一种改良的肝星状细胞分离方法

目的改良肝星状细胞（HSC）的分离方法,为深入研究肝纤维化的发生机制奠定基础。方法采用Ⅳ型胶原酶和链酶蛋白酶原位消化肝脏,在1．040～1．060g／mL范围内多重多次密度梯度离心分离HSC,台盼蓝染色测定细胞存活率,328nm紫外光激发HSC自发蓝绿色荧光法和Desmin细胞免疫荧光法鉴定HSC及检测HSC纯度。Desmin和n—SMA细胞免疫荧光方法鉴定HSC静息和活化状态。结果每只大鼠肝脏HSC得率为（3～5）×10^7个,HSC存活率在95％以上,纯度在90％以上,体外培养14d后活化的HSC纯度几乎达100％。结论在1．040～1．060g／mL密度范围内采用多重多次密度梯度离心方法,避免了HSC分离过程中因密度配比误差及HSC自身密度相对不均一性所导致的细胞流失,建立了一种改良的肝星状细胞分离方法,HSC得率和纯度更加稳定,达到国外文献报道水平。     

姜黄素激活PPARγ下调肝星状细胞α-SMA和Ⅰ型胶原的基因表达

[目的]研究姜黄素抑制大鼠肝星状细胞（HSC）活化作用与过氧化物酶体增殖因子活化受体γ（PPARγ）信号转导途径之间的关系。[方法]肝脏原位灌流酶消化、Nycodenz密度梯度离心方法分离培养大鼠HSC，并使用姜黄素对细胞进行相应处理，采用四甲基偶氮唑蓝（MTT）比色分析法检测姜黄素对细胞增殖的影响。收集裂解细胞并抽提细胞总RNA，采用半定量RT-PCR检测姜黄素处理对α平滑肌肌动蛋白（α-SMA）和Ⅰ型胶原的基因表达水平的影响。[结果]姜黄素在10-50μmol／L范围内呈剂量依赖性抑制体外培养大鼠传代HSC的增殖，且能在基因水平显著降低α-SMA（P〈0．05）和Ⅰ型胶原的基因表达水平（P〈0．01）。这些作用均可以被PPARγ特异性阻断剂GW9662阻断。[结论]姜黄素抑制HSC增殖、活化和合成Ⅰ型胶原的作用依赖于PPARγ信号转导途径的激活。     

一种简易分离大鼠肝脏库普弗细胞的方法

目的建立一种经济、简单、稳定的大鼠肝脏库普弗细胞(KCs)分离方法。方法采用前灌流液(D-Hanks)和胰酶消化液两步法原位灌注大鼠肝脏,低速离心去除肝细胞,Percoll分离液不连续密度梯度离心法和选择性贴壁法纯化KCs,将培养7 d的KCs采用吞噬latex-beads实验及ED1免疫细胞化学法鉴定并检测其纯度。结果每只大鼠肝脏KCs的得率为(1.2～1.8)×107个,细胞存活率达95%,KCs纯度近100%。结论此新方法分离获得的KCs细胞得率和纯度较高且较稳定,同时较传统方法胶原酶的使用相比,此方法更经济实用。     

花生四烯酸和亚油酸刺激的Kupffer细胞对肝星状细胞增殖的影响

目的：探讨花生四烯酸，亚油酸及其刺激的Kupffer细胞(KC)对大鼠肝星状细胞(HSC)增殖的影响。方法：用链霉蛋白酶和胶原酶原位灌流，Nycodenz密度梯度离心分离大鼠HSC和KC，应用免疫组织化学、吞噬功能试验、电镜等方法进行鉴定，制备花生四烯酸和亚油酸刺激的KC培养的上清液(KCCM)，并以MTT比色法观察花生四烯酸和亚油酸及其刺激的KCCM对HSC增殖效应。结果：花生四烯酸和亚油酸浓度分别在25μg／ml和50～100μg／ml时对HSC增殖有促进作用(P<0．01)，50～100μg／ml花生四烯酸对HSC增殖有抑制作用，倒置显微镜观察可见HSC细胞轮廓非常明显，胞质粗糙，内有颗粒沉积；与正常和KCCM组相比，KCCM 花生四烯酸组和KCCM 亚油酸组对HSC增殖有促进作用(P<0．01)；KCCM 花生四烯酸组促HSC增殖作用较KCCM 亚油酸组明显(P<0．05)；KCCM组高于正常组．但统计学无差异（P>0．05)。结论：花生四烯酸和亚油酸在一定浓度下可促进HSC增殖，高浓度对HSC有毒性作用，花生四烯酸和亚油酸及其刺激的KCCM可促进HSC增殖，其可能通过脂质过氧化作用而与脂肪肝肝纤维化的发生有关。     

大鼠肝贮脂细胞,肝细胞的同时分离和培养

目的:建立肝脏细胞的共培养和研究同一个体不同细胞的代谢,深入探讨肝纤维化的细胞机制。方法与结果:采用胶原酶肝脏原位灌流,消化肝脏将细胞分散,通过离心初步分得肝实质细胞和非实质细胞,肝实质细胞采用49.2％淋巴细胞分离液行密度梯度离心,获得精制肝细胞;非实质细胞经进一步酶消化,再用18％Nycodenz行密度梯度离心,得到纯化的贮脂细胞。肝细胞得率为5×10~7/肝～10×10~7/肝,存活率>85％,贮脂细胞得率为2×10~7/肝～4×10~7/肝,存活率>98％,纯度>95％。经本方法同时分得的两种细胞在得率,纯度和活率方面与单独分离的细胞相近,且具经济、简便、稳定的特点。结论:建立了同时分离培养肝细胞和贮脂细胞的方法。     

特异性小干扰RNA对晚期糖基化终产物受体介导肝星状细胞激活和胶原生成的抑制作用

目的 探讨特异性小分子干扰RNA对晚期糖基化终产物受体（RAGE）介导肝星状细胞（HSC）激活和胶原生成的影响。方法 构建RAGE特异性siRNA表达载体，经脂质体转染入HSC—T6细胞，以空白和转染非特异性siRNA表达载体pGCsi-C为对照，分别用实时荧光定量PCR和Western印迹法检测各组HSC—T6细胞RAGE、α平滑肌肌动蛋白（α—SMA）和Ⅰ型胶原基因及蛋白的表达。结果 与空白对照组和pGCsi—C组相比，转染特异性siRNA表达载体pGCsi—R1的HSC—T6细胞RAGE mRNA和相对分子质量50×10^3、46×10^3的RAGE蛋白表达分别下调79．65％、78．04％（F=26．005，P〈0．01），56．09％、54．93％（F=365．185，P〈0．01）和63．67％、59．67％（F=386．07，P〈0．01），同时α—SMAmRNA和蛋白、Ⅰ型胶原mRNA和蛋白的表达也受到明显抑制，分别为空白对照组和pGCsi—C组的57．53％、65．50％（F=20．913，P〈0．05）、58．48％、62．80％（F=56．592，P〈0．05）、71．16％、74．23％（F=18．091，P〈0．05）和71．91％、76．09％（F=17．299，P〈0．05）。结论 RAGE特异性siRNA可在细胞内稳定表达，并能有效抑制HSC的激活和Ⅰ型胶原生成。     

重组转化生长因子β3基因对大鼠肝星状细胞Ⅰ型胶原表达的影响

目的观察转化生长因子D3基因（TGFβ3）对大鼠肝星状细胞株（HSC—T6）Ⅰ型胶原合成的影响。方法TGFβ3表达质粒[pcDNA3．1（＋）-TGFβ31和TGFβ1表达质粒[pcDNA3．1（＋）-TGFD11的构建。通过脂质体介导方法，将pcDNA3．1（＋）-TGFβ1、pcDNA3．1（＋）-TGFβ3分别及共同转染体外培养的HSC—T6细胞，荧光定量PCR法及Westernblot法分别检测转染后TGFβ1、TGFD3、Ⅰ型胶原mRNA及蛋白质的表达。将pcDNA3．1（＋）-TGFD1转染HSC—T6细胞，经G418筛选建立高表达TGFD1的HSC～T6细胞克隆，pcDNA3．1（＋）-TGFD3转染克隆细胞，荧光定量PCR法检测转染后TGFβ3、TGFβ1及Ⅰ型胶原mRNA的表达，Westernblot法检测TGFβ1、Ⅰ型胶原蛋白的表达情况。结果构建的pcDNA3．1（＋）TGFD3、pcDNA3．1（＋）-TGFD1质粒可转染HSC—T6细胞，转染率28．2％。pcDNA3．1（＋）TGF侈3转染细胞后，Ⅰ型胶原mRNA及蛋白的表达较空白组及对照组增加，以72h增高最为明显（P〈0．05）；共转染组Ⅰ型胶原mRNA及蛋白质的表达较pcDNA3．1（＋）-TGFβ1转染组明显降低（P〈0．05）。TGF侈3转染克隆细胞后，TGFD1mRNA表达较克隆组无明显改变（P〉0．05），而蛋白质表达明显下降（P〈0．05），Ⅰ型胶原mRNA及蛋白质表达均较克隆组明显降低（P〈0．05）。结论TGFD3基因转染正常培养的HSC—T6细胞，增加Ⅰ型胶原的表达；转染高表达TGFβ1的克隆组HSC—T6细胞，Ⅰ型胶原表达明显降低，提示TGFβ3对肝纤维化的发生有抑制作用。     

晚期非小细胞肺癌同步放化疗与单纯放疗的疗效对比

目的研究同步放化疗对不能手术的晚期非小细胞肺癌的疗效及毒副反应。方法72例不能手术的晚期非小细胞肺癌患者被随机分成同步放化疗组（同步组）和单纯放疗组（单放组），两组放疗方法相同，均采用常规分割放疗，每次210Gy，每周5次，肿瘤灶总剂量为60-70Gy，同步组在放疗同时给予足叶乙甙、卡铂化疗。结果同步组和单放组的有效率分别是77．8％和58．3％，经统计学处理差异无显著性（P〉0．05），两组的完全缓解率分别是27．8％和8．3％，同步组明显高于单放组（P〈0．05）。同步组和单放组的中位生存时间分别为14个月和9个月（P〈0．05），1年生存率分别为55．1％和30．1％，同步组优于单放组（P〈0．05）。同步组的远处转移率（52．8％）明显低于单放组（83．3％）（P〈0．05）。两组间毒副反应差异无显著性（P〉0．05）。结论同步放化疗对不能手术的晚期非小细胞肺癌有较好的疗效，值得进一步临床研究。     

大鼠肝脏细胞同步分离与培养方法的建立

目的建立一种同时分离大鼠肝细胞(parenchyma cell,PC)、肝星状细胞(hepatic stellate cell,HSC)、肝窦内皮细胞(liver sinusoidal endothelial cell,SEC)与库普弗细胞(Kupffer cell,KC)的操作方案。方法采用EDTA/胶原酶两步灌注法获取肝细胞悬液,通过低速离心首先分离出PC组分,然后通过链蛋白酶E去除非实质细胞(nonparenchymal cell,NPC)中的PC,最后通Nycodenz和Percoll溶液进行梯度离心分离并纯化HSC、SEC和KC,体外培养并观察各细胞表型和功能的变化。结果 PC、HSC、SEC和KC平均每克组织产量分别为(26±9.2)×10~6、(1.5±0.2)×10~6、(7.4±1.5)×10~6和(3.1±0.9)×10~6,活力分别为(93.1±2.3)%、(90.1±3.4)%、(96.2±2.1)%和(98.2±1.7)%,纯度分别为(98.5±1.2)%、(95.1±2.5)%、(93.6±3.6)%和(98.1±1.4)%;HSC在体外培养过程中逐渐丧失维生素A,转化为肌成纤维样(myofibroblast,MF)细胞;SEC在体外培养第3天出现凋亡,至7天凋亡达到顶峰,随后少量存活的细胞在体外增殖,至培养14天时形成典型的"铺路石"形态;培养14天的SEC保留清道夫功能,但丧失窗孔结构;KC可在体外增殖,培养至14天仍具有吞噬功能,表达特征性标志CD68。结论本方法可同时分离大鼠肝脏细胞,细胞数量、纯度和活力可满足下游实验。     

不同基因型慢性乙型肝炎患者外周血辅助性T细胞分化和树突状细胞的功能

目的探讨不同基因型的慢性乙型肝炎（乙肝）患者体内辅助性T细胞（Th）和树突状细胞（DCs）功能差别。方法检测29例不同基因型慢性乙肝患者外周血Th细胞因子（IL-12、IL-2、IL-4）的表达，并对其中20例不同基因型患者进行外周血DC的分离和体外诱导培养，观测其形态和数量变化，检测DC分泌的IL-12含量，对数据应用t检验进行统计学分析。结果慢性乙肝患者与正常对照组比较，外周血IL-12、IL-2、IL-4表达明显升高（t=2．9272，P〈0．01；P〈0．01；P〈0．05）。C型患者组与D型患者组比较，丙氨酸转氨酶（ALT）、IL-12、IL-2、Th1／Th2明显升高（t=2．4626，P〈0．05；P〈0．05；P〈0．05；P〈0．01），IL-4明显降低（P〈0．05）。慢性乙肝患者外周血的单核细胞经培养和细胞因子诱导后，可获得成熟的具有典型形态的DC。慢性乙肝患者组与正常对照组比较，DC数量明显减少（t=5．4186，P〈0．01）。DC分泌的IL-12水平明显降低（t=3．265，P〈0．01）。不同基因型患者之间DC的数量与DC分泌IL-12差异无统计学意义。结论慢性乙肝患者体内存在Th分化的不平衡；C型慢性乙肝患者体内Th1优势应答，肝脏炎症较重，治疗效果及预后较差。     

Mechanism of red blood cell aging: Relationship of cell density and cell age

The human red cell has a life span of 120 days. The mechanism that determines cell removal from the circulation with such precision remains unknown. Most studies of red cell aging have been based on analysis of cells of progressively increasing age separated by density. The relationship between red cell age and density has been recently challenged, and the hypothesis has been put forward that cell death is not the result of a progressive deterioration of essential cell constituents. This theory was based on preliminary observations in transient erythroblastopenia of childhood, which could not later be confirmed. When the relationship between cell aging and increasing density is critically reviewed, it appears to be based on firm experimental evidence, confirmed by in vivo demonstration of decreasing survival of cells of increasing age. Analysis of studies using buoyant density gradients reveals that this technique can easily distinguish the single exponential slope of decline for those cell components that change progressively throughout the red cell life span from the biphasic decline of those that decrease drastically at the reticulocyte-mature red cell transition. The view that the aging of the red cell and its removal from the circulation result from a progressive series of events during the 120 days of its life span appears to be the most consistent with the available data. Density separation, validated by much experimental evidence, remains a most useful technique for the study of the mechanism of aging of the red cell. © 1993 Wiley-Liss, Inc.     

细胞自噬与结肠癌Lovo细胞迁移和侵袭的相关性研究

目的探讨不同自噬状态对结直肠癌Lovo细胞迁移和侵袭能力的影响。 方法将Lovo细胞分为自噬增强组、正常细胞组和3-甲基嘌呤自噬抑制组。用激光共聚焦显微镜观察绿色荧光颗粒,用Q-PCR检测Beclin1 mRNA表达水平,透射电镜观察自噬溶酶体,Transwell实验评价Lovo细胞迁移与侵袭能力。 结果绿色荧光颗粒数以自噬增强组最多,其次为正常细胞组,而自噬抑制组最少。Beclin1 mRNA表达量自噬增强组为(1.23±0.12)个,正常细胞组为(1±0.13)个,自噬抑制组为(0.98±0.1)个。自噬增强组可见大量的自噬溶酶体,明显多于正常细胞组和自噬抑制组。迁移实验细胞计数：自噬增强组高于正常细胞组(138.0±16.7与90.7±12.9,P＝0.026)和自噬抑制组(138.0±16.7与92.7±26.7,P＝0.030)。侵袭实验细胞计数：自噬增强组高于正常细胞组(147.0±13.0与99.0±20.5,P＝0.028)和自噬抑制组(147.0±13.0与95.7±25.6,P＝0.021)。 结论结肠癌Lovo细胞自噬增强促进肿瘤细胞迁移和浸润,可能是局部浸润和远处转移的机制之一。     

Red cell rheology in stomatocyte-echinocyte transformation: roles of cell geometry and cell shape

The influence of the shape of the red blood cell during stomatocyte- echinocyte transformation on its deformability was studied by microsieving through pores with diameters of 2.6, 4.5, and 6.9 micron. A stomatocytic transformation was produced by chlorpromazine (0.02, 0.1, and 0.5 mmol/L) and an echinocytic transformation by sodium salicylate (7.5, 30, and 120 mmol/L). For spherostomatocytes, an increase in filtration resistance through 2.6 and 4.5 micron pores was observed, whereas for spheroechinocytes, a decrease in filtration resistance through 2.6 micron pores was found. Larger pores (6.9 micron) were not sensitive to those shape changes. The changes in deformability can be explained by the fact that the surface area of (sphero)-stomatocytes decreased, whereas that of (sphero)-echinocytes increased; the cell volume remained essentially constant. Echinocytes produced by 24-hour adenosine triphosphate depletion differed from drug- induced echinocytes: they had an increased cell volume at constant surface area and consequently an increased filtration resistance through 2.6- and 4.5-micron filter pores. Shape changes with spicule formation are therefore not a homogeneous entity, and cell geometric factors (eg, surface area and volume) must be assessed with care. The viscosity of red cell suspensions at a hematocrit level of 45% was higher for drug-induced echinocytes than discocytes or stomatocytes at all shear rates tested. We conclude that the normal discocyte represents an optimum shape for the flow in vivo since a stomatocytic transformation could impair the passage through the microcirculation (decrease in cell filterability) and an echinocytic transformation could impair the flow in larger vessels (increase in blood viscosity).     

Liver cell adenoma and liver cell adenomatosis

During the last three decades liver cell adenoma and liver cell adenomatosis have emerged as new clinical entities in hepato-logical practice due to the widespread use of oral contraceptives and increased imaging of the liver. On review of published series there is evidence that 10% of liver cell adenomas progress to hepatocellular carcinoma, diagnosis is best made by open or laparoscopic excision biopsy, and the preferred treatment modality is resection of the liver cell adenoma to prevent bleeding and malignant transformation. In liver cell adenomatosis, the association with oral contraceptive use is not as high as in solitary liver cell adenomas. The risk of malignant transformation is not increased compared with solitary liver cell adenomas. Treatment consists of close monitoring and imaging, resection of superficially located, large (>4 cm) or growing liver cell adenomas. Liver transplantation is the last resort in case of substantive concern about malignant transformation or for large, painful adenomas in liver cell adenomatosis after treatment attempts by liver resection.     

胆红素对肝星状细胞-T6增殖及细胞周期的影响

[目的]观察胆红素对肝星状细胞(HSC)-T6增殖及细胞周期影响.[方法]将培养细胞分成正常组和胆红素不同浓度(10 μmol/L、30 umol/L、50 /μmol/L、70 μmol/L、100 μmol/L)干预组,采用MTT法观察胆红素对HSC-T6增殖的影响,流式细胞仪观察各组细胞周期的变化.[结果]①不同浓度胆红素对HSC-T6均有促进增殖作用,且呈一定的量效关系,与正常组比较差异有统计学意义(P＜0.05);②10 μmol/L、50 μmol/L、100 μmol/L浓度胆红素作用HSC-T6后,G0/G1期减少,S期增加,G2/M期增加,与正常组比较均P＜0.05.[结论]胆红素对HSC-T6均有促进增殖作用.     

Properties of the mantle cell and mantle cell lymphoma.

The major lymphoid inhabitant of the follicular mantle is the mantle cell, an immunologically na?ve B cell. It is the putative cell of origin of mantle cell lymphoma (MCL), the cells of which have similar morphologic, immunophenotypic, and molecular characteristics to the normal B lymphocytes of the mantle zone. In the past year a number of advances have been made in the biology of the normal mantle cell, its interactions with the other constituents of the follicular and mantle zone microenvironments, and the development of neoplasia in this cell population. In addition, new developments in diagnostic molecular pathology have been used to more readily identify cases of MCL. The authors summarize these new advances in the understanding of the biology of the mantle cell and newer ancillary techniques in the diagnosis of lymphomas arising from this cell type.     

Establishment and characterization of a mantle cell lymphoma cell line

A mantle cell lymphoma (MCL) cell line (JeKo-1) was established from peripheral blood mononuclear cells of a patient with a large cell variant of MCL showing leukaemic conversion. JeKo-1 cells were Epstein-Barr virus negative and showed a B-cell phenotype with IgM⁺, IgD⁺, CD3^?, CD5⁺, CD10^?, CD19⁺, CD20⁺ and CD23^?; they overexpressed cyclin Dl, Bcl-2, c-Myc and Rb proteins. Bcl-1/J_H gene rearrangement was confirmed by polymerase chain reaction, although karyotypic analysis showed 40/41 chromosomes devoid of apparent t(11;14)(q13;q32) translocation. JeKo-1 cells were highly tumourigenic in SCID mice.     

Kinetics of dendritic cell chimerism and T cell chimerism in allogeneic hematopoietic stem cell recipients

Dendritic cells (DC) as potent antigen-presenting cells (APC) and T cells as effector cells play an essential role in the pathophysiology of both graft-versus-host (GvH) and graft-versus-leukemia (GvL) reactions after transplantation. Therefore, we determined the kinetics of DC and T-cell chimerism establishment after allogeneic hematopoietic cell transplantation (AHCT) in a group of 144 patients, using fluorescence-activated cell sorting (FACS) or magnetic cell sorting (MACS) followed by FISH or STR-PCR analysis for chimerism evaluation. In all, three cell lines investigated (CD3(+) T cells, CD11c(+) DC1 and CD123(+) DC2), we found a rapid and consistent establishment of complete donor chimerism (CDC) in over 70% of all patients during the first 6 weeks after AHCT. The rate of patients with CDC increased significantly over time within the first year after transplantation. A related donor (P=0.004) as well as an underlying lymphatic leukemia (P=0.03) were found to be significantly associated with development of MC in T cells. No significant correlation between DC or T cell chimerism and GvHD or relapse was detected. Our results thus demonstrate a fast and stable CDC in DC1, DC2 and T cells after AHCT that continuously increases over time in nearly all patients.     

Mixed small cell and non-small cell lung cancer

Seventeen (10 percent) of 176 patients with small-cell carcinoma of the lung seen at this hospital since 1976 proved to have mixed small-cell and non-small-cell tumors. The presence of a mixed lung cancer was established prior to chemotherapy or irradiation in nine patients. Eight were initially diagnosed as pure small-cell carcinoma but proved to have a mixed tumor at either surgery or autopsy. Of the 17 patients, eight received chemotherapy, and four had a partial response. Six of the 40 autopsies performed on patients with small-cell lung cancer demonstrated intrathoracic tumor which was histologically mixed. Extrathoracic metastases in these patients were heterogeneous and included pure small-cell, pure non-small-cell, and mixed histologic type. We conclude that mixed small-cell and non-small-cell lung cancers are relatively frequent and carry important prognostic and therapeutic implications. Clinical management of patients with small-cell lung cancer should therefore be flexible and tailored to the potential for histologic diversity. Mixed lung cancer in previously untreated patients suggests a common endodermal origin for small-cell and non-small-cell pulmonary tumors.     

Smooth muscle cell and endothelial cell growth factors

Blood vessels are composed basically of two cell types, vascular endothelial cells (ECs) and vascular smooth muscle cells (SMCs), whose proliferation in vivo is tightly regulated. A number of growth regulatory polypeptides have been identified that stimulate the proliferation of vascular cells. This article reviews briefly the structural properties and biologic activities of the best-characterized vascular cell growth factors. A fuller understanding of the properties of vascular cell growth modulators is an important element in delineating the proliferative events that are associated with vascular injury; with SMC hyperplasia such as occurs in restenosis following angioplasty, in atherosclerosis, and in hypertension; and with angiogenesis, both physiologic and pathologic.