pcDNA3.1/hIL-18真核表达载体的构建及表达

目的 :构建重组真核表达质粒pcDNA3.1/IL 18,并在哺乳动物细胞COS 7和Rlc310中进行瞬时和稳定性表达。方法 :从含hIL 18基因的中介载体 pGEM TEasy( pGEM T/hIL 18)中 ,以限制性内切酶酶切方法获得目的片段 ,克隆入真核表达质粒 pcDNA3.1( )中。以脂质体法转染COS 7和Rlc310细胞 ,用RT PCR检测IL 18mRNA的水平 ,免疫组化染色法检测蛋白表达。结果 :构建了hIL 18基因的重组真核表达质粒pcDNA3.1/IL 18,并可在哺乳动物细胞中瞬时、稳定表达 ,获得了可稳定表达hIL 18基因的Rlc310细胞株。结论 :pcDNA3.1/IL 18的构建及表达 ,为IL 18抗肿瘤作用的研究奠定了基础     

人乳头瘤病毒18型L1-E6、L1-E7嵌合基因表达载体的构建及表达

目的 构建HPV 18 L1－E6，L1－E7嵌合基因的表达载体，并在CHO细胞中表达。方法 克隆HPV18 L1－E6和L1－E7基因，插入中介载体pGEMT-Easy中并测序鉴定。采用PCR定点突变法，突变L1－E6，L1－E7基因序列中与转化作用相关的位点，分别与L1基因连接后插入真核表达载体pVAX1，构建真核表达质粒pVAX-1L1 E6Mxx,L1E7Mxx。用磷酸钙沉淀法，转染CHO细胞，以抗HPV－18L1，抗E6和抗E7特异性单克隆抗体（mAb)做ELISA和免疫细胞化学法检测。结果 ELISA检测显示，转染各种pVAX1-LIE6Mxx-L1E7Mxx融合蛋白表达质粒的细胞提取物的P－N值均＞2．1；免疫细胞化学检测，在胞浆，胞核可见棕黄色颗粒。结论 我建的pVAX1-L1E6Mxx-E7Mxx融合蛋白质表达质粒，可在转当细胞内表达相应的L1－E6Mxx和L1－E7Mxx蛋白，为今后进行DNA疫苗的研究奠定了基础。     

THE INFLUENCE OF HUMAN SINGLE CHAIN INTELEUKIN-12 GENE TRANSDUCTION ON THE BIOIOGICAI BEHAVIOR OF HEPATOMA 7721 CELLS

Objective. To investigate the anti-tumor effects of human single chain interleukin-12 (hscIL-12).``Method. pcDNA/ hscIL-12 recombinant was transfected into human hepatic carcinoma cells (7721 cells)by lipofectin method. The 7721/hscIL-12 cells which secrete hscIL-12 stably, were obtained via G418 selection, and in vitro the influence of hscIL-12 gene transduction on the growth of tumor cells was evaluated by cell cycle analysis. In vivo, genetically engineered 7721 cells (7721/hscIL-12, 7721/pcDNA) and parental cells were implanted into BALB/c nude mice, respectively. 7721/pcDNA and 7721/hscIL-1 2 groups were divided into two sub-groups on day 8: one was administered with hPBL twice, 6 days at interval; the other was given equal volume of PBS. Mice were sacrificed on day 26, and spleens and tumors were taken out for histologic assay.``Results. hscIL-12 produced stably by 7721/hscIL-12 cells had bioactivity, and it was proved by Western blot, immunocytochemistry, and in situ hybridization. In vitro, compared with 7721 and 7721/pcDNA, the 7721/hscIL-12 grew much more slowly. FACS assay showed apparent Gl arrest of 7721/hscIL-12 cells. In animal experiment, on day 8 after inoculation, the tumors of 7721 and 7721/pcDNA group were up to 5 ～ 7mm,while those of 7721/hscIL-12 group were 2 ～4mm. When treated with hPBL, the tumor of 7721/hscIL-12 group disappeared completely. Histologically, the tumors from 7721/hsclL-12 without hPBL treatment had numerous lymphocyte infiltration, the tumor cells displayed depression looking, atrophy, focal necrosis and apoptosis, whereas the tumors of 7721 and 7721/pcDNA groups grew thrivingly.``Conclusion. hscIL-12 transduced 7721 cells could induced significant antitumor immune response which resulted in tumor regression totally when the hPBL was inoculated, and also hscIL-12 has certain effects on mice immune svstem. These findings suggest that hscIL-12 and hscIL-12 gene therapy might have promising prospects in clinical application.     

Bacterially produced human B7-1 protein encompassing its complete extracellular domain maintains its costimulatory activity in vitro

Ｏｂｊｅｃｔｉｖｅ　Ｔｏｉｎｖｅｓｔｉｇａｔｅｗｈｉｃｈｏｆｔｈｅｔｗｏｉｍｍｕｎｏｇｌｏｂｕｌｉｎ (Ｉｇ ) ｌｉｋｅｄｏｍａｉｎｓ ,ｉｍｍｕｎｏｇｌｏｂｕｌｉｎｖａｒｉａｂｌｅｒｅｇｉｏｎｈｏｍｏｌｏｇｏｕｓｄｏｍａｉｎＩｇＶ (ｈＢ7 1ＩｇＶ) ,ｏｒｉｍｍｕｎｏｇｌｏｂｕｌｉｎｃｏｎｓｔａｎｔｒｅｇｉｏｎｈｏｍｏｌｏｇｏｕｓｄｏｍａｉｎＩｇＣ (ｈＢ7 1ＩｇＣ)ｏｎｈｕｍａｎＢ7 1ｍｏｌｅｃｕｌｅｃｏｎｔａｉｎｔｈｅｒｅｃｅｐｔｏｒｂｉｎｄｉｎｇｓｉｔｅｓ ,ａｎｄｔｏｅｖａｌｕａｔｅｉｆｔｈｅＢ7 1ｍ…     

碘缺乏病区硒与胎儿脑海马区原癌基因表达

目的　研究微量元素硒 (Se)与胎儿脑海马区神经元原癌基因c-fos蛋白表达之间的关系。方法　在陕西省缺碘 (I)低硒区 (宜君县 )缺碘高硒区 (紫阳县 )及对照区 (临潼县 )共采集 5 1例脐血进行微量元素Se、I的检测 ,并在以上 3地共解剖孕 2 0～ 37周的水囊引产胎儿胎脑 2 0例 ,通过免疫组化学法检测胎脑海马区神经元原癌基因c -fos蛋白产物的表达。结果　 3地I水平无显著性差异 ,Se水平有显著性差异 (P <0 0 5 ) ,以紫阳为最高 ,宜君为最低 ;紫阳c-fos蛋白表达较强 ,与宜君相比有显著性差异 (P <0 0 5 )。结论　Se的水平可能影响胎脑海马区神经元c -fos蛋白的表达 ,并进而影响胎儿神经系统及智能的发育     

伴有软骨基质分泌的乳腺化生性癌临床病理学观察

目的分析伴有软骨基质分泌的乳腺化生性癌的临床病理学特征、免疫组化标记特点及其组织起源。方法收集5例伴软骨基质分泌的乳腺化生性癌的临床及随访资料,观察组织病理形态学及免疫组化标记,并对相关文献进行复习。结果5例患者年龄37～48岁,组织标本切面灰黄或灰褐色,结节状,界限尚清。镜下见肿瘤细胞围绕软骨粘液样或透明软骨样基质呈巢状,条索状排列,密集的瘤细胞可聚集于周边,中央区细胞稀疏,周边肿瘤细胞向基质移行中无梭形细胞肉瘤样成份。免疫组化结果显示,5例均同时表达CK、Vim及S-100,但不表达ER、PR、Her-2。结论伴有软骨基质分化的乳腺化生性癌是一种罕见肿瘤,具有特征性的病理组织形态和免疫表型,临床过程与普通非特异性浸润性乳腺癌无异。     

不同佐剂增强小鼠黑色素瘤瘤苗抗瘤细胞免疫效应的比较

 目的 评价5种不同配方佐荆在低免疫原性小鼠黑色素瘤瘤苗保护性免疫中的细胞免疫增强作用。方法 实验组用5种不同配方的佐剂（FCA，FCA＋IL-2＋GM-CSF，FIA＋IL-2＋GM-CSF，FIA＋SWZY，FIA＋SWZY＋IL-2＋GM-CSF）和照射灭活的D5黑色素瘤细胞制成瘤苗免疫小鼠，对照组免疫用单纯灭活D5黑色素瘤细胞。末次免疫后3天各组取半数动物检测DTH反应、NK细胞活性及特异性CTL活性，剩余半数动物接种活D5黑色素瘤细胞，3周后再次检测上述各免疫学参数。结果 与对照组相比，经免疫后的各实验组DTH反应、NK细胞活性、CTL活性均明显升高（P〈0．05），但成瘤后随肿瘤增大，则呈下降趋势。结论 5种佐剂配方制成的瘤苗均能诱导免疫小鼠对低免疫原性肿瘤的细胞免疫应答。其中佐剂SWZY和FCA增强免疫的效果相当，而毒副作用较小，可望成为一种人类肿瘤免疫治疗的新型佐剂。