血管内皮生长因子定向转染诱导缺血心肌血管生成的研究

目的观察血管内皮生长因子（VEGF）质粒直接心肌注射对兔急性心肌缺血后侧支循环形成的影响。方法建立兔左冠状动脉结扎的急性心肌缺血模型，取缺血边缘区以先期构建的真核表达质粒pcDNA3／VEGF165和真核载体pcDNA3的DNA分别多点心肌注射，给药后2、4周取材分别行逆转录-聚合酶链反应（RT-PCR）和蛋白浓度分析、苏木素-伊红（HE）染色、VEGF染色。结果治疗组VEGF165 mRNA含量较对照组明显增多；蛋白浓度分析转染后VEGF165显著增多，高峰出现于实验后2周；治疗组缺血心肌新生血管数目明显多于对照组。结论VEGF165外源性基因能在转染心肌细胞后成功表达，促进了缺血心肌血管新生和侧支循环形成。     

白细胞介素-3基因疗法对骨髓细胞粘附分子表达的影响

以大剂量化疗后骨髓功能严重损伤、进行同基因骨髓移植小鼠为实验模型，观察白细胞介素－３基因治疗后实验小鼠骨髓细胞粘附分子表达水平。实验结果示，白细胞介素－３基因治疗小鼠骨髓细胞表达淋巴细胞功能相关抗原分子－１及免疗球蛋白粘附分子－１水平为２７．４６％和２２．３５％，明显高于未经白细胞介素－３基因治疗单纯骨髓移植组的１４％和１２．１７％。表明白细胞介素－３基因疗法能显著加快骨髓移植后骨髓细胞免疫功能恢复过程。     

Duchenne肌营养不良研究进展

Duchenne肌营养不良 (DMD)是儿童时期常见的严重肌营养不良 ,为X连锁隐性遗传病 ,病因为基因突变所致抗肌萎缩蛋白先天缺陷 ,主要病理过程为肌肉纤维进行性变性、坏死、脂肪填充 ,可累及多系统。本文就DMD的基因诊断、临床诊断及治疗方面近年来国内外研究进展情况综述如下。1　DMD的基因诊断1 987年由美国哈佛大学的Kunkel博士克隆了DMD致病基因 ,DMD基因长约 2 30 0Kb ,有 80个外显子 ,Xp2 1基因产物为抗肌萎缩蛋白 ,是 1 4Kb长的mR NA编码 ,主要位于骨骼肌的质膜面起细胞骨架作用 ,抗肌萎缩蛋白的基因缺失、重复或点突变 ,引起…     

基因沉默与RNA干扰

生物体经过漫长的进化和自然选择后逐渐形成了对异常基因活动进行监控和防御的能力,这种天然免疫机制在最近几年的研究中得到了进一步认识。所谓异常基因活动是指病毒基因侵入和复制,转座子和转基因表达。正常基因活动几乎不产生双链RNA(double-stranded RNA,dsRNA),而异常基     

联合应用IL-2和IL-3的基因疗法增强骨髓移植后骨髓细胞毒活性及抗肿瘤效果的实验研究

目的：探讨联合应用成纤维细胞介导ＩＬ２和ＩＬ３的基因疗法对骨髓移植（ＢＭＴ）后荷瘤小鼠抗肿瘤作用的效果及机理。方法：将分别转染ＩＬ２基因及ＩＬ３基因的ＮＩＨ３Ｔ３细胞以单独或联合方式移植至ＢＭＴ的荷瘤小鼠腹腔内８ｄ后，取出小鼠骨髓细胞检测杀伤活性的变化以及ＩＬ２受体的表达，并观察荷瘤小鼠的存活期。结果：ＩＬ３基因治疗虽然可加速ＢＭＴ后造血重建过程，但对骨髓细胞的ＮＫ、ＬＡＫ活性具有一定的抑制作用；ＩＬ２基因治疗可明显提高骨髓细胞的杀伤活性；联合应用基因治疗后荷瘤小鼠骨髓ＮＫ、ＬＡＫ活性及骨髓细胞ＣＤ２５表达升高，明显延长大剂量化疗后接受ＢＭＴ的荷瘤小鼠的存活期。结论：联合应用ＩＬ２和ＩＬ３的基因疗法能协同提高骨髓细胞ＩＬ２受体表达水平，提高骨髓细胞毒活性，增强ＢＭＴ后的抗肿瘤效果。     

体内直接导入转基因IL—2包装细胞的抗瘤作用

利用构建分泌人ＩＬ－２的逆转录病毒的包装细胞，直接体内注射，观察了对Ｂ１６小鼠黑色素瘤生长影响及转基因效果。狭缝印迹分析结果显示，Ｂ１６细胞在体内获得ＩＬ－２ｍＲＮＡ表达。同时，肿瘤生长受到抑制，表现在小鼠成瘤与存活时间延长，免疫功能也获得增强，ＩＦＮ－γ水平升高。本结果与本室以往报道的体外基因转移的动物实验结果相符，提示直接体内注射病毒包装细胞可转移外源基因及显示抑癌作用。     

血管内皮生长因子受体启动子驱动重组腺病毒双自杀基因系统选择性杀伤大肠癌细胞

OBJECTIVE: To observe the selective killing effect of adenovirus (Ad)-mediated double suicide gene driven by kinase domain-containing receptor(KDR) promoter on human colorectal cancer LoVo cells and human umbilical vein endothelial ECV304 cells. METHODS: The plasmid pAdEasy-KDR-CDglyTK was transfected into 293 packaging cells for amplification of the infectious Ad and used to infect the KDR-producing cells (ECV304 and LoVo) and the KDR-nonproducing cells (LS174T) respectively. The three cells were treated with the prodrugs 5-flurocytosine (5-FC) and ganciclovir (GCV) at different concentrations after infection. The killing effects of the fusion gene system on the cells were evaluated. The distribution of cell cycle was detected by flow cytometry. RESULTS: The infection rates of the recombinant Ad were similar among the 3 cells, gradually increasing with the increment of multiplicity of infection (MOI) and reaching 100% with the MOI of 200. The LoVo cells and ECV304 cells infected with Ad-KDR-CDglyTK were highly sensitive to both of the prodrugs (P>0.1), whereas the infected LS174T cells failed to exhibit similar sensitivity (P<0.001). The killing effect of CD/TK fusion gene on the target cells was much stronger than that of either suicide gene (P<0.001). The cell cycle of LoVo cells was arrested at G1 phase. CONCLUSION: The CD/TK fusion gene system driven by KDR promoter can selectively kill KDR-expressing human colorectal cancer LoVo cells and endothelial cells.     

基因治疗帕金森病的实验研究

本研究把Ⅰ型人酪氨酸羟化酶(human tyrosine hydroxylase type Ⅰ;HTH_1)cDNA连接到逆转录病毒载体LNSX上,构建成真核表达载体 LNSHTH_1,然后通过in vivo途径转染帕金森病模型鼠的纹状体细胞。外源的HTH_1基因在宿主脑内得到了表达,并使其病理行为改善约70％。这种新的基因治疗帕金森病的方式,有其独特的优越性。     

放射线联合p53基因及内皮抑素治疗小鼠前列腺癌皮下移植瘤

Objective To test the hypothesis that p53 gene therapy combined with endostatin can enhance tumor response to radiation therapy of RM-1 mouse xenograft prostate cancer and to investigate its mechanism. Methods A mouse prostate cancer model was established. Then mice with xenograft tumor were randomly divided into group A (control), B (radiation), C (radiation and rAdp53), D (radiation and rh-endostatin) and E (radiation and rAdp53 and rh-endostatin). On day 1, rAdp53 was injected intra-tumorously with 1 × 1010 vp per animal to group C and E. From day 1 to 14, rh-endostatin was given 15 mg/kg intraperitoneally daily to group D and E. On day 4 single fraction of 15 Gy was given to tumors in groups B, C, D and E. Normal saline was injected intra-tumorously or intraperitoneaUy accordingly as control. No treatment was done to group A. Tumor volume was measured daily. Samples were collected on Days 5, 10 and 15. Ki67, CD31, p53 and VEGF were detected by means of immunohistochemistry. Results (1) Radiation alone, radiation combined with intra-tumorous injection of Adp53 and/or intraperitoneal injection of rh-endostatin resulted in tumor growth arrest of RM-1 cells in vivo (P = 0.000). Radiation combined with both rAdp53 and rh-endostatin was the most effective treatment (P < 0.05). (2) All the four treatment groups had a decreased expression of mutant type P53 (P = 0.000). The expression of Ki67 in groups B and C were equal (P 0.05) and increasing (P = 0.000), respectively. Group D had a up-down-up curve (P < 0.05), but group E had a up-down one. On day 5 the expresion of VEGF in group E was the lowest (P < 0.05). An increased expression of MVD compared with the control was shown, and MVD in groups C, D and E were always higher than that in the control (P < 0.05). Conclusions The limitation of radiotherapy could be overcome by combination with beth p53 gene therapy and endostatin on the growth of mouse prostate cancer cell. Radiation, rAdp53 and endostatin have their own role but they can be interacted with each other.