重组腺病毒对体外培养耳蜗Corti器的转染特性

目的:腺病毒载体是在内耳基因治疗的实验研究中应用最广泛的载体之一,但载体的靶向性不明显。通过重组腺病毒携带报告基因EGFP转染体外培养耳蜗器官,观察各型细胞的转染特性。方法:实验于2004-08/2006-01在美国纽约州立大学布法罗分校听力耳聋研究中心完成。①实验材料:Fisher大鼠来源于布法罗大学的实验动物中心。AdV/EGFP是Buffalo大学Dr.Lee惠赠。②实验方法:选取出生3d的大鼠分离获取耳蜗组织。使用携带报告基因-EGFP的重组腺病毒(AdV/EGFP),以1×107,1×108,1×109,2×109VP/mL作用耳蜗,于感染后6,12,24,48h和3d,固定组织,观察感染特性。分离出生3d的大鼠耳蜗Corti器,基底膜分离后,立即进入2mL滴度为1×109VP/mL,含2mmol/L的庆大霉素病毒溶液,作用3h后平铺到胶粒上,加入含2mmol/L庆大霉素的生长培养基,作用24h后收获样品。③实验评估:采用TRITC-labeledphaloidin组织化学染色,荧光显微镜观察绿色荧光蛋白EGFP和TRIC在耳蜗细胞中的表达定位。结果:①重组腺病毒可转染离体培养耳蜗组织中的各种类型细胞,外沟细胞感染率最高,其次为齿间细胞。仅有很少部分的毛细胞和螺旋神经元被感染。②在病毒滴度在107～109VP/mL的范围内,重组腺病毒感染耳蜗具有剂量依赖性,但大于这个滴度并不会增加感染效率,而且高浓度的重组病毒可造成毛细胞和外沟细胞破坏。③EGFP最早于感染后6h出现,48h达到高峰,外源基因高表达至少可维持7d。④庆大霉素损伤所有的毛细胞后,重组腺病毒能够感染多数的Deiters’细胞,齿间细胞的感染效率高于腺病毒对正常耳蜗组织的感染。结论:重组腺病毒转染效率在耳蜗各种类型细胞明显不同,重组腺病毒优先感染耳蜗支持细胞。

软骨源性形态发生蛋白1转基因细胞片的初步探讨

目的探讨软骨源性形态发生蛋白1（cartilage．derivedmorphogeneticprotein1，CDMPl）转基因细胞片的构建及其活性鉴定。方法取1月龄新西兰白兔骨髓分离培养BMSCs，取第3～6代细胞进行实验。实验分为3组，A组：腺病毒（adenovirus，Ad）．巨细胞病毒（cytomegalovirus，CMV）-人CDMP1（humanCDMPl，hCDMP1）-内部核糖体进入位点（internalribosomeentrysite，IRES）．增强型绿色荧光蛋白（enhancedgreenfluorescentprotein，EGFP）转染BMSCs，B组：Ad-CMV-EGFP（空载体腺病毒）转染BMSCs，C组：未转染的BMSCs。倒置荧光显微镜观察3组细胞荧光表达情况，MTT法检测3组细胞增殖活力。将3组处于对数生长期的细胞接种于温度敏感性6孔板获得细胞片，行形态学及HE染色观察，RT-PCR及Westemblot检测3组细胞片hCDMP1和II型胶原mRNA及蛋白表达，阿利新蓝染色检测糖胺聚糖（glycosaminoglycans，GAG）表达。结果倒置荧光显微镜下观察示转染细胞在72h表达明亮荧光，转染效率达90％；MTT法检测示，3组细胞生长曲线基本呈S形，培养1～9d吸光度（A）值比较差异均无统计学意义（P〉0．05）。通过温度敏感性6孔板体外可成功收获完整的三维立体细胞片结构，RT-PCR及Westemblot检测示，A组细胞片中hCDMP1和II型胶原mRNA及蛋白均呈阳性表达，B、C组均为阴性。HE染色和阿利新蓝染色示，A组纤维组织丰富，细胞外基质较多，蓝色异染颗粒多；B、C组细胞外基质相对较少，无明显特异性蓝染颗粒；A组GAG阳性染色面积明显低于B、C组，灰度值明显高于B、C组（P〈0．05）。结论hCDMPl基因转染的BMSCs细胞片能表达II型胶原和GAG，具有成软骨活性，其克服了传统组织工程中细胞利用率低、支架异质性等缺点，有望构建出致密的组织工程软骨，为软骨组织工程进一步发展提供了新思路。

Inhibitory effect of adenoviral vector-mediated AT2R gene transfection on neointimal hyperplasia

Objective: To investigate the effect of adenoviral vector-mediated AT2R gene transfection on neointimal hyperplasia after rat carotid artery balloon injury. Methods :AT2R gene was transferred into rat carotid arteries by recombinant adenovirus pAd-AT2R after the establishment of rat carotid balloon injury restenosis model. The arteries were harvested on the 14th day after gene transfer. The efficiency of trans-gene delivery was measured by the expression of adenovirus-encoding green fluorescent protein (GFP) under fluorescent microscope. The expression of AT2R and PCNA (proliferating cell nuclear antigen) was e-valuated by RT-PCR, immunocytochemistry, immunofluorescence staining, confocal microscopy, respectively. The ratio of intimal to medial area (I/M) was quantified with images and determined by an image analysis system. Results: GFP-positive area in adventitia, media and the forming neointima was about 40%. Adenoviral delivery of rat AT2R gene up-regulated AT2R expression in balloon-injured rat carotid and reduced PCNA expression and I/M significantly in neointima(P<0. 01). Double immunofluorescence labeling of AT2R and PCNA also showed that AT2R gene transfer inhibited VSMCs proliferation in neointima. Conclusion: AT2R gene transfer may be a novel promising therapy to limit neointimal hyperplasia.

Adenovirus-mediated PTEN gene transfection suppresses growth and promotes chemosensitivity of endometrial carcinoma

Objective： To determine the potential of sustained transgene expression by intratumoral injection of Ad-PTEN in the nude mouse model of endometrial carcinoma. Methods and Results： We constructed recombinant adenovirus carrying the wild-type PTEN gene （Ad-PTEN）. RL95-2 cells, an endometrial carcinoma cell line lacking PTEN function, was infected with Ad-PTEN and showed increased expression of PTEN and chemosensitivity to doxorubicin, decreased proliferation rate, and elevated apoptosis and Go/G1 arrest. Furthermore, the tumorigenicity of these cells was also completely suppressed. These results indicated that gene therapy with Ad-PTEN could significantly inhibit the endometrial carcinoma xenografts growth in nude mice by intratumoral injection, induce apoptosis of tumor cells, and reduce expression of proliferating cell nuclear antigen （PCNA）. Immunohistochemistry analysis also showed that the expression of progesterone receptors （PR） in Ad-PTEN treated tumor cells were induced, while P-glycoproteins （P-gp） and estrogen receptors （ER） decreased significantly. Conclusion： PTEN may play an important role in the development of endometrial carcinoma. Our findings cast new lights for treatment ofendometrial carcinoma.

Adenovirus-mediated TGF-β1 gene transfer to human degenerative lumbar intervertebral disc cells

Objective To test the possibility of modification of human degenerative lumbar disc cells by the exogenous growth factor gene, transforming growth factor β_1 (TGF-β_ 1) cDNA, and the expression of the encoded protein. Methods Nucleus pulposus samples were surgically obtained from 8 patients with degenerat ive lumbar disc disease. The cells were cultured and directly infected by two a denoviral constructs, Ad/CMV-EGFP containing the enhanced green fluorecence pro tein (EGFP) gene (marker gene) and Ad/CMV-TGF-β_1 containing the potentially therapeutic TGF-β_1 gene. Transgene expression was analyzed by fluorescence production and immunohistochemical staining (Ad/CMV-TGF-β_1). Results Culture cells transducted by Ad/CMV-EGFP showed specific green fluorescence und er the fluoroscope and expression sustained for at least 4 weeks. When infe cted by Ad/CMV-TGF-β_1, approximally 30% of cultured cells were staind brown (+) with TGF-β_1 staining. Conclusion This study established the strategy of delivering a potentially therapeutic gene , TGF-β_1, by using an adenoviral vector to human degenerative lumbar interve rtebral disc cells.

Effect of adenovirus-mediated gene transfection of vascular endothelial growth factor on survival of random flaps in rats

Objective: To evaluate the effect of local application of vascular endothelial growth factor ( VEGF ) via adenovirus-mediated gene transfer on survival of full thickness flaps selected randomly in rats.Methods: Thirty Sprague-Dawley rats weighing 480-520 g were used in this study. A dorsal flap (8 cm × 2 cm) in full thickness with the pedicle located at the level of the iliac crest was designed. Then the rats received 1 012 pfu replication-deficient recombinant adenovirus carrying VEGF ( AdCMV-VEGF group, n = 10 ), 1012 pfu recombinant β-galactosidase adenovirus ( AdCMV-Gal group, n = 10) and 1 ml saline (saline group, n = 10), respectively, in the distal two thirds of the proposed flap by means of subdermal injection at 8 different locations. Three days after treatment, the flaps were elevated as originally designed and sutured back in situ. The survival rate of the flaps was evaluated on day 7 after operation.Results: The survival rate of the flaps in the AdCMV-VEGF group increased significantly as compared with those of the AdCMV-Gal group (P < 0.01) and the saline group ( P < 0.01). Immunohistochemical staining showed that VEGF was expressed in the survival flaps injected with AdCMV-VEGF. Histological analysis showed that more granulation tissues and angiogenesis were observed in the AdCMV-VEGF group than those in the AdCMV-Gal and the saline groups.Conclusions: Local application of adenovims-mediated VEGF165 cDNA may efficiently improve the survival of ischemic skin flaps.