p53 Protein expression in laryngeal squamous cell carcinomas bearing wild-type and mutated p53 gene

We performed an immunohistochemical analysis to investigate the expression of p53 protein in a panel of 18 laryngeal squamous cell carcinomas, 15 primary tumours and three in relapse, previously analysed by us for the presence of p53 gene mutations. Dysplastic and/or normal surrounding mucosa was evaluated in 15 different tumours. The results of our study are the following: (1) expression of p53 protein was observed in one out of five tumours positive for p53 gene mutations (20%) and in 10 out of 13 (80%) negative cases; (2), p53 protein over-expression was frequently observed in normal and/or dysplastic mucosa surrounding either wild-type (7/11) or mutated p53 tumours (2/4); (3), p53 immunoreactive cells showed a pattern of distribution in normal and mildly/moderately dysplastic mucosa (basal layers), different from that in severely dysplastic mucosa (whole thickness). These data further support the hypothesis that p53 protein over-expression may be a marker of the earliest phases of multistep tumorigenesis in laryngeal squamous cell carcinoma.     

Effects of exposure to air pollution on blood coagulation

BACKGROUND: Consistent evidence has indicated that air pollution increases the risk of cardiovascular diseases. The underlying mechanisms linking air pollutants to increased cardiovascular risk are unclear. OBJECTIVES: We investigated the association between the pollution levels and changes in such global coagulation tests as the prothrombin time (PT) and the activated partial thromboplastin time (APTT) in 1218 normal subjects from the Lombardia Region, Italy. Plasma fibrinogen and naturally occurring anticoagulant proteins were also evaluated. METHODS: Hourly concentrations of particulate (PM10) and gaseous pollutants (CO, NO2, SO2, and O3) were obtained from 53 monitoring sites covering the study area. Generalized additive models were applied to compute standardized regression coefficients controlled for age, gender, body mass index, smoking, alcohol, hormone use, temperature, day of the year, and long-term trends. RESULTS: The PT became shorter with higher ambient air concentrations at the time of the study of PM10 (coefficient = -0.06; P < 0.05), CO (coefficient = -0.11; P < 0.001) and NO2 (coefficient =-0.06; P < 0.05). In the 30 days before blood sampling, the PT was also negatively associated with the average PM(10) (coefficient = -0.08; P < 0.05) and NO2 (coefficient = -0.08; P < 0.05). No association was found between the APTT and air pollutant levels. In addition, no consistent relations with air pollution were found for fibrinogen, antithrombin, protein C and protein S. CONCLUSIONS: This investigation shows that air pollution is associated with changes in the global coagulation function, suggesting a tendency towards hypercoagulability after short-term exposure to air pollution. Whether these changes contribute to trigger cardiovascular events remains to be established.     

天蚕素A-蛙皮肽杂合肽基因的表达及产物抗菌活性测定

目的 研究天蚕素A-蛙皮肽杂合肽基因[CA（1-8）-MA（1-12）]抗菌肽的抗菌活性。方法 将天蚕素A-蛙皮肽杂合肽基因抗菌肽的人工基因克隆到具有自切割功能的表达载体pTYB12上，并在大肠杆菌（E.coli)BL21(DE3)中进行表达，表达产物用几丁质树脂进行了亲和吸附，自切割后洗脱得到抗菌肽，进行透析以去除盐类物质，最后用大肠杆菌与金黄色葡萄球菌进行活性检测。结果 天蚕素A-蛙皮肽杂合肽[CA（1-8）-MA（1-12）]对大肠杆菌与金黄色葡萄球菌均有一定的抗菌活性。结论 天蚕素A-蛙皮肽杂合肽具有抗菌活性。其抑瘤活性的研究还有待于进一步研究。     

P16、Cyclin D1蛋白表达与胰腺癌生物学行为

目的 探讨P16、CyclinD1蛋白表达水平与胰腺癌发生、发展及预后的关系。方法 应用免疫组织化学SP法结合微波抗原修复法对53例原发性胰腺癌和42例胰腺良性病变及正常组织中P16蛋白及CyclinD1蛋白进行检测。结果 胰腺癌组织、胰腺良性病变和正常组织中P16蛋白的阳性率分别为66．04％和87．71％（P＜0．05）；CyclinD1表达的阳性率分别为52．83％和23．81％（P＜0．05），P16及CyclinD1蛋白表达与胰腺癌的组织分化、病理分期及术后生存期密切相关。结论 P16及CyclinD1蛋白在胰腺癌中的表达可判断胰腺癌的预后。     

电离辐射调控脂质体介导的p16基因在HeLa细胞中的表达

目的探讨不同剂量γ射线照射后诱导脂质体介导的p16基因在HeLa细胞中的表达及抗癌作用。方法用脂质体Lipofectamin介导重组质粒Egr-p16转染人HeLa细胞，采用RT-PCR的方法检测了。Coγ射线照射转染后的人HeLa细胞剂量效应和时程变化，用细胞计数检测细胞增殖的变化，用流式细胞术检测细胞周期的变化。结果研究证实0．5～8Gv照射后p16的转录水平高于对照，在2～4Gy照射后达到峰值，2Gy照射后2—24h高于对照组，在照射后4h达到峰值，然后逐渐下降。细胞生长曲线显示体外稳定转染联合。Coγ射线照射对HeLa细胞的增殖具有明显的抑制作用。细胞周期变化显示转染后的HeLa细胞经过照射后G0／G1期比例呈现剂量依赖性的下降，而S期则出现剂量依赖性的增加，出现明显的S期阻滞，G2／M期在2Gy出现明显的阻滞，在5和10Gy时逐渐下降，但是仍然高于对照组。结论^60Coγ射线可诱导转染的HeLa细胞p16转录水平的增强，同时在细胞增殖上出现明显的变化。     

梅毒螺旋体外膜蛋白Gpd的基因型分析及其重组蛋白的免疫原性鉴定

目的　构建梅毒螺旋体 (Treponemapallidum ,Tp)外膜蛋白Gpd基因的原核表达载体 ,检测其表达产物的免疫原性 ,并比较梅毒螺旋体各菌株Gpd基因序列的同源度。方法　从TpNichols株基因组模板中PCR扩增Gpd基因 ,与GenBank登录的序列做blast比较 ,定向克隆构建原核表达重组体pET2 8b( + ) -Gpd ,转入大肠杆菌ril表达菌 ,SDS -PAGE分析重组蛋白的表达 ,Western -blot检测重组蛋白的免疫原性。结果　载体上所连目的基因片段序列与GenBank登录的Nichols株Gpd基因序列完全一致 ,同其他病原性密螺旋体菌株登陆序列比较同源度为 98%～ 1 0 0 %。SDS -PAGE检测诱导产物显示有一Mr约为 41kDa的特异蛋白带 ,免疫印迹技术检测其能与梅毒阳性标准血清反应。结论　Tp原核表达重组体pET2 8b( + ) -Gpd成功构建 ,且能够在ril表达菌中融合表达 ,为进一步研究该蛋白的生物学功能奠定了一定的实验基础。     

MMP-2和Kiss-1在膀胱癌中的表达及意义

目的:研究基质金属蛋白酶2(MMP-2)和肿瘤转移抑制因子Kiss-1在膀胱癌中的表达以及与肿瘤侵袭的关系.方法:采用免疫组织化学S-P法检测40例膀胱癌和10例正常膀胱组织标本中MMP-2和Kiss-1的表达,并与临床病理参数进行比较.结果:膀胱癌MMP-2阳性率57.5%,表达与肿瘤分期、分级密切相关(P＜0.05).Kiss1阳性率为42.5%,表达随肿瘤分期、分级升高呈下降趋势.MMP-2与Kiss-1的表达无显著相关性(P＞0.05).结论:MMP-2和Kiss-1的表达水平对于评价膀胱癌的预后具有重要意义.     

人IL-10基因重组复制缺陷型腺病毒DNA的构建

目的 构建人IL-10基因的重组腺病毒并进行体外表达和检测。方法从质粒pcDNA3IL-10的CMV启动子下游完整切下IL-10 cDNA的片段,将其定向插入Gateway载体,在克隆酶的作用下将阳性克隆质粒转入目的腺病毒载体。PacI酶线性化IL-10腺病毒DNA后阳离子质脂体转染293A细胞,获得人IL-10的复制缺陷型重组腺病毒。体外感染人胰腺癌细胞株Bxpc-3和大鼠胰腺细胞株AR-42J;Western blot检测人IL-10的表达。结果 成功地构建人IL-10重组腺病毒,病毒滴度达2×109PFU/mL。体外感染的细胞株均检测到IL-10的表达。结论 构建复制缺陷型重组腺病毒能够介导IL-10的基因表达,为细胞因子的抗炎冶疗奠定实验基础。     

宣威昆明两地肺癌ras基因表达及突变的比较研究

目的 :比较ras基因在宣威、昆明两地肺癌中遗传变异的差异性 .方法 :采用免疫组化染色和PCR -SSCP -DNA直接测序技术 ,对 4 5例宣威地区肺癌和 4 5例昆明地区肺癌石蜡包埋组织标本中ras蛋白的表达及k -ras基因 12密码子突变进行检测 .结果 :5 5例 (5 5 / 90 )ras蛋白表达阳性 ,其中宣威肺癌组 31例 ,昆明肺癌组 2 4例 ,2 8例 (2 8/ 90 )检测到k -ras基因 12密码子突变 .对ras基因在两组肺癌中的表达及突变进行比较分析表明 :ras蛋白表达及k -ras基因 12密码子突变在宣威肺癌组中高于昆明肺癌组 ,但差异无统计学意义 (P >0 0 5 ) ,ras蛋白在Ⅰ期肺癌病例以及腺癌病例中的表达 ,宣威肺癌组均高于昆明肺癌组 ,差异有统计学意义 (P =0 0 4 5 ,χ2 =4 5 72 ,P =0 0 32 ) .结论 :ras基因的激活是肺癌发生过程中常见的分子生物学事件 ;宣威地区肺癌与昆明地区肺癌相比 ,ras蛋白在Ⅰ期病例以及腺癌病例中的表达存在着差异 .k -ras基因 12密码子在宣威地区肺癌中的突变与昆明地区肺癌相比无明显特异性     

An Integrated Technique for Identification of Differential Genes Expressed in Patients with Cancer

Summary: To develop a method for identification of differential gene expression between different cell populations, several convenient techniques of molecular biology, including subtractive hybridization, suppression PCR, T/A cloning and sequencing, were used to identify genes expressed differentially in CD45^- and CD45^- cells isolated from U266 cell line of multiple myeloma. Our results showed that the levels of abundant genes scale down 20 times through subtractive hybridization.Plasmid DNA from CD45^- cell clones was hybridized with forward or backward cDNA probes synthesized from CD45^- and CD45 cells, respectively. A few of differentially expressed genes reconfirmed by RT-PCR were identified from 500 expressed clones of CD45^- cells. It is concluded that a strategy for gene expression identification developed from conventional molecular biological methods can be used in different laboratories.