实时荧光定量PCR检测大鼠VEGF mRNA方法的建立及初步应用

目的建立大鼠血管内皮生长因子（vascular endothelial growth factor,VEGF）实时荧光定量PCR检测方法,对健康大鼠不同组织中VEGF mRNA水平进行检测分析。方法跨内含子设计引物和TaqMan探针,扩增片段TA克隆入pGEM-T载体,制成标准品,分析其特异性、灵敏性和重复性;提取健康大鼠不同部位组织（肺、心、肝脏、肾脏、脑、胃、肠和颌下腺）总RNA,分析其VEGF mRNA表达水平。结果测序及电泳条带证实PCR反应的特异性,批内CV为2.0%～6.7%,批间CV值为3.5%～10.6%,r〉0.99;肺组织中VEGF mRNA表达最高,与其他任意组相比差异均具有统计学意义（P〈0.05）,其它各组织间差异无统计学意义。结论本研究成功建立了检测大鼠VEGF mRNA的实时荧光定量PCR方法,具有高灵敏、高特异、高重复性的特点;健康大鼠不同部位组织中VEGF mRNA的表达水平分析为以后研究疾病大鼠模型表达水平奠定了基础。     

miRNA-125a Taqman实时荧光定量PCR检测方法的建立及初步应用

目的 建立一种用于肝癌早期诊断和监测的血清miRNA-125a Taqman实时荧光定量PCR检测方法。方法 采用Trizol法提取肝癌患者血清中总RNA,以肝癌发生具有代表性的miRNA-125a基因为检测序列,分别设计逆转录引物、扩增引物和Taqman探针; 构建和制备miRNA-125a重组质粒标准品,建立血清miRNA-125a Taqman 实时荧光定量PCR检测方法。结果 成功构建了miRNA-125a重组质粒载体,制备了miRNA-125a重组质粒标准品,建立了血清miRNA-125a Taqman实时荧光定量PCR扩增体系。经实验验证,所设计引物具有较好的特异性,引物的最低检测限为78.125 copies/μl; 对17例肝癌患者血清miRNA-125a的检测结果在(1.45×10²～3.52×10⁵)copies/μl之间。结论 血清miRNA-125a Taqman实时荧光定量PCR检测方法的建立,为原发性肝癌早期诊断和监测提供一种新的分子诊断定量方法。     

人类Gfi1基因SYBR Green I实时荧光定量PCR检测方法的建立

目的 建立测定人类Gfi1mRNA表达量的SYBR Green I实时荧光定量PCR方法,为进一步研究新基因的功能奠定基础.方法 以Gfi1基因为靶基因设计引物.构建携带GficDNA的质粒plox-Gfi1,经过纯化,质粒浓度测定,拷贝数的计算及10倍的梯度稀释制备标准品.应用ABI stepone PCR仪对Cfi1mRNA进行实时荧光定量PCR检测,建立标准曲线和熔解曲线.结果 建立了Cfi1mRNA表达的实时荧光定量PCR方法,本法线性范围为6.36×102～6.36×108copies/μl,线性相关系数y2为0.996,熔解曲线为单峰,Tm值为(89.98±0.22)℃,标准品的循环阈值批内变异系数和批间变异系数分别为1.35%～3.61%和1.49%～3.42%.结论 本研究建立的Gfi1mRNA表达实时荧光定量PCR检测方法速度快,灵敏度高,特异性强,重复性及稳定性好,可用于Gfi1基因的定量检测.     

逆转录实时荧光定量PCR检测p73 mRNA在结肠癌的表达

目的:建立一种实时定量PCR方法检测p73mRNA的表达水平,探讨其与结肠癌发生、发展的可能关系。方法:采用SYBRGreenⅠ逆转录实时荧光定量PCR检测39例结肠癌组织和其癌旁组织中p73mRNA的表达。结果:p73基因在结肠癌肿瘤组织的表达水平高于其在癌旁组织的表达(P<0.01);表达水平与肿瘤的恶性程度、分化及淋巴结转移显著相关(P<0.05)。结论:p73的高表达可能与结肠癌的发生、发展及预后有联系。     

多重实时定量PCR检测BCR-ABL mRNA方法的建立

目的建立一种快速、可靠的实时定量PCR（RQ-PCR）检测BCR-ABL mRNA的方法。方法应用LightCycler技术,设计在同一PCR反应管内可扩增b3a2、b2a2、ela2几种常见的BCR-ABL转录本和b3a3、b2a3等少见转录本,并对RQ-PCR主要要素进行优化,评价优化后RQ-PCR的敏感性、特异性和可靠性。结果建立的RQ-PCR方法可检出10^3健康人单个核细胞中的1个K562细胞,最低可检出100个拷贝BCR—ABL分子。BCR—ABL和ABL的循环域值（C1）值（拷贝数）批内变异系数分别为0．68％（12．93％）和0．59％（8．60％）,批间变异系数分别为1．14％（18．89％）和1．01％（12．72％）。结论RQ-PCR可作为评价慢性粒细胞白血病（CML）疾病进展、预后判断和骨髓移植后微量残留白血病监测的灵敏、可靠的方法。     

实时荧光定量PCR检测乳腺癌组织中KLK4 mRNA表达

目的探讨KLK4基因在人乳腺癌组织中表达的临床意义。方法用实时荧光定量PCR(FQ-PCR)的方法检测39例乳腺癌组织和25例正常组织中KLK4 mRNA的表达,以及mRNA表达量与雌激素受体(ER)、孕激素受体(PR)、CerbB-2及肿瘤的转移情况进行相关性分析。结果正常乳腺组织和癌组织KLK4 mRNA的相对表达水平分别为0.0120±0.0044和0.0272±0.0067,两者差异显著(P<0.01);乳腺癌组织中KLK4的相对表达水平在ER( )和ER(-)组分别为0.0269±0.0070和0.0276±0.0067;在PR( )和PR(-)组分别为0.0270±0.0065和0.0275±0.0081;在CerbB-2( )和CerbB-2(-)组分别为0.0270±0.0064和0.0301±0.0074;在肿瘤有、无转移组分别为0.0258±0.0061和0.0276±0.0066,各组间KLK4mRNA表达水平均无显著性差异(P>0.05)。结论在乳腺癌组织中KLK4 mRNA的表达水平显著高于正常乳腺组织,但在肿瘤是否转移,雌激素受体、孕激素受体及CerbB-2阳性和阴性组间无显著性差异。     

实时荧光定量PCR检测布鲁氏菌核酸DNA的应用评价

目的探讨实时荧光定量PCR检测布鲁氏菌核酸DNA浓度的检测范围和检测健康人全血中核酸DNA的Ct本底值。方法采用试剂盒提取纯菌、健康人和临床患者血液中的核酸DNA,应用实时荧光定量PCR进行检测。结果经检测,40份健康人群血液标本Ct值平均值为37.0,标准差为1.9,Ct值95%置信区间为33.0 ~ 38.8。 将布鲁氏菌核酸DNA标准品倍比稀释（56.2 ng ~ 0.026 5 fg）,实时荧光定量PCR检测灵敏度为6.7 fg。结论实时荧光定量PCR检测纯菌核酸DNA的灵敏度相当于2个基因组DNA拷贝数,但用于检测血液标本尚待进一步研究。     

基于TaqMan探针的幽门螺杆菌实时荧光定量PCR检测方法的建立

目的探讨基于TaqMan探针的幽门螺杆菌23SrDNA基因片段的实时荧光定量PCR检测的有效方法。方法根据幽门螺杆菌23SrDNA序列设计引物和探针,构建质粒标准品,建立实时荧光定量PCR体系并进行方法学评价,并对2012年5月至10月收集的100份临床样本进行检测同时应用免疫组化及银染方法进行比较分析。结果建立了检测23SrDNA的实时荧光定量PCR方法：107-102copies/ul范围内均有＂S＂型扩增曲线,最低检测浓度为102copies/ul,标准曲线相关系数为1。对临床其它消化道病原体不出现特异性的扩增曲线。对100份样本进行检测,实时荧光定量PCR能检出31份阳性,而免疫组化方法能检出25份阳性,银染方法能检出28份阳性。结论实时荧光定量PCR方法可成功检测幽门螺杆菌,且该方法具有灵敏度和特异性高及重复性好等优点。     

乙型肝炎病毒核酸的实时荧光定量PCR检测及其临床意义

目的建立实时荧光定量PCR(rea l-tim e fluorescence quan titative PCR,RFQ-PCR)检测HBV-DNA含量的方法,探讨其在乙型肝炎预防、诊断、治疗及判断病情等方面的临床应用价值。方法在HBV S基因高保守区设计了特异性的引物和探针,利用T aqM an探针的基本原理,PCR扩增目的片段并实时检测产物的荧光强度,根据标准品建立的标准曲线,由软件自动计算出待测样本中HBV-DNA的准确含量。结果220例临床样本的检测结果显示,乙肝大三阳患者HBV-DNA阳性率(98.8%)显著高于其它各组(P<0.01),病毒平均含量为7.52±0.43 cop ies/m l;小三阳患者HBV-DNA阳性率低于大三阳组(P<0.01),但平均病毒含量与大三阳组无差异(P>0.05),高达7.41±0.26 cop ies/m l;单独HB sA g阳性和单独HB sA b阳性组HBV-DNA阳性率分别为56.4%,19.2%,平均病毒含量分别为5.35±0.32 cop ies/m l,4.02±0.31 cop ies/m l;80例献血员血清仍有3例HBV-DNA阳性,其病毒含量低于其它各组。结论RFQ-PCR检测HBV-DNA含量比EL ISA法具有更好的灵敏度和特异性,能准确反映HBV在体内的复制情况,对临床上抗病毒药物的选择和判断疾病的预后意义重大。     

实时荧光定量PCR检测DHX32 mRNA方法的建立及初步应用

目的建立实时荧光定量PCR(FQ-RT-PCR)检测DHX32 mRNA的方法并研究其在结直肠癌中的表达。方法用Taq-Man探针建立检测DHX32 mRNA的FQ-RT-PCR方法,评价其特异性、重复性及扩增效率,并用该法检测DHX32 mRNA在结直肠癌中的表达水平。结果批内变异系数1.1%～1.9%,批间变异系数1.2%～2.5%;扩增效率为0.85,相关系数为0.9990。癌组织DHX32 mRNA表达水平中位数为1.90×10-3(四分位间距5.47×10-3),明显高于癌旁组织0.09×10-3(四分位间距1.41×10-3),两者差异有统计学意义(P<0.05)。DHX32 mRNA表达水平与结直肠癌癌栓形成、淋巴结转移、组织学分级以及Dukes分期关系密切(P<0.05)。肿瘤的恶性程度越高,DHX32 mRNA表达水平越高。结论FQ-RT-PCR检测DHX32 mRNA的方法特异性好,重复性高,操作简单,无污染。DHX32 mRNA表达上调可能在结直肠癌发生、发展中起重要作用,其表达水平可作为结直肠癌恶性程度的评价指标。     

Advanced glycation end products cause epithelial-myofibroblast transdifferentiation via the receptor for advanced glycation end products (RAGE)

Tubulointerstitial disease, a prominent phenomenon in diabetic nephropathy, correlates with decline in renal function. The underlying pathogenic link between chronic hyperglycemia and the development of tubulointerstitial injury has not been fully elucidated, but myofibroblast formation represents a key step in the development of tubulointerstitial fibrosis. RAGE, the receptor for advanced glycation end products (AGEs), induces the expression of TGF-beta and other cytokines that are proposed to mediate the transdifferentiation of epithelial cells to form myofibroblasts. Here we report specific binding of (125)I-AGE-BSA to cell membranes prepared from a rat proximal tubule cell line and show that the binding site was RAGE. AGE exposure induced dose-dependent epithelial-myofibroblast transdifferentiation determined by morphological changes, de novo alpha smooth-muscle actin expression, and loss of epithelial E-cadherin staining. These effects could be blocked with neutralizing Ab's to RAGE or to TGF-beta. Transdifferentiation was also apparent in the proximal tubules of diabetic rats and in a renal biopsy from a patient with type 1 diabetes. The AGE cross-link breaker, phenyl-4,5-dimethylthiazolium bromide (ALT 711) reduced transdifferentiation in diabetic rats in association with reduced tubular AGE and TGF-beta expression. This study provides a novel mechanism to explain the development of tubulointerstitial disease in diabetic nephropathy and provides a new treatment target.     

Targeting the receptor for advanced glycation end products (RAGE) in type 1 diabetes

Type 1 diabetes (T1D) is one of the most common chronic diseases manifesting in early life, with the prevalence increasing worldwide at a rate of approximately 3% per annum. The prolonged hyperglycaemia characteristic of T1D upregulates the receptor for advanced glycation end products (RAGE) and accelerates the formation of RAGE ligands, including advanced glycation end products, high-mobility group protein B1, S100 calcium-binding proteins, and amyloid-beta. Interestingly, changes in the expression of RAGE and these ligands are evident in patients before the onset of T1D. RAGE signals via various proinflammatory cascades, resulting in the production of reactive oxygen species and cytokines. A large number of proinflammatory ligands that can signal via RAGE have been implicated in several chronic diseases, including T1D. Therefore, it is unsurprising that RAGE has become a potential therapeutic target for the treatment and prevention of disease. In this review, we will explore how RAGE might be targeted to prevent the development of T1D.     

Receptor for advanced glycation end products (RAGE) and glyoxalase I gene polymorphisms in pathological pregnancy

ObjectivesThe aim of the study was to analyze polymorphisms of receptor for advanced glycation end products (RAGE) gene, and glyoxalase I gene and soluble RAGE, sRAGE, in physiological and pathological pregnancy.Design and methodsPolymorphisms of RAGE gene (? 429 T/C, ? 374 T/A, 557 G/A, 2184 A/G) and glyoxalase I gene (A419C) and sRAGE serum levels were determined in 284 women with pathological and physiological pregnancy.ResultsNo differences in distribution of genotype and allelic frequencies of studied polymorphisms were found. GA genotype of RAGE 557 G/A polymorphism (known as Gly82Ser) is associated with lower sRAGE serum levels in healthy pregnant women compared to GG genotype (483 ± 104 vs. 692 ± 262 pg/mL, p = 0.008). sRAGE correlates negatively with ALT in patients with pregnancy intrahepatic cholestasis (r = ? 0.536, p = 0.05).ConclusionsWe did not show any association of RAGE and glyoxalase I gene polymorphisms with pathological pregnancy, however further studies are needed to confirm the results.     

人微小病毒B19荧光定量聚合酶链式反应检测方法的建立

目的 建立可同时检测各型人微小病毒B19 (HPV B19)的荧光定量聚合酶链式反应(pCR)方法.方法 运用在线工具MAFFT比对分析3种基因型的HPV B19代表病毒株NS1区域序列,针对保守区序列设计特异性的引物和探针.建立检测HPV B19实时荧光定量PCR方法,并对该方法的敏感性和特异性进行评估.结果 1×1...     

Receptor for advanced glycation end products (RAGE) regulates sepsis but not the adaptive immune response

While the initiation of the adaptive and innate immune response is well understood, less is known about cellular mechanisms propagating inflammation. The receptor for advanced glycation end products (RAGE), a transmembrane receptor of the immunoglobulin superfamily, leads to perpetuated cell activation. Using novel animal models with defective or tissue-specific RAGE expression, we show that in these animal models RAGE does not play a role in the adaptive immune response. However, deletion of RAGE provides protection from the lethal effects of septic shock caused by cecal ligation and puncture. Such protection is reversed by reconstitution of RAGE in endothelial and hematopoietic cells. These results indicate that the innate immune response is controlled by pattern-recognition receptors not only at the initiating steps but also at the phase of perpetuation.     

实时荧光定量PCR技术的理论研究及其医学应用

背景:实时荧光定量PCR是指在PCR反应体系中加入荧光基团,利用荧光信号累积实时监测整个PCR进程,最后通过标准曲线对未知模板进行定量分析的方法.目的:对实时荧光定量PCR的理论进行研究并探讨其在医学方面的应用和进展.方法:以real.time fluorescence quota PCR,theorem,application为检索词,检索PubMed数据库(2000-01/2008-12).以实时荧光定量PCR,原理,应用为检索词,检索万方数据库(2000-0112008-12),清华同方中文系列数据库(2000-01/2008-12).文献检索语种限制为英文和中文.以细胞因子和肿瘤耐药基因的表达为评价指标.纳入实时荧光定量PCR技术的方法学研究和实时荧光定量PCR技术的医学应用研究.排除重复性研究和Meta分析.结果与结论:实时荧光定量PCR技术不仅实现了PCR从定性到定量的飞跃,而且与常规PCR相比,它具有特异性更强、灵敏度高、重复性好、定量准确、自动化程度高、全封闭反应等优点,成为分子生物学研究中的重要工具.实时荧光定量PCR的应用范围非常广泛,包括mRNA表达的研究、DNA拷贝数的检测、单核苷酸多态性的测定等,已广泛应用于医学临床,它能对结核分枝杆菌、乙型、丙型肝炎、爱滋病病毒、淋球菌、沙眼衣原体等病原体进行准确的定量检测.其定量范围极宽,无需做梯度稀释,特异性更强,克服了假阳性.由于传统的PCR技术不能准确定量,使其在实际应用方面受到很大限制.因此,对PCR产物进行准确定量,尤其是病毒性病原的动态监控,成为迫切需要.