过氧化氢导致线粒体DNA编码细胞色素C氧化酶基因突变与其编码蛋白结构与功能关系的研究

目的探讨过氧化氢(H2O2)导致线粒体DNA(mtDNA)编码细胞色素C氧化酶(COX)基因损伤(突变)后对其编码蛋白结构与功能的影响及两者之间的关系.方法采用生物信息学技术,运用计算机同源模建的方法模拟人天然COX亚单位和其突变体的空间结构,并利用已有的实验数据,结合模建的结果进行了结构、功能的分析、研究.结果在COX Ⅰ的三维结构中突变的部位(残基297～306)位于分子的最外部,而该区域参与了与B亚基(相当于COXⅡ亚基)的作用,该部位的突变使突变部位附近残基的侧链结构环境发生了极大的变化,导致附近的电荷性质、亲疏水性质和空间特性出现变化,这些变化对酶复合物的形成有直接的影响;COXⅡ突变后,组成分子结构的"椅靠"部分和"椅面"部分之间的夹角略有增大,而"椅靠"和"椅面"之间的夹角的变化将会影响整个酶分子的稳定性.结论H2O2导致的mtDNA编码COX Ⅰ、COXⅡ亚基基因突变使其编码的COX蛋白空间结构发生改变,这种改变是其功能改变(如酶蛋白活性下降)的主要原因.     

中国仓鼠肺成纤维细胞和HeLa细胞DNA氧化损伤的自身修复能力与比较

目的:研究不同氧化相关因素对中国仓鼠肺成纤维细胞(CHL)和HeLa细胞DNA损伤的自身修复情况.方法:将CHL细胞和HeLa细胞用不同氧化相关因素处理一定时间[CHL细胞:过氧化氢(H2O2)25 min,重铬酸钾(K2Cr2O7)105 min,阿霉素(Dox)75 min;HeLa细胞:H2O2 25 min,K2Cr2O7 105 min],随后立即去毒培养0、0.5、1、2、3 h,以碱性单细胞凝胶电泳技术检测DNA链断裂情况.结果:①CHL细胞经H2O2、K2Cr2O7、Dox作用后引起DNA链断裂,去毒培养1 h链断裂修复明显(P<0.01);去毒培养2～3 h,前两毒剂的损伤组完全修复,而Dox组链断裂仍高于未损伤组;②HeLa细胞经H2O2、K2Cr2O7作用后引起DNA链断裂,去毒培养0.5 h链断裂明显修复(P<0.01),去毒培养1 h则完全修复;③CHL细胞和HeLa细胞损伤后修复的拖尾率与修复时间的回归系数显著不同(P<0.05).结论:两种细胞在氧化性DNA损伤后均迅速启动自身修复,但HeLa细胞比CHL细胞有更快的修复能力;同时这两种细胞由Dox所致损伤修复能力均较H2O2、K2Cr2O7所致的差.     

Biochemical and biological activity of the anthracycline analog, 4-demethyl-6-0-methyl-doxorubicin

Summary The chromophore-modified derivative of doxorubicin, 4-demethyl-6-0-methyl-doxorubicin, has been tested for antitumor activity in a range of experimental murine tumor systems. In contrast to the inactive 6-0-methyl derivative of daunorubicin, 4-demethyl-6-0-methyl-doxorubicin provided antitumor effects comparable to that of the parent compound. In addition, detailed DNA-interaction studies showed that the doxorubicin derivative retains the ability to bind DNA by the intercalation mechanism. However, the binding affinity was appreciably reduced following structural modification in the anthraquinone chromophore. On the basis of the proposed models of intercalation, these results could be rationalized in terms of steric influence of the bulky methoxy group. The results of this study are in agreement with the correlation already observed between DNA binding and relative antitumor activity of anthracyclines.     

Demonstration of human papillomavirus types 6 and 11 in juvenile laryngeal papillomatosis by in-situ DNA hybridization

A study is reported in which an in situ hybridization technique for the demonstration of human papillomavirus (HPV) employing a biotin--streptavidin polyalkaline phosphatase complex has been successfully applied to formalin-fixed paraffin-processed tissue obtained from ten patients with juvenile laryngeal papillomatosis. In all cases, positive results were obtained for either HPV type 6 or 11. Normal vocal cord epithelium was negative.     

JX-1对雄性小鼠生殖细胞致突变作用的研究

本文采用小鼠精子试验和小鼠睾丸DNA合成抑制试验,检测了JX-1对雄性生殖细胞的致突变作用。实验结果表明JX-1对小鼠精子总数、精子活动率及精子畸形率均无统计学意义的增加,与小鼠睾丸DNA合成抑制试验阴性反应一致。作者认为JX-1对雄性生殖细胞为一种非诱变剂。     

DNA双链断裂残留损伤与癌细胞辐射敏感性研究

目的 了解不同组织来源癌细胞株和人体肿瘤组织原代细胞的DNA双链断裂损伤修复的个体差异性,探寻预测癌细胞辐射敏感性的生物指标。方法 ⁶⁰Co γ射线照射诱发DNA损伤,脉冲电场凝胶电泳检测DNA双链断裂损伤修复,细胞克隆形成能力法检测细胞辐射敏感性。结果 8个不同组织来源癌细胞株的辐射敏感性有较大的差异(D₀为0.65~2.15 Gy),不同细胞株20 Gy γ射线照射诱发产生的DNA双链断裂原初损伤有一定的差别,但与细胞辐射抗性无相关性。辐射敏感细胞SX-10的DNA双链断裂修复缺陷发生在早期快速修复相,而A2780细胞的修复缺陷是发生在晚期慢速修复相。20 Gy照射修复2 h后DNA双链断裂残留量与细胞辐射敏感性指标D₀或SF₂值有显著的相关性。不同个体患者脑肿瘤组织原代细胞之间,辐射诱发DNA双链断裂的修复反应存在明显差异,修复2 h后残留损伤的个体差异性分布类似于癌细胞株。结论 DNA双链断裂残留损伤与癌细胞辐射抗性有显著相关性,可作生物指标预测肿瘤组织细胞对放射治疗的反应性。     

癌胚抗原DNA疫苗对大肠癌特异性细胞免疫的激活效果

目的检测以质粒pIRES为载体构建的带有全序列癌胚抗原(CEA)基因的核苷酸疫苗对机体特异性抗肿瘤免疫反应的激活效果。方法将CEA基因片段连接于真核表达质粒pIRES中,用肌肉注射方法接种核酸疫苗;检测CEA在小鼠肌肉组织中的表达情况及其对小鼠脾细胞CEA特异性细胞免疫反应的激活效果。结果小鼠经肌肉注射质粒后,免疫组化证实该核酸疫苗可在体内有效表达CEA;分子免疫检测显示注射后小鼠特异性淋巴细胞增值反应明显并且伴有自然杀伤细胞NK活性显著增高。结论实验所构建的核酸疫苗pIRESCEA可在体外及小鼠体内高效表达并表现出良好的细胞免疫原性。     

肾母细胞瘤RASSF1A基因启动子区甲基化研究

目的了解肾母细胞瘤RASSF1A基因启动子区甲基化情况。方法手术获取9例肾母细胞瘤及瘤旁残留肾组织,提取DNA后用亚硫酸氢钠处理,巢式PCR扩增RASSF1A基因启动子区DNA片段,测序,检测启动子区甲基化情况。结果发现1例16个CpG岛位点中有10个存在甲基化(10/16),相应瘤旁残留肾组织无甲基化(0/16);在另1例瘤组织中检测到12个CpG岛位点存在甲基化(12/16),但仅有8个CpG岛位点与上例相同,另外4个位点不同。结论肾母细胞瘤中RASSF1A基因启动子区存在甲基化,与瘤旁残留肾组织甲基化情况有差别。     

Epstein-Barr virus DNA in Reed-Sternberg cells of Hodgkin''s disease is frequently associated with CR2 (EBV receptor) expression

We studied 44 cases of Hodgkin's disease for the presence of Epstein-Barr virus (EBV) DNA, its localization and the expression of the EBV receptor on the tumour cells. EBV DNA was found in 52% (16/31) of the Hodgkin's lymphomas using the polymerase chain reaction. With a very sensitive non-radioactive DNA in situ hybridization technique in combination with immunohistochemistry for CD 30 or CD 15 antigens, EBV DNA was localized to Reed-Sternberg cells and its mononuclear variants. The relationship between the presence of EBV DNA and the expression of the EBV-receptor CR2 (CD 21) on Reed-Sternberg cells was studied using the same techniques and two different monoclonal anti-CD 21 antibodies. CR2 could be detected on a substantial number of the Reed-Sternberg cells in EBV DNA positive Hodgkin's lymphomas (9/12; 75%), whereas in EBV negative cases positivity with anti-CD 21 was rare (1/13; 8%). The results indicate that CR2 expression on Reed-Sternberg cells and the presence of EBV DNA sequences are frequently associated in Hodgkin's lymphomas.