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新生大鼠原代软骨细胞分离和培养方法的改进

吉林大学学报（医学版） 2024年第5期50卷 1438-1449页

作者：杨丹聃陈骄阳王馨珩赵泽彤潘莹薛百功高长曌吉林大学基础医学院病原免疫细胞遗传学实验中心吉林长春130021 吉林大学中日联谊医院皮肤科吉林长春130033 吉林大学基础医学院细胞学系吉林长春130021 吉林大学中日联谊医院放疗科吉林长春130033

目的:探讨新生大鼠原代软骨细胞分离和培养的改进方法,以建立高效经济的体外软骨细胞培养体系。方法:从新生大鼠关节中分离原代软骨细胞,分为过夜消化(OD)组和快速消化(RD)组进行分离,OD组软骨细胞采用Ⅱ型胶原酶过夜消化,RD组软骨细胞... 详细信息

目的:探讨新生大鼠原代软骨细胞分离和培养的改进方法,以建立高效经济的体外软骨细胞培养体系。方法:从新生大鼠关节中分离原代软骨细胞,分为过夜消化(OD)组和快速消化(RD)组进行分离,OD组软骨细胞采用Ⅱ型胶原酶过夜消化,RD组软骨细胞采用预消化的物理化学消化相结合的手段分离细胞。采用含0%(空白组1)、1%、2%、4%和10%胎牛血清(FBS),0(空白组2)、0.1、0.2、0.4、0.8、1.0、2.0 g·L^(-1)维生素C(VC)和0(空白组3)、0.5、1.0、2.0、4.0、8.0、10.0μg·L^(-1)聚乳酸-羟基乙酸共聚体(PLGA)纳米粒子的改良培养液培养软骨细胞。将杜氏改良Eagle培养基F12营养混合液(DMEM/F12)与含不同浓度FBS、VC和PLGA的培养液分别混合,并按照各成分浓度进行相应分组。采用细胞计数仪计数各组细胞并检测各组细胞存活率和直径,采用甲苯胺蓝特异性染色法检测各组细胞形态表现,采用CCK-8法检测各组细胞增殖活性,采用细胞黏附实验检测各组细胞黏附率,采用Hoechst/碘化丙碇(PI)染色检测各组细胞凋亡情况,采用MTT法检测改良培养液培养后各组细胞增殖活性,将细胞分为DMEM/F12+10%FBS组(对照组)、DMEM/F12+1%FBS组、DMEM/F12+1%FBS+0.4 g·L^(-1)VC+1μg·L^(-1)PLGA组,采用实时荧光定量PCR(RT-qPCR)法检测改良培养液培养后各组细胞中性别决定区域Y框转录因子9(SOX9)、Ⅱ型胶原α1链(Col2A1)、Ⅹ型胶原α1链(Col10A1)和基质金属蛋白酶13(MMP13)mRNA表达水平,采用免疫荧光染色检测改良培养液培养后各组细胞中Ⅱ型胶原(COLⅡ)和SOX9表达情况。结果:OD组原代软骨细胞存活率小于RD组,细胞平均直径大于RD组。OD组原代软骨细胞形态较大,呈梭形,大多数细胞出现伪足;RD组原代软骨细胞形态较小,大多数细胞呈菱形,仅部分细胞出现伪足。2组原代软骨细胞经甲苯胺蓝特异性染色均显色明显,但RD组消化时间较短,软骨细胞实际培养时间较OD组缩短9~13 h,原代软骨细胞形态更为幼稚。OD组原代软骨细胞在培养24 h时增殖较为缓慢,培养48 h时增殖速度升高,较培养12 h时增殖活性明显升高(P<0.01)。RD组原代软骨细胞在培养24 h时增殖稍缓,培养48 h时增殖速度加快,较培养12 h时增殖活性明显升高(P<0.01);培养24和48 h时,与OD组比较,RD组原代细胞增殖速度升高(P<0.05)。RD组软骨凋亡细胞数少于OD组,2组均无坏死软骨细胞。大鼠软骨细胞增殖活性随着培养液中FBS浓度升高而升高,与空白组1比较,培养液中含1%、2%、4%和10%FBS时,软骨大鼠细胞增殖活性明显升高(P<0.05)。与空白组2比较,培养液中含0.2~1.0 g·L^(-1)VC时大鼠软骨细胞增殖活性明显升高(P<0.05),其中含0.4 g·L^(-1)VC时大鼠软骨细胞增殖活性最高(P<0.01)。与空白组3比较,培养液中含1~4μg·L^(-1)PLGA时,大鼠软骨细胞增殖活性明显升高(P<0.05),其中含1μg·L^(-1)PLGA时大鼠软骨细胞增殖活性最高(P<0.05)。与DMEM/F12+10%FBS组比较,DMEM/F12+1%FBS组大鼠软骨细胞中SOX9 mRNA和COL2A1 mRNA表达水平均明显升高(P<0.05或P<0.01)。与DMEM/F12+10%FBS组比较,DMEM/F12+1%FBS+0.4 g·L^(-1)VC+1μg·L^(-1)PLGA组大鼠软骨细胞中SOX9 mRNA和COL2A1 mRNA表达水平明显升高(P<0.01)。免疫荧光染色,荧光显微镜下DMEM/F12+10%FBS组部分软骨细胞中出现COLⅡ绿色荧光信号和SOX9红色荧光信号,荧光强度弱;DMEM/F12+1%FBS组大多数软骨细胞中出现COLⅡ绿色荧光信号和SOX9红色荧光信号,荧光强度明显强于DMEM/F12+10%FBS组;DMEM/F12+1%FBS+0.4 g·L^(-1)VC+1μg·L^(-1)PLGA组软骨细胞中均出现COLⅡ绿色荧光信号和SOX9红色荧光信号,荧光强度较DMEM/F12+10%FBS组和DMEM/F12+1%FBS组明显升高。DMEM/F12+1%FBS组软骨细胞中COLⅡ和SOX9蛋白表达量明显高于DMEM/F12+10%FBS组,DMEM/F12+1%FBS+0.4 g·L^(-1)VC+1μg·L^(-1)PLGA组软骨细胞中COLⅡ和SOX9蛋白表达量明显高于DMEM/F12+10%FBS组。结论:改良后的大鼠原代软骨细胞分离和培养方法可以弥

关键词：软骨细胞原代细胞培养体外技术条件培养基血清

超声在小儿永存第5主动脉弓诊断评估中的临床价值

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中国超声医学杂志 2024年第3期40卷 336-339页

作者：贺新建魏九茹崔云陈娇阳张帅陶曙光赵宁任哲河北省儿童医院超声诊断科河北省小儿心血管重点实验室河北省儿童健康与疾病临床医学研究中心河北省妇幼保健中心胎儿保健科河北省儿童医院心脏外科

目的探讨超声在小儿永存第5主动脉弓(PFAA)诊断评估中的临床指导价值。方法收集超声诊断且经电子计算机断层扫描血管造影(CTA)或手术证实的11例PFAA患儿资料，分析不同分型的超声特征并进行评估。结果 11例超声诊断与CTA或手术结果均... 详细信息

目的探讨超声在小儿永存第5主动脉弓(PFAA)诊断评估中的临床指导价值。方法收集超声诊断且经电子计算机断层扫描血管造影(CTA)或手术证实的11例PFAA患儿资料，分析不同分型的超声特征并进行评估。结果 11例超声诊断与CTA或手术结果均相符。2例右位PFAA合并血管环或法洛四联证，9例左位PFAA均未合并复杂畸形。Weinberg A型4例：1例右位双腔主动脉弓(无血流梗阻)合并法洛四联证及迷走左锁骨下动脉，行法洛四联证根治术；2例左位双腔主动脉弓无梗阻者未手术；1例左位双腔主动脉弓PFAA远端闭锁伴主动脉缩窄，行主动脉缩窄纠治术。B型7例：第4主动脉弓离断伴PFAA缩窄6例，其中4例重度缩窄左心室收缩功能减低者行急诊手术；1例发现后早期行PAFF缩窄纠治术；1例放弃治疗；1例不伴缩窄者只行房间隔缺损修补术。补充1例C型胎儿病例，追踪失访。结论重度缩窄导致左心收缩功能减低是小儿PFAA最常见的危害之一，需急诊手术，右位PFAA易合并血管环及其他复杂畸形。超声在PFAA的诊断评估中具有重要价值。

关键词：先天性心脏病永存第5主动脉弓超声

An Efficient Probe for Bacterial Nitroreductase Imaging and Detection Based on NanoLuc-Furimazine Bioluminescent Pair

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Chinese Journal of Chemistry 2024年第12期42卷 1373-1380页

作者： Ximeng Shi Yumeng Wang jiaoyang Yu Yating Yang Zecheng Jin Hanghang Wang Dalei Wu Wei chen Jianming Guo Yinan Zhang Jiangsu Key LaboratoryforFunctional Substances of Chinese Medicine School of PharmacyNanjing Universityof Chinese MedicineNanjingJiangsu210023China Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization School of PharmacyNanjing University of ChineseMedicineNanjingJiangsu210023China Clinical Research Center the Second Hospital of NanjingAffiliated Hospital to Nanjing University of Chinese MedicineNanjingJiangsu210003China Key Laboratory of Resources Biology and Biotechnology in Western China Ministry of EducationCollege of Life SciencesNorthwestUniversityXi'anShaanxi710069China Helmholtz International Lab State Key Laboratoryof Microbial TechnologyShandong UniversityQingdaoShandong 266237China

Comprehensive SummaryThe detection of critical endogenous species, such as bacteria in microenvironments in the body, requires better imaging tools for visualization and monitoring of biological events. Bioluminescence imaging is the most popular strategy for obtaining real-time in living cells and organisms. Herein, we introduced a nitroaryl group on the C-3 position and a hydroxy group at the C-6 phenyl ring on furimazine to report the first bioluminescent probe (7) based on NanoLuc-furimazine bioluminescent pair for the detection of nitroreductase in bacteria. The probe, which possessed up to 560-fold intensity increase with a low detection limit of 16 ng/mL of nitroreductase, has the most efficient uncage efficiency in comparison with other bioluminescent congeners, thus enabling highly selective and sensitive visualization of NTR activity in a panel of clinical priority pathogens. Additionally, imaging of the recombinant strain as well as the NTR from mouse feces indicated the potential of this probe in the application of different mouse disease models.

关键词： Nitroreductase NanoLuc-furimazine Biosensors Analytical methods Luminescence Imaging agents

Clinical phenotype features and genetic etiologies of 38 children with progressive myoclonic epilepsy

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Acta Epileptologica 2020年第1期2卷 111-125页

作者： Jing Zhang Ying Yang Xueyang Niu jiaoyang chen Wei Sun Changhong Ding Lifang Dai Liping Zhang Qi Zeng Yi chen Xiaojuan Tian Xiaoling Yang Taoyun Ji Zhixian Yang Yanling Yang Yuwu Jiang Yuehua Zhang Department of Pediatrics Peking University First HospitalNo.1 of Xian Men StreetXicheng DistrictBeijing 100034China. Department of Neurology Xuanwu HospitalCapital Medical UniversityNo.45 of Changchun StreetXicheng DistrictBeijing 100034China. Department of Neurology Beijing Children’s HospitalCapital Medical UniversityNo.56 of South Lishi RoadXicheng DistrictBeijing 100045China. Department of Pediatrics Xuanwu HospitalCapital Medical UniversityNo.45 of Changchun StreetXicheng DistrictBeijing 100034China.

Background:Progressive myoclonic epilepsy(PME)is a group of neurodegenerative diseases with genetic heterogeneity and phenotypic similarities,and many cases remain unknown of the genetic *** study is aim to summarize the clinical features and study the genetic causes of PME ***:Sanger sequencing of the target gene,Next Generation Sequencing(NGS)panels of epilepsy,trio-based Whole Exome Sequencing(WES)and detection of cytosine-adenine-guanine(CAG)repeat number were used to investigate the genetic causes of PME ***:Thirty-eight children with PME whose seizure onset age ranged from 3 months to 12 years were collected from February 2012 to November 2019 in three hospitals in Beijing,*** seizure types included myoclonic seizures(n=38),focal seizures(n=19),generalized tonic-clonie seizure(GTCS)(n=13),absence seizures(n=4),atonic seizures(n=3),epileptic spasms(n=2)and tonic seizures(n=1).Twenty-seven cases were sporadic and 11 had family members *** PME-related genes were identified in 30 out of 38(78.9%)patients who had either recessively inherited or de novo heterozygous *** these 30 cases,there were 12 cases(31.6%)of neuronal ceroid lipofuscinoses(the causing gene contains TPP1,PPT1,CLN5,CLN6 and MFSD8),two cases of sialidosis(the causing gene is NEU1),two cases of neuronopathic Gaucher disease(the causing gene is GBA),one case of spinal muscular atrophy-progressive myoclonic epilepsy(the causing gene is ASAH1),four cases of KCNC1 mutation-related PME,four cases of KCTD7 mutation-related PME,two cases of TBC1D24 mutation-related PME,one case of GOSR2 related PME,and two of dentatorubral-pallidoluysian atrophy(the causing gene is ATN1).In total,13 PME genes were identified in our *** etiology was not clear in eight ***:PME is a group of clinically and genetically heterogeneous *** diagnosis was clear in 78.9%of PME *** of genetic testing methods could inc

关键词： Progressive myoclonic epilepsy Genotype Phenotype

癫痫伴肌阵挛-失张力发作的分子遗传学研究进展

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中华儿科杂志 2019年第5期57卷 384-387页

作者：陈娇阳张月华北京大学第一医院儿科 100034

癫痫伴肌阵挛-失张力发作(EMAS)发病率低,包括多种全面性发作类型,预后个体差异大。目前认为遗传因素在EMAS发病中起主要作用,文献报道已发现8个致病基因与EMAS发病有关,分别为SCN1A、SCN1B、SCN2A、CHD2、SLC2A1、SLC6A1、SYNGAP1和ST... 详细信息

癫痫伴肌阵挛-失张力发作(EMAS)发病率低,包括多种全面性发作类型,预后个体差异大。目前认为遗传因素在EMAS发病中起主要作用,文献报道已发现8个致病基因与EMAS发病有关,分别为SCN1A、SCN1B、SCN2A、CHD2、SLC2A1、SLC6A1、SYNGAP1和STX1B基因,现对该病的分子遗传学研究进展进行综述。

关键词：分子遗传学发作类型肌阵挛张力癫痫 SCN1A 发病率个体差异

Cancer stem cells: advances in knowledge and implications for cancer therapy

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Signal Transduction and Targeted Therapy 2024年第8期9卷 3242-3304页

作者： Xianjing Chu Wentao Tian jiaoyang Ning Gang Xiao Yunqi Zhou Ziqi Wang Zhuofan Zhai Guilong Tanzhu Jie Yang Rongrong Zhou Department of Oncology Xiangya HospitalCentral South UniversityChangsha 410008China Department of Dermatology Xiangya HospitalCentral South UniversityChangsha 410008China xiangya Lung Cancer Center Xiangya HospitalCentral South UniversityChangsha 410008China National Clinical Research Center for Geriatric Disorders Xiangya HospitalCentral South UniversityChangshaHunan Province 410008China

Cancer stem cells(CSCs),a small subset of cells in tumors that are characterized by self-renewal and continuous proliferation,lead to tumorigenesis,metastasis,and maintain tumor *** continues to be a significant global disease *** the past,surgery,radiotherapy,and chemotherapy were the main cancer *** technology of cancer treatments continues to develop and advance,and the emergence of targeted therapy,and immunotherapy provides more options for patients to a certain ***,the limitations of efficacy and treatment resistance are still *** review begins with a brief introduction of the historical discoveries,original hypotheses,and pathways that regulate CSCs,such as WNT/β-Catenin,hedgehog,Notch,NFkB,JAK/STAT,TGF-β,PI3K/AKT,PPAR pathway,and their *** focus on the role of CSCs in various therapeutic outcomes and resistance,including how the treatments affect the content of CSCs and the alteration of related molecules,CSCs-mediated therapeutic resistance,and the clinical value of targeting CSCs in patients with refractory,progressed or advanced *** summary,CSCs affect therapeutic efficacy,and the treatment method of targeting CSCs is stll difficult to *** regulatory mechanisms and targeting biomarkers of CSCs is currently the mainstream idea.

关键词： Cancer maintain knowledge