Efficient RT-PCR on platelet mRNA after long-term storage

We have developed a procedure permitting RT-PCR from mRNA even after a long-term storage (1 year) of platelet samples in ethanol (EtOH-platelets) at −80°C. To validate our method, we have analysed the human platelet alloantigen system (HPA-1) which is coded by β₃ mRNA. We have also demonstrated the efficiency of amplification of part of the coding region for (i) α_IIb subunit mRNA, (ii) α_v subunit mRNA, and (iii) the seven transmembrane domain thrombin receptor mRNA.     

腺病毒介导的NT-3基因在骨髓间质干细胞中的表达

目的:研究腺病毒介导的NT-3基因在培养的大鼠骨髓间质干细胞(mesenchymal stem cells,MSCs)中的表达.方法:在293细胞中培养扩增NT-3重组腺病毒(adenovirus vector for NT-3,Ad-NT-3),测定病毒滴度,然后用Ad-NT-3感染传代培养的MSCs,RT-PCR技术检测NT-3基因的表达.结果:Ad-NT-3扩增后获得了较高滴度的病毒,MSCs经Ad-NT-3感染后有NT-3 mRNA的转录.结论:腺病毒介导的NT-3基因可转入培养的MSCs并高效表达,为NT-3基因治疗的研究奠定了基础.     

结缔组织生长因子在脑胶质瘤组织中的表达和肿瘤级别的关系

目的通过检测人脑胶质瘤患者肿瘤组织中结缔组织生长因子的表达水平,探讨其和肿瘤级别的关系及作用机制。方法采用荧光实时定量聚合酶链式反应(RT-PCR)法检测结缔组织生长因子(CTGF)mRNA在47例脑胶质瘤组织和25例正常脑组织中的表达水平,统计分析表达水平和肿瘤级别之间的关系。结果CTGF在胶质瘤Ⅰ-Ⅱ级(高分化组)及胶质瘤Ⅲ~Ⅳ级(低分化组)中的表达均明显高于正常脑组织(P<0.01),而且在Ⅲ~Ⅳ级胶质瘤中的表达量显著高于胶质瘤Ⅰ~Ⅱ级(P<0.01),说明随着肿瘤级别的升高CTGF表达也增强。结论CTGF在胶质瘤组织中高表达,而且表达水平和恶性程度有密切联系。CTGF的检测可作为胶质瘤恶性程度判断的参考,为从基因水平上探讨胶质瘤的生物学行为、预后及治疗提供新的思路。     

原发性肝癌患者外周血GPC-3mRNA的表达与微转移的关系

目的探讨原发性肝细胞癌（HCC）患者外周血的Glypican-3（GPC-3）mRNA检测用于诊断HCC早期微转移的可行性。方法采用巢式逆转录聚合酶链反应（RT-PCR）技术检测39例原发性肝细胞癌（HCC）、19例肝炎和肝硬化、10例转移性肝癌、10例肝血管瘤患者、19例健康献血员外周血GPC-3mRNA水平。结果HCC患者外周血中，GPC-3mRNA的检出率为62％（24／39），GPC-3mRNA的检出率与肿瘤直径77％（17／22）、癌结节数目67％（19／24）、临床TNM分期76％（19／25）、门静脉癌栓90％（9／10）、远处转移100％（6／6）等临床参数密切相关。GPC-3mRNA在转移性肝癌、肝炎和肝硬化、肝血管瘤患者及健康献血员外周血中均未检出。结论巢式RT—PCR检测HCC患者外周血单核细胞GPC-3mRNA是一种早期发现原发性肝细胞癌血道播散的可靠而又敏感的实验方法。     

Pre-Implantation Multiple Cytokine mRNA Expression Analysis of Donor Lung Grafts Predicts Survival After Lung Transplantation in Humans

While current donor selection with clinical findings is generally effective, the imprecise nature of the assessment forces clinicians to remain on the conservative side. A reliable biological marker would assist donor selection and would improve donor organ utilization. We collected biopsies from 169 donor lungs before implantation. Expression levels of IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma and IL-1beta were measured by quantitative real-time RT-PCR (qRT-PCR). Seventeen cases died within 30 days after transplantation. No donor factor was significantly associated with 30-day mortality. Univariate analysis of the 84 cases for development of the prediction model showed that IL-6, IL-8, TNF-alpha and IL-1beta were risk factors for mortality and IL-10 and IFN-gamma were protective factors. We analyzed the cytokine expression ratios of risk to protective cytokines. A stepwise logistic regression for 30-day mortality demonstrated that a model containing the ratio of IL-6/IL-10 was the most predictive (p = 0.0013). When applied to the remaining 85 cases for validation, the test of model fit was significant (p = 0.039). Using the cytokine ratio, we were able to define three risk groups with striking differences in survival (p = 0.0003). Multi-cytokine analysis of the donor lung graft with qRT-PCR shows significant promise as a strategy to biologically evaluate the donor lung prior to implantation.     

中药何首乌饮对衰老大鼠卵巢组织胰岛素样生长因子Ⅰ及胰岛素样生长因子结合蛋白3表达的影响

目的 探讨何首乌饮抗大鼠卵巢组织衰老的机理,为抗女性性腺的衰老研究提供理论指导和实验依据.方法 将60只SD大鼠随机分为3组:正常组、模型组、预防组.采用D-半乳糖连续腹腔注射法制造雌性大鼠亚急性衰老模型,预防组在造模的同时以中药方剂何首乌饮灌胃.应用原位杂交方法检测各组大鼠卵巢组织胰岛素样生长因子Ⅰ(IGF-Ⅰ)蛋白的表达,用RT-PCR方法检测各组大鼠卵巢组织胰岛素样生长因子结合蛋白3(IGFBP3)基因的表达情况.结果 何首乌饮明显抑制了衰老大鼠卵巢组织中IGFBP3基因表达的上调趋势(P＜0.05),且明显提高了衰老大鼠卵巢组织中IGF-Ⅰ蛋白的表达(P＜0.05).结论 何首乌饮通过影响衰老大鼠卵巢中细胞因子IGF-Ⅰ、IGFBP3的表达,起到抗大鼠性腺衰老的作用.     

Rh phenotype prediction by DNA typing and its application to practice

The complexity of the RHD and RHCE genes, which is the greatest of all blood group systems, confounds analysis at the molecular level. RH DNA typing was introduced in 1993 and has been applied to prenatal testing. PCR-SSP analysis covering multiple polymorphisms was recently introduced for the screening and initial characterization of partial D. Our objective is to summarize the accrued knowledge relevant to the approaches to Rh phenotype prediction by DNA typing, their possible applications beyond research laboratories and their limitations. The procedures, results and problems encountered are highly detailed. It is recommended that DNA typing comprises an analysis of more than one polymorphism. We discuss future directions and propose a piecemeal approach to improve reliability and cost-efficiency of blood group genotyping that may eventually replace the prevalent serology-based techniques even for many routine tasks. Transfusion medicine is in the unique position of being able to utilize the most extensive phenotype databases available to check and develop genotyping strategies.