脂多糖刺激树突状细胞前后TLR2、3、4基因表达的检测

目的 检测脂多糖作用于树突状细胞前后TLR2、3、4基因的表达情况。方法 从外周血分离单核细胞，加入rhGM-CSF及rhIL-4，培养的第6天，实验组加入脂多糖刺激24h，对照组不加。分别提取细胞总RNA，行半定量RT-PCR，产物进行琼脂糖凝胶电泳分析。结果 单核细胞经GM-CSF及IL-4诱导7d后的DC表达较高水平TLR2、3、4。脂多糖刺激24h后，TLR2、3、4表达下降，TLR4几乎测不到。结论 树突状细胞在受脂多糖刺激前后TLRs的表达有显著变化，推测和它的功能成熟有关。     

脊髓损伤后热休克蛋白27、表皮脂肪酸结合蛋白和金属蛋白酶组织抑制因子-1的基因表达及甲基强的松龙对其表达的影响

目的研究脊髓损伤后热休克蛋白27(HSP27)、表皮脂肪酸结合蛋白(FABPs)和金属蛋白酶组织抑制因子-1(TIMP-1)的基因表达及甲基强的松龙(MP)对其表达的影响.方法SD大鼠30只.随机分为假手术组、单纯脊髓损伤组(损伤组)及脊髓损伤+大剂量MP治疗组(MP组),每组10只.应用改良的Allen's打击法致T8脊髓损伤.MP组大鼠伤后即刻从尾静脉内注射大剂量MP(30mg/kg).损伤后24h切取损伤平面上下0.5cm的脊髓组织,进行RT-PCR反应,检测HSP27、FABPs和TIMP-1的基因表达.结果术后24h假手术组HSP27、FABPs和TIMP-1的基因表达相对丰度分别为0.0643±0.0152、0.6413±0.1005和0.7091±0.0577;损伤组上述三个因子的表达升高,分别为1.0013±0.3861、1.2187±0.2851和0.8971±0.1092,与假手术组比较差异有显著性(P＜0.01、P＜0.05、P＜0.05);MP组上述三个因子的表达继续升高,分别为1.2858±0.1384、1.7122±0.1766和1.2081±0.1093,与损伤组比较差异有显著性(P＜0.05、P＜0.01和P＜0.01).结论脊髓损伤后,邻近损伤处的脊髓组织中HSP27、FABPs及TIMP-1的基因表达显著增高,大剂量MP能进一步促进三个因子表达,发挥组织保护作用.     

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.     

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.     

“分步给药”在低氧易感患者无痛苦胃镜检查中安全性的探讨

目的 探讨在低氧易感患者无痛苦胃镜检查中采用分步给药的安全性和镇静/镇痛效果.方法 接受胃镜检查的180例患者,其中有鼾症、或咳嗽、咳痰症状的患者120例单双号随机入选Ⅰ组(分步给药组)和Ⅱ组(普通组),60例无鼾症、咳嗽、咳痰症状的患者为Ⅲ组(常规给药组),Ⅰ组采用分步给药法,Ⅲ组采用常规给药法对患者实施胃镜检查前的镇静/镇痛术,Ⅱ组则进行普通胃镜检查,分析2种镇静法对SBP、DBP、SpO2、HR、清醒时间、患者反应、患者对检查过程的感受以及是否愿意再次接受检查的影响,评价分步给药法在低氧易感患者无痛苦胃镜检查中的安全性和镇静/镇痛效果.结果 Ⅰ组、Ⅲ组检查中SBP、DBP变化无明显差异(均P＞0.05),Ⅰ组检查前、中SpO2的变化小于Ⅲ组(均P＜0.01);Ⅰ组进镜时咽部受刺激产生的恶心咽吐、流涎、咳嗽、躁动反应多于Ⅲ组(均P＜0.01),但较Ⅱ组少(均P＜0.01);Ⅰ组对胃镜检查的评价与Ⅲ组无明显差异(P＞0.05),与Ⅱ组相比有明显差异(P＜0.01).结论 分步给药法是一种适用于低氧易感患者无痛胃镜检查的安全、有效的镇静/镇痛方法.