腺病毒载体介导胸苷激酶基因/ACV对大鼠脑胶质瘤的离体和活体杀伤作用

建立缺陷型重组腺病毒载体介导的胸苷激酶基因治疗系统（Adtk／ACV）,进行离休和活体治疗大鼠C6脑胶质瘤研究．将质粒pAdtk与pJM17共转染腺病毒包装细胞──293细胞,同源重组后制备纯化的Adtk并以PCR证实;Adtk对离休C6细胞的杀伤作用随着Adtk滴度和ACV剂量增加而增强,并有旁观者效应,而未转染C6细胞和AdLacZ转染C6细胞均未被杀伤;扫描电镜下见Adtk／ACV处理的细胞呈明显病理改变;ACV可有效杀伤Adtk／C6细胞．在大鼠脑立体定向仪引导下于右额叶接种2×106个C6细胞,分别于第3,6,8和10天原位注射Adtk,同时腹腔注射ACV（100mg／（kg·d-1））,3d组、6d组、8d组大鼠成活期在90d以上,组织学检查未发现肿瘤细胞．10d组成活期（28．5±4．6）d,而C6未治疗组和AdLacZ／ACV治疗组成活期分别为（1．8±3．1）d和（14．0±2．2）d．本文建立的Adtk／ACV在离体和活体水平对大鼠C6脑胶质瘤有强烈的杀伤作用,效果确实,应用方便,ACV价格便宜,有潜在临床应用价值．     

Thymidine kinase gene mutation leads to reduced virulence of pseudorabies virus

To explore correlation between the tk gene structure of pseudorabies virus (PRV) and its virulence, to study the effect of the gene mutation on PRV biological properties, and to investigate mechinism of reduced virulence, thymidine kinase (TK)-deficient mutant of pseudorabies virus strain Hubei (PRV HB) was isolated by selection for resistance to 5-bromodeoxyuridine. The tk genes of PRV HB and its TK^― mutant were cloned and sequenced. 1587 base pairs of the tk gene and flanking regions of wild-type (wt) virus were sequenced, which included an open reading frame (ORF) of 1098 bp encoding a protein of 366 amino acids. The ORF contained two 137-bp repeated sequences, which were connected by an adenosine. 1458 bp of the tk and flanking regions of TK^― mutant were sequenced. Analysis of the tk gene sequence of TK^― mutant indicated that one of 137 bp repeated sequence and the connecting adenosine in the tk gene of the wt virus was deleted and a repeated sequence of 8 nucleotides (GCGCGCC) was inserted. All other nucleotides of TK^―mutant were identical to that of wt virus. Deletion and insertion of the nucleotide sequence resulted in a frameshift and a premature chain termination, and the resultant TK protein was not active. Analysis of the amino acid sequence revealed that TK protein of PRV HB contained the conserved consensus sequence of herpesviral TKs and an additional conserved-DHR-motif. The results of this work also indicated that TK^― mutant was genetically stable. Compared to PRV HB, virulence of TK^― mutant was greatly decreased. Mice vaccinated with TK^― mutant were completely protected against a lethal challenge with virulent PRV (HB).     

Induction of the synthesis of thymidine kinase by aldosterone in the kidney of the immature male rat

Summary Thymidine kinase activity was studied in kidneys from immature male rats after administration of aldosterone. Kinetic studies showed that the enzyme activity reached its maximum level 24 h after aldosterone injection. That increase was specific for aldosterone and could be related to the synthesis of new molecules of enzyme.     

Mechanism of age-dependent involution in embryonic chick notochords

To study the possible mechanism of the age-dependent involution of the notochord, isolated mesenchymefree notochords of chick embryos were cultured in vitro and compared with their counterparts in vivo. Two different aspects were evaluated: (1) DNA synthesis measured by [³H]thymidine incorporation and visualized by autoradiography and (2) cell death quantified by counting the number of pyknotic nuclei. The results demonstrate that [³H]thymidine uptake by notochords shows an age-dependent decrease in vitro as well as in vivo. The number of [³H]thymidine-labelled notochord cells, however, is higher in vitro than in vivo. At the same time, there is an age-dependent increase in pyknosis in the notochord in vivo and in vitro. So, during the aging process, the number of both pyknotic nuclei and of [³H]thymidine-labelled nuclei suggest a high turnover of notochord cells in vitro. From these results, we can conclude that the process of involution in aging notochord seems to be controlled by a programmed intrinsic process, which might be influenced partially by the microenvironment in vivo.     

乳腺癌特异性多肽介导生物大分子体内外效应

探讨乳腺癌特异性多肽PI携带外源性生物大分子靶向抗肿瘤的作用.将分离纯化获得的融合蛋白PI-EGFP与靶细胞MDA-MB-231体外共培养,探讨融合蛋白与靶细胞的结合能力;将此融合蛋白经尾静脉及肿瘤局部注射入荷瘤裸鼠体内,探讨其在肿瘤部位的聚集程度;利用分子克隆技术构建重组原核表达载体pET-28a(+)-pI-tk,诱导表达、分离纯化、鉴定获得的融合蛋白PI-HSV-TK,将不同浓度的融合蛋白与MDA-MB-231细胞共培养,经更昔洛韦(ganciclovir,GCV)作用后,探讨PI-HSV-tk对细胞的靶向杀伤效应.荧光显微镜下观察,在靶细胞内可检测到绿色荧光信号,经尾静脉及局部注射融合蛋白PI-EGFP后,在不同组织器官可见不同强度的荧光信号,在尾静脉注射组的肾脏和肿瘤部位可检测到荧光信号,而局部注射组仅在肿瘤部位可检测到;成功构建了重组原核表达载体pET-28a(+)-pI-tk;分离纯化获得高效表达的PI-TK融合蛋白,SDS-PAGE电泳及Western blotting鉴定融合蛋白的表达正确;CCK-8法及流式细胞仪检测显示,GCV对转导融合蛋白的MDA-MB-231细胞有杀伤作用,IC50值为152.64μg/mL.乳腺癌特异性转导多肽能携带生物大分子进入靶细胞,并携带具有杀伤效应的物质,使其发挥靶向治疗作用,为进一步探讨该多肽作为靶向性载体奠定实验基础和理论依据.     

成年小白鼠新生神经元的观察

将 3[H]标记的胸腺嘧啶核苷 (3[H]-thymidine)注入成年小白鼠腹腔 ,大脑中的神经细胞有丝分裂 ,可通过放射自显影在新生神经细胞中以银粒方式显现 3[H]标记 ,在光镜下观察脑切片中的银粒细胞即为新生神经元 .本实验利用放射自显影的方法 ,在成年小白鼠的海马和嗅球区域观察到新生神经元 .     

齐多夫定的合成工艺改进

胸昔与羧酸反应成酯，保护伯醇羟基，然后经成“氧桥”，上“叠氮”，最后脱保护得齐多夫定，收率为66％．本法工艺简单，产率高，便于生产．     

DNA synthesis in exocrine and endocrine pancreas after partial hepatectomy in Syrian golden hamsters

Summary ³H-thymidine autoradiography showed an enhanced DNA synthesis, in acinar and islet cells of pancreas after partial hepatectomy in syrian golden hamsters. A significant nuclear labeling index of acinar cells was observed between 48 and 84 h and reached control levels by 120 h. An increased labeling index of islet cells was also observed, however, this increase was not statistically significant. These results indicate growth factor(s) produced after partial hepatectomy is capable of inducing DNA synthesis in pancreas.This work is supported by NIH grant CA 36043.     

齐多夫定合成改进

以胸苷为原料,5′位醇羟基用对甲氧基苯甲酸成酯保护,然后与偶氮二甲酸二乙酯和三苯基膦反应成"氧桥",再与叠氮化钠反应引入"叠氮"基团,最后脱保护得齐多夫定,总收率为67%.本法在最后一步脱保护基后用碱性去离子水除杂,用热正丁醇纯化产物,操作简单,适宜工业化生产.