指数均较各对照组显著减少;实验组血清CTLA4Ig水平较Ad-CTFLA4Ig对照组显著升高.结论 负载Ad-CTLA4Ig的受者未成熟DC可减少腺病毒中和抗体,维持CTLA4Ig的稳定表达,从而延长大鼠移植肾的存活时间.     

腺病毒载体转染局部血管的安全性研究

目的 观察腺病毒载体转染局部血管,外源性基因的表达能否局限于转染部位,而不出现于其他部位,亦即观察腺病毒载体转染局部血管的安全性。方法 用可溶性支架将Ａｄｖ５－ＣＭＶ或Ａｄｖ５－ＣＭＶ／ＬａｃＺ质粒导入１６只大鼠颈动脉吻合口。术后７天,取对照组和治疗组全部已经行吻合的颈动脉以及治疗组的升主动脉、心、脑、肝、肺、脾、肾、行β－半乳糖苷酶活性测定和组织化学染色。结果 治疗组劲动脉β－半乳糖苷酶表达量为     

Bicistronic adenovirus-mediated gene transfer of CTLA4Ig gene and CD40Ig gene result in indefinite survival of islet xenograft

Blockade of CD40-CD154 costimulatory pathway in mice and primates with anti-CD154 monoclonal antibodies results in prolonged survival of vascularized organs and islet grafts. CD40Ig, a recombinant fusion protein comprised of the extracellular domain of human CD40 molecule in frame fused with the site-mutated human IgG1 Fc region, abrogated the cognate interaction of CD40-CD154 pathway by binding the CD154 molecule. In this study, replication-defective adenovirus containing the CD40Ig gene was prepared by homologous recombination and used to infect freshly isolated islets from LEW rats (RT-1(1)) in vitro using a titered dose. The islet transfectants (500 per recipient) were transplanted under the left kidney capsule of streptozocin-rendered diabetic C57BL/6 mouse recipient (H-2(b)). The mean survival time of AdCD40Ig-transfected islet grafts was significantly prolonged, while mock-infected grafts and AdEGFP-transfected grafts were rejected in normal fashion. Additionally, dose-dependent prolongation of islet graft survival was observed in mice receiving AdCD40Ig-transfected grafts. In conclusion, local production of Cd40Ig via adenoviral-mediated gene transfer induced dose-dependent prolongation of LEW --> Balb-c islet xenografts.     

Additive efficacy of CTLA4Ig and OX40Ig secreted by genetically modified grafts

BACKGROUND: The use of systemic immunosuppressive drugs have been paramount in the success in transplantation, but there are serious deleterious effects. Genetic modification of grafts to secrete immunomodulators locally may be a way to reduce the need for systemic immunosuppression. METHODS AND RESULTS: An insulinoma cell line, NIT, having the nonobese diabetic (NOD) genotype but also expressing the SV40 large T Ag, was transfected with CTLA4Ig or OX40Ig in an attempt to block signals in the costimulatory/adhesion pathways. The extracellular domains of these molecules have been fused to the Fc of IgG2c derived from the NOD mouse strain. This resulted in secreted and dimerized proteins. SV40 T Ag is potent at inducing graft rejection. Test and control transfectants were transplanted subcutaneously into young NOD mice to determine whether secretion of CTLA4Ig and OX40Ig would promote survival of the insulinoma graft. In immunodeficient mice, cell growth was similar for all transfectants. However, in immunocompetent NOD mice, the survival/growth of test grafts was significantly better than that of controls. By combining test transfectants, we found that graft survival was enhanced in an additive and significant fashion. In vitro, there was a significant reduction in immune responses-compared with control-when purified fusion proteins were added to mixed leukocyte reaction cultures. CONCLUSIONS: We conclude that blockade of individual costimulatory/adhesion signals by graft manipulation can contribute to transplantation success and that blockade of combinations of signals in these pathways enhances this success. Successful immunomodulation by the graft itself can be achieved.