二乙酰二脱水卫矛醇对小鼠白血病L1210细胞增殖的影响

目的　研究二乙酰二脱水卫矛醇 (1 ,2 :5 ,6 dianhydro 3 ,4 diacetylgalactitol,DADAG)的抗脑白血病作用及机制。方法　用小鼠脑内移植瘤模型、MTT法、DNA掺入法、流式细胞仪和Westernblot法 ,观察DADAG对小鼠脑内移植瘤和体外白血病L1 2 1 0 细胞的作用 ,并探讨作用机制。结果　DADAG对DBA/ 2小鼠脑内移植白血病L1 2 1 0 有明显的抑制作用 ;对体外白血病L1 2 1 0 细胞同样有很强的抗增殖作用 ,其IC50 值为 2 4 6mg·L- 1 。DADAG不可逆地抑制L1 2 1 0 细胞内DNA的生物合成。DADAG 2 4mg·L- 1 处理L1 2 1 0 细胞 6h后 ,细胞发生G2 /M周期阻滞 ,2 4h后达最高峰。细胞周期素B1 蛋白水平在DADAG处理 2 4h后开始下降 ,而磷酸化的细胞周期依赖性激酶CDK1在DADAG处理 6h后开始上调 ,并呈时间依赖性。结论　DADAG的抗脑白血病作用与其抑制白血病细胞的增殖密切相关     

Discussing options between patients and health care professionals in genetic diagnosis: ethical and legal criteria

The specific characteristics of genetic data lead to ethical-legal conflicts in the framework of genetic diagnosis. Several international organisations, including UNESCO and the Council of Europe, have enacted rules referring to the use of genetic information. This paper discusses possible legal and ethical criteria that could be used in genetic testing.     

一种较好的RNA分离制备方法及其在人胚RNA提取中的应用

以钒基核苷酸复合物为核酸酶抑制剂,从人胚组织中制备总RNA,所得产品几乎不含DNA和蛋白质,经过分离Poly(A)~ mRNA,证明制品有合成cDNA的完整功能,方法比较简捷,适于大量制备以满足分离mRNA 之需,并讨论了此法的优缺点。     

增强子Ⅰ对乙型肝炎病毒DNA疫苗免疫应答的影响

目的研究乙型肝炎病毒(HBV)自身增强子Ⅰ(enhancerⅠ,ENHⅠ)对HBVDNA疫苗免疫应答的影响。方法采用PCR法以HBVadr亚型全基因DNA序列为模板分别扩增表面抗原(HBsAg)和HBsAg-ENHⅠ基因片段,重组到载体VR1012中,构建两种HBVDNA疫苗,转染COS-7细胞及HepG2细胞并免疫BALB/c小鼠。采用蛋白印迹、ELISA、ELISPOT等方法检测其在COS-7和HepG2细胞内的表达及小鼠的体液及细胞免疫应答效果。结果转染的HepG2和COS-7细胞均表达HBsAg;连接ENHⅠ的HBVDNA疫苗转染HepG2细胞后HBsAg表达量明显升高,两种疫苗转染COS-7细胞表达HBsAg无明显差异;免疫小鼠后第2周产生HBsAb及HBsAg特异性细胞毒T淋巴细胞(CTL),两种疫苗免疫产生的HBsAb及HBsAg特异性CTL无明显差异。结论ENHⅠ可使HBVDNA疫苗转染HepG2细胞表达HBsAg明显增加,对转染COS-7细胞表达HBsAg及接种BALB/c小鼠引起的免疫应答无明显影响。     

CAD基因原核表达载体的构建及表达产物的活性鉴定

目的构建pCool—GST—ICAD／CAD的表达载体，并存大肠杆菌BL21（DE3）中表达具有生物学活性的CAD核酸酶？方法用PCR扩增CAD基因，将扩增产物克隆入pCool—GST—ICAD载体中构建出pCool—GST—ICAD／CAD表达载体。经酶切和电泳鉴定正确后，在大肠杆菌BL21（DE3）中诱导表达，表达产物经亲和层析、离子交换层析及凝胶过滤等方法分离纯化。最后用SDS—PAGE和DNA降解实验进行鉴定。结果构建了pCool—GST—ICAD／CAD原核表达载体，重组载体转化后表达出毫克级水平的GST—ICAD／CAD蛋白复合体。经分离纯化后得到纯度很好的CAD—ICAD篮白复合体，在SDS—PAGE电泳上旱现清晰的两条蛋白带。经DNA降解实验证明纯化所得的CAD蛋白具有非特异性降解DNA的核酸酶作用。结论成功制备了具有生物学活性的CAD核酸酶，为进一步研究细胞凋亡的作用机制提供了有效的制剂。     

一种适用于聚合酶链反应的常见念珠菌基因组DNA提取方法的研究

目的介绍一种简单、快速的真菌DNA提取方法,提高真菌DNA提取效率,减少毒性和污染性,以适应临床研究需要。方法同时用溶细胞酶结合Biospin真菌基因组DNA提取试剂盒和十六烷基三甲基溴化铵(CTAB)法,提取白色念珠菌、热带念珠菌、克柔念珠菌和净平滑念珠菌基因组DNA,用A260/A280的比值检测DNA的纯度并计算质量浓度,同时行聚合酶链反应(PCR)以评价其可靠性。结果溶细胞酶结合Biospin真菌基因组DNA提取试剂成功提取所用真菌基因组DNA,其纯度及质量浓度能满足PCR反应的要求。结论用溶细胞酶结合Biospin真菌基因组DNA提取试剂提取DNA,简单、快速、高效,可用于PCR反应。     

Sperm chromatin integrity in young men with no experiences of infertility and men from idiopathic infertility couples

Damage to the genetic component of spermatozoa seems to play the main role in a majority of cases where current approaches fail to reveal the specific cause of male infertility. In this study, we compared semen quality in men assigned to two defined groups: men from couples with unexplained infertility – idiopathic infertility (A) and young men with no experiences of infertility (B). All samples were examined by standard ejaculate analysis and sperm chromatin structure assay (SCSA). Sperm chromatin damage was significantly higher in men from group A than in those from group B. Similar results were obtained by comparison of men from group A (all men were normozoospermic) with normozoospermic men from group B. According to these results, we can suppose that chromatin disorders may be the causal factor of subfertility or infertility in some of these men. No evidence for a strong association between chromatin disorders and standard parameters of ejaculates was found. We failed to confirm a relationship between smoking and sperm quality in men from any of the investigated groups. SCSA is a method that facilitates the identification of infertile men who otherwise show normal semen variables.     

Priming with a very low dose of DNA complexed with cationic block copolymers followed by protein boost elicits broad and long-lasting antigen-specific humoral and cellular responses in mice

Cationic block copolymers spontaneously assemble via electrostatic interactions with DNA molecules in aqueous solution giving rise to micellar structures that protect the DNA from enzymatic degradation both in vitro and in vivo. In addition, we have previously shown that they are safe, not immunogenic and greatly increased antigen-specific CTL responses following six intramuscular inoculations of a very low dose (1 μg) of the vaccine DNA as compared to naked DNA. Nevertheless, they failed to elicit detectable humoral responses against the antigen. To gain further insight in the potential application of this technology, here we show that a shorter immunization protocol based on two DNA intramuscular inoculations of 1 μg of DNA delivered by these copolymers and a protein boost elicits in mice broad (both humoral and cellular) and long-lasting responses and increases the antigen-specific Th1-type T cell responses and CTLs as compared to priming with naked DNA. These results indicate that cationic block copolymers represent a promising adjuvant and delivery technology for DNA vaccination strategies aimed at combating intracellular pathogens.     

PCR-RSSO基础上HLA-Ⅰ、Ⅱ基因分型的研究

目的通过对PCR-DNA技术的分析,探讨人类白细胞抗原(HLA)基因分型方法。方法采用PCR反向序列特异性寡核苷酸(polymerase chain reaction-reverse sequence specific oligonucleotide,PCR-RSSO)杂交技术,建立改良半量扩增体系全自动HLA-I、Ⅱ等位基因分型方法,进行了635份血液标本HLA-A、B、C、DR、DQ等位基因分型,其中166份DNA同时采用序列特异性引物技术(PCR-SSP)和手工全量扩增体系PCR-RSSO技术。对全自动半量PCR-RSSO、PCR-SSP、手工PCR-RSSO 3种方法做两两比较。结果全自动半量PCR-RSSO的分型成功率为98.4%(3 124/3 175),PCR-SSP为98.8%(656/664),手工PCR-RSSO为88.3%(733/830)。经χ2检验,全自动半量PCR-RSSO与PCR-SSP的分型成功率无统计学差异,与手工PCR-RSSO有显著差异(P<0.05)。结论PCR-RSSO可识别HLA-Ⅰ,Ⅱ共706个等位基因,覆盖WHO命名委员会2000年公布的936个等位基因的75.43%;对706个HLA等位基因的分型均为中~高分辨率,有分辨纯合子等位基因的能力;易长期保存书面的实验原始资料,即杂交条;具有成本低、劳动强度低、省时和DNA消耗量少等优点。PCR-RSSO适合于造血干细胞移植和建立造血干细胞及脐带血干细胞库的组织配型。