Mechanisms of the mechanically activated ion channel Piezo1 protein in mediating osteogenic differentiation of periodontal ligament stem cells via the Notch signaling pathway

目的 探究机械激活性离子通道压电蛋白Piezo1通过Notch信号通路介导人牙周膜干细胞（hPDLSC）成骨分化作用机制。方法 选取自2016年1月1日—2018年1月1日就诊于北京儿童医院正畸科的8~14岁儿童因阻生而拔出的年轻恒牙的牙周膜组织为细胞来源,采用酶消化法对hPDLSC进行提取。采用免疫组织化学染色法检测角蛋白、波形蛋白的表达和流式细胞术对hPDLSC的标志物CD146和STRO-1进行鉴定。构建和筛选siRNA-Piezo1基因干扰载体和Piezo1基因过表达质粒。应用Flexcell 4000T机械牵张应力仪器构建体外牵张力学hPDLSC细胞模型。实验分成5组：siRNA干扰组、过表达组、空白对照组、牵张应力组和阴性对照组。荧光定量聚合酶链反应（RT-qPCR）检测Piezo1、Notch1和成骨基因碱性磷酸酶（ALP）、Runt相关基因2（Runx2）、骨钙素（OCN）和骨涎蛋白（BSP）的表达。Western blot检测成骨标志蛋白ALP和Runx2的表达。Fluo-3 AM探针检测细胞内钙离子含量。结果 hPDLSC波形蛋白染色阳性,角蛋白染色阴性。流式细胞仪检测hPDLSC标志物STRO-1表达阳性,CD146表达阳性。空病毒载体、siRNA-Piezo1干扰序列和Piezo1过表达载体序列均可以通过慢病毒转染hPDLSC,而且转染效率较高,均在90%。逆转录聚合酶链反应结果显示,siRNA干扰组、过表达组、空白对照组、牵张应力组和阴性对照组Piezo1 mRNA的表达水平差异有统计学意义（F=9.573,P<0.05）;过表达组Piezo1 mRNA的表达水平明显高于siRNA干扰组,差异有统计学意义（q=3.893,P<0.05）;牵张应力组Piezo1 mRNA的表达水平明显高于空白对照组,差异有统计学意义（q=2.006,P<0.05）。Notch1和成骨基因ALP、Runx2、OCN和BSP的mRNA表达具有同样的趋势。Western blot检测结果显示siRNA干扰组、过表达组、空白对照组、牵张应力组和阴性对照组成骨标志蛋白ALP蛋白表达差异有统计学意义（F=11.207,P<0.001）;过表达组ALP蛋白的表达水平明显高于siRNA干扰组,差异有统计学意义（q=2.991,P<0.05）;牵张应力组ALP蛋白的表达水平明显高于空白对照组,差异有统计学意义（q=3.007,P<0.05）。Runx2蛋白表达具有同样趋势。细胞内钙离子检测结果显示,过表达组和牵张应力组细胞内钙离子荧光强度明显高于siRNA干扰组。结论 机械牵张应力可以促进Piezo1蛋白的表达,以Ca²⁺为第二信使,激活Notch1信号通路,激活ALP、Runx2、OCN和BSP的表达,促进hPDLSC成骨分化。而siRNA-Piezo1干扰质粒可以阻断这一进程,反之,Piezo1的过表达质粒可以促进hPDLSC成骨分化进程。     

An observational study assessing the pathways to care among treatment seeking users of natural opiates

Abstract

Background: Natural opiate users constitute a large proportion of opioid dependent individuals in India, and enjoy socio-cultural sanction in certain parts of the country. However, no study has assessed the pathways to care among this population in India.

Objective: To assess the pathways to care among treatment-seeking natural opiate dependent individuals.

Method: This cross sectional, explorative study was conducted at a tertiary care drug treatment centre located in North India. A total of 125 male participants aged >18?years, seeking treatment for natural opiate dependence from our outpatient clinic were included. A semi-structured proforma and WHO mental health encounter form was applied to assess socio-demographic, treatment details and pathways to care.

Results: The mean age was 46.17 (±11.98) years. Poppy husk (phukki/doda/posht) was the most common primary natural opiate used (84%). First point of treatment contact was addiction psychiatrist (n?=?90; 72%) in majority. First time treatment seeking was either by self-referral (60.8%) or referral by relatives and friends (24.8%) with mean time lag of 18.63?years after the onset.

Conclusion: Natural opiates dependent patients seek treatment late in the course of their illness, often directly from a tertiary addiction treatment centre. Barriers to seek treatment needs to be addressed.     

小柴胡汤治疗癫痫的网络药理学研究

目的:通过中药网络药理学技术研究小柴胡汤治疗癫痫的活性成分、作用靶标、靶标的相互作用关系、靶标的生物学功能及其作用的通路,探讨其可能的作用机制。方法:在中药系统药理学数据库和分析平台上筛选出小柴胡汤的活性成分,并根据活性成分检索其成分作用的靶标。利用在线文本挖掘服务器(GeneCards)和人类孟德尔遗传数据库(OMIM)查询与癫痫相关的靶标,并使用在线软件venny 2.1 对癫痫相关的靶标与小柴胡汤作用的靶标取交集,得到小柴胡汤治疗癫痫的潜在靶标156个。将潜在靶标导入Cytoscape软件,初步获得“成分-靶标”关系网络;同时基于clusterProfiler包对潜在靶标进行基因本体(GO)功能富集分析、基因组百科全书(KEGG)通路富集分析。并根据KEGG富集分析结果,构建“潜在靶标-KEGG通路”关系网络。此外,为了解靶标之间的相互作用关系,把潜在靶标导入STRING在线数据库,构建蛋白-蛋白相互作用网络(PPI网络),将关系网络编排导入Cytoscape软件进行拓扑结构分析获得核心子网络。结果:小柴胡汤的活性成分有162种,主要包括:槲皮素、汉黄芩素、黄芩苷、山柰酚、人参皂苷Rh2、异鼠李素、柚皮素、木蝴蝶素等;作用的靶标有156个。其主要作用的途径有:IL-17信号通路、细胞凋亡、C型凝集素受体信号通路、内分泌的阻力等。结论:小柴胡汤治疗癫痫体现了中药复方多成分、多靶标、多通路共同作用的特点。本研究进一步拓宽了小柴胡汤在神经系统疾病方面的应用,为进一步的实验研究提供了理论基础。     

STANDARDIZED CLINICAL PATHWAYS FOR ESOPHAGECTOMY ARE NOT A REALITY IN BRAZIL,EVEN WITH A HIGH PREVALENCE OF ESOPHAGEAL CANCER AND ACHALASIA

Background:

The adoption of standardized protocols and specialized multidisciplinary teams for esophagectomy involve changes in routines with the implantation of expensive clinical practices and deviations from ingrained treatment philosophies.

Aim:

To evaluate the prevalence of standardized protocols and specialized multidisciplinary teams in São Paulo state, Brazil.

Methods:

Institutions that routinely perform esophagectomies in São Paulo were contacted and questioned about the work team involved in the procedure and the presence of standardized routines in the preoperatory care.

Results:

Fifteen centers answered the questionnaire: 10 (67%) public institutions and five (33%) private. There were seven (47%) medical schools, six (40%) with a residency program and two (13%) nonacademic institutions. The mean number of esophagectomies per year was 23. There was a multidisciplinary pre-operative team in nine (60%). There was a multidisciplinary postoperative team in 11 (73%). Early mobilization protocol was adopted in 12 (80%) institutions, early feeding in 13 (87%), routinely epidural in seven (47%), analgesia protocol in seven (47%), hydric restriction in six (40%), early extubation in six (40%), standardized hospitalization time in four (27%) and standardized intensive care time in two (13%).

Conclusion:

The prevalence of standardized protocols and specialized teams is very low in Sao Paulo state, Brazil. The presence of specialized surgeons is a reality and standardized protocols related directly to surgeons have higher frequency than those related to other professionals in the multidisciplinary team.     

自噬——脑缺血损伤中的双刃剑

近来研究表明,自噬可能成为卒中治疗过程中的新靶点,但是自噬在脑缺血过程中的激活是否提升神经元存活率一直存在争议,关于其对于脑缺血后神经元发挥的作用是保护还是加重损伤尚不明确。本文主要综述脑缺血后自噬在缺血/低氧损伤中可能发挥的双重作用及其可能的信号通路。     

基于系统生物学整合技术挖掘结直肠癌形成中的核心通路和驱动基因

目的 探索结直肠癌(Colorectal carcinoma, CRC)形成中的核心通路和关键基因;方法 利用Meta分析技术从以往5项CRC发生相关转录组学研究中筛选癌及癌旁差异表达基因;ComBat方法合并5项研究的癌组织基因表达谱数据,针对上述差异表达基因用PCIT软件进行共表达网络构建;CFinder软件揭示该共表达网络中存在的核心亚网络,并用Gather软件确定主要核心亚网络所富集的生物学功能;以重要核心亚网络（或通路）为重点,联合节点基因在CRC中的表达变化方向和相应染色体区域扩增或缺失的信息,发现亚网络功能形成中的候选驱动基因。结果 Meta分析转录组学研究共发现差异表达基因2073个,其中在5项研究中癌组织一致上调1174个,一致下调899个,这些基因在CRC样本中形成的共表达网络包括798个基因节点和1462条边,存在22个核心亚网络;最大的核心亚网络由77个基因和436条边组成,功能涉及细胞周期和增殖信号调控,UBE2C、MYBL2、FAM83D、AURKA、TPX2等11个基因被预测为该信号功能的驱动基因。结论 细胞周期和增殖信号通路是结直肠癌发生中的核心通路,UBE2C和AURKA等11个基因是该核心通路的驱动基因。