神经营养素-3重组腺病毒促进脊髓背根神经节的存活

目的：观察神经营养素－3（neruotrophin3,NT－3)重组腺病毒（adorsal root ganglia ,DRG）细胞存活的促进作用,方法：原代培养DRG,加入不同感染增殖率（multiplicity of infection,MOI)的Ad-NT-3,观察DRG细胞的形态,MTT法测定DRG细胞的增殖活性,结果：当加入MOI为10和50的Ad-NT－3时,形态学发现,DRG细胞数目增加,突起变长,尤其以3d后表现更为明显,DRG细胞的增殖活性显增加（P＜0．01）,当MOI为100时1d和3d,DRG细胞数目明显减少,无突起或变短,部分细胞死亡,MTT法测定DRG细胞的增殖活性均显降低（P＜0．01）,结论：NT－3基因能通过腺病毒介导表达NT－3蛋白,促进体外培养DRG细胞的生活。     

腺相关病毒介导的人内皮抑素基因转染对肝细胞性肝癌生长的抑制作用

目的　研究腺相关病毒介导的人内皮抑素基因转染对肝细胞性肝癌的抑制作用。方法　用含人内皮抑素的腺相关病毒(r AAV2 /EGFP- Endo)转染肝癌细胞Hep3B,通过流式细胞仪检测其转染率。MTT法检测转染细胞的培养液上清对人脐静脉内皮细胞ECV30 4增生的抑制作用。Hep3B细胞接种裸鼠建立移植瘤模型,分别瘤内注射r AAV2 /EGFP- Endo(2×10 1 0 v.g.)、r AAV2 /EGFP或生理盐水,3周后检测裸鼠血液中内皮抑素的表达及移植瘤的体积和微血管密度(MVD)。结果　流式细胞仪结果显示重组腺相关病毒体外对Hep3B细胞的转染率为6 2 .5 % ;转染内皮抑素基因的Hep3B细胞的培养液上清能显著抑制ECV30 4细胞的生长(P<0 .0 1)。移植瘤内注射r AAV2 /EGFP- Endo后,荷瘤裸鼠血清中内皮抑素浓度为(82 .6 4±7.5 4 ) μg/L,移植瘤的体积和微血管密度均明显低于对照组(P<0 .0 1)。结论　腺相关病毒介导的人内皮抑素基因转染能够有效抑制人肝细胞性肝癌的生长。     

腺病毒介导的PTEN和p16基因对前列腺癌细胞系的作用

[目的]探讨腺病毒介导的PTEN和p16基因蛋白对抑制前列腺癌细胞增殖和调控其凋亡的作用.[方法]构建携带人PTEN和p16基因的腺病毒载体,转染体外培养的前列腺癌细胞系PC-3,采用RTPCR、Westem b1ot检测目的基因的表达.通过细胞生长试验、流式细胞仪检测PC-3转染前后细胞增殖和凋亡的变化.[结果]病毒滴度 Ad-PTEN为1.8×107 pfu/ml、Ad-p16为1.2×1010 pfu/ml,RT-PCR检测可见PTEN-mRNA(462bp)和p16-mRNA(520bp)表达,Western blot检测有PTEN蛋白(60KD)和p16蛋白(16KD)特异表达,并可明显抑制PC-3细胞的增殖,诱导其凋亡,联合基因治疗组与单基因组相比差异显著.[结论]PTEN和p16可明显的抑制前列腺癌细胞株PC 3的增殖,增加细胞的凋亡,转染携带PTEN和p16的重组腺病毒载体有望成为治疗前列腺癌的有效方法.     

重组腺病毒介导的人p27kip1基因高表达对体外培养的大鼠主动脉血管平滑肌细胞迁移的影响

目的　探讨重组腺病毒介导的p2 7kip1基因及其蛋白产物高表达对血管平滑肌细胞 (VSMCs)迁移的抑制作用。方法　将含人p2 7kip1cDNA的重组腺病毒 (Adhp2 7kip1)及含 β 半乳糖苷酶基因的重组腺病毒 (AdLacZ)在体外转染原代大鼠主动脉VSMCs,用Westernblot及Boyden趋化小室检测外源性p2 7kip1蛋白在细胞内的表达及对VSM Cs迁移的影响。结果　转染后 2 4h ,Adhp2 7kip1转染的VSMCs内p2 7kip1蛋白高表达 ,而AdLacZ转染的VSMCs内仅显示极低水平的内源性p2 7kip1蛋白表达 ;Boyden趋化小室检测显示未转染的VSMCs、AdLacZ及Adhp2 7kip1转染的VSMCs血清诱导后的迁移细胞数分别为 139± 2 6、10 6± 16及 6 8± 14。结论　外源性p2 7kip1基因及蛋白产物在VSMCs内高表达可显著抑制VSMCs的迁移     

简化的两步法细菌内同源重组高效制备腺病毒质粒

目的 :应用简化的两步法细菌内同源重组高效制备腺病毒质粒。方法 :对细菌内同源重组法进行改进和简化 ,先构建含腺病毒基因组质粒pAdEasy 1的BJ5 183细菌 ,筛选出链霉素和氨苄青霉素抗性菌落 ,继而应用氯化钙法制作BJ5 183pAdEasy 1感受态细菌。用PmeⅠ酶使转移质粒pAdtrack CMV TK线性化 ,和BJ5 183pAdEasy 1感受态细菌混合进行转化 ,在含 12 5 μg mL卡那霉素的LB琼脂平皿上培养 ,筛选出卡那霉素抗性的细菌进行质粒抽提纯化 ,获得重组腺病毒质粒。结果 :卡那霉素抗性细菌有 2种 ,一种是含pAdeasy CMV TK(约 34kb) ,另一种含pAdtrack CMV TK(约 10kb) ,两者可经琼脂糖电泳加以鉴别。构建重组腺病毒质粒的成功率达 90 % (9 10 )。结论 :简化的两步法细菌内同源重组是一种简便易行、快速高效的腺病毒质粒构建方法     

Modulation of glioma cell invasion and motility by adenoviral gene transfer of PAI-1

A number of studies have emphasized the role of PAI-1 as an important regulator of tumor cell invasion and metastasis. The hallmark of primary tumors of the central nervous system and glioblastomas in particular is the diffuse invasion into the normal brain tissue. Since PAI-1 is expressed in such tumors, we studied the effect of adenoviral-mediated transfer of the PAI-1 gene in regulating the in vitro invasiveness of D54Mg glioma cells into Matrigel, and into fetal rat brain aggregates. Treatment of D54Mg cells with 50 MOI (multiplicity of infection) of the replication defective vector AdCMVPAI-1 increased PAI-1 expression 23-fold compared to control vectors, and the invasion through Matrigel was reduced by 67%. The motility of the cells was reduced by 58% compared to controls (indicating that inhibition of motility was the principal effect of PAI-1 in these cells). The ability of D54Mg tumor spheroids to invade fetal rat brain aggregates was not reduced by the PAI-1 gene transfer. The results show that overexpression of PAI-1 can inhibit glioma cell motility and invasion through extracellular matrix (ECM) components, like laminin and collagen, but does not inhibit tumor cell invasion in a three-dimensional invasion assay, simulating normal brain tissue having a different ECM and interstitial composition. The different results obtained in the two invasion assays reflect the complex biological effects of the uPA/PAI-1 system, and questions a simplistic view of PAI-1 as an inhibitor of brain tumor invasion. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pathogenic and pathological characteristic of new type gosling viral enteritis first observed in China

AIM: To study the purifying method and characteristics of new gosling viral enteritis virus (NGVEV),the etiological agent of new gosling viral enteritis (NGVE) which was first recognized in China, as well as the pathomorphological development in goslings infected artificially with NGVEV. METHODS: (1)NGVEV virions were purified by the procedure of treatment with chloroform and ammonium sulfate precipitation, dialysis to remove the sulfate radical and ammonium ion and separation by gel filtration chromatography, and SDS-PAGE. (2)Forty-2-day-old White Sichuan goslings were orally administered with NGVEV and 24 hr later 2 birds were randomly selected and killed at 24hr intervals until death occurred. Specimens(duodenum, ileum, liver, heart, kidney, spleen, lung, proventriculus, pancreas, esophagus, and the intestinal embolus) were taken until all birds in this group died and were sectioned and stained with hemotoxylin and eosin and studied by light microscope. RESULTS: NGVEV shared the typical characteristics of Adenovirus and which structural proteins consisted of 15 polypeptides. Necrosis and sloughing of the epithelial cells covering the villus tips of the duodenum were first observed in goslings 2 days postinfection artificially with NGVEV. With the progress of infection, this lesion rapidly occurred in the epithelium at the base of the villus and with infiltration of the inflammatory cells, the jejunum tended to be involved. With the intensification of mucosa necrosis and inflammatory exudation of the small intestine, fibrinonecrotic enteritis was further developed and embolus composed of either intestinal contents wrapped by pseudo-membrane or of the mixture of fibrous exudate and necrotic intestinal mucosa were observed in the middle-lower part of the small intestine. This structure occluded the intestinal tract and made the intestine dilated in appearance. The intestinal glandular cells underwent degeneration, necrosis and might be found sloughed into the lumen. Hemorrhage and hyperemia could be observed on the lung and kidney. Epithelial cells of the renal tubular underwent degeneration. In some cases, granular degeneration and fatty degeneration could be found in the liver and in some cases at a later stage of this disease the epithelial cells of trachea and proventriculus might be found sloughed. In some cases at an early stage of this disease, cardiac hyperemia and hemorrhage could be observed. Esophagus, pancreas and brain were found normal. Analyses and comparisons between the pathologic lesions of NGVE and Gosling Plague (GP) were available in this paper as well. CONCLUSION: (1)NGVEV is adenovirus. (2)Pathological characteristic could be as the data for NGVE diagnosis.     

Flt3配体修饰小鼠肝癌疫苗的制备及其体内抗肿瘤活性观察

目的:以小鼠Flt3配体(murine flt3 ligand, mFL)的重组腺病毒载体(AdmFL)体外感染小鼠肝癌细胞株Hepa1-6制备肝癌疫苗,并观察其体内抗肿瘤活性.方法:腺病毒载体体外感染Hepa1-6细胞,48 h后通过载体所携带的绿色荧光蛋白(GFP)的表达检测感染的效率;并用ELISA方法检测第0、2、4、6、8天培养上清中mFL的表达量;每天进行细胞计数(连续14 d),观察细胞的体外生长曲线;取5×106个修饰后的Hepa1-6细胞接种C57BL/6小鼠,4周后于原接种部位的对侧再接种2×106个野生型Hepa1-6或EL4细胞.结果:AdmFL能有效感染靶细胞Hepa1-6,培养上清中mFL表达量在第2天最高,达到(71.1±6.3) ng/ml,感染后Hepa1-6的体外生长未受到明显抑制;修饰后Hepa1-6细胞的体内成瘤性下降;4周后再接种Hepa1-6细胞,肿瘤生长速度明显降低;而再接种的EL4细胞呈渐进性生长,与对照组相比无显著差异.结论:腺病毒介导的Flt3配体基因修饰后的肝癌细胞的体内成瘤性下降,并能激发特异性免疫保护反应,是有效的肝癌疫苗.