人肝癌细胞器官特异性转移与基质金属蛋白酶分泌的关系

目的　探讨不同转移潜能的人原发性肝癌细胞系基质金属蛋白酶 (MMPs)分泌与器官特异性转移的关系。方法　将C5 7BL/6小鼠的肺、脾组织粗提物与高低转移潜能的人肝癌细胞系MHCC97 H和MHCC97 L共培养 ,明胶酶谱法检测其分泌上清MMPs表达水平 ,并用荧光免疫染色法比较MMP 9的表达。结果　在无血清培养液培养条件下 ,MHCC97 H和MHCC97 L细胞均分泌较少MMP 9(2 .1± 0 .2 ,1.4± 0 .1) ,不分泌MMP 2 ;但MHCC97 H细胞分泌活性型MMP 9(17.4± 2 .2 )显著高于MHCC97 L细胞 (6.7± 0 .2 ,P <0 .0 0 1)。与肺组织粗提物孵育后 ,MHCC97 H和MHCC97 L细胞分泌MMP 9、活性型MMP 9和MMP 2均显著高于无血清培养液条件下MMPs分泌 (P <0 .0 0 1) ;MHCC97 H细胞分泌MMP 9(18.8± 1.2 )、活性型MMP 9(10 0 .1± 1.1)、MMP 2 (2 2 .4± 1.3 )均显著高于MHCC97 L细胞 (7.8± 0 .3 ,40 .8± 2 .2 ,8.2± 0 .4,P <0 .0 0 1)。脾组织粗提物孵育后的MHCC97 H和MHCC97 L细胞不分泌MMPs。荧光免疫细胞化学法检测MMP 9表达位于MHCC97 H细胞核周和细胞浆 ,而在MHCC97 L细胞未见明显的MMP 9表达。结论　不同转移潜能的人肝癌细胞分泌MMPs差异与其侵袭转移能力相关。肺组织粗提物促进不同转移潜能的人肝癌细胞分泌MMPs可能与其器官特异性     

免疫抑制剂对肝癌细胞增殖、运动侵袭的影响

目的探讨免疫抑制剂对肝癌细胞增殖、运动侵袭能力的影响,为肝癌肝移植术后免疫抑制剂的选用提供指导。方法用裸鼠人高转移肝癌模型LCI-D20,研究环孢素A(CsA)、他克莫司(FK506)、雷帕霉索(RPM)对肿瘤生长及自发肺转移的影响。采用扫描电镜、噻唑蓝比色法 (MTT)、流式细胞仪、Boyden小室等方法分别研究各免疫抑制剂对人肝癌高转移细胞株 MHCC97H增殖、凋亡、细胞周期、运动及侵袭的影响。结果体内实验中,RPM抑制了LCI-D20 模型移植瘤的生长[(0．76±0．38)g vs(2．09±0．75)g,P<0．05]及肺转移的发生(2／7 vs．7／7, P<0．05);CsA组肺部转移灶的数目明显多于对照组(6±2 vs．4±1,P<0．05);FK506组肺转移发生率虽较对照组减少,但差异无统计学意义。CsA及FK506均对移植瘤的生长无影响。体外实验中,RPM抑制了MHCC97H增殖,并使细胞周期停滞于G_0-G_1期。RPM及FK506抑制了 MHCC97H的运动、侵袭(P<0．05),而CsA则促进了MHCC97H的运动、侵袭(P<0．05)。结论 CsA促进了肝癌的转移;FK506不促进肝癌的转移;而RPM则抑制了肝癌细胞的增殖及转移。     

天花粉蛋白抑制肝细胞癌肺转移的实验研究

目的观察天花粉蛋白(TCS)抑制肝癌肺转移及可能机制。方法应用LCI-D20模型观察荷瘤裸鼠肺转移;采用MHCC97H细胞株观察TCS对瘤细胞粘附、运动及侵袭的干预。结果TCS组肺转移率为41·7%(5/12),对照组为100%(20/20),差别有显著统计学意义(P<0·01)。MHCC97H与纤连蛋白粘附力强,粘附百分比为74·3%。TCS降低粘附力,作用呈剂量依赖性。80μg/ml组粘附率仅相当于干预前的47·8%。MHCC97H运动力强,未干预组穿膜细胞数47·67±4·03/200倍视野。TCS抑制瘤细胞运动,作用呈剂量依赖性。10μg/ml组穿膜细胞数仅相当于干预前的51·0%(24·33±1·50/200倍视野)。MHCC97H侵袭力强,未干预组穿膜细胞数34·90±4·86/200倍视野。TCS抑制瘤细胞侵袭,作用呈剂量依赖性。5μg/ml组穿膜细胞数仅相当于干预前的53·8%(18·77±4·81/200倍视野),抑制明显。结论TCS通过干扰MHCC97H与细胞外基质粘附、抑制MHCC97H运动和侵袭力,减少LCI-D20肺转移。     

绿色荧光高/低转移人肝癌细胞株MHCC97-HG/LG的建立及鉴定

目的建立稳定自发绿色荧光的高、低转移人肝癌细胞株并明确鉴定。方法脂质体转染法将绿色荧光蛋白(GFP)基因分别转染至高、低转移人肝癌细胞株MHCC97-H／L内,经 G418反复筛选并单克隆化,得到两株新细胞株MHCC97-HG／LG,对新细胞株GFP表达稳定性、生物学特性、体内外侵袭转移能力及肿瘤血管生成等进行鉴定。结果 MHCC97-HG／LG在体外培养或裸鼠体内接种36 d后均能稳定表达绿色荧光。MHCC97-HG肝、肺转移率均为100％,较 MHCC97-LG及其父系体内外侵袭转移能力显著增强(P<0．01),其原位肿瘤微血管密度较 MHCC97-LG显著增加[(121．0±15．9)／HP vs(87．0±14．2)／HP,P<0．01]。体视荧光显微镜可实时观察裸鼠体内MHCC97-HG／LG种植瘤生长、转移及血管形成情况。结论 MHCC97-HG／LG及其动物模型在肝癌转移机制、肿瘤血管生成等诸多研究领域可能有较广阔的应用前景。     

不同转移潜能人肝癌单克隆细胞株的分离和建立

目的 从高转移潜能的人肝癌细胞系中分离出不同转移潜能的单克隆细胞株。方法 采用有限稀释法,对高转移潜能人肝细胞癌细胞系MHCC97进行单克隆培养,筛选不同转移潜能的克隆细胞株,测定目标克隆株的生长速度、核型、AFP分泌情况,体外侵袭能力、裸鼠接种肺转移率。结果 筛选出高、低两个不同转移潜能的单克隆细胞株,分别命名为MHCC97-H和MHCC97-L,前者肺转移率为100%,后者为40%;MHCC97-H的细胞倍增时间为34.2h,MHCC97-L为60.0h;流式细胞分析显示,MHCC97-H与MHCC97-L相比,前者G0-G1期细胞比例低,S期、G2-M期细胞比例高;人工基底膜穿透能力前者明显强于后者。结论 本实验证实了MHCC97癌细胞系的异质性。这两个单克隆细胞株对于肝癌生物学行为的比较研究有一定的使用价值。     

抗黏附多肽对人肝癌高转移细胞系MHCC97侵袭转移的抑制作用

目的　研究抗黏附多肽GRGDS、YIGSR对人肝癌高转移细胞系MHCC97侵袭转移的抑制作用。方法　选择GRGDS、YIGSR作为抗黏附多肽 ,选具有高转移潜能的人肝癌细胞系MHCC97为靶细胞。用体外细胞 基质黏附实验检测两种多肽对MHCC97细胞对所包被的纤维连接蛋白 (FN)的黏附抑制作用 ;用体外细胞侵袭实验检测两种多肽对MHCC97细胞的侵袭抑制作用。结果　GRGDS、YIGSR对MHCC97细胞对FN黏附的抑制作用呈时间剂量依赖。当药物浓度达 2 0 0mg/L时 ,黏附抑制率达 60 %。两种多肽分别与细胞共育 48h后 ,均显示出明显的侵袭抑制能力。将细胞接种于裸鼠后给予GRGDS或YIGSR治疗 ,与对照组比较 ,有明显的肺重减轻、肺转移灶减少 (P <0 .0 5 )。结论　抗黏附多肽GRGDS、YIGSR对人肝癌高转移细胞系MHCC97具有明显抑制黏附、抑制侵袭转移的作用。     

趋化因子受体CXCR4在肝细胞癌表达及肺转移中作用的研究

目的 以人肝细胞癌高、低肺转移潜能细胞株MHCC97-H、MHCC97-L为研究对象,研究趋化因子受体CXCR4在肝细胞癌肺转移过程中的作用和意义.方法 RT-PCR和Western blotting比较MHCC97-H、MHCC97-L细胞株CXCR4 mRNA及蛋白质水平的表达.CXCR4配体SDF-1α及肺提取物趋化实验和抗体抑制实验观察SDF-1α和裸鼠肺组织匀浆液对MHCC97-H的趋化迁移作用.结果 趋化因子受体CXCR4在MHCC97-H细胞株表达低于低肺转移潜能细胞株MHCC97-L,蛋白质水平与mRNA水平一致.SDF-1α对MHCC97-H趋化实验表明在1～100 ng/ml浓度范围内均有趋化作用,在浓度为50 ng/L时趋化作用最显著(P＜0.05).肺提取物亦发现对MHCC97-H有趋化作用,作用强于MHCC97-L及DMEM组(P＜0.05).但加入CXCR4抗体后其趋化作用并未明显下调.结论 CXCR4在肝癌组织及细胞表达并可能参与肝癌肺转移,但并非肝癌细胞趋化转移的主要受体分子.     

抑制黏着斑激酶表达对人肝癌细胞转移潜能的作用

目的研究抑制人肝癌细胞MHCC97-H中黏着斑激酶（FAK）表达对细胞黏附、侵袭和骨架重塑的影响。方法利用脂质体转染FAKsiRNA至MHCC97-H细胞内。Western blot法检测RNA干扰前后MHCC97．H细胞中FAK蛋白的表达水平。黏附试验和侵袭试验分别检测MHCC97．H细胞黏附和侵袭能力在干扰前后的变化。明胶酶谱试验检测MHCC97-H细胞分泌MMPs的变化。用激光共聚焦显微镜观察免疫荧光标记细胞的骨架重塑的变化。结果特异性FAKsiRNA明显抑制MHCC97．H细胞中FAK表达。干扰后MHCC97-H细胞与细胞外基质纤维连接蛋白的黏附率为35．8％,低于对照组的57．3％（P〈0．05）。干扰后MHCC97-H细胞穿透Tanswell小室的数目由对照组的（31．3±2．6）个减少至（14．5±3．1）个（P〈0．05）。干扰后MHCC97-H细胞分泌MMPs的量明显减少。FAK表达抑制影响骨架蛋白Vinculin在PDGF—BB诱导细胞骨架重塑中分布,阻碍片状伪足形成,延迟黏着斑形成时间。结论FAK表达受到抑制后,通过减少MMPs分泌和影响细胞骨架重塑可降低MHCC97-H细胞黏附、侵袭能力。     

基因芯片筛选人肝癌细胞株MHCC97中血管生成拟态相关基因

目的 通过观察三维培养条件下高、低转移人肝癌细胞株MHCC97-H、MHCC97-L形成血管生成拟态(VM)的差异,探讨VM形成与细胞外基质和粘连分子相关机制.方法 建立MHCC97-H、MHCC97-L三维培养体系,倒置显微镜下观察血管样结构形成差异.以人脐静脉内皮细胞、无转移人肝癌细胞株Hep3B及正常人肝细胞株HL-7702作对照.应用细胞外基质和粘连分子基因芯片筛选MHCC97-H和MHCC97-L中差异表达的基因,并采用RT-PCR和Western blot验证芯片的结果.组间比较采用两样本t检验.结果 三维培养24h,MHC097-H形成的血管样结构长度为(474±16)mm/cm2,MHCC97-L为(320±41)mm/cm2,两者比较,差异有统计学意义(t=6.119,P＜0.05).Hep3B及HL-7702均不形成血管样结构.在113个细胞外基质和粘连分子相关基因中,MHCC97-H表达较MHCC97-L上调的有7个,下调的有3个.选取差异性表达的腱糖蛋白-C和细胞外基质蛋白1经RT-PCR及Western blot验证,结果与基因芯片结果基本一致.结论 高转移人肝癌细胞株MHCC97-H体外形成VM能力明显较低转移人肝癌细胞株MHCC97-L强,其原因可能与MHCC97-H差异表达某些细胞外基质和粘连分子相关基因有关.     

hTcf-4基因在肝癌细胞系中的表达及其突变的研究

目的　研究hTcf 4基因在肝癌细胞系中的表达及突变的情况 ,以探讨hTcf 4基因与肝癌发生及侵袭转移的关系。方法　用逆转录 聚合酶链反应 (RT PCR)检测 5种肝癌细胞系和肺癌细胞系、乳腺癌细胞系、胃癌细胞系及正常肝细胞中hTcf 4基因表达 ,并用聚合酶链反应 单链构像多态性分析 (PCR SSCP)方法检测肝癌细胞系中hTcf 4外显子 1、4、9、1 5的突变情况。结果　hTcf 4mRNA在不同细胞系中均有表达。但不同细胞系的表达水平差异有显著性 (P <0 .0 5)。低转移肝癌细胞系HepG2、Hep3B、SMMC 772 1、BEL 740 2的表达水平分别为0 .70 5± 0 .0 50 ,0 .745± 0 .0 65 ,0 .71 0± 0 .0 78,0 .746± 0 .0 84,均高于正常肝细胞 0 .452± 0 .0 38(P <0 .0 0 1 )及肺癌细胞系 0 .531± 0 .0 83、乳腺癌细胞系 0 .578± 0 .0 64、胃癌细胞系 0 .50 3± 0 .0 33等非肝癌细胞系 (P <0 .0 5)。而高转移肝癌细胞系MHCC97表达水平为 0 .92 4± 0 .0 74,又高于低转移肝癌细胞系(P <0 .0 1 )。 5种肝癌细胞系中 ,只有MHCC 97hTcf 4基因第 1 5外显子处存在点突变。结论　hTcf 4基因高表达与突变可能与肝癌发生及侵袭转移有关     

Presenteeism and Absenteeism Before and After Total Hip and Knee Arthroplasty

BackgroundAbsenteeism is costly, yet evidence suggests that presenteeism—illness-related reduced productivity at work—is costlier. We quantified employed patients’ presenteeism and absenteeism before and after total joint arthroplasty (TJA).MethodsWe measured presenteeism (0-100 scale, 100 full performance) and absenteeism using the World Health Organization’s Health and Work Performance Questionnaire before and after TJA among a convenience sample of employed patients. We captured detailed information about employment and job characteristics and evaluated how and among whom presenteeism and absenteeism improved.ResultsIn total, 636 primary, unilateral TJA patients responded to an enrollment email, confirmed employment, and completed a preoperative survey (mean age: 62.1 years, 55.3% women). Full at-work performance was reported by 19.7%. Among 520 (81.8%) who responded to a 1-year follow-up, 473 (91.0%) were still employed, and 461 (88.7%) had resumed working. Among patients reporting at baseline and 1 year, average at-work performance improved from 80.7 to 89.4. A Wilcoxon signed-rank test indicated that postoperative performance was significantly higher than preoperative performance (P < .0001). The percentage of patients who reported full at-work performance increased from 20.9% to 36.8% (delta = 15.9%, 95% confidence interval = [10.0%, 21.9%], P < .0001). Presenteeism gains were concentrated among patients who reported declining work performance leading up to surgery. Average changes in absences were relatively small. Combined, the average monthly value lost by employers to presenteeism declined from 15.3% to 8.3% and to absenteeism from 16.9% to 15.5% (ie, mitigated loss of 8.4% of monthly value).ConclusionAmong employed patients before TJA, presenteeism and absenteeism were similarly costly. After, employed patients reported increased performance, concentrated among those with declining performance leading up to surgery.     

The German emergency and disaster medicine and management systemdhistory and present

As well for optimized emergency management in individual cases as for optimized mass medicine in disaster management, the principle of the medical doctors approaching the patient directly and timely, even close to the site of the incident, is a long-standing marker for quality of care and patient survival in Germany. Professional rescue and emergency forces, including medical services, are the “Golden Standard” of emergency management systems. Regulative laws, proper organization of resources, equipment, training and adequate delivery of medical measures are key factors in systematic approaches to manage emergencies and disasters alike and thus save lives. During disasters command, communication, coordination and cooperation are essential to cope with extreme situations, even more so in a globalized world. In this article, we describe the major historical milestones, the current state of the German system in emergency and disaster management and its integration into the broader European approach.     

Differences in Osteocyte Density and Bone Histomorphometry Between Men and Women and Between Healthy and Osteoporotic Subjects

Bone defects related to osteoporosis develop with increasing age and differ between males and females. It is currently thought that the bone remodeling process is supervised by osteocytes in a strain-dependent manner. We have shown an altered response of osteocytes from osteoporotic patients to mechanical loading, and osteocyte density is reduced in osteoporotic patients, which might relate to imperfect bone remodeling, leading to lack of bone mass and strength. Hence, information on osteocyte density will contribute to a better understanding of bone biology in males and females and to the assessment of osteoporosis. Osteocyte density as well as conventional histomorphometric parameters of trabecular bone were determined in cancellous iliac crest bone of healthy postmenopausal women and men and of osteoporotic women and men. Osteocyte density was higher in healthy females than in healthy males and lower in osteoporotic females than in healthy females. Bone mass was reduced in osteoporotic patients, both male and female. In females, trabecular number was reduced, whereas in males, trabecular thickness was reduced and eroded surface was increased. There were no correlations between the parameter groups bone architecture, bone formation, bone resorption, and osteocyte density. These results are consistent with impaired osteoblast function in osteoporotic patients and with a different mechanism of bone loss between men and women, in which osteocyte density might play a role. The reduced osteocyte numbers in female osteoporotic patients might relate to imperfect bone remodeling leading to lack of bone mass and strength. M. G. Mullender and S. D. Tan contributed equally to this work.     

肝内胆管囊腺瘤和囊腺癌的CT、MRI及病理表现

目的探讨肝内胆管囊腺瘤和囊腺癌的CT、MRI和病理特点。方法回顾性分析经手术病理证实的6例肝内胆管囊腺瘤和2例肝内胆管囊腺癌的影像及临床病理资料，将病变的影像表现与其病理大体形态及组织学表现作对照分析。结果6例肝内胆管囊腺瘤，女4例、男2例；2例肝内胆管囊腺癌均为女性病人；8例病人平均年龄55岁。所有病灶均表现为多房囊性肿块，肿瘤囊腔各分房内常为多种液体成分，在CT上可表现为不同密度、在MRI上可表现为不同信号强度。囊内出现多发大小不等的壁结节在胆管囊腺癌内更常见，囊内有分隔但无壁结节只见于胆管囊腺瘤。在7例CT扫描中，4例胆管囊腺瘤和1例胆管囊腺癌可见囊壁或分隔上钙化，囊壁、囊内分隔及囊内结节均为轻、中度延迟增强。肿瘤中出现卵巢样间质见于3例胆管囊腺瘤和1例胆管囊腺癌，且均为女性病人。结论肝内胆管囊腺瘤和囊腺癌是肝脏不常见的囊性肿瘤，影像上多房、囊内有分隔且各分房囊内密度或信号不一致，高度提示肝内胆管囊腺瘤或囊腺癌的诊断，如囊内伴有多发大小不等的结节，则进一步提示囊腺癌的可能。但影像学表现不能区分肿瘤中有无卵巢样间质。