Significance of beta-catenin and pRB pathway components in malignant ovarian germ cell tumours: INK4A promoter CpG island methylation is associated with cell proliferation

To clarify the mechanisms underlying cell cycle promotion in malignant germ cell tumours of the ovary (MGCTOs), beta-catenin and components of the pRB pathway, cyclin D1 and p16, were analysed in relation to cell proliferation. Immunohistochemically, p16 protein was not expressed in a number of MGCTOs (9 of 42 tumours: 21.4%) and was associated with p16 gene (INK4A) promoter 5'-CpG islands methylation. Amplification of the cyclin D1 gene (CCND1) was detected in a small number of MGCTOs (5 of 42 tumours: 13.5%). Reduced expression of p16 due to promoter methylation correlated significantly with increased cell proliferation as evidenced by Ki-67 labelling index (p < 0.001) and mitotic index (p < 0.01). In some tumour types, nuclear localization of beta-catenin has been reported to be associated with beta-catenin gene (CTNNB1) mutation, cyclin D1 overexpression, and increased cell proliferation. Nuclear localization of beta-catenin, which was observed in MGCTOs other than dysgerminoma, was not associated with cyclin D1 expression and increased cell proliferation, but appeared to be related to tumour differentiation. Furthermore, CTNNB1 mutations were not detected in any of the MGCTOs examined. Our results suggest that reduced expression of p16 due to INK4A promoter methylation is one of the principal factors that promote cell proliferation in MGCTOs. Thus, p16 may be a novel target for gene therapies to treat MGCTOs.     

A new microcellular cytotoxicity test based on calcein AM release

We present a microtest for cell-mediated immunity, based on the use of the Tarasaki tray and calcein AM vital dye. The number of target cells needed has been reduced to 500 per test with a corresponding tenfold reduction in the number of effector cells needed. Results were read at the rate of 1 second per test using a fluorimeter attached to a microscope. Each reaction was also confirmed visually with the use of ethidium bromide as a counterstain for dead cells. The calcein AM dye used to stain the living cells was shown to have a low spontaneous leakage rate—less than 15% in 4 hours at 37°C. Dilutions of targets stained by calcein AM had a linear relationship with measured fluorescence values. NK cells, LAKs, and CTLs were readily detectable by this microtest. Quantitation of killing and kinetic analysis was readily performed with this test system. A significant positive correlation to ⁵¹Cr-release results was found. We conclude that the microtest should find wide application in studies of cell-mediated immunity.     

铀作业人员的微循环改变

对铀作业者１９９例及无毒物接触史的正常人１０１例行甲襞微循环检测。结果，铀作业人员甲襞微循环有改变，其加权积分值大于对照组。主要表现在管袢数减少（Ｐ＜０．０１）、管径增粗（Ｐ＜０．０１）、毛细血管运动增加（Ｐ＜０．０１）、乳丛增多（Ｐ＜０．０１）。通过对铀致微循环改变的机理试以分析，提出这种微循环改变是铀中毒的病理表现，亦是铀中毒的重要发病环节；提示铀中毒治疗中应注重改善微循环。     

rhGM-CSF作为成人乙肝疫苗无(弱)应答者免疫佐剂初探

Objective To investigate the effect of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) as adjuvant on immune response in adults of non-and hyporesponders to hepatitis B vaccine. Methods Those who were once immunized with recombined yeast gene hepatitis B vaccine more than one standard scheme in two years and negative for hepatitis B markers were randomly sorted as group A and group B. 33 adults of group A were given hepatitis B vaccine 10 μg each time. The immune procedure was O, 1 and 6month. 34 adults of group B were given rhGM-CSF 300 μg for the first day, then 10 μg each time for routine immune. The blood samples were collected before the first injection and in 1, 2 and 8 months (T1, T2, TS)following the first injection to test Anti-HBs. Results Anti-HBs positive conversion rates of group A and B at T8 was 39.39% and 64.71% respectively(P=0.038). Anti-HBs levels of group B at TI, T2, T8 were(113.85±198.56) mIU/ml, (312.40±349.44) mIU/ml, (427.74±411. 58) mIU/ml (P=0.001). There was significant difference between group A and B in T8 Anti-HBs levels(P=0.010). Conclusion Better immune response was found in the group of rhGM-CSF with hepatitis B vaccine. So rhGM-CSF can induce the immune respond to hepatitis B vaccine.     

阴沟肠杆菌敏感株与诱导耐药株药敏异同分析

目的 应用体外诱导耐药的方法,模拟临床上不合理使用抗生素过程,通过对阴沟肠杆菌敏感株与诱导耐药株药敏异同分析,为临床合理应用抗生素提供依据.方法 取临床分离对头孢他啶敏感的阴沟肠杆菌10株.应用不同浓度梯度头孢他啶逐级诱导成为耐药株,比较分析10株敏感茵和诱导后耐药菌药敏结果.结果 临床分离株大部分对头孢菌素类抗生素敏感:经头孢他啶诱导后,时大部分头孢菌素、青霉素类抗生素及氨曲南耐药,只对头孢吡肟仍保持敏感率达90%.此外.对亚胺培南,阿米卡星,复方新诺明,环丙沙星仍保持诱导前的敏感性.结论 长时间、不规范应用低于正常杀茵剂量的抗生素容易诱导出细菌的耐药性,临床抗感染治疗合理选择抗生素和足量地应用抗茵药物非常必要.     

白塞氏病相关抗原的重组表达及抗原性初步分析

目的：探讨Kinectin的抗原性，为下一步研究Kinectin在白塞氏病中的意义奠定基础。方法：从人Hep2细胞抽提RNA，利用RT—PCR扩增能覆盖Kinectin全长的3个片段，其位置分别相当于氨基酸22—510(kin—5)，503-994(kin-M)和920-1346(kin-3)。将扩增的PCR片段克隆到pET42—a( )载体中，诱导表达的融合蛋白用白塞氏病病人血清进行Western blot分析。结果：构建的3个表达Kinectin部分片段载体(pET／kin—5、pET／kin-M和pET／Kin-3)具有正确的阅读框架，DNA测序符合率为99％，能在大肠杆菌中表达相应的肽段。8份阳性血清中，用Western blot分析发现，6份血清与kin-M反应，5份血清与kin-3反应，只有一份血清与kin-5有反应。结论：成功地构建表达覆盖Kinectin全长的3个载体，并初步发现Kinectin的抗原性可能主要位于其中间段和羧基端。     

不同导联系统记录体表心室碎裂电位效果对比研究

本文分别应用标准１２导联系统，Ｆｒａｎｋ正交导联系统和改良Ｆｒａｎｋ正交导联系统记录健康人３６例、陈旧性心肌梗塞４２例的心室碎裂电位，通过分析这些导联系统中相互垂直或近似垂直的导联上ＶＦＰ的形态，数量关系，发现改良Ｆｒａｎｋ正导联系统记录ＶＦＰ较其它导联系统更科学，合理，且操作方便。     

西洋参茎叶皂甙对 CPHD病人外周淋巴细胞增强与调节作用

目的 :为了开发西洋参在临床医学方面的应用 ,提高CPHD病人的各项免疫指标。方法 :选择 4 0例CPHD病人 ,分成用药组 2 0例 ,对照组 (非用药组 ) 2 0例 ,通过流式细胞术检测病人外周血中T淋巴细胞亚群中各亚群所占的比例来反映机体的细胞免疫状态。结果 :通过两组临床研究观察 ,用药组疗效优于对照组 (P <0 0 5 )。西洋参茎叶皂甙可直接作用于淋巴细胞分化成熟过程 ,具有增强和调节CPHD病人机体免疫功能的作用。结论 :合理应用西洋参茎叶茎叶皂甙对改善CPHD病人免疫功能低下和紊乱状态 ,具有重要临床意义。     

pp~(60c-src(+))在神经生长端的表达及其意义

目的　探索 pp60c-src( + ) 在神经生长端的表达特征及其生理意义。方法　用免疫细胞化学方法检测 pp60c-src( + ) 在初代培养鸡胚脊神经节细胞内的分布特征。结果　pp60c -src( + ) 的免疫活性分布在神经细胞的细胞膜下、核周体、神经突起 ;在生长端体部和丝状伪足 ,pp60c -src( + ) 的免疫活性较强 ,少数免疫活性较弱。在伸长的突起上有时可见 pp60c-src( + ) 免疫活性分布在串珠样膨体上。结论　pp60c-src( + ) 参与生长端的运动和生长 ;在生长端分化、神经生长过程中 ,pp60c-src( + ) 的调控作用具有时空特异性     

Hsp65与HBV多表位融合基因的表达及其免疫应答研究

目的 用基因工程的方法构建并表达一种由 Hsp65、通用辅助T细胞表位(PADRE)和HBV表面抗原与核心抗原的多个表位组成的乙型肝炎治疗性疫苗(简称为Hsp65-HBV).方法 采用PCR方法人工合成由PADRE和HBV的多个表位组成的基因片段,将此片段克隆入原核表达质粒pET28a/Hsp65后.利用大肠杆菌进行融合蛋白的表达.用纯化后的蛋白免疫nalb/c 小鼠.将小鼠脾细胞用HBsAg 装载的P815细胞在体外刺激5 d后,用流式细胞仪检测IFN-γ 细胞的频率.用ELISA方法检测小鼠血清中的特异性抗体(IgG1和IgG2a)的产生.结果 由Hsp65、通用辅助T细胞表位(PADRE)和HBV的多个表位组成的融合蛋白能在大肠杆菌中进行高水平的表达,能诱导小鼠产生IgG2a亚型的特异性抗体,并能诱导 HBV 特异性的 IFN-γ 的淋巴细胞的产生.结论 获得了大肠杆菌表达的由Hsp65、通用辅助T细胞表位(PADRE)和HBV的多个表位组成的融合蛋白,并能被机体以MHC-Ⅰ途径递呈,为检测本融合蛋白是否能激发出HBV特异性CTL反应奠定了基础.