携带Fas基因重组腺病毒治疗瘢痕疙瘩的体内实验研究

目的 研究携带人Fas基因的两种重组腺病毒对瘢痕疙瘩的体内治疗效果。方法 构建瘢痕疙瘩裸鼠模型,应用携带Fas基因的常规腺病毒Ad—Fas（T）和细菌内重组腺病毒Ad—Fas（B）注射及Fas单克隆抗体（FasMcab）辅助对植入裸鼠皮下的瘢痕疙瘩组织进行治疗。通过大体观察、常规病理及电镜观察检测瘢痕疙瘩组织的变化。结果 单纯使用腺病毒注射的瘢痕疙瘩组织块体积仅轻度缩小。前期注射Ad—Fas（B）或Ad—Fas（T）后,应用FasMcab作为后续治疗,瘢痕疙瘩组织块均明显缩小,HE染色证实瘢痕疙瘩组织结构遭到破坏,电镜观察发现细胞凋亡证据。结论 重组腺病毒Ad—Fas（B）及Ad—Fas（T）的瘢痕疙瘩基因治疗效果令人满意。为瘢痕疙瘩的治疗提供了新途径。     

携带Fas基因重组腺病毒治疗瘢痕疙瘩的体外研究

目的 应用成功制备的携带人Fas基因的两种重组腺病毒，感染瘢痕疙瘩(keroid，K)成纤维细胞(fibroblasts，FB)，使其稳定高效地表达以替换原有无功能的Fas蛋白，并恢复正常的重建后Fas信号传导通道。方法 腺病毒感染KFB后，检测及比较两种腺病毒转染前后，KFB内Fas蛋白的表达变化、蛋白功能以及细胞增殖-凋亡状况。结果 腺病毒感染后的KFB均能稳定提高Fas蛋白的表达，并且在Fas单克隆抗体的诱导下可以发生明显的细胞凋亡。结论 ①构建的两种重组腺病毒Ad-Fas在体外实验中能有效地提高KFB蛋白的表达，并可以重建原来阻断的死亡信号传导通道；②细菌内重组腺病毒体外治疗效果更佳；⑧再次估证了Fas基因突变与K之间的联系，为K基因治疗展示了一条新途径。     

重组腺病毒介导Fas基因转染瘢痕疙瘩细胞并诱导其凋亡的实验研究

目的　制备含人Fas基因的重组腺病毒 ,感染瘢痕疙瘩成纤维细胞后 ,使其稳定高效地表达以替换原有无功能的Fas蛋白 ,并恢复正常的重建后Fas信号传导通道。方法　Fas基因自PcDNA3 1 Fas质粒中切取 ,应用PDC315载体系统构建携带Fas基因的重组腺病毒 ,感染瘢痕疙瘩成纤维细胞 ,检测Fas蛋白的表达及功能。结果　成功地构建了携带Fas基因的重组腺病毒 ,感染后瘢痕疙瘩成纤维细胞能稳定表达的有功能的Fas蛋白。结论　利用重组腺病毒转染可以重建被阻断的瘢痕疙瘩成纤维细胞Fas死亡信号传导通道 ,并再次估证了Fas基因突变与瘢痕疙瘩之间的联系 ,为瘢痕疙瘩基因治疗展示了一条新途径     

重组腺病毒介导mda-7/IL-24基因与阿霉素联合治疗裸鼠肝癌

目的 将重组腺病毒介导的mda-7/IL-24(melanoma differentiation-associated gene-7)与阿霉素(adriamycin,ADM)联合治疗裸鼠肝癌,探索基因治疗和化疗相结合治疗肿瘤的新方法.方法 构建携带人mda-7/IL-24的重组腺病毒载体(Ad-mda-7),单独使用该载体或ADM以及两者联合使用对实验性肝癌裸鼠进行治疗,观察抗肿瘤效果.结果 成功构建重组腺病毒并实现体内表达,Ad-mda-7+ADM联合治疗组裸鼠平均生存时间为(83.8±4.82)d,与其它3组相比生存期明显延长(P＜0.01);Ad-mda-7+ADM组裸鼠肝癌生长大小与单独使用ADM或Ad-mda-7相比明显缩小(P＜0.01).结论 重组腺病毒介导mda-7/IL-24联合阿霉素对裸鼠人肝癌转移模型有明显的抗肿瘤作用及协同效应,可望用于肝癌基因治疗的研究.     

RNA干扰技术阻抑热休克蛋白对瘢痕疙瘩生成的影响

目的 利用RNA干扰(RNAi)技术,通过热休克蛋白47重组质粒(HSP47 siRNA)和脂质体的混合液,对瘢痕疙瘩裸鼠动物模型的体内干预后,分析HSP47基因在瘢痕疙瘩生成中的作用.方法 构建裸鼠瘢痕疙瘩动物模型,于第16天腹腔麻醉后在实验组瘢痕疙瘩内注射质粒、脂质体混合液0.25 ml,对照组裸鼠腹腔注射磷酸缓冲液(PBS)0.25 ml,原笼饲养,7 d回收标本,分别做mRNA水平检测,胶原蛋白水平检测以及免疫组织化学检测.结果 实验组 HSP47 mRNA表达明显降低,且实验组总胶原含量,Ⅰ型胶原蛋白 mRNA表达与对照组比较也明显降低,差异均有统计学意义(P＜0.05).结论 利用RNAi技术,通过过HSP47 siRNA表达载体特异性沉默瘢痕疙瘩中HSP47基因表达后,瘢痕疙瘩中胶原蛋白的合成和分泌均能得到明显抑制,表明HSP47基因具有促进瘢痕疙瘩生成作用,为抑制瘢痕疙瘩提供新的靶点.     

增生性瘢痕和瘢痕疙瘩裸鼠模型的研制

目的：复制瘢痕动物模型，旨在从形态学和组织学上验证该模型的可靠性，为瘢痕的基础研究开辟一条新途径。方法：将增生性瘢痕和瘢痕疙瘩小组织块分别植入裸鼠皮下，携带一段时间（５～１２０天），且与原瘢痕作光镜和电镜对照。结果：术后裸鼠全部成活。植入物无变性坏死，且无异性细胞浸润，并保持其原有的胶原形态，透射电镜也证实细胞特性保持不变。结论：增生性瘢痕和瘢痕疙瘩植入裸鼠体内能保持其原有的组织学结构特征，裸鼠用作瘢痕动物模型是可行的、可靠的。     

手术切除加激素注射治疗胸前瘢痕疙瘩

目的：探讨胸前瘢痕疙瘩的有效治疗方法。方法：采用手术切除加瘢痕区激素注射治疗64块胸前瘢痕疙瘩。结果：64块瘢痕疙瘩变为线状或萎缩性瘢痕，随诊1～6年，均获良好控制。结论：手术切除加激素辅助治疗胸前瘢痕疙瘩安全高效，易为多数医生和患者所接受。     

保留表皮的瘢痕疙瘩裸鼠模型的建立及观察

目的选择一种合理的建立瘢痕疙瘩裸鼠模型的方法,能保持瘢痕疙瘩的生物学特性,为瘢痕疙瘩的实验研究和临床研究提供理想的动物模型。方法分别采用去表皮瘢痕疙瘩组织移植和保留表皮瘢痕疙瘩组织移植两种方法制作瘢痕疙瘩的裸鼠模型,观察各两组瘢痕疙瘩组织体积变化、持续时间等生物学特性变化规律,HE染色观察组织学变化情况,采用t检验分析两组模型组织体积变化结果。结果在组织移植后第2～8周,保留表皮组瘢痕疙瘩组织块较去表皮组瘢痕疙瘩组织块的增生明显增快(p     

《中国修复重建外科杂志》(2005年)第19卷文题索引

B瘢痕进一步重视病理性瘢痕发生机制的研究(付小兵等)1:1瘢痕疙瘩中皮肤附件结构破坏与瘢痕增生的关系(姜笃银等)1:15 p53基因第72位密码子多态与瘢痕疙瘩易感性研究(卓阳等)1:28病理性瘢痕中热休克蛋白47的表达与胶原沉积相关性研究(陈俊杰等)1:31携带F as基因重组腺病毒治疗瘢痕疙瘩的体外研究(鲁峰等)1:35肌成纤维细胞在病理性瘢痕形成中的机制(刘勇等)1:39实时荧光定量RT-PCR检测病理性瘢痕中热休克蛋白47mRNA的表达(陈俊杰等)8:613C创面愈合瘦素对体外大鼠成纤维细胞增殖与胶原合成的影响(李培兵等)1:20苯丙酸诺龙对大鼠成纤维细…     

钙离子拮抗剂局部注射治疗病理性瘢痕

病理性瘢痕(增生性瘢痕和瘢痕疙瘩)的治疗在整形外科一直非常棘手，长期以来，首选激素类药物局部注射治疗，但长期使用副作用大，亦易产生耐药性。我们自1999年3月开始对不宜使用或适宜使用康宁克通-A但效果不佳的瘢痕疙瘩患者，选用瘢痕内注射异搏定治疗，取得满意效果。     

Ex vivo bone morphogenetic protein-9 gene therapy using human mesenchymal stem cells induces spinal fusion in rodents

OBJECTIVE: Ex vivo gene therapy with the use of human mesenchymal stem cells (hMSCs) and bone morphogenetic protein (BMP) genes provides a local supply of precursor cells and a supraphysiological dose of osteoinductive molecules that may promote bone formation in patients with inadequate hMSC populations because of age, osteoporosis, metastatic bone disease, iatrogenic depletion, or other metabolic derangements. This study was undertaken to evaluate the efficacy of ex vivo gene therapy with the use of hMSCs and the BMP-9 gene to promote spinal fusion in the rat. METHODS: Sixteen athymic nude rats were treated with hMSCs transduced with recombinant, replication-defective Type 5 adenovirus containing the cytomegalovirus promoter and either the BMP-9 (Ad-BMP-9) or the beta-galactosidase (Ad-beta-gal) gene. Ad-beta-gal served as the control. Each animal received a percutaneous, paraspinal injection of 10(6) hMSCs transduced with 50 plaque-forming units/cell adenovirus in the lumbar region, with Ad-BMP-9 on the left and Ad-beta-gal on the right. At 8 weeks postinjection, computed tomographic scans of the lumbosacral spine were obtained, and the lumbosacral spine specimens were examined histologically. RESULTS: Both computed tomographic studies and histological analysis clearly demonstrated large volumes of ectopic bone at the Ad-BMP-9-transduced hMSC injection sites, resulting in successful spinal fusion and no evidence of nerve root compression or local or systemic toxicity. The contralateral regions that were treated with Ad-beta-gal-transduced hMSCs showed no evidence of osteogenesis. CONCLUSION: The results of this study suggest that hMSC and BMP-9 ex vivo gene therapy may be useful in inducing spinal fusion as well as other related procedures and certainly warrants further clinical development.     

Effect of bone morphogenetic protein-12 gene transfer on mesenchymal progenitor cells

Recombinant adenovirus mediated human bone morphogenetic protein-12 gene transfer induced tendon and cartilage-like tissue formation in vivo. The recombinant adenovirus with the human bone morphogenetic protein-12 gene was constructed, and mature human bone morphogenetic protein-12 expression mediated by adenovirus gene transfer was detected by specific antibody. Unlike bone morphogenetic protein-2 gene transfer, bone morphogenetic protein-12 gene transferred mesenchymal progenitor cell line C3H 10T1/2 showed no change of alkaline phosphatase activity, which is the mark of cell differentiation into osteoblastic phenotype. Injection of bone morphogenetic protein-12 gene transferred C3H 10T1/2 cells into nude mice thigh muscles induced tendon and cartilage-like tissue formation. The results indicate bone morphogenetic protein-12 has different effects on mesenchymal progenitor cell differentiation, and it may influence the cell differentiation into a nonosteoblast lineage.     

人骨形态发生蛋白-2基因转染人脂肪源性基质细胞的异位成骨作用

目的探讨腺病毒介导的人骨形态发生蛋白-2(Adv-hBMP-2)基因转染人脂肪源性基质细胞(ADSCs)的可行性及其成骨作用。方法从人脂肪组织中提取间充质细胞，分别转染β-半乳糖苷酶(β-gal)基因和hBMP-2基因，通过X-gal染色明确转染效率．-免疫沉淀 Western blot法和ELISA法检测BMP-2表达，确定BMP-2表达量、表达时间及其相互关系，流式细胞仪观察基因转染对细胞凋亡的影响，并将转基因细胞注入裸鼠股部肌内行X线及组织学检查。结果X-gal染色显示转染效率达91％。免疫沉淀 Western blot法和ELISA法显示转染hBMP-2的ADSC具有较高且稳定的BMP-2的表达，20d内表达量无明显减退。流式细胞表明腺病毒转染后细胞凋亡率上升，但上升幅度不大。裸鼠肌内注射转基因细胞后2周即显示有异位骨形成，4周明显增多，对照组无骨组织形成。结论ADSC是较好的基因治疗载体细胞，转染hBMP-2后可以诱导裸鼠体内骨形成。．     

抗血管生成对瘢痕疙瘩成纤维细胞增生和凋亡的影响

目的 探讨人工抗血管生成对瘢痕疙瘩成纤维细胞的增生和凋亡的影响,为瘢痕疙瘩的抗血管治疗提供实验依据.方法 手术切除人瘢痕疙瘩1条,将其分成30块,分别种植于裸鼠皮下,其中24块成活,分成3组,应用促血管内皮生长因子(VEGF组)、血管内皮抑制素(Endostar组)和0.9%的氯化钠溶液(对照组)干预瘢痕疙瘩微血管数量...     

Polyethylene glycol (molecular weight 400 DA) vehicle improves gene expression of adenovirus mediated gene therapy

PURPOSE: A significant limitation of adenoviral mediated suicide gene therapy is poor gene distribution in vivo. The choice of vehicle has been demonstrated to affect the level of adenoviral delivered gene transduction. We examined the hypotheses that 1) adenovirus suspended in PEG400 improves gene expression in the na?ve canine prostate model, 2) improved transgene expression with PEG400 results in improved tumor control and 3) vehicle affects the initial adenoviral spread from a single intratumor injection. MATERIALS AND METHODS: The magnitude and volume of gene expression were measured 24 hours following intraprostatic injection of adenovirus suspended in PEG400 (12.5% weight per volume) or saline as vehicle. Tumor growth delay was measured in mice bearing human tumor xenografts following the injection of adenovirus in PEG400 and saline. The initial spread of adenovirus was measured by confocal microscopy following a single injection of fluorescently labeled adenoviral particles in human tumor xenografts using each vehicle. RESULTS: Adenovirus suspended in PEG400 provided an average of twice the level of gene expression in the canine prostate and significantly better tumor control relative to saline in preclinical tumor models (p = 0.046 and 0.036, respectively). The initial spread of adenovirus with PEG400 was superior to that of adenovirus in saline and the latter was largely limited to the needle tract. CONCLUSIONS: Adenoviral gene therapy vectors suspended in PEG400 results in improved tumor control because of greater initial adenoviral spread, and the increased volume and magnitude of gene expression in vivo.     

Genetically targeted radiotherapy of head and neck squamous cell carcinoma using the sodium-iodide symporter (NIS)

BACKGROUND: Gene therapy that uses delivery of the sodium-iodide symporter (NIS) gene followed by radioiodide administration has been proposed as a novel form of radiotherapy for nonthyroidal cancers. METHODS: In vitro [(125)I] iodide accumulation and efflux from cells was determined after treatment with an NIS-expressing adenovirus (Ad-NIS). A clonogenic survival assay and tumor growth experiment that used athymic mice were used to demonstrate the in vitro and in vivo cytotoxicity of Ad-NIS treatment and [(131)I] iodide delivery. RESULTS: Head and neck squamous cell carcinoma (HNSCC) cell lines treated with Ad-NIS exhibit significant amounts of radioiodide accumulation and retention. In vitro HNSCC cell survival was significantly diminished after NIS gene delivery followed by administration of [(131)I] iodide. Moreover, NIS gene transfer/[(131)I] iodide administration dramatically attenuated HNSCC tumor formation in athymic mice. CONCLUSIONS: Our data demonstrate the feasibility of genetically targeted radiotherapy by use of the NIS gene as a possible therapeutic intervention in head and neck cancer.     

Adenovirus mediated gelsolin gene therapy for orthotopic human bladder cancer in nude mice

PURPOSE: Gelsolin is an actin regulatory protein that is undetectable or reduced in human bladder tumors compared with normal epithelial cells. Whether the over expression of gelsolin could inhibit tumor growth was investigated in an orthotopic bladder cancer nude mouse model using recombinant adenovirus encoding wild-type gelsolin (Ad-GSN). MATERIALS AND METHODS: The 2 human bladder cancer cell lines KU-7 and UMUC-2 were transduced with Ad-GSN in vitro. Flow cytometric analysis was done to examine the cell cycle after transducing the adenovirus. Cell growth was compared with control groups of these cells transduced with adenovirus containing the Escherichia coli beta-galactosidase gene Ad-betagal. In vivo KU-7 cells were introduced into the bladder of nude mice (day 0), followed by 3 injections into the urethra (days 2 to 4) with Ad-GSN or Ad-betagal (1 x 10 pfu). At 8 days after initial adenovirus exposure (day 10) each bladder was sectioned and stained, and the mass of the tumor was digitally determined. RESULTS: Bladder cancer cell growth (KU-7 and UMUC-2) was inhibited after these cells were transduced with Ad-GSN in vitro. Based on flow cytometric analysis over expression of gelsolin may cause these cells to arrest or delay at the G2/M phase of the cell cycle. In the orthotopic bladder cancer model the mass of the tumor was approximately 90% less in Ad-GSN treated animals than in controls. CONCLUSIONS: Ad-GSN provides a significant tumor suppressive effect on human bladder cancer cells in this orthotopic nude mouse model. Adenovirus mediated over expression of gelsolin may be useful therapy for human bladder cancer.