荧光短片段多重PCR在脊髓性肌萎缩症SMN1基因突变检测中的应用

目的 设计特异性扩增引物,建立荧光短片段多重PCR技术检测SMN1基因拷贝数变异的方法,验证方法的检测性能并初步应用于基因拷贝数检测。方法 选取107例来自神经系统疾病生物样本库的人外周血DNA样品（包括SMN1基因0拷贝30例、1拷贝47例、2拷贝30例）,设计5’端带有FAM荧光基团的SMN1基因7号外显子引物以及三对内参基因引物,利用荧光短片段多重PCR技术,使用基因分析仪片段分析多重PCR的产物,并与相应的MLPA检测结果进行比较。结果 特异性扩增SMN1基因7号外显子及内参基因的引物可实现目的片段的扩增,通过计算可得到0拷贝、1拷贝、2拷贝的样本结果。对107例DNA样本的SMN1基因拷贝数荧光短片段多重PCR的检测结果进行分析,实验结果均与MLPA检测结果相一致。结论 使用荧光标记多重PCR反应检测SMN1拷贝数可重复性好,精确度高,与MLPA得到的结果高度一致,具有便捷、准确、成本低的优势,可满足临床高准确性的检测要求,可用于SMA新生儿携带者的大规模筛查。     

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。     

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。     

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。     

Influence of ABCB1 polymorphisms and serum concentrations on venlafaxine response in patients with major depressive disorder

Background: The pharmacokinetics and the pharmacodynamics of antidepressants show large inter-individual variations which result in unpredictable clinical responses.

Aim: The aim of the study was to examine the effect of ABCB1 polymorphisms and the serum concentrations on the efficacy and tolerability of venlafaxine in patients with major depressive disorder (MDD).

Methods: Fifty-two outpatients who met the Diagnostic and Statistical Manual of Mental Disorders Fourth Edition (DSM-IV) criteria for MDD were recruited for the study. The severity of depression was assessed using the 17-item Hamilton Rating Scale for Depression scale (HDRS₁₇) and tolerability was assessed based on a query regarding side-effects for 6 weeks. The ABCB1 C3435T/A and G2677T/A polymorphisms were genotyped by PCR/RFLP and steady-state serum venlafaxine concentrations were measured by high-performance liquid chromatography.

Results: Patients with the TT genotype for the C3435T and the TT/TA genotype for the G2677T/A polymorphism showed significantly higher frequencies in venlafaxine-induced akathisia. This relationship was not observed for efficacy. As regards serum venlafaxine concentrations, patient groups showed no significant differences in efficacy and tolerability.

Conclusion: The results suggest that individuals with the TT-TT/TA genotypes for the C3435T-G2677T/A polymorphisms of ABCB1 may be pre-disposed to a risk of akathisia.     

Utilization of a two-standard system in real-time PCR for quantification of gene expression in the brain

In this study, we applied for real-time PCR the two-standard system that we had worked out previously for PCR with gel-detection of products. Genomic DNA of a known concentration was used as external standard and mRNA of the DNA-dependent RNA-polymerase II was used as internal standard. It was shown that PCR with gel-detection of products and real-time PCR provide similar results and demonstrate almost identical accuracy and repeatability when the two-standard system is used. With the help of the both methods and using the two-standard system we have confirmed the link between the genetically determined freezing reaction in mice and reduced 5-HT_1A receptor mRNA level in the midbrain. We have also found that the genetically determined freezing reaction in mice is not connected with changes in Tph2 gene expression.     

MRP1基因表达与耐药性癫痫的相关性研究

目的研究MRP1基因表达与耐药性癫痫的相关性。方法选取90例符合诊断标准的癫痫患者,其中药物敏感患者50例,癫痫耐药患者40例。采用实时荧光定量PCR方法检测癫痫患者外周血中MRP1基因mRNA的表达水平。结果耐药性癫痫组MRP1基因mRNA表达量(9.52±1.38)高于药物敏感组(1),差异有统计学意义(P<0.05)。结论癫痫患者外周血中MRP1的表达水平,可作为诊断耐药性癫痫的参考指标。     

Copy number assessment of SMN1 based on real-time PCR with high-resolution melting: fast and highly reliable testing

BackgroundSpinal muscular atrophy (SMA) is a neuromuscular disease mainly caused by the absence of both copies of the survival motor neuron 1 (SMN1) gene. Multiple regions recommended population-wide SMA screening to quantify the copy number of SMN1. SMN1 diagnostic assays for the simplified procedure, high sensitivity, and throughput continue to be needed.MethodsReal-time PCR with high-resolution melting for the quantifying of the SMN1 gene exon 7 copies and exon 8 copies were established and confirmed by multiplex ligation-dependent probe amplification (MLPA). The diagnosis of 2563 individuals, including SMA patients, suspected cases, and the general population, was tested by real-time PCR. The results were compared with the gold standard test MLPA.ResultsIn this study, the homozygous and heterozygous deletions were detected by real-time PCR with a high-resolution melting method with an incidence of 10.18% and 2.26%, respectively. In addition, the R-value distribution (P > 0.05) among 8 replicates and the coefficient of variation (CV < 0.003) suggested that the real-time PCR screening test had high reproducibility. High concordance was obtained between real-time PCR with high-resolution melting and MLPA.ConclusionsThe real-time PCR based on high-resolution melting provides a sensitive and high-throughput approach to large-scale SMA carrier screening with low cost and labor.     

BRIP1在胶质瘤中的表达及临床意义

目的 探讨乳腺癌易感基因相互作用蛋白1(BRIP1)在胶质瘤中的表达及其与病人预后的相关性.方法 从GEO数据库GPL570平台选取三个BRIP1基因表达谱芯片(GSE4290、GSE50161和GSE74195)分析胶质瘤BRIP1的表达情况.使用TC-GA下载有关胶质瘤BRIP1表达、生存率和临床特征的数据,多因素...     

脑胶质瘤FBLIM1的表达及临床意义

目的 探讨FBLIM1在脑胶质瘤中的表达及临床意义。方法 采用R软件分析UCSC数据库中癌症基因组图谱联合基因型组织表达数据集（TCGA、TARGET、GTEx）中的662例胶质瘤和1 157例正常脑组织的FBLIM1表达水平。利用TCGA数据库698例胶质瘤mRNA-seq及临床数据分析FBLIM1表达与胶质瘤临床特征的关系,用Cox比例回归风险模型分析胶质瘤生存预后的影响因素,采用Kaplan-Meier法分析TCGA数据库及中国脑胶质瘤基因组图谱（CGGA）数据库共计1 975例胶质瘤的FBLIM1表达与生存预后的关系。结果 胶质瘤FBLIM1表达水平较正常脑组织明显增高（P<0.05）,且肿瘤WHO分级越高,FBLIM1表达水平越高（P<0.05）;胶质瘤FBLIM1表达与IDH基因状态、1p/19q联合缺失、WHO分级及病人年龄显著相关（P<0.05）;FBLIM1过表达为胶质瘤病人生存预后不良的独立危险因素（OR=1.444;95% CI 1.032~2.020;P<0.05）;生存曲线分析显示FBLIM1高表达的胶质瘤病人中位总生存期较低表达病人明显缩短（P<0.05）。结论 胶质瘤FBLIM1呈高表达,与病人生存预后不良相关。