基于UHPLC-QqQ-MS的四逆散水提液中16种主要化学成分的定量研究

目的 建立同时测定四逆散水提液中16种化学成分含量的方法。方法 应用UHPLC-QqQ-MS技术,采用负离子模式进行扫描,以MRM配对离子方式进行定量。结果 柴胡皂苷A、柴胡皂苷B₂、柴胡皂苷C、柴胡皂苷D、橙皮素、儿茶素、甘草苷、甘草酸、甘草素、绿原酸、没食子酸、芍药苷、芍药内酯苷、异甘草素、异绿原酸A、橙皮苷在负离子模式下响应良好。结论 本实验所建立的同时测定四逆散水提液中16种化学成分含量的分析方法专属性强、快速、灵敏且准确可靠,在完成含量测定的同时又可以准确给出化合物质量数信息,从而达到成分鉴别的效果,该方法可以为四逆散水提液的全面质量控制提供技术支持。      

Effect and Mechanism of Ganoderma lucidum Polysaccharides on Human Fibroblasts and Skin Wound Healing in Mice

Objective: To investigate the effects of Ganoderma lucidum polysaccharides(GL-PS) on human fibroblasts and skin wound healing in Kunming male mice and to explore the putative molecular mechanism. Methods: Primary human skin fibroblasts were cultured. The viability of fibroblasts treated with 0, 10, 20, 40, 80, and 160 μg/mL of GL-PS, respectively were detected by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-Htetrazolium bromide(MTT). The migration ability of fibroblasts treated with 0, 10, 20, and 40 μg/mL of GL-PS were measured by transwell assay. The secretion of the C-terminal peptide of procollagen type Ⅰ(CICP) and transforming growth factor-β1(TGF-β1) in the cell supernatant was tested by enzyme-linked immunosorbent assay. The expression of β-catenin was detected by Western blot. Furthermore, the Kunming mouse model with full-layer skin resection trauma was established, and was treated with 10, 20, and 40 mg/mL of GL-PS, respectively as external use. The size of the wound was measured daily, complete healing time in each group was recorded and the percentage of wound contraction was calculated. Results: Compared with the control group, 10, 20, and 40 μg/mL of GL-PS significantly increased the viability of fibroblasts, promoted the migration ability of fibroblasts, and up-regulated the expressions of CICP and TGF-β1 in fibroblasts(P0.05 or P0.01). The expression of β-catenin in fibroblasts treated with 20 and 40 μg/mL of GL-PS was significantly higher than that of the control group(P0.01). Furthermore, after external use of 10, 20, and 40 mg/mL of GL-PS, the rates of wound healing in mice were significantly higher and the wound healing time was significantly less than the control group(P0.05 or P0.01). Conclusion: A certain concentration of GL-PS may promote wound healing via activation of the Wnt/β-catenin signaling pathway and up-regulation of TGF-β1, which might serve as a promising source of skin wound healing.     

改良可视双腔支气管导管塑形方法在胸科手术单肺通气中的应用

目的 观察改良可视双腔支气管导管（Visual Double-Lumen Tube,VDLT）塑形方法在胸外科手术单肺通气中的应用效果。方法 选取2020-08至2021-08我院胸外科择期手术单肺通气患者60例,美国麻醉医师协会（American Society of Anesthesiologists,ASA）分级I～Ⅲ级,年龄24～80岁,性别不限,BMI 19～28 kg/m2。采用随机数字表法将患者分为传统塑形组（Tsaditional Shaping,TS组）和改良塑形组（Isproved Shaping,IS组）,每组30例。记录麻醉诱导前（T1）、麻醉诱导后即刻（T2）、插管后1 min(T3)的心率（Heart Rate,HR）及平均动脉血压（Mean Arterial Pressure,MAP）;记录插管时间、插管次数、支气管定位情况;记录术后咽喉部疼痛、声音嘶哑等并发症情况。结果 TS组插管时间（67.1±23.8）s明显长于IS组（38.9±10.0）s;TS组插管次数1.3±0.4次明显多于IS组1.0±0.0次（P<0.05）,两组可视双腔支气管导管支...     

不同灸法治疗原发性痛经的临床研究进展

目的:通过对近十年不同灸法治疗原发性痛经的临床研究文献进行整理分析,了解各种灸法治疗原发性痛经的临床疗效,以期择优选择适宜灸法治疗原发性痛经。方法:以"灸""原发性痛经""功能性痛经"为主题词,在中英文数据库上检索近十年(2010年1月—2020年2月)灸法治疗原发性痛经临床研究文献。结果:临床上灸法治疗原发性痛经的方法众多,主要以温和灸、温针灸、热敏灸为主,应用灸法治疗原发性痛经最多的证型是寒凝血瘀型(或寒湿凝滞型),但大部分临床研究未进行辨证给予艾灸治疗痛经以及同种证型痛经应用不同灸法的疗效差异未进行比较。结论:各种灸法治疗原发性痛经的临床疗效均较显著,但疗效差异不详,故今后要开展更多高质量的临床研究不同灸法治疗痛经之间疗效差异,明确不同证型痛经所适宜的灸法以提高临床疗效。     

老年人群klotho G-395A、F352V与C370S基因多态性及组合与其血清klotho蛋白水平的相关性研究

目的:探讨重庆市汉族老年人群Klotho G-395A、F352V与C370S 3个位点的单核苷酸多态性(Single nucleotide polymorphism,SNP)及其组合与血清klotho蛋白水平的相关性。方法:随机选择汉族老年人390例,采用等位基因特异性引物PCR技术检测Klotho G-395A、F352V与C370S SNP,酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)检测其血清klotho蛋白水平。结果:Klotho G-395A、F352V与C370S SNP各等位基因型携带者其血清klotho蛋白水平均无显著性差异(P>0.05)。基因组合分析研究发现Klotho G-395A、F352V与C370S SNP各组合等位基因型携带者其血清klotho蛋白水平差异性较大,其中Klotho G-395G+F352F+C370S携带者较Klotho G-395G+F352F+C370C携带者其血清klotho蛋白水平明显增高(P=0.056)。结论:Klotho G-395A、F352V与C370S SNP各等位基因型携带者其血清klotho蛋白水平无显著性差异,而组合等位基因型研究发现Klotho G-395G+F352F+C370S携带者较Klotho G-395G+F352F+C370C组合等位基因型携带者血清klotho蛋白水平增高有显著性趋势。     

不同处理时间50 Hz 3.6 mT正弦交变磁场对人脐带干细胞增殖与成骨性分化的研究

目的：研究50 Hz 3.6 mT不同处理时间的正弦交变电磁场（Sinusoidal electromagnetic field,SEMFs）对体外培养人脐带干细胞（Human umbilical cord stem cells,HUCSC）增殖与成骨性分化的影响。方法：体外分离培养HUCSC,传代后随机分为6组。用频率50 Hz,3.6 mT的SEMFs分别每天处理HUCSC 0.0（对照组）、 0.5、1.0、1.5、2.0 h和2.5 h,倒置相差显微镜观察细胞形态,MTT法测定细胞增殖,在磁场处理后的15 d 和17 d 分别用茜素红和vonkossa对钙化结节进行染色,在磁场处理后的第4天和第6天 PCR检测胶原Ⅰ（Collagen-Ⅰ）和骨形态发生蛋白（Bone morphogenetic protein-2,BMP-2）mRNA表达量的变化,在磁场处理后的10、12、14 d 和 16 d测定ALP活性。结果：1.0、1.5、2.0 h和2.5 h组促进HUCSC增殖;磁场处理后的7~9 d细胞出现钙化结节;在SEMFs处理后的第14天和第16天0.5 h组和1.0 h组碱性磷酸酶（Alkaline phosphatase,ALP）活性显著高于对照;SEMFs处理组能增加HUCSC钙化面积,其中尤以0.5 h和1.0 h最为明显;SEMFs能增加Collagen-Ⅰ和BMP-2 mRNA表达量,其中尤以0.5 h和1.0 h组作用最为明显。结论：50 Hz,3.6 mT 1.0、1.5、2.0 h和2.5 h促进HUCSC增殖,同时SEMFs组能促进体外培养HUCSC成骨性分化,尤以处理0.5 h和1.0 h促进成骨性分化最为明显。     

股骨近端髓内钉与动力髋螺钉在股骨粗隆部骨折治疗中临床疗效的对比分析

[目的]评价股骨近端髓内钉(PFNA)与动力髋螺钉(DHS)在股骨粗隆骨折治疗中的临床疗效.[方法]将应用PFNA(39例)与DHS(36例)治疗的75例患者,从术中情况、术后并发症以及术后髋关节功能的恢复情况对两种治疗方法的临床疗效进行比较分析.[结果]DHS的手术失血量高于PFNA(P＜0.05),同时其术后并发症也较PFNA明显增多(P＜0.05),髋关节功能Harris评分优秀率也低于PFNA治疗组(P＜0.05).[结论]PFNA在治疗上较DHS具有操作简单、创伤小、骨折固定牢固、并发症少及术后患者可早期离床负重等优点,是一种治疗股骨粗隆间骨折的理想方法,值得临床推广使用.     

补肾行气活血法对骨髓基质干细胞诱导成软骨细胞作用的影响

[目的]观察补肾行气活血法对诱导骨髓基质干细胞(BMSCs)成软骨细胞作用的影响.[方法]将传至第3代的BMSCs分为阴性对照组和治疗组,分别采用软骨细胞诱导培养基加体积分数为10%的空白对照血清和诱导培养基加体积分数10%的骨炎定含药血清培养诱导成软骨细胞,观察软骨细胞的生长状况及细胞表型特征.[结果]治疗组在细胞贴壁、生长形态及成软骨特性方面明显优于对照组,细胞表型的免疫组化染色均为阳性.[结论]以补肾行气活血法为组方依据的骨炎定含药血清既可促进BMSCs的增殖,又可促进诱导的软骨细胞分化.     

Individual and joint effects of borderline ankle-brachial index and high plasma total homocysteine on all-cause death in hypertensive adults

BACKGROUNDThe cardiovascular hazards of total homocysteine (tHcy) are long known. In addition, despite the acknowledgment on the importance of low ankle-brachial index (ABI) (< 0.9), borderline ABI (0.91-0.99) was once commonly overlooked. This study aims to explore the independent and joint effect of tHcy level and borderline ABI on all-cause death in hypertensive population.METHODSThis study included 10,538 participants from China H-type Hypertension Registry Study. ABI was described into two groups: normal ABI (1.00-1.40) and borderline ABI. tHcy level was also divided into two groups: < 15.02 and ≥ 15.02 μmo/L. Four groups were analyzed, using COX proportional hazard regression model, separately and pairwise to observe the independent and joint effect on all-cause death.RESULTSA total of 126 (1.2%) deaths were observed in the 1.7 years follow-up time. Borderline ABI has a higher predicted risk of death than normal ABI (HR = 1.87, 95%CI: 1.17-3.00) after adjusting for potential covariates. Compare with tHcy level < 15.02 μmo/L (low tHcy), those with tHcy ≥ 15.02 μmo/L (high tHcy) had higher risk to event outcome (HR = 1.99, 95% CI: 1.30-3.05). According to the cumulative hazard curve, group with borderline ABI and high tHcy level has significantly higher altitude and larger increasing rate over follow-up period compare to other groups. Among those with borderline ABI, participants with high tHcy had higher death risk than those with low tHcy, nevertheless, no significant different between borderline and normal ABI among those with low tHcy levels.CONCLUSIONSBorderline ABI and tHcy level both have independent predictive value on all-cause death. The combined group of borderline ABI and high tHcy has highest risk factor of outcomes, which suggested the mutual additive value of borderline ABI and tHcy. More attention should be given to the importance of borderline ABI in hypertensive population, especially with elevated tHcy level.

Homocysteine (Hcy) is a sulfur-containing, non-proteinogenic amino acid synthesized through the transmethylation of amino acid methionine from one-carbon metabolism. Elevated plasma total homocysteine (tHcy) level is associated with endothelial dysfunction, increased blood coagulation, and metabolic disturbance, promoting cardiovascular diseases, stroke, and coronary artery disease.^[1,2] Notably, patients with high Hcy levels and concomitant hypertension were suggested to be at particularly higher risk.^[3] Moreover, increasing studies have explored a positive association between advanced Hcy level with all-cause mortality. According to a recent dose-response meta-analysis, for each 5-μmol/L increment of tHcy levels, the risk for all-cause mortality increased by 33.6%.^[4]The ankle-brachial index (ABI) is an effective, well-established measure that is commonly used in the diagnosis of peripheral artery disease (PAD),^[5] meanwhile was well studied as an important indicator of atherosclerosis and CVD events.^[6] Although ankle-brachial index (ABI) ≤ 0.90 has been recognized as the threshold value for abnormal/low ABI, which was proven to increase the risk of all-cause mortality,^[7] a study from the American Heart Association has suggested ABI between 0.91 and 1.00 should be considered as “borderline area” in terms of cardiovascular risks,^[8] considering of prior probability and sensitivity of ABI calculation. Emerging studies have aimed to explore the predictive value of borderline ABI,^[9-11] however, controversy remains because of limited and inconsistent data. The current study aimed to explore the individual and joint effect of borderline ABI and tHcy on all-cause mortality among hypertensive adults. Although ABI level ≤ 0.90 has been and is going to remain significant in clinical practice, we believe broader concern should be placed on borderline ABI, especially for its value in risk differentiation and identification. To the best of our knowledge, there are no similar previous studies.     

增殖细胞核抗原突变体的稳定高表达对HeLa细胞顺铂敏感性的影响

目的 建立稳定高表达增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)或突变体PC-NA(mutant PCNA,mPCNA)目的蛋白的宫颈癌细胞系HeLa细胞,观察PCNA在顺铂诱导的DNA损伤修复中的作用.方法 采用显性负突变策略,通过构建重组人PCNA或mPCNA的真核表达质粒pCDNA3.1/V5-His A-PCNA(His-PCNA)和pCDNA31/V5-His A-mPCNA(His-mPCNA),稳定转染到HeLa细胞中,G418筛选建立稳定高表达目的蛋白的HeLa细胞系;Western印迹法检测蛋白的表达情况;MTT法测定顺铂损伤后不同细胞系的细胞存活率.结果 真核表达质粒经酶切、测序分析与实验设计完全一致;稳定建系后,Western印迹结果显示在相应位置可见清楚的目的条带;MTT结果显示稳定高表达突变体PCNA的细胞系与对照组相比,其细胞存活率呈明显下降趋势.结论 成功构建了真核表达质粒His-PCNA和His-mPC-NA;建立了稳定高表达该质粒的HeLa细胞系;稳定高表达突变体PCNA的细胞系对顺铂损伤更敏感.