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(2000-01/2008-12),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。     

荧光定量PCR方法检测脑脊液中结核分枝杆菌DNA的意义

目的 研究应用实时荧光定量PCR法检测脑脊液中结核分枝杆菌DNA（TB-DNA）对结核性脑膜炎的临床诊断意义.方法 采用实时荧光定量PCR方法、抗酸染色和改良罗氏培养三种方法,对136例结核性脑膜炎患者及64例非结核性脑膜炎患者的脑脊液标本进行检测并对结果进行比较.结果 荧光定量PCR方法的敏感性显著高于抗酸染色和改良罗氏培养,阳性率分别为59.56%、3.68%、7.8%（P〈0.01）.结论 荧光定量PCR方法具有较高的特异性和敏感性,具有简便、快速的特点,可作为结核病诊断的常规方法,对结核性脑膜炎的诊断具有重要指导意义.     

SYBR荧光实时定量PCR检测不同病理类型颅咽管瘤ICAM-1 mRNA表达

目的定量研究细胞间粘附分子基因(ICAM—1 mRNA)在不同病理类型颅咽管瘤的表达差异及意义。方法收集30例经手术治疗的颅咽管瘤标本,采用SYBR荧光实时定量PCR法检测 ICAM-1 mRNA在肿瘤组织的表达,并对表达结果行统计学分析。结果造釉细胞型颅咽管瘤 ICAM-1mRNA表达量为(62．18±6．43)×103 copies／μg,鳞状乳头型颅咽管瘤ICAM-1 mRNA表达量为 (1．13±0．17)×103 copies／μg,造釉细胞型颅咽管瘤ICAM-1 mRNA表达量显著性高于鳞状乳头型颅咽管瘤(P<0．01)。结论两种病理类型颅咽管瘤ICAM—1 mRNA表达存在显著性差异,此差异性可能与两种病理类型颅咽管瘤不同的肿瘤炎症有关。     

参照多重PCR片段分析法进行实时荧光PCR法对ABCB1三等位基因测定结果读取方法的确定

目的 为解决实时荧光PCR法对三等位基因测定结果无法正常读取的问题,通过参照多重PCR片段分析法,确定实时荧光PCR法读取方法,实现临床对ABCB1三等位基因准确、简便且价格低廉的检测。方法 收集西安市精神卫生中心2020年3月-2021年3月DNA样本2 794例,抽取5%作为实验样本,分别进行实时荧光PCR法和多重PCR片段分析法测定。根据PCR曲线Ct值的比较以及多重PCR片段分析法碱基峰位强度的比较,对比分析两种方法的判读结果,对其中报告结果不相同的样本进行数据核查并确定PCR读取方法。结果 共抽取139例样本,其中存在120例等位基因及19例三等位基因,实时荧光PCR法和多重PCR片段分析法对等位基因的检测结果完全一致。根据多重PCR片段分析结果,对19例三等位基因制定了实时荧光PCR法的读取方法：扩增曲线图中,当∣∣Ct._G-Ct._T∣-∣Ct._G-Ct._A∣∣<3,分别读取两组碱基Ct值小的碱基,将其组合形成判读结果;当∣∣Ct._G-Ct._T     

实时荧光定量PCR技术在结核性脑膜炎早期诊断中的应用

目的 探讨荧光定量PCR技术在结核性脑膜炎早期诊断中的应用价值.方法 应用荧光定量PCR技术检测84例患者脑脊液,并与传统方法比较.结果 结核分枝杆菌DNA检出阳性率与涂片检出率、结核菌培养阳性率、LAM抗体检出率差异有统计学意义（P〈0.05）.42例结核性脑膜炎患者组ADA值升高21例,对照组5例,其敏感性与结核分枝杆菌DNA的阳性率差异无统计学意义（P＞0.05）,但其特异性（90%）低于定量PCR法（100%）,差异有统计学意义（P〈0.05）.结论 实时荧光定量PCR方法与其他传统方法比较,在结核性脑膜炎的早期诊断中有一定优势.     

实时荧光定量聚合酶链反应检测腓骨肌萎缩症1A型基因重复

腓骨肌萎缩症（Charcot-Marie-Tooth disease,CMT）,是神经系统常见的遗传性疾病之一,根据电生理和病理学研究将其分成两大组：CMT1（脱髓鞘型）和CMT2（神经元型或称轴索型）.该病是一组遗传异质性疾病,目前已发现30个基因座位与不同类型的CMT相关,其中有11型已明确了致病基因,发病率最高的是常染色体显性遗传的CMT1A.我们已先后建立起短串联重复序列分析（STR）和聚合酶链反应（PCR）酶切法检测特异性融合片段来诊断CMT1A基因重复.STR结合银染的方法因有便宜、省时和所需DNA数量少等优点曾被广泛应用,但主要缺点是受杂合率的限制,如果被检测者为纯合子,则无法得出结论;再者,实验结果不稳定,银染重复性不理想.而PCR酶切法的阳性率则较低.因此,为提高基因重复检测的阳性率,并适于在临床应用,我们应用实时荧光定量PCR技术,以SYBRGreen Ⅰ荧光染料为标记物,采用标准曲线的相对定量方法检测了18例CMT1A患者的基因重复,现报道如下.     

SYBR Green实时定量PCR检测人原发脑胶质瘤中REV3和REV7基因的表达

目的 运用实时定量PCR检测跨损伤修复基因REV3和REV7在人脑原发脑胶质瘤组织中的表达水平,探讨其和肿瘤级别之间的关系.方法 采用SYBR Green实时定量聚合酶链式反应(RT-PCR)法检测跨损伤修复基因REV3和REV7的mRNA在85例原发胶质瘤(Ⅱ级20例、Ⅲ级20例和Ⅳ级45例)和14例正常脑组织中的表达水平,统计学分析表达水平和肿瘤级别之间的关系.结果 与正常组织相比,REV3和REV7在各病理级别胶质瘤中均表达上调(P＜0.05);并且REV3在Ⅳ级胶质瘤中的表达比在Ⅱ级和Ⅲ级中都要高(P＜0.05).秩相关分析表明:REV3的表达量与胶质瘤病理分级呈正相关性(r=0.454,P＜0.001).结论 REV3和REV7在胶质瘤组织中均高表达,而且REV3表达水平和恶性程度有密切联系.     

实时荧光定量RT-PCR检测肥胖大鼠胰岛细胞CB1的基因表达

目的定量分析大麻素受体1(CB1)基因在成年肥胖大鼠和正常大鼠胰岛细胞的表达情况。方法采用高脂饲料喂养成年雄性SD大鼠,同批正常非肥胖对照组采用常规饲料喂养20周,分离纯化2组大鼠的胰岛细胞,通过TaqMan实时荧光定量RT-PCR法检测肥胖大鼠和对照大鼠胰岛细胞mRNA的表达水平。结果肥胖大鼠胰岛细胞中CB1的mRNA表达水平显著高于正常对照组(比值为16.7:1,P<0.05)。结论利用高脂饲料喂养20周的肥胖大鼠胰岛内CB1的表达水平显著增加,肥胖时主要表达于胰岛B细胞,CB1表达水平的变化可能与2型糖尿病的发病相关。     

实时荧光定量RT-PCR法建立版纳微型猪近交系中厚皮创面转化生长因子表达体系*◆

背景：版纳微型猪近交系能较好的模拟人取皮区创面,构建动物模型,检测与创面愈合及瘢痕形成密切相关的转化生长因子的表达。 目的：观察创面愈合过程中转化生长因子β1基因的表达情况。 方法：利用版纳微型猪近交系4~6月龄猪构建了皮肤创面愈合动物模型,通过提取皮肤创面总RNA,设计特异引物,对与创面愈合相关密切的转化生长因子β1基因进行了RT-PCR扩增。纯化目的片段并与pMD18-T载体连接,转化宿主菌DH5α,提取重组质粒DNA,并经酶切、PCR和测序鉴定,计算重组质粒原液拷贝数浓度并制备梯度浓度标准品,进行实时荧光定量PCR。 结果与结论：建立的转化生长因子β1基因mRNA表达实时荧光定量PCR检测方法特异性较好,检测灵敏度可达103 拷贝/µL,线性范围达103~109拷贝/µL,阈值循环数与PCR体系中起始模板量的对数值之间存在良好的线性关系(R2=0.988),扩增效率高(E=107.433%)。利用该检测体系检测了45份样品,效果良好,该方法可为研究TGF-β1基因在创面愈合过程中的作用机理奠定分子生物学基础。     

人mGluR1基因mRNA实时荧光定量PCR方法的建立

目的建立代谢型谷氨酸受体1(mGluR1)基因mRNA表达水平的Taq Manreal-time PCR检测方法。方法以β-actin为内参基因,根据GenBank中人mGluR1及β-actin基因序列,分别设计了两套特异性引物和TaqMan探针,接着对反应的退火温度、引物浓度、探针浓度、Mg2 浓度进行优化,然后以优化的条件建立相对定量标准曲线,并对该方法的稳定性进行分析。结果mGluR1及β-actin基因的real-time PCR扩增效率分别为99.7%和100.0%;相对定量标准曲线的CT值线性范围分别为8.1～30.9和11.9～32.1,相关系数分别为0.999及1.000;批内及批间变异系数<6.4%。结论本研究所建立的针对mGluR1 mRNA表达水平的Taqman real-time PCR检测方法具有扩增效率高、稳定性好等特点,为进一步探索mGluR1的功能及其mRNA表达水平的变化和各种疾病发生、发展的相关性提供了方法学基础。     

Randomised,double blind,multicentre, placebo controlled study of sulodexide in the treatment of venous leg ulcers

Sulodexide, a highly purified glycosaminoglycan, was investigated for treatment of venous leg ulcers. Patients (n = 235) undergoing local treatment including wound care and compression bandaging, were randomised to receive either sulodexide or matching placebo for three months. Primary study endpoint was complete ulcer healing after 2 months; secondary endpoints were ulcer healing at 3 months and the time-course changes of ulcer areas. The proportion of patients with complete ulcer healing was higher with sulodexide at 2 months (p = 0.018) and 3 months. The "number needed to treat" to obtain one additional patient healed with sulodexide was 7 at 2 months and 5 at 3 months. The changes in ulcer surface area with time were significant for sulodexide only (p = 0.004). Fibrinogen significantly decreased in sulodexide patients (p = 0.006). In conclusion, sulodexide associated with local treatment proved to be effective and well tolerated in the management of venous leg ulcers.     

阿尔茨海默病患者外周血APP mRNA水平的变化

目的建立荧光定量PCR方法检测Alzheimer病(AD)患者外周血中淀粉样蛋白前体 (APP)的 mRNA水平,并探讨该基因在AD患者外周血中的表达及意义。方法根据APP的基因序列,设计并合成引物和荧光标记探针。将PCR扩增目的片段用AT克隆的方法克隆入T载体,重组质粒经筛选、鉴定后,作为阳性模板,用于标准曲线的制定和样品检测。用该方法检测30例AD患者和 23例正常老年人对照组外周血中APP基因的mRNA水平。结果应用重组质粒制作的定量曲线循环阈值与模板浓度具有良好的线性关系。AD组APP基因平均表达水平为(2．54×105±1．53×105) copies/μgRNA,高于对照组(6．03×104±7．58×105)copies／μgRNA(P<0．001)。结论荧光定量PCR检测AD患者APP mRNA水平的方法较常规PCR技术更为简便、快速、准确。用该法测得APP在AD患者外周血中的mRNA水平有所增加。     

人mGluR1基因mRNA实时荧光定量PCR方法的建立

目的建立代谢型谷氨酸受体1（mGluR1）基因mRNA表达水平的TaqMan real-time PCR检测方法。方法以争actin为内参基因，根据GenBank中人mGluR1及β-actin基因序列，分别设计了两套特异性引物和TaqMan探针，接着对反应的退火温度、引物浓度、探针浓度、Mg^2＋浓度进行优化，然后以优化的条件建立相对定量标准曲线，并对该方法的稳定性进行分析。结果mGluR1及争actin基因的real-time PCR扩增效率分别为99．7％和100．0％；相对定量标准曲线的CT值线性范围分别为8．1～30．9和11．9～32．1，相关系数分别为0．999及1．000；批内及批闻变异系数〈6．4％。结论本研究所建立的针对mGluR1 mRNA表达水平的Taqman real—time PCR检测方法具有扩增效率高、稳定性好等特点，为进一步探索mGluR1的功能及其mRNA表达水平的变化和各种疾病发生、发展的相关性提供了方法学基础。     

Deep venous thrombosis of the leg due to psychiatric stupor

Abstract  We report the cases of two patients with psychiatric stupor who developed venous thrombosis. A 29-year-old schizophrenic woman had been hospitalized in psychiatric institutions three times because of stupor associated with auditory hallucinations and thought blocking. These symptoms recurred and she was admitted to our hospital with deep venous thrombosis of her left leg. The other patient was a 67-year-old woman with depression. She had also suffered from insomnia. Following admission to our hospital, she developed a depressive stupor complicated by deep venous thrombosis of her left leg. Both cases were treated with sodium heparin and urokinase, and completely resolved. It is well known that dehydration, infection and decubitus ulcers are important physical complications of psychiatric stupor, but there have been few reports of deep venous thrombosis as a physical complication of stupor.     

Peripheral neuropathy in chronic venous insufficiency

Chronic venous insufficiency (CVI) of the lower legs may cause tissue damage, but involvement of peripheral nerves is uncertain. We examined 30 patients with CVI and 20 healthy controls using motor and sensory nerve conduction studies, vibration testing and thermotesting, quantitative sudomotor axon-reflex test, and laser Doppler flowmetry. Subjects with possible confounding factors for peripheral neuropathies were excluded. Prolongation of distal motor latency of the peroneal nerve (median, 5.4 versus 4.5 ms; P = 0.02), increased limits for warm (9.60 degrees C versus 5.20 degrees C; P = 0.016) and cold detection (3.45 degrees C versus 1.55 degrees C; P = 0.016) and reduced vibration sense (2.8925 versus 1.1075; P < 0.008) were found. The results demonstrate a disturbance of A-alpha fibers, A-beta fibers, A-delta fibers, and thermoafferent-C fibers, possibly induced by ischemia due to venous microangiopathy and increased endoneurial pressure. Analogous to neuropathic ulcers in diabetes, the CVI-associated neuropathy may also be a cofactor in the development of venous ulcers.     

慢性静脉压增高状态下脑微循环的动态研究

目的 研究慢性静脉压增高状态下脑微循环的动态变化。方法 选择Wistar雄性大鼠16只(对照组A n=6;实验组B n=10),全身麻醉下行右侧颈总动脉与右侧颈外静脉端端吻合。利用激光多普勒扫描技术测定吻合前-后以及2周后吻合部结扎前-后脑表25处局部脑血流(ICBF)和局部脑血流量(ICBV)的变化,区域脑血流(rCBF)和区域脑血流量(rCBV)分别用各自的25个数据的中位数表示。A组:单纯右颈总动脉结扎;B组:右颈总动脉与右颈外静脉端端吻合。结果 A组的两侧大脑半球以及B组的左侧大脑半球吻合前后rCBF、rCBV的变化无统计学意义,B组的右侧大脑半球吻合前后rCBF rCBV也无统计学意义,但2周后rCBF显著降低(p<0.05),rCBV显著增高(P<0.05),吻合部结扎后rCBF即刻增高(P<0.05),rCBV即刻降低(P<0.05):结论 一侧颈总动脉-颈外静脉吻合模型对研究慢性静脉压增高状态下脑缺血的研究具有实用价值;在慢性静脉压增高状态下结扎动静脉短路后,脑灌注压迅速上升,CBF得到改善。     

Hexachlorophene-induced degeneration of adrenergic nerves: Application of quantitative image analysis to Falck-Hillarp fluorescence histochemistry

Summary Possible toxic effects of hexachlorophene (HCP) on sympathetic adrenergic nerves were studied using Falck-Hillarp fluorescence histochemistry on whole-mounts of albino rat irides. HCP dissolved in 5 l DMSO, or DMSO alone, was injected into the anterior chamber of the eye. HCP caused a dose-dependent degeneration of adrenergic nerves, first observable after 7 g and profound after 21 g. One and 3 days after 35 g of HCP there was an almost total loss of adrenergic nerves. Regeneration from remaining non-terminal axons led to an almost complete reformation of the adrenergic nerve plexus after 18 days. The results demonstrate a new aspect of hexachlorophene neurotoxicity, degeneration of peripheral adrenergic nerve terminals and suggest that neurotoxic actions on thin unmyelinated fiber systems should be looked for also in the central nervous system (CNS).Supported by the Swedish Council for Planning and Coordination of Research (project Chemical Hazards in the Environment), The Swedish Medical Research Council 14X-01305, 14P-5867, Magnus Bergvalls Stiftelse, and Karolinska Institutets Fonder     

The measurement of adenosine and estrogen receptor expression in rat brains following ovariectomy using quantitative PCR analysis

In our laboratory we have developed a quantitative-polymerase chain reaction (Q-PCR) strategy to examine the differential expression of adenosine receptor (ADOR), A(1), A(2A), A(2B) and A(3), and estrogen receptors (ER) alpha and beta. Brain and uterine mRNA were first used to optimise specific amplification conditions prior to SYBR Green I real time analysis of receptor subtype expression. SYBR Green I provided a convenient and sensitive means of examining specific PCR amplification product in real time, and allowed the generation of standard curves from which relative receptor abundance could be determined. Real time Q-PCR analysis was then performed, to examine changes in receptor expression levels in brains of adult female Wistar rats 3-month post ovariectomy. Comparison with sham-operated age-matched control rats demonstrated both comparative and absolute-copy number changes in receptor levels. Evaluation of both analytical methods investigated 18S rRNA as an internal reference for comparative gene expression analysis in the brain. The results of this study revealed preferential repression of ADORA(2A) (>4-fold down) and consistent (>2-fold) down-regulation of ADORA(1), ADORA(3), and ER-beta, following ovariectomy. No change was found in ADORA(2B) or ER-alpha. Analysis of absolute copy number in this study revealed a correlation between receptor expression in response to ovariectomy, and relative receptor subtype abundance in the brain.