目的 观察低氧对p53RFP基因表达及启动子活性的影响,探索低氧反应元件(HRE)是否参与了低氧对p53RFP基因启动子活性的调控。方法 将HEK293细胞置于低氧条件下培养,实时定量PCR及Western blot 检测p53RFP mRNA及蛋白表达水平;通过定点突变技术构建HRE突变型p53RFP启动子双荧光素酶报告基因载体并转染HEK293细胞,分别置于常氧和低氧条件下培养,双荧光素酶报告基因检测系统检测启动子活性。结果 低氧处理不同时间点p53RFP mRNA 表达水平均显著增加,差异有统计学意义(F=96.493,P<0.001);低氧组p53RFP 及HIF-1α蛋白表达量均呈时间依赖性递增。成功构建了HRE 突变型p53RFP 启动子双荧光素酶报告基因载体,双荧光素酶报告基因检测显示,与常氧组相比,低氧条件下野生型p53RFP基因启动子活性增加(t=-19.504,P<0.001),HRE单突变或双突变后启动子活性均较野生型降低(F=160.891,P<0.001)。结论 低氧可诱导p53RFP基因表达上调;成功构建了HRE 突变型p53RFP 双荧光素酶报告基因载体,HRE 位点可能在低氧调控p53RFP基因启动子活性中发挥关键作用。

Objective To investigate the effect of hypoxia on p53RFP gene expression and p53RFP promoter activity, and explore whether hypoxia responsive element (HRE) is involved in the regulation of p53RFP gene promoter activity by hypoxia. Methods HEK293 cells were cultured under normoxia and hypoxia conditions, p53RFP expressions at mRNA and protein levels were detected by real-time PCR and Western blot respectively. The HRE mutant p53RFP promoter dual luciferase reporter gene vector was constructed by site-directed mutagenesis technology and transfected into HEK293 cells under normoxia and hypoxia conditions. The promoter activities were detected by dual luciferase reporter system. Results Compared with the normoxia group, the expression of p53RFP mRNA in the hypoxia group increased significantly at different time points, and the difference was statistically significant(F=96.493,P<0.001); the expression of p53RFP and HIF-1α protein in the hypoxia group increased in a time-dependent manner. The luciferase reporter vectors containing p53RFP with mutant HRE were successfully constructed. The dual luciferase reporter gene assay showed that the activity of the wild-type p53RFP gene promoter was significantly increased under hypoxic conditions compared to normoxic condition(t=-19.504,P<0.001),and the promoter activity of p53RFP with HRE single mutation or double mutation were both significantly lower than that of wild type under hypoxic condition (F=160.891,P<0.001). Conclusions p53RFP gene expression was induced by hypoxia; the p53RFP promoter with mutant HRE dual luciferase reporter gene vectors were successfully constructed, and the HRE locus may play a key role in the hypoxia regulation of p53RFP gene promoter activity.

目的 探讨慢性阻塞性肺疾病(COPD)并发低氧血症患者抢救中使用小剂量肾上腺素及布地奈德的价值。方法 纳入50例COPD并发低氧血症患者研究(2018年4月—2021年4月),按双盲法分为对照组(n=25,采用布地奈德雾化吸入治疗)、观察组(n=25,在对照组基础上采用肾上腺素治疗),统计2组抢救成功率、临床指标、预后效果。结果 (1)抢救成功率:观察组(96.00%)高于对照组(76.00%),组间对比P<0.05。(2)临床指标:观察组PaCO₂(43.29±4.92 mmHg)低于对照组,PaO₂(86.77±8.25 mmHg)、SpO₂(92.14±2.82%)、pH(7.43±0.12)、FVC(2.41±0.28 L)、FEV1(1.72±0.72 L)、FEV1/FVC(70.95±8.22%)高于对照组,组间对比P<0.05。(3)预后效果:观察组气喘(3.22±1.08 d)、哮鸣音(5.21±1.11 d)消失时间及住院时间(9.61±2.24 d)短于对照组,组间对比P<0.05。结论 小剂量肾上腺素联合布地奈德在COPD并发低氧血症治疗中效果确切,可提高抢救成功率,亦可改善其肺功能及血气指标,值得临床参考。

Objective To explore the value of low dose epinephrine and budesonide aerosol inhalation in the rescue of patients with chronic obstructive pulmonary disease(COPD) complicated with hypoxemia. Methods Fifty patients with COPD complicated with hypoxemia(April 2018 to April 2021)were enrolled and divided into control group(n=25,treated with budesonide aerosol inhalation)and observation group(n=25,treated with epinephrine additionally)according to double-blind method.The rescue success rate,clinical indicators and prognosis of the two groups were statistically analyzed. Results (1)The success rate of rescue of the observation group(96.00%)was higher than that of the control group(76.00%,P<0.05). (2)Clinical indicators:PaCO₂(43.29 1±4.92 mmHg)in the observation group was lower than that of the control group,PaO₂(86.774±8.25 mmHg), SpO₂(92.14±2.82%), pH(7.43±0.12), FVC(2.41±0.28 L), FEV1 (1.72±0.72 L), FEV1/FVC (70.95±8.22%)were higher than that in the control group(P<0.05). (3)Prognosis effect:asthma duration in the observation group(3.22±1.08 d),wheezing disappeared time(5.211±1.11 d)and hospitalization time(9.611±2.24 d)were shorter than that of the control group(P<0.05). Conclusions Low dose epinephrine combined with budesonide in the treatment of COPD complicated with hypoxemia has definite effect, which can improve the success rate of rescue,also improve lung function and blood gas index. It is worthy of clinical reference.

目的 采用生物信息学方法预测低氧预处理人胎盘绒毛膜间充质干细胞环状RNAs相对应的miRNA及其靶基因,并分析靶基因所参与的生物学过程和信号通路。方法 用Arraystar公司的商业软件为环状RNAs预测其相对应的miRNAs,分别用targetScan7.1和mirdbV5数据库预测miRNAs的靶基因,并取两个预测结果的合集,应用在线网站http://www.geneontology.org和http://www.genome.ad.jp/kegg对靶基因进行功能富集分析和信号通路富集分析。结果 功能富集分析表明,circRNAs的靶基因主要涉及到细胞发育、细胞分化和细胞发育调控。东京基因和基因组百科全书信号通路富集分析表明肿瘤中转录失控和有丝分裂原激活蛋白激酶(MAPK)信号通路最有意义,而且分析发现MAPK信号通路为核心通路。本研究表明,低氧预处理使得间充质干细胞中部分circRNAs的表达量发生差异性变化。结论 低氧预处理人胎盘绒毛膜间充质干细胞环状RNAs同低氧预处理间充质干细胞的生物学特性变化密切有关,为了解低氧预处理影响间充质干细胞特性发生变化的分子机制提供新思路。

Objective To predict the miRNA and its target genes of circular RNAs in hypoxia- preconditioned human palcenta chorionic mesenchymal stem cells using bioinformatics, and analyze the biological process and signaling pathway. Methods Arraystar's commercial software was used to predict the corresponding miRNAs of circular RNAs. The target genes of miRNAs were predicted by targetScan7.1 and mirdbV5 databases respectively, and an intersection of two prediction results was obtained. The online databases http://www. geneontology.org and http://www.genome.ad.jp/kegg performed functional enrichment analysis and signal pathway enrichment analysis of target genes. Results Functional enrichment analysis indicated that the target genes of circRNAs mainly involved cell development, cell differentiation and cell development regulation. The signal enrichment analysis of the Tokyo Gene and Genome Encyclopedia indicates that transcriptional misregulation in cancer and mitogen-activated protein kinase (MAPK) signaling pathway are most meaningful, and the MAPK signaling pathway is found to be the core pathway. This study showed that hypoxic preconditioning caused significant changes in the expression of mesenchymal stem cell circRNAs. Conclusion The changes of circular RNAs in hypoxia-preconditioned human placental chorionic mesenchymal stem cell is closely related to the biological characteristics of hypoxia-preconditioned mesenchymal stem cells. This study provides a new idea for understanding the molecular mechanism of hypoxic preconditioning affecting the changes of biological characteristics in mesenchymal stem cells.

目的 评价实时荧光定量PCR分析低氧性肺动脉高压大鼠肺动脉平滑肌基因表达时12个候选内参基因表达的稳定性,获得最适合的内参基因。方法 以低氧性肺动脉高压大鼠肺动脉平滑肌为研究对象,选择文献报道的常用12种内参基因为候选内参基因,利用geNorm和NormFinder程序分析实时荧光定量PCR数据,筛选出最适内参基因。结果 12个候选内参基因在低氧性肺动脉高压大鼠肺动脉平滑肌表达稳定性由强到弱顺序为:TBP>B2M>HPRT1>HMBS>RPL13a>18sRNA>PPIA>ACTB>GUSB>TFRC>GAPDH>PGK1,平均表达稳定度(M值)均<0.5,geNorm和NormFinder评估后推荐使用TBP和B2M一起作为该研究时的内参基因。结论 同时使用TBP和B2M是实时荧光定量PCR分析低氧性肺动脉高压大鼠肺动脉平滑肌基因表达的最适合内参基因,为低氧性肺动脉高压相关基因研究提供最优内参基因。

Objective To compare and select the suitable reference genes in real-time quantitative PCR analysis of rat pulmonary artery smooth muscle cells mRNA expression level of pulmonary hypertension. Methods To choose appropriate reference gene, the expression of twelve commonly use housekeeping genes were examined in rat pulmonary artery smooth muscle cells of hypoxia-induced pulmonary hypertension by using geNorm and NormFinder programs. Results The expression consistency of 12 genes was (from high to low): TBP>B2M>HPRT1>HMBS>RPL13a>18sRNA>PPIA>ACTB>GUSB>TFRC>GAPDH>PGK1. The average expression stability(M) values of them were low than 0.5. TBP and B2M reference genes were recommended to use in the same condition. Conclusion TBP and B2M reference genes were the most suitable combination of the reference genes for real-time quantitative PCR analysis in rat pulmonary artery smooth muscle cells of hypoxia-induced pulmonary hypertension.