目的 构建靶向人Akt基因的shRNA真核表达载体,通过RNAi下调Akt基因在结肠癌Lovo细胞系中的表达,观察对结肠癌细胞生长的影响.方法 将靶向人Akt基因的两条shRNA(Akt1-U6-1#;Aktl-U6-2#)表达序列克隆到入门载体pENTRTM/U6的黏性末端,测序鉴定后与目的表达载体Plentio/Block-iT DEST Vector进行LR位点特异性重组；分别将入门载体与目的表达载体转染入Lovo细胞,对细胞株行RT-PCR和Western blotting检测,观察siRNA对靶基因Akt的抑制效果.结果 DNA测序证实pENTRTM/U6-TF-shRNA入门载体和Plentio/Block-iT DEST目的表达载体为阳性克隆,转染Lovo细胞后,Akt基因在mRNA和蛋白水平的表达量受到明显抑制.结论 成功构建了靶向人Akt的shRNA表达载体,且该载体在体外能有效抑制Lovo细胞Akt基因表达,为进一步研究Akt的功能和肿瘤的基因治疗奠定了实验基础.%Objective To investigate the association of forced expiratory volume in 1 second (FEV1) and the maximum peak expiratory flow (PEF) with small airway function in asthmatic children of different ages and genders. Methods This cross-sectional study was conducted among 619 asthmatic children with disease remission aged 3 to 13 years. The children were divided into 3 age groups, namely 3 to 5 years group (314 cases), 6 to 9 years group (207 cases) and 10 to 13 years group (98 cases), and their respiratory physiological parameters such as FEV1 and PEF were measured. Results Of the airway function parameters, PEF showed the highest abnormality rate (>85%) in these asthmatic children. In male and female asthmatic children aged 6 to 9 years, abnormalities in forced expiratory flow rate 25% (MEF25) showed the highest frequency (56% and 63%, respectively). In 3-5 years and 10-13 years groups, MEF25 abnormalities were the most frequent in male children (43% and 71%, respectively), whereas abnormalities in MEF50 were the most common in female children (33% and 69%, respectively). FEV1 and PEF were positively correlated to all the parameters of small airway functions in these asthmatic children (r>0.5, P<0.01) except for MEF25 in female asthmatic children aged 3 to 5 years (r=0.19, P=0.168; r=0.086, P=0.535). Conclusion In asthmatic children, FEV1 and PEF are positively correlated to the parameters of small airway function with only the exception of MEF25 in female children aged 3 to 5 years, suggesting the value of FEV1 in the diagnosis of asthma in children.
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