Quantitative Detection of Interleukin 8 Gene Expression in Lung Cancer by Real-time Polymerase Chain Reaction

Objective To develop a method for absolute quantification of interleukin 8 (IL—8) mRNA by using real-time polymerase chain reaction (PCR). Methods The IL —8 mRNA and protein expression in 2 human lung cancer cell lines, H460 and A549, were evaluated by real-time PCR and ELISA. The IL-8 mRNA expression in 9 cases of normal lung tissue and 44 cases of non-small cell lung cancer (NSCLC) were examined. Results The IL—8 mRNA copy number in a given sample can be measured by real-time PCR. The gene expression of IL—8 is correlated with its protein secretion. The normalized value of IL—8 expression was 4.87±1.69 (copies/ 10⁴ GAPDH copies) in normal lung tissue and 17.04 ±23.96 in NSCLC, respectively. The difference between these two groups is statistically significant (P=0.002). Using 9.74 and 19.48 as cut-off points for positive expression and overexpression of IL—8, 52.3%(23/44cases) of NSCLC were found to express an increased level of IL-8, among which 29.5% (13/ 44cases) were defined as positive expression and 22.7%(10/44cases) as overexpression. Statistical analysis indicated that IL—8 overexpression was significantly increased in female cancers, squamous carcinoma, and in late stages of disease (P<0.05). Conclusion The IL—8 gene expression can be determined by a real-time PCR technique. IL -8 overexpression is correlated with gender, histopathology and stages of the disease.     

实时荧光聚合酶链反应定量检测LASIK术后兔角膜纤维连接蛋白1的表达

目的　定量检测纤维连接蛋白 1(fibronectin 1,FN1)在LASIK术后不同时段角膜中的表达水平 ,探讨FN1与LASIK术后早期伤口愈合的关系。方法　 2 0只新西兰白兔 ,左眼按近视 4D行LASIK术 ,右眼作对照组。分别于术后 4、4 8h ,1、2周取角膜 ,荧光半定量PCR方法检测中央手术区及周边非手术区角膜FN1mRNA的表达。结果　LASIK术后 4h左眼手术区FN1的mRNA表达水平比右眼相应对照区明显升高 ,4 8h降低至对照组水平 ,其后随时间的延长无明显改变。结论　实时荧光定量PCR定量检测FN1在角膜中的表达结果提示 ,FN1参与LASIK术后角膜瓣与基质床的粘附过程     

多重实时PCR快速同时检测沙门菌和志贺菌

目的 建立改良分子信标，多重实时PCR同时检测沙门菌和志贺菌的快速方法，应用于食源性致病菌的快速诊断。方法 根据GenBank公布的沙门菌侵袭性基因invA和ssaR基因，分别设计一对引物和改良分子信标探针，用同色荧光标记，用于同体系检测沙门菌。志贺菌根据ipaH基因的保守序列，设计引物和改良分子信标探针，加入沙门菌检测体系中，建立三重实时PCR一改良分子信标检测体系，应用于同时对沙门菌、志贺菌食物中毒的快速诊断和门诊肠道致病菌的检测。结果 改良分子信标一多重实时PCR反应体系DNA灵敏度为69～93fg/μl。菌液灵敏度为32～64CFU／ml或1～2CFu／PCR反应体系，无交叉反应。该反应体系同时检测134株沙门菌和67株志贺菌，均出现特异的荧光信号，两种细菌检测互不干扰。对细菌性食物中毒样本等共1100份同时进行沙门菌和志贺菌检测，569份沙门菌实时PCR阳性，其中551份沙门菌培养阳性；42份志贺菌实时PCR阳性，其中41份志贺菌培养阳性。从样品处理到检测结果仅需时间2h至1d。结论 改良分子信标-多重实时PCR检测体系快速、灵敏度高，特异性强，可用于沙门菌和志贺菌食物中毒的快速诊断，伤寒、痢疾等肠道传染病的初筛及预防医学门诊的健康人群体检，为食源性疾病的分子流行病学调查提供新的检测手段。     

同种异基因大鼠子宫移植细胞因子表达的研究

目的研究大鼠子宫移植中γ干扰素(IFNγ)、转化生长因子β1(TGFβ1)和白细胞介素-10(IL-10)的表达及其与排斥反应的关系。方法建立大鼠(SD→W istar)子宫异位移植模型,分为正常子宫对照组、同基因子宫移植组和异基因子宫移植组。各组大鼠分别于术后第2~6天切取移植物进行病理学检查、实时荧光定量PCR检测细胞因子mRNA表达。结果移植后第3天,异基因组组织病理表现出排斥反应并进行性加重;同基因组移植子宫无排斥现象;与对照组相比,同基因组中3种细胞因子mRNA表达均无明显差异(P>0.05);与对照组和同基因组相比,异基因组中除TGFβ1移植后第3天表达无明显差异(P>0.05),其余细胞因子均明显升高(P<0.01)。3种细胞因子表达于移植后第4或第5天达到高峰(P<0.01);IFNγ升高幅度为IL-10的4倍,TGFβ1的16倍。结论细胞因子IFNγ、TGFβ1、IL-10在SD→W istar大鼠子宫移植的急性排斥反应中有一定作用,其中以IFNγ的作用最为明显。     

B型呼吸道合胞病毒荧光定量RT-PCR检测

目的建立B型人呼吸道合胞病毒（respiratory syncytial virus，RSV）的快速荧光定量RT-PCR检测方法。用于B型RSV实验室早期诊断与病毒核酸的定量分析。方法利用B型RSVN基因保守区设计引物和TaqMan-MGB（minor groove binder，DNA小沟结合物）探针，优化反应体系和反应条件；并对方法进行特异性、灵敏度和重复性评价；同时以10倍梯度稀释的质粒为标准品建立荧光定量RT-PCR的标准曲线，构建定量分析模型。并用该方法对100份临床呼吸道样本进行检测。结果该方法与其他呼吸道病毒均无交叉反应，检测灵敏度达1TCID50／ml 50％组织培养物感染剂量病毒滴度，临床样本中B型RSV的检测阳性率达18％。结论B型RSV荧光定量RT-PCR检测方法快速、敏感、特异，适用于B型RSV的早期诊断和核酸定量分析。     

白背叶根抗鸭乙型肝炎病毒的实验研究

目的:观察白背叶根体内抗鸭乙型肝炎病毒(duck hepatitis B virus,D-HBV)的作用。方法:采用PCR技术筛选D-HBV阳性3日龄广州麻鸭40只。将麻鸭随机分为空白对照组,白背叶根高、中、低剂量组和拉米夫定组,分别给予相应药物进行干预。用药前、用药第7、14、21天及停药第7天,取各组麻鸭静脉血,采用实时定量荧光PCR技术检测血清D-HBV DNA含量;治疗前后取肝脏组织进行病理组织学观察。结果:拉米夫定组于用药后,血清病毒水平迅速降低,但停药后存在反跳现象。白背叶根高、中剂量组分别于治疗第14、21天其血清病毒含量明显下降,低剂量组治疗期间未见明显的抑制病毒作用。停药后,白背叶根高、中剂量组与空白对照组比较仍表现出一定的病毒抑制作用。白背叶根高剂量组对肝脏炎症的改善作用较拉米夫定组明显。结论:白背叶根有抑制体内D-HBV复制的作用,其作用大小与剂量及用药时间相关;其治疗作用较拉米夫定弱,但作用维持时间长,且用药较为安全。     

通过PC机串口采集动态实时心电信号

介绍了网上心电实时监护系统，应用ATmega8型8位微处理器芯片实现了10位精度的心电AD转换．通过RS232行接口向PC机传送数据，并介绍这2部分的软、硬件设计思想。     

Molecular monitoring of tumour load kinetics predicts disease progression after non-myeloablative allogeneic stem cell transplantation in multiple myeloma.

BACKGROUND: Non-myeloablative allogeneic stem cell transplantation followed by immunomodulatory therapies is considered a potentially curative approach in the treatment of multiple myeloma and most effective in a minimal residual disease setting. PATIENTS AND METHODS: The aim of this study was to find the most sensitive real-time PCR assay (TaqMan), based on the IGH rearrangement, to quantify the tumour load of 11 patients with multiple myeloma after non-myeloablative allogeneic transplantation. Patient-allele specific primers (ASO) and the TaqMan probe were derived from CDR2 and CDR3 hypervariable regions of IGH, while consensus primers were located within the FR3 and FR4/JH regions. Four different approaches of primer combinations were tested. RESULTS: ASO-forward and -reverse primers together with the clone-specific TaqMan probe were the most sensitive approach compared with the JH (P=0.071) or the FR3 consensus primer (P <0.001). The detection limit amounted to 1/10(4)-1/10(5) cells. Consecutively, 120 samples from 11 patients prior and post allogeneic transplantation were analysed. Three patients reached complete clinical remission accompanied by molecular remission. Disease progression or relapse was seen in six patients. In five, molecular progressive disease was detected prior to the clinical diagnosis of progression or relapse. CONCLUSION: Patient-specific real-time IGH-PCR provides the opportunity for earlier treatment intervention.     

RT-3DE定量评价心梗患者PCI术前后左室功能的变化

为探讨实时三维超声心动图(RT-3DE)定量评估心梗患者经皮冠状动脉介入治疗(PCI)术前后左室收缩功能及同步性参数的变化,选取100例接受PCI治疗的心肌梗死患者和100例正常对照组,应用RT-3DE技术对正常对照组和心梗患者PCI术前及术后1个月测量左心室收缩功能及同步性参数并进行比较。心梗患者PCI术前组左心室各功能参数值均劣于正常对照组,而术后1个月左心室各参数值均较术前明显改善。PCI术前、术后应用RT-3DE能够准确定位、定量反映左心室收缩功能及运动同步性,对心肌梗死的临床诊断及疗效评估具有重要价值。     

实时细胞电子分析技术用于水蛭、三七质量评价的初步研究

采用实时细胞电子分析技术测定水蛭、三七不同样本的时间剂量依赖性细胞反应曲线(TCRPs),对不同产地样本的TCRPs进行分析,并对生物活性强弱进行排序.同时,采用中国药典2010年版中的化学检测法和凝血酶滴定法分别对水蛭、三七各样本进行同步分析,比较不同检测方法得出结果的相关性.可见以实时细胞电子分析技术为基础的细胞生物活性测定结果与另两法所得结果基本一致.说明本技术能反映中药生物活性,有望为中药质量标准研究提供一种新方法.