三七片DNA条形码分子鉴定及方法学考察

目的为研究DNA条形码技术在中成药鉴定中的应用,以三七片为研究对象,对方法的适用性、专属性与精密度进行考察。方法收集15批次市售三七片样品,考察三七片DNA提取条件,并对"中药材DNA条形码分子鉴定法指导原则"中PCR扩增、序列获得、结果判定等方法适用性进行确认;收集三七、人参、西洋参,制作三七片及其掺伪品,考察方法的专属性和重现性。结果三七片取样量100 mg,56℃水浴8 h所获得DNA的质量浓度平均值为60.7 ng/μL,PCR扩增、序列获得和结果判定均可获成功;三七、人参和西洋参的ITS2序列长度均为230bp,三七与人参、三七与西洋参的序列间均存在7个稳定的SNP位点,自制三七片和掺伪三七片均可成功获得ITS2序列,不同比例三七与人参、三七与西洋参的测序峰图在SNP位点处呈现相应峰高比的SNP套峰具备专属性;重复性、中间精密度和重现性考察符合《中国药典》2015年版(通则9101)相关要求。结论 ITS2序列作为DNA条形码能够稳定、准确鉴定三七片的原料药材,具备良好的专属性和精密度,三七片DNA条形码分子鉴定法将为保障三七片临床用药安全提供新的技术手段,并对《中国药典》其他收载单方制剂的鉴定提供参考。     

Rociletinib (CO-1686) enhanced the efficacy of chemotherapeutic agents in ABCG2-overexpressing cancer cells in vitro and in vivo

Overexpression of adenosine triphosphate (ATP)-binding cassette subfamily G member 2 (ABCG2) in cancer cells is known to cause multidrug resistance (MDR), which severely limits the clinical efficacy of chemotherapy. Currently, there is no FDA-approved MDR modulator for clinical use. In this study, rociletinib (CO-1686), a mutant-selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), was found to significantly improve the efficacy of ABCG2 substrate chemotherapeutic agents in the transporter-overexpressing cancer cells in vitro and in MDR tumor xenografts in nude mice, without incurring additional toxicity. Mechanistic studies revealed that in ABCG2-overexpressing cancer cells, rociletinib inhibited ABCG2-mediated drug efflux and increased intracellular accumulation of ABCG2 probe substrates. Moreover, rociletinib, inhibited the ATPase activity, and competed with [¹²⁵I] iodoarylazidoprazosin (IAAP) photolabeling of ABCG2. However, ABCG2 expression at mRNA and protein levels was not altered in the ABCG2-overexpressing cells after treatment with rociletinib. In addition, rociletinib did not inhibit EGFR downstream signaling and phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK). Our results collectively showed that rociletinib reversed ABCG2-mediated MDR by inhibiting ABCG2 efflux function, thus increasing the cellular accumulation of the transporter substrate anticancer drugs. The findings advocated the combination use of rociletinib and other chemotherapeutic drugs in cancer patients with ABCG2-overexpressing MDR tumors.     

温阳补肾法对高原类风湿关节炎患者血清25羟维生素D3水平的影响

目的:观察温阳补肾法对高原类风湿关节炎患者血清25羟维生素D3水平的影响。方法:选取2015年7月至2017年8月青海省中医院收治的高原类风湿关节炎患者132例作为研究对象,随机分为对照组和观察组,每组66例。对照组应用常规西医治疗,观察组在对照组治疗基础上应用温阳补肾法治疗,2组均连续治疗3个月。比较2组治疗后临床疗效,统计2组治疗前后主要临床症状与体征、健康状况评定量表(HAQ)评分和DAS28评分,检测2组治疗前后实验室检查指标、血清25羟维生素D3以及血液流变学指标水平。结果:治疗后,观察组临床治疗总有效率显著高于对照组(P 0. 05); 2组关节肿胀指数、关节疼痛指数、关节压痛指数、晨僵时间、关节功能分级和20 m步行时间均较治疗前显著降低(P 0. 05),且观察组均显著低于对照组(P 0. 05); 2组HAQ评分、DAS28评分、类风湿因子(RF)、C反应蛋白(CRP)和红细胞沉降率(ESR)水平均较治疗前显著降低(P 0. 05),且观察组均显著低于对照组(P 0. 05); 2组血清25羟维生素D3水平均较治疗前显著升高(P 0. 05),且观察组显著高于对照组(P 0. 05); 2组红细胞电泳指数、红细胞压积、血浆黏度、全血低切黏度、全血高切黏度水平均较治疗前显著降低(P 0. 05),且观察组均显著低于对照组(P 0. 05)。结论:温阳补肾法治疗高原类风湿关节炎,疗效满意,同时能明显增加血清25羟维生素D3水平。     

CDC25B and CDC25C overexpression in nonmelanoma skin cancer suppresses cell death

Non‐melanoma skin cancer frequently results from chronic exposure to ultraviolet (UV) irradiation. UV‐induced DNA damage activates cell cycle arrest checkpoints through degradation of the cyclin‐dependent kinase activators, the cell division cycle 25 (CDC25) phosphatases. We previously reported increased CDC25A in nonmelanoma skin cancer, but CDC25B and CDC25C had not been previously examined. Consequently, we hypothesized that increased expression of CDC25B and CDC25C increases tumor cell proliferation and skin tumor growth. We found that CDC25B and CDC25C were increased in mouse and human skin cancers. CDC25B was primarily cytoplasmic in skin and skin tumors and was significantly increased in the squamous cell carcinoma (SCC), while CDC25C was mostly nuclear in the skin, with an increased cytoplasmic signal in the premalignant and malignant tumors. Surprisingly, forced expression of CDC25B or CDC25C in cultured SCC cells did not affect proliferation, but instead suppressed apoptosis, while CDC25C silencing increased apoptosis without impacting proliferation. Targeting CDC25C to the nucleus via mutation of its nuclear export sequence, however, increased proliferation in SCC cells. Overexpression of CDC25C in the nuclear compartment did not hinder the ability of CDC25C to suppress apoptosis, neither did mutation of sites necessary for its interaction with 14‐3‐3 proteins. Analysis of apoptotic signaling pathways revealed that CDC25C increased activating phosphorylation of Akt on Ser⁴⁷³, increased inhibitory phosphorylation of proapoptotic BAD on Ser¹³⁶, and increased the survival protein Survivin. Silencing of CDC25C significantly reduced Survivin levels. Taken together, these data suggest that increased expression of CDC25B or CDC25C are mechanisms by which skin cancers evade apoptotic cell death.     

灰毡毛忍冬LmC3H1基因的克隆及表达模式与绿原酸含量相关性分析

目的:以灰毡毛忍冬为材料,克隆对-香豆酸3-羟化酶(LmC3H1)基因,进行生物信息学和表达模式分析,结合绿原酸含量,研究推测灰毡毛忍冬LmC3H1基因的功能。方法:通过逆转录聚合酶链式反应(RT-PCR)和RACE技术克隆LmC3H1基因的全长c DNA序列,对该序列进行生物信息学分析,并利用实时荧光定量PCR(Real-time PCR)和HPLC分别测定灰毡毛忍冬茎、叶及不同花期花中LmC3H1的相对表达量及绿原酸含量。结果:克隆得LmC3H1(Gen Bank:MN177695)基因,开放阅读框(ORF)长度为1 533 bp,编码510个氨基酸,推测其分子式为C_(2618)H_(4134)N_(718)O_(727)S_(22),相对分子质量为58 005.32,等电点8.92,为亲水性蛋白,定位于叶绿体中,具有跨膜区域LLLIPAVLFLISLVYPLI,含有细胞色素P450的保守结构域CYTOCHROME_P450(422-433 aa);Real-time PCR结果显示,LmC3H1在灰毡毛忍冬茎、叶及不同花期花有不同程度的表达,其中在花发育阶段,白色花蕾期相对表达量最高,花蕾初期及白色开花期次之;白色花蕾期花与茎、叶比,花的相对表达量最高,叶的最低;HPLC结果显示,从绿白色花蕾期到金黄色开花期绿原酸含量呈上升趋势,金黄色开花期含量最高,不同器官中,花中绿原酸最高,茎最低。结论:克隆得到灰毡毛忍冬LmC3H1基因,推测LmC3H1可能参与灰毡毛忍冬花绿原酸的生物合成。该研究为进一步研究该基因的功能及探究灰毡毛忍冬绿原酸生物合成和调节机制提供了依据,同时为遗传改良灰毡毛忍冬品质奠定了基础。     

Was It Worth Introducing Health Economic Evaluation of Innovative Drugs in the French Regulatory Setting? The Case of New Hepatitis C Drugs

Objective

This paper constitutes the first attempt to draw lessons from the recent uptake of health economic evaluation of innovative drugs in the French regulatory framework.