POLE2对非小细胞肺癌细胞侵袭迁移能力的影响

目的 探讨POLE2对非小细胞肺癌（NSCLC）细胞侵袭迁移能力的影响。方法 采用实时荧光定量PCR（qRT-PCR）检测人支气管上皮样细胞BEAS-2B和NSCLC细胞A549、SPC-A1中POLE2的表达水平。构建POLE2过表达细胞模型,qRT-PCR和蛋白质印迹法（Western blot）验证转染效率。采用Cell Counting Kit-8（CCK-8）实验检测NSCLC细胞的增殖能力,Transwell实验检测NSCLC细胞的侵袭迁移能力,qRT-PCR检测MMP9的表达水平。结果 人NSCLC细胞SPC-A1、A549中POLE2的表达水平显著高于人支气管上皮样细胞BEAS-2B（P<0.05）。过表达POLE2促进了A549细胞的增殖和侵袭迁移,上调了MMP9的表达（P<0.05）。结论 过表达POLE2可能通过上调MMP9促进NSCLC细胞的侵袭迁移。     

FDX1在胶质瘤中的表达及其预后意义

目的 探讨铁氧还原蛋白1（FDX1）在胶质瘤中的表达水平和预后意义。方法 采用免疫组织化学法检测我院神经外科40例恶性胶质瘤样本和20例外伤性脑组织样本中FDX1的表达水平,分析其与患者临床病理因素和预后的关系。结果 FDX1在恶性胶质瘤组织中的蛋白表达水平显著高于外伤性脑组织（P<0.01）;FDX1的表达与恶性胶质瘤病理分级呈正相关（P<0.01）;Kaplan-Meier生存分析显示FDX1高表达组患者总生存率显著低于低表达组患者（P<0.01）。Cox多因素回归模型显示FDX1表达强度（相对危险度为2.003,P<0.01）和肿瘤分级（相对危险度为2.279,P<0.01）是恶性胶质瘤患者预后的独立危险因素。结论 FDX1高表达与恶性胶质瘤的发生发展有关,对患者总体生存时间有显著影响,可能是胶质瘤患者预后不良的重要指标。     

肝癌、肝硬化中自然杀伤细胞的免疫组织化学研究

目的: 研究原发性肝细胞癌、肝硬化、正常肝组织中自然杀伤(nature killer,NK)细胞数量、分布及其与患者预后的关系.方法: 原发性肝细胞癌60例,单纯性肝硬化62例,正常肝组织23例,以SP免疫组化法检测NK细胞.结果: ①癌中与癌旁组织的NK细胞计数明显高于肝硬化(P<0.01),癌中NK细胞计数高于正常肝组织(P<0.05).②肝癌组织学分级与NK细胞数量无明显关系.③癌中NK细胞随着临床分期的发展有下降的趋势(P<0.05).④15月内转移复发组癌中和癌旁的NK细胞计数均明显低于无转移复发组(P<0.01).结论: NK细胞计数可能是反映机体抗肿瘤免疫状态和判断患者预后的重要指标.     

特异结合唾液酸化LewisX抗原DNA适配子抑制肝癌细胞株HepG2体外侵袭转移

目的 观察特异结合唾液酸化LewisX (SLeX)抗原DNA适配子抑制HepG2细胞与E-选择素黏附能力及其体外抑制HepG2细胞浸润转移能力.方法 采用黏附实验、Transwell体外侵袭实验,检测该适配子对HepG2细胞与E选择素黏附及对HepG2细胞体外侵袭的影响.结果 该DNA适配子可有效抑制HepG2细胞与E-选择素黏附,黏附细胞数随适配子浓度的增加而减少(P＜0.01),20 nmol浓度的适配子与单克隆抗体CSLEX-1效果相似;Transwell侵袭实验中,5、10、20nmol适配子组的侵袭细胞数分别为159.00±3.27、142.00±5.50、115.00±5.07,与对照组(178.00±4.64)比较,差异有统计学意义(P＜0.01).结论 特异结合唾液酸化LewisX抗原DNA适配子可以抑制HepG2细胞与E-选择素黏附,阻断Lewis-selectin途径,抑制HepG2细胞体外侵袭转移.     

三氧化二砷抑制肝癌细胞侵袭转移的实验研究

目的了解三氧化二砷（As2O3）对肝癌HepG2细胞体外黏附、侵袭和迁移的抑制作用。方法采用MTT法观察As2O3对HepG2细胞黏附能力的影响,以Transwell检测As2O3对HepG2细胞迁移、侵袭能力的影响。结果As2O3作用后30、60、90、120min时的细胞黏附率较对照组均明显下降,不同时间组与对照组之间差异均有显著性（P〈0.05）;As2O3作用后游走及侵袭穿膜细胞数也较对照组明显下降（P〈0.01）。结论As2O3可明显抑制HepG2细胞细胞黏附、迁移和侵袭的能力。     

微侵袭血肿清除术对高血压脑出血患者术后神经功能恢复及并发症的影响

目的 分析微侵袭血肿清除术对高血压脑出血患者术后神经功能恢复及并发症的影响。方法 选取2017年6月至2020年5月期间我院38例高血压脑出血患者,按照手术方式不同将患者分为微侵袭组（n=12）和大骨瓣组（n=26）。微侵袭组给予微侵袭血肿清除术治疗;大骨瓣组采用基底节区改良翼点入路,骨窗大小约为6 cm×8 cm。比较两组患者临床疗效、手术情况、临床神经功能缺失量表（NDS）评分、日常生活能力量表（ADL）评分以及并发症发生情况。结果 微侵袭组患者总好转率为91.67%,高于大骨瓣组的73.07%,差异无统计学意义（P>0.05）;微侵袭组手术时间、术中出血量明显小于大骨瓣组（P<0.05）,两组血肿清除率、住院时间比较无明显差异（P>0.05）;手术后3个月,两组患者NDS评分均明显低于本组手术前（P<0.05）,ADL评分均明显高于本组手术前（P<0.05）,微侵袭组患者NDS评分明显低于大骨瓣组（P<0.05）,ADL评分明显高于大骨瓣组（P<0.05）;两组患者颅内感染、肺部感染以及再出血并发症总发生率对比无统计学差异（P>0.05）。结论 与传统大骨瓣开颅术相比,微侵袭血肿清除术治疗高血压脑出血更加安全有效,患者术后神经功能恢复更好,并发症更少。     

FOS样抗原1在头颈鳞癌中的表达及临床意义

目的 探讨FOS样抗原1（FOSL1）蛋白在头颈鳞癌中的表达及其与临床病理特点及患者预后的关系。方法 使用UALCAN数据库预测FOSL1在头颈鳞癌（HNSC）中的表达情况,收集98例头颈鳞癌患者,采用免疫组化法检测头颈鳞癌及其癌旁正常组织中FOSL1蛋白的表达,并分析其表达与临床特征的关系。结果 UALCAN数据库及免疫组化结果均显示FOSL1蛋白在头颈鳞癌中的相对表达量明显高于癌旁正常组织（P<0.05）;FOSL1的表达与头颈鳞癌的大小、淋巴结转移、病理分级及HPV状态密切相关（P<0.05）。Kaplan-Meier分析显示FOSL1高表达组HNSC患者总积生存率明显低于低表达组HNSC患者（P< 0.05）。结论 FOSL1蛋白参与头颈鳞癌的发生发展,FOSL1的表达水平有助于头颈鳞癌恶性程度的评价及预后的判断。     

PCNA与Caspase-3在胆囊癌组织中的表达及其临床意义

目的 探讨增殖细胞核抗原(PCNA)和半胱氨酸天冬氨酸蛋白酶3(Caspase-3)在胆囊癌组织中的表达及临床意义.方法 采用免疫组化检测42例胆囊癌组织和10例慢性胆囊炎组织石蜡切片中PCNA和Caspase-3的表达水平,并分析与临床病理特征之间的关系.结果 (1)PCNA的阳性表达率在胆囊癌及慢性胆囊炎组分别为64.29%和30‰两组间差异有显著性(P<0.05);Caspase-3在胆囊癌及慢性胆囊炎组分别为30.95%和70%,两组间差异有显著性(P<0.05).(2)胆囊癌组织中,PCNA和Caspase-3表达水平在性别、年龄方面差异无统计学意义(P>0.05),但与淋巴结转移、癌组织分化程度及病理TNM分期有关(P<0.05).(3)PCNA和Caspase-3的表达呈负相关(rs=-0.62,P<0.01).结论 PCNA和Caspase-3的表达在胆囊癌组织中呈负相关,且两者表达水平与胆囊癌淋巴结转移、癌组织分化程度及病TNM分期有关,提示PCNA和Caspase-3是反映胆囊癌生物学行为的重要指标.     

胃癌组织中HIF-1α,CD68及CD206在胃癌及癌旁组织中的表达及临床意义

目的 分析低氧诱导分子-1α（HIF-1α）及肿瘤相关巨噬细胞（TAM）相关抗原CD68、CD206在胃癌及癌旁组织中的表达情况。方法 利用免疫组化技术检测43例胃癌和癌旁组织中HIF-1α、CD68、CD206的表达状态,计算三种蛋白表达的阳性率及阳性细胞数,揭示其与临床因素的相关性。结果 HIF-1α、CD68、CD206的阳性表达率分别为58.1%、69.8%、51.2%,均高于癌旁组织（P<0.05）。胃癌、癌旁组织中每个视野下CD68⁺细胞数分别为（39.7±7.6）、（8.5±2.8）个;CD206⁺阳性细胞数分别为（32.0±9.2）、（3.4±1.8）个;HIF-1α⁺细胞数（22.9±5.6）、（2.1±1.2）个;组间比较差异均有统计学意义（P<0.01）。胃癌组织中CD206⁺细胞数与HIF-1α⁺细胞数表达呈正相关（P<0.01,R²=0.641）。三种蛋白的表达与胃癌病理分期、淋巴结转移明显相关（P<0.05）。结论 胃癌组织中CD68、CD206、HIF-1α表达率及阳性细胞数明显增加,且CD206与HIF-1α表达呈正相关。胃癌缺氧区域对M2型巨噬细胞有趋化作用,促进胃癌发生发展。     

血晶素对大鼠70%肝脏切除后再生的影响

目的 探讨血晶素(Hemin)对大鼠70%肝脏切除术后肝脏再生的影响.方法 复制大鼠70%肝脏切除模型,随机分成血晶素治疗组和生理盐水对照组,在术后第1天、第2天、第3天和第7天测定比较肝重/体重、血清肿瘤坏死因子α(TNF-α)、肝脏组织中血晶素加氧酶1(HO-1)含量及肝细胞核增殖蛋白抗原(PCNA)表达指数.结果 术后第7天治疗组肝重/体重明显高于对照组(P<0.05),第3天开始肝细胞 PCNA表达指数较对照组升高,差异有统计学意义(P<0.01).治疗组术后肝脏组织中HO-1浓度比对照组高,血清TNF-α的含量比对照组低(P<0.05).结论 血晶素大鼠肝脏70%切除术后肝脏再生的速度明显提高,这可能跟HO-1表达升高有关.     

血管内皮生长因子-C shRNA对肝癌HepG2细胞增殖及侵袭能力的影响

目的 检测靶向血管内皮生长因子-C(vasenlar endothelial growth factor-C,VEGF-C)shRNA质粒载体对肝癌HepG2细胞增殖及侵袭能力的影响.方法 构建VEGF-C shRNA质粒载体,脂质体转染方法转入肝癌HepG2细胞.通过倒置荧光显微镜及流式细胞仪检测细胞的转染率;RT-PCR及Western blot检测转染细胞内VEGF-C mRNA及蛋白的表达变化;MTT法检测细胞增殖抑制率;人工基底膜体外侵袭实验检测细胞侵袭能力.结果 VEGF-C shRNA稳定转染后,肝癌HepG2细胞内VEGF-C mRNA及蛋白表达水平显著下降;VEGF-C shRNA对肝癌HepG2细胞具有明显的增殖抑制作用,其抑制增殖效应呈时间依赖性;VEGF-C shRNA可有效抑制肝癌HepG2细胞的人工基底膜体外侵袭能力,抑制率为51.54%.结论 VEGF-C在肝癌增殖、侵袭转移中发挥重要作用;通过RNA干扰技术实现VEGF-C沉默,在肝癌的基因治疗中具有较好的应用前景.     

曲格列酮对肝癌HepG2细胞生长和分化的影响

目的 探讨过氧化物酶体增殖物活化受体(peroxisome proliferator-activated receptor, PPAR)γ激动剂曲格列酮对肝癌HepG2细胞生长和分化的影响.方法 以曲格列酮处理体外培养的肝癌HepG2细胞,MTT法检测细胞增殖,流式细胞仪检测细胞周期,分化标记物E-钙黏蛋白和清蛋白分别用免疫细胞化学和溴甲酚绿法检测,化学发光法检测肿瘤标记物AFP.Western blot检测细胞周期蛋白Dl、c-myc蛋白的表达.结果 曲格列酮以浓度依赖性方式抑制肝癌细胞生长,使细胞周期明显阻滞于G0/G1,并诱导E-钙黏蛋白表达.经曲格列酮处理后,清蛋白分泌量显著增加,AFP明显下降,细胞周期蛋白Dl、c-myc蛋白表达水平下降.结论 曲格列酮可诱导肝癌细胞分化,抑制其生长,其机制可能涉及下调细胞周期蛋白D1和c-myc蛋白表达.     

Rac1基因沉默对髓母细胞瘤细胞侵袭移动的影响

目的 观察Rac1基因沉默后对髓母细胞瘤细胞侵袭移动的影响.方法 用逆转录-聚合酶链反应(RT-PCR)和Western blot方法检测Rac1 mRNA和蛋白在髓母细胞瘤Daoy细胞株中的表达;通过转染Rac1 shRNA观察Rac1基因沉默后Daoy细胞骨架的变化,再转染Rac1 N17和Rac1 L61,观察Rac1基因缺失和表达增强后对Daoy细胞侵袭、移动的影响.结果 Rac1 mRNA和蛋白在Daoy细胞株中均有高表达;Rac1基因沉默后Daoy细胞交联的F-actin网和细胞膜伪足形成减少;单层细胞划痕24 h后,Rac1 shRNA组、Rac1 N17组、对照质粒(control)组细胞迁移数分别是86±4、97±6、198±7;Rac1 shRNA和Rac1 N17两组分别与control组比较,差异均有统计学意义(P<0.01).在侵袭实验中,Rac1 shRNA组、Rac1 N17组、对照质粒组细胞侵袭数分别是30±4、45±6、78±7;Rac1 shRNA组和Rac1 N17两组与对照组比较,差异均有统计学意义(P<0.01).结论 Rac1基因与Daoy细胞骨架形成密切相关,RNA干扰沉默Rac1基因可以抑制Daoy细胞的侵袭移动.     

丙型肝炎病毒核心蛋白反式激活基因2表达产物的亚细胞定位

目的研究HCV核心蛋白反式激活基因2(TAHCCP2)在细胞内的表达及表达产物的亚细胞定位。方法构建HBeAgTP基因的绿色荧光蛋白表达质粒pEGFP-C1-TAHCCP2,转染HepG2细胞,24h后荧光显微镜下观察表达蛋白的亚细胞定位。结果成功构建出TAHCCP2基因的绿色荧光蛋白表达质粒pEGFP-C1-TAHCCP2,其表达的蛋白定位于细胞核。结论TAHCCP2基因可表达TAHCCP2蛋白且定位于细胞核。     

NF-κB、Caspase-3在腺苷诱导人HepG2细胞凋亡中的作用

目的 研究NF-κB、Caspase-3在腺苷(ADO)体外诱导人肝癌HepG2细胞凋亡中的作用.方法 将不同浓度的ADO(0.1～5 mmol/L)作用于HepG2细胞,采用MTT法测定ADO抑制细胞增殖的时间效应和剂量效应.2.0 mmol/L ADO单用或联合NF-κB抑制剂吡咯烷二硫氨基甲酸(PDTC,100 μmol/L)作用HepG2细胞12 h、24 h,采用Hoechst 33342荧光染色法及流式细胞术(FCM)观察细胞凋亡及细胞周期,Western blot技术检测NF-κB蛋白表达;荧光比色法测定Caspase-3酶活性.结果 ADO对HepG2细胞生长具有显著抑制作用,并呈一定的量效和时效关系.药物作用24 h、48 h的IC50分别为2.52 mmol/L和1.89 mmol/L.ADO单独处理HepG2细胞12 h和24 h或联合PDTC处理后,细胞凋亡率分别为8.30%、22.32%;20.18%、30.89%,均显著高于对照组(0.81%,P<0.001);ADO作用HepG2细胞后荧光显微镜观察到细胞凋亡的形态学改变,FCM分析药物处理组显示典型特征性的亚二倍体凋亡峰(sub-G1),细胞生长周期阻滞于G0/G1期;同时伴有Caspase-3活性显著升高(P<0.05).ADO处理后显著增加了NF-κB蛋白表达(P<0.05);PDTC有效抑制了NF-κB表达,同时增加了 Caspase-3活性及ADO的细胞毒作用(P<0.05).结论 ADO诱导了HepG2细胞凋亡并活化Caspase-3.抑制NF-κB活性可通过Caspase-3途径增强ADO的细胞毒作用.     

Regulation of osteoclast apoptosis and motility by small GTPase binding protein Rac1.

The role of Rac1 in osteoclast survival and bone-resorbing activity was examined using adenovirus vector expression systems. Rac1 is critically involved in M-CSF receptor signaling and mediates survival signaling primarily through PI3K/Akt pathways. Rac1 also plays a significant role in bone resorptive activity, probably by regulating the motility of osteoclasts. INTRODUCTION: Rac1 is a member of Rho family small G-proteins, and recent studies have revealed that it mediates anti-apoptotic signals in some types of cells. Rac1 is reported to be required for the cytoskeletal organization and bone-resorbing activity of osteoclasts, but their roles in osteoclast survival and function are not fully elucidated. MATERIALS AND METHODS: We constructed the adenovirus vector carrying cDNA of either the dominant negative Rac1 (Rac1(DN)) or constitutively active Rac1 (Rac1(CA)) gene, and osteoclast-like cells (OCLs) generated in mouse co-culture system were infected with these viruses. To examine the role of Rac1 in osteoclast survival and function, we performed pit formation assays, survival assays, and Western blotting, including an activated-Rac1 pull-down assay using adenovirus-infected OCLs. To further clarify the mechanism of Rac1 regulation in osteoclast survival, some specific inhibitors and adenovirus vectors of signal transduction molecules were used. To quantify membrane movement before and after macrophage colony-stimulating factor (M-CSF) treatment, OCLs expressing either enhanced green fluorescent protein (EGFP) or Rac1(DN) were recorded with a time-lapse video microscope. RESULTS: Adenovirus vector-mediated dominant negative Rac1 (Rac1(DN)) expression significantly reduced pit formation, and promoted their apoptosis. M-CSF rapidly activated Rac1, and the prosurvival effect of M-CSF for OCLs was abrogated by Rac1(DN) overexpression. Constitutively active Rac1 enhanced OCL survival, which was completely suppressed by phosphatidylinositol 3'-kinase (PI3K) inhibitors, whereas a Mek inhibitor had only partial effect. Rac1(DN) also partially blocked the activation of Akt induced by the overexpressing catalytic subunit of PI3K. Using time-lapse video microscopy, we found that Rac1(DN) expression reduced membrane ruffling and the spreading of OCLs in response to M-CSF. CONCLUSIONS: Small guanosine triphosphatase (GTPase) Rac1 is critically involved in M-CSF receptor signaling and mediates survival signaling of osteoclasts primarily by modulating PI3K/Akt pathways. Rac1 also plays a significant role in the bone resorptive activity of cells, probably by regulating the motility of osteoclasts.     

表柔比星+5-氟尿嘧啶、奥沙利铂对人肝癌HepG2细胞增殖及凋亡的影响

目的观察表柔比星、奥沙利铂、5-氟尿嘧啶(5-FU)三种药物联合对人肝癌细胞株HepG2生长的影响,并探讨其可能发生的机制。方法设立空白对照组、表柔比星组、5-FU+奥沙利铂组、表柔比星+奥沙利铂+5-FU组。经MTT实验方法确认各药物48 h的IC50做为联合用药的浓度分别为表柔比星(3.44μmol/L)、5-FU(1.76 mmol/L)、奥沙利铂(6.89mmol/L),实时荧光定量PCR实验方法测定各组间Bcl-2基因、Bax基因表达的变化,Western blot实验方法测定各组间Bcl-2蛋白、Bax蛋白表达的变化。结果各用药组均够抑制HepG2细胞增殖、诱导细胞凋亡、增加Bax mRNA及Bax蛋白的表达,同时降低Bcl-2mRNA及Bcl-2蛋白的表达,且表柔比星+奥沙利铂+5-FU组的作用优于表柔比星组、奥沙利铂+5-FU组,差异有统计学意义(P0.01)。结论表柔比星+奥沙利铂+5-FU组能够明显抑制HepG2细胞的增殖,其联合作用可能通过促进Bax基因的表达,抑制Bcl-2基因的表达,从而加速肿瘤细胞的凋亡。     

Rac1蛋白调控表皮干细胞迁移促进创面愈合的研究

目的 观察Rac1蛋白对表皮干细胞(ESC)迁移运动的调控作用,为完善创面愈合的基础理论以及指导临床应用提供参考.方法 将慢病毒空载体FUGW单独和分别与Rac1蛋白抑制型突变体Rac1T17N、Rac1蛋白活化型突变体Rac1Q61L融合后转染入ESC,按照随机数字表法分为3部分进行如下实验.(1)将ESC分别接种于Ⅰ型胶原溶液(20 μg/mL)、Ⅳ胶原溶液(20 μg/mL)或纤连蛋白溶液(10 μg/mL)包被的24孔细胞培养板,采用CytoTox 96 比色试剂盒检测ESC对不同基质的黏附率.(2)选取上述黏附于Ⅳ型胶原的1000个ESC,四甲基异硫氰酸罗丹明标记的鬼笔环肽染色,激光扫描共聚焦显微镜下观察黏附细胞的形态延展并比较面积大小.(3)选用Transwell小室,上室加入ESC、下室中加入含基质细胞衍生因子1(SDF-1)的限定性角质形成细胞无血清培养液(以不加SDF-1的培养液为对照),倒置相差显微镜下观察ESC的趋化能力,结果以细胞迁移变化率表示.(4)将ESC接种于6孔细胞培养板孵育12 h,加入含4 μg/mL丝裂霉素C的培养液继续孵育2 h,于单层贴壁细胞划痕,6、12 h后统计剩余划痕宽度百分率.对数据进行t检验.结果 与转染FUGW的ESC比较,转染Rac1Q61L的ESC对Ⅰ型胶原的黏附率明显增加(t＝5.302,P<0.05),转染Rac1T17N的ESC对Ⅰ型胶原(t＝13.741,P<0.05)、Ⅳ型胶原(t＝15.676,P<0.05)及纤连蛋白(t＝8.256,P<0.05)的黏附率均明显下降.激光扫描共聚焦显微镜下观察,与转染FUGW的ESC比较,转染Rac1Q61L的ESC面积明显增大,细胞边缘有层板状伪足伸出;转染Rac1T17N的ESC面积显著缩小.在趋化因子SDF-1作用下,与转染FUGW的ESC比较,转染Rac1Q61L的ESC迁移变化率升高43.4%,转染Rac1T17N的ESC迁移变化率下降78.0%;无SDF-1作用时,与转染FUGW的ESC比较,转染Rac1T17N的ESC迁移变化率下降55.2%,转染Rac1Q61L的ESC迁移变化率未见明显变化(升高1.7%).划痕后6、12 h,转染Rac1Q61L的ESC剩余划痕宽度百分率分别为(39±9)%、(6±5)%,低于转染FUGW的ESC[(43±5)%,t=1.027,P>0.05;(18±7)%,t=4.389,P<0.05];划痕后6、12 h,转染Rac1T17N的ESC剩余划痕宽度百分率分别为(81±9)%、(71±11)%,明显高于转染FUGW的ESC(t值分别为11.386、11.726,P值均小于0.05).结论 Rac1蛋白可通过影响ESC的黏附、延展以及趋化能力调控细胞迁移,并可能因此参与ESC促进创面愈合的进程.

Abstract：

Objective To investigate modulatory role of Rac1 protein in epidermal stem cell (ESC) migration during wound healing,in order to provide a reference for enriching basic theory of wound healing and guiding clinical application. Methods Constitutively active mutant of Rac1 protein (Rac1Q61L) or dominant negative isoform of Rac1 protein (Rac1T17N) was transfected into ESC using a retroviral vector FUGW,and retroviral vector FUGW transfected into ESC in singles was used as blank control. The cells were divided into 3 parts according to the random number table and treated as follows. First,equal numbers of cells were inoculated into 24-well plates coated with collagen Ⅰ (20 μg/mL),collagen Ⅳ (20 μg/mL) or fibronectin (10 μg/mL). Cells adhered to above matrices were quantitated using CytoTox 96 colorimetric kit. Second,1000 cells adhered to collagen Ⅳ,after being stained with tetramethyl rhodamine isothiocyanate-phalloidin,were collected for observation of cell morphology and comparison of spreading area under confocal laser scanning microscope. Third,ESC with density of 2×105 cells per well were placed in upper compartment of Transwell chamber,DK-SFM culture medium alone or that containing stromal cell derived factor 1 (SDF-1) was added into lower compartment of Transwell chamber. Migration of ESC was observed using inverted phase contrast microscope,and the result was denoted as migration rate. Lastly,ESC with density of 7.5×105 cells per well was inoculated into 6-well plates for 12 hours,and treated with 4 μg/mL mitomycin C for 2 hours. The remaining scratch width of monolayer was respectively measured 6 hours or 12 hours after scratching to calculate the percentage of remaining scratch width. Data were processed with t test.Results Compared with that of blank control,the number of Rac1Q61L-transfected cells adhered to collagen Ⅰ was significantly increased (t＝5.302,P<0.05),while the number of Rac1T17N-transfected cells adhered to collagen Ⅰ,Ⅳ,and fibronectin were all obviously decreased (with t value respectively 13.741,15.676,8.256,P values all below 0.05). Confocal laser scanning microscope showed that spreading area of Rac1Q61L-transfected ESC (with laminate pseudopodia on edge) and Rac1T17N-transfected ESC was respectively larger and smaller as compared with that of blank control. With SDF-1 effect,the migration rate of Rac1T17N-transfected ESC was decreased by 78.0% and Rac1Q61L-transfected ESC was increased by 43.4% as compared with that of blank control. Without SDF-1 effect,the migration rate of Rac1T17N-transfected ESC was decreased by 55.2%,while the migration rate of Rac1Q61L-transfected ESC was close to that of blank control. Six or 12 hours after scratching,the percentage of remaining scratch width in Rac1Q61L-transfected ESC was lower as compared with that in blank control[(39±9)% vs. (43±5)%,(6±5)% vs. (18±7)%,with t value respectively 1.027,4.389,with P value respectively above and below 0.05],while that in Rac1T17N-transfected ESC[(81±9)%,(71±11)%,respectively]was obviously higher as compared with that in blank control (with t value respectively 11.386,11.726,P values all below 0.05). Conclusions Rac1 protein may control the migration of ESC by regulating its adhesion,spreading,and chemotaxis,and it plays an active role in wound healing accelerated by ESC.     

Iron availability and complex stability of iron hydroxyethyl starch and iron dextran a comparative in vitro study with liver cells and macrophages.

BACKGROUND: Intravenous iron (IVI) therapy is required in patients with end-stage renal disease (ESRD) under chronic haemodialysis (HD). In this in vitro study we investigated the availability and stability of iron hydroxyethyl starch (iron-Hes) compounds in THP-1 cells (macrophage phenotype) and liver cells (HepG2 cells) and compared it with the well-known iron dextran. METHODS: The uptake and release of these iron formulations by THP-1 cells (macrophage phenotype) and HepG2 cells were investigated with atomic absorption spectrometry (AAS). Ferritin was measured by ELISA. HepG2 cells were used to investigate effects of IVI on the intracellular labile iron pool (LIP), which was measured by using the fluorescent calcein assay. The amount of redox-active iron within the iron formulations was assayed using dichlorofluorescein as fluorescent probe. RESULTS: All iron preparations were taken up, stored in ferritin and released again by macrophages and HepG2-cells. This study shows that the availability and stability of iron-HES formulations in vitro are comparable with the well-known iron dextran compounds. CONCLUSIONS: Our results indicate that these new iron formulations have a good stability and availability in vitro and are comparable with the well-known iron dextran complexes.     

线粒体融合素基因2对肝癌细胞的增殖及对化疗敏感性的影响

目的：探讨线粒体融合素基因2(mfn2)对肝癌细胞的增殖及对化疗敏感性的影响。 方法：实验分3组：重组质粒pEGFPmfn2转染HepG2细胞为实验组,空质粒pEGFP–N2转染HepG2细胞为阴性对照组,HepG2细胞为空白对照组。RT-PCR和Western Blot 分别检测转染后各组细胞mfn2 mRNA和蛋白水平的表达。采用细胞计数法和MTT法检测mfn2基因对肝癌细胞增殖的影响;流式细胞术检测转染后细胞周期的分布情况;MTT法检测转染mfn2基因对化疗敏感性的影响。 结果：转染48 h 后,转染mfn2组细胞生长开始受到明显抑制,明显低于空白对照组和转染空载体组,差异均有显著性(P< 0.05);转染pEGFPmfn2组G0/G1期所占比例为(83.2±1.5)%,明显高于转染空质粒pEGFP组与空白对照组G0/G1期所占比例,差异均有显著性 (P<0.05)。实验组HepG2细胞对化疗药物5-氟尿嘧啶的敏感性增强。 结论：mfn2对肝癌细胞具有增殖抑制作用,其机制可能与细胞周期阻滞有关;mfn2可增强化疗药物的敏感性。