用原位杂交和RNA斑点杂交法检测人肝细胞癌和癌旁肝中甲胎蛋白mRNA

用生物素标记AFP cDNA探针作原位杂交,检测了12例人肝细胞癌和癌旁肝冰冻切片和石蜡组织切片中的AFP mRNA,同时分别抽提了3例人肝癌和癌旁肝总RNA作斑点杂交.结果提示癌和癌旁肝组织中均有AFP基因的过量表达,AFP mRNA在癌内分布比较均一,而在癌旁肝组织中则比较多样,可散在分布、小灶性分布或成片状分布,从而证实不仅肝癌细胞可以自己合成AFP,而且癌旁肝细胞也可以自己合成AFP。     

应用免疫酶技术进行大鼠肝组织内甲胎蛋白的定位

甲胎蛋白(AFP)在肝癌免疫诊断中已得到广泛应用,为了进一步深入了解 AFP 在正常及病理情况下的发生发展规律,除对血清 AFP 的含量进行动态观察外,尚须了解其产生部位、产生时间、产生后的分布以及与肝癌细胞分化程度的关系等等。大鼠是研究肝损害与癌变的较好的动物,我们采用二乙基亚硝胺(DENA)在大鼠身     

大鼠甲胎蛋白的分离纯化和抗体制备

用聚丙烯酰胺凝胶电泳(PAGE)分离纯化大鼠羊水中的甲胎蛋白(AFP)具有简便、省时的特点。将微量AFP注入淋巴结内,制备高效价的AFP抗体。比较四种固定液对AFP免疫组织化学染色的影响,示丙酮-甲醛固定液能很好保存AFP的抗原性,具有阳性颗粒强、背景染色淡的优点,故是AFP免疫组织化学的最佳固定液。     

黄曲霉毒素B_1对大鼠肝脏毒性作用的病理学研究

用雄性Wistar大鼠,体重130～150 g,实验组31只,对照组10只,连续喂黄曲霉毒素B_1结晶品45周,每只大鼠受毒总剂量3.88 mg,分别在第8、9、11、12、13、14、15、16个月处死,每次处死给药组3只,对照组1只,第17个月后自然死亡7只。肝组织切片用HE染色;对28只大鼠肝组织进行了AFP免疫酶染色的形态学观察,15只鼠作了电镜观察,其结果:(1)光镜观察的主要表现为肝细胞的水样变性、嗜酸性变、嗜碱性改变、脂肪变性以及肝细胞的灶性坏死。其应答反应显示卵圆细胞和小胆管增殖、肝细胞的再生及炎细胞浸润。上述改变为慢性非特异性中毒性肝炎;(2)电镜观察主要改变有肝细胞核仁均质化,核样体形成,线粒体肿胀和基质均质化,继而形成空泡和灶性坏死;(3)85.71％(24/28)宿主肝细胞AFP免疫酶染阳性,多呈全胞浆型分布,阳性程度多为弱至中等度。AFP阳性肝细胞呈散在或灶性分布。这一改变表明,是由于AFB_1对肝细胞毒性损害所产生的返祖现象。     

肝癌患者血清甲胎蛋白含量的细胞学基础及其临床意义的研究

肝癌患者血清甲胎蛋白(AFP)含量明显差异的原因是临床迫切需要了解的实际问题。为此,本研究应用DNA含量流式细胞仪,AFP免疫酶组化标记和大鼠血清AFP含量的ELISA测定技术,从动物肝癌模型和临床研究两方面进行了探讨。用二乙基亚硝胺诱发Wistar大鼠肝癌的实验表明,随着诱癌过程中肝硬变与肝癌的相继出现,肝组织中AFP阳性细胞的数量不断增多,血清AFP含量则相应增高。     

胃癌并发肝转移病人的甲胎蛋白研究(附一例尸检报告)

原发性胃癌并发肝转移病人血清中出现较高水平的甲胎蛋白(AFP)极为少见,虽然国外自Bourreille(1970)首次报告后,见有少数报告,但进一步研究其AFP的产生及其定位的报道更少。现将一例原发性胃癌并发肝转移伴有较高水平AFP的尸检病例,用AFP免疫酶标记定位的方法,对胃癌及转移性肝癌内AFP的定位和分布进行了检测,并对AFP的产生和定位问题进行探讨。     

甲胎蛋白对体外白细胞间介素-2产生的抑制作用

本文报道了用MLA-144 T细胞瘤株的培养、大鼠混合淋巴细胞反应及刀豆素A(ConA)刺激淋巴细胞等三种方式制备白细胞间介素-2(IL-2)。以~3H-胸苷掺入效应细胞值作为测定IL-2活性的指标,分别测定甲胎蛋白(AFP)对IL-2产生的影响,与无AFP对照相比较,观察到AFP对体外三种方式产生IL-2均有明显的抑制作用。     

HGF对大鼠和人骨髓细胞诱导AFP和白蛋白的作用

目的: 比较体外HGF诱导大鼠和人骨髓干细胞向肝细胞转分化的作用.方法: SD大鼠骨髓单个核细胞分为单纯IMDM/F12培养基对照组(C1组)和肝细胞生长因子组(H1组,加HGF 20 μg/L).人骨髓单个核细胞分为单纯IMDM/F12培养基对照组(C2组)和肝细胞生长因子组(H2组,加HGF 20 μg/L).分别留取培养10,20 d的细胞,用AFP和白蛋白作为细胞标志,用免疫组化和Western Blotting方法,观察HGF对骨髓细胞转化的诱导作用.结果: 人和大鼠骨髓细胞培养后10,20 d的H1和H2组,AFP免疫组化和Western Blotting染色阳性;用单纯IMDM/F12培养基培养(C1和C2)10～20 d后,AFP表达阴性.人和大鼠新鲜骨髓细胞白蛋白免疫组化可见少量阳性染色细胞,H1和H2组培养后10,20 d,用免疫组化和Western Blotting法检测,可见白蛋白表达增强.结论: HGF可诱导体外培养的人和大鼠骨髓细胞表达AFP 和白蛋白.     

AFP-L3、HSP70及AFP水平在大鼠肝癌发展中的动态变化

目的：通过动态检测大鼠肝癌模型造模各阶段血清中AFP-L3、HSP70和AFP水平的变化,对其在肝癌早期诊断中的价值做综合评价。方法：SD大鼠CCl4橄榄油溶液腹壁皮下注射,食用白酒溶液代替饮用水,建立大鼠肝硬化基础上的肝癌模型。从造模第6周起,每2周心脏采血1次,ELISA法检测大鼠血清中AFP-L3、HSP70和AFP水平,并取部分肝组织行病理学检验造模进度。结果：造模第8周发生肝硬化,第10周发生不典型增生及早期肝癌结节。AFP-L3和HSP70在造模第10周即显著升高,其阳性率分别为60%和70%,两者联合检测的阳性率为90%,HSP70在肝硬化阶段也有升高,AFP-L3没有显著变化。AFP水平在造模第12周才有显著升高,阳性率为60%,肝硬化阶段也略有升高,肝癌早期无显著变化。AFP-L3/AFP比值在造模第10周即显著升高（35.5%）,其敏感性和特异性分别达70%和100%。结论：成功建立了大鼠肝硬化基础上的肝癌模型,传统指标AFP对极早期的肝癌不敏感,特异性不高,而AFP-L3和HSP70均较AFP敏感,两者联合可提高检测的敏感性,AFP-L3和AFP-L3/AFP比值均较AFP特异。     

测定大鼠血清甲胎蛋白的酶联免疫吸附法

用自制的甲胎蛋白(AFP)抗体和酶标AFP抗体,建立大鼠血清AFP定量的酶联免疫吸附法(ELISA)即双抗体夹心法。对方法的特异性和重复性进行了研究。测定时在酶标抗体溶液中加入0.5％的牛血清白蛋白可减少塑料板小孔对酶标抗体的非特异吸附。用本法对127例正常雄性Wistar成鼠血清AFP进行了测定,结果为30.35±31.34ng/ml,正常值范围应在115.25ng/ml以下。     

体外诱导人骨髓间质干细胞分化为肝细胞样细胞

目的:探讨人骨髓间质干细胞(MSCs)在体外特定条件下是否可以转化为肝细胞样细胞.方法:分离培养人骨髓间质干细胞,采用肝细胞生长因子(hepatocyte growth factor, HGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)及CCl4损伤的小鼠肝脏共培养等诱导方式,在不同时间点分别用RT-PCR和荧光定量PCR检测肝细胞特异性基因的表达,免疫组织化学染色检测肝细胞标志. 结果: 在HGF和bFGF诱导第7天时甲胎蛋白(alpha fetal protein,AFP)、细胞角蛋白18(cytokeratin-18, CK18)和色氨酸2,3-双加氧酶(tryptophan 2,3-dioxygenase,TDO)基因表达阳性,CK18和TDO的表达随诱导时间延长增高;第7天诱导细胞组织化学染色AFP、白蛋白(ALB)、肝细胞核因子(hepatocyte nuclear factor,HNF)、肝细胞特异性抗原(HSA)和CK18表达阳性.与损伤小鼠肝组织共培养21 d后细胞表达AFP和TDO.结论:HGF和bFGF联合诱导以及与CCl4损伤肝组织共培养的方法可体外促进人MSCs分化为肝细胞样细胞,可为肝脏疾病或肝损伤的干细胞治疗提供细胞来源.     

A novel diagnostic marker, p28(GANK) distinguishes hepatocellular carcinoma from potential mimics

PURPOSE. To investigate the sensitivity, specificity, and spatial distribution of the product of p28 gene (p28(GANK) protein) in human hepatocellular carcinoma (HCC) and nonhepatocellular carcinomas in relation to immunostaining with Cytokeratin 18 (CK18), alpha-fetoprotein (AFP), and Hepatocyte paraffin 1 (HepPar1). METHOD. In this retrospective study, formalin-fixed paraffin-embedded tissues from 24 HCCs, five intrahepatic cholangiocarcinomas (ICC), five combined hepatocellular cholangiocarcinomas (C-HCC-CC) and mine metastatic hepatic carcinomas (MHC) were immunostained for p28(GANK) as well as CK18, AFP and HepPar1. Only cases with more intensified staining in carcinoma contrast to the adjacent liver tissues were accepted as positive. RESULT. In HCC, p28(GANK) was expressed restrictively in hepatocytes of both para-lesion and carcinoma liver tissues, while absent in the bile duct epithelial cells, Kupffer cells, and other interstitial cells. The positive staining of p28(GANK) was noted in 16 (66.7％) specimens of HCC and three (60.0％) specimens of C-HCC-CC, and no specific lesion staining was found in all tested specimens of ICC and MHC. Sensitivity and specificity for hepatocyte-originated carcinoma were, respectively, 65.5％ and 100％ for p28(GANK), 79.3％ and 85.2％ for CK18, 20.7％ and 100％ for AFP, 79.3％ and 92.0％ for HepPar1. CONCLUSION. The hepatocytic staining for p28(GANK) is clearly useful in differentiating hepatocyte-originated carcinoma from non-HCC. p28(GANK) may be used as an ancillary marker for the diagnosis of HCC.     

胚胎干细胞向肝细胞定向诱导分化的体外实验研究

目的 探讨胚胎干细胞体外定向诱导分化为肝细胞的条件和机制。方法 选用Balb／c小鼠胚胎干细胞,经过拟胚体发育分化5d后开始定向诱导培养,将拟胚体转入白明胶处理的培养板中贴壁生长,在不同时间段分别在细胞培养基中添加酸性成纤维细胞生长因子(aFGF)、肝细胞生长因子(HGF)等细胞生长因子进行肝细胞定向诱导。细胞分化过程的形态学变化用倒置显微镜追踪观察;用放免法对细胞培养上清中抗小鼠甲胎蛋白(AFP)和抗小鼠白蛋白(ALB)进行动态检测,用免疫细胞化学法检测肝细胞分化标记物AFP,ALB,CK8,CK18,并计算其阳性表达率(肝细胞分化率)。结果 胚胎干细胞在重组小鼠白血病抑制因子(LIF)存在的情况下保持未分化生长状态,撤去LIF后很快发育为拟胚体并继续分化。在第8天检测到上清中AFP出现,浓度为3．4n/ml,ALB在第11天开始检测到,浓度为0．2μg／ml,随培养时间延长,AFP和ALB浓度逐渐增加。第8天分化细胞开始出现AFP阳性,第10天出现CK8、CK18阳性,第11天出现ALB阳性,阳性细胞结构符合正常肝细胞结构特点。计算分化细胞中ALB阳性细胞率,得到肝细胞的分化率为30％。结论 胚胎干细胞经过拟胚体发育阶段,在特定培养条件和aFGF、HGF、等生长因子的作用下,可定向分化为肝细胞,这种细胞分化系统有望成为肝细胞替代疗法中的新型肝细胞来源。     

利用人脐带血干细胞制备人鼠肝组织嵌合体模型

目的利用人脐带血干细胞研究制备HBV感染人鼠嵌合小鼠模型。方法将人脐血干细胞,经尾静脉分两次注射到裸鼠体内。分别于第7,14,21天取肝组织,进行AFP免疫组织化学检测,分析人肝细胞在裸鼠体内嵌合生长情况,并进行乙肝病毒感染实验,定量检测感染后小鼠血清中AFP、ALB。结果实验组小鼠在第7、14、21天肝脏内AFP持续阳性表达,AFP表达于细胞质内。HBV感染后小鼠肝脏HBsAg组化显示密集成片的阳性细胞。实验组和对照组表达差异显著(P<0.05)。结论将人脐血干细胞移植裸鼠肝脏内可以存活并分化成人肝细胞形成嵌合鼠,并能被HBV感染。     

共培养诱导人骨髓多能成体祖细胞分化为肝细胞样细胞

目的探索人骨髓来源多能成体祖细胞（ZHJ-MAPC）在与人肝细胞系L02在体外共培养条件下诱导分化为肝细胞的可行性。方法（1）间接共培养：将ZHJ-MAPC和人肝细胞系L02仅培养液相通行间接共培养。分别于培养第1、3、5、7天应用免疫细胞化学法鉴定ZHJ-MAPC的白蛋白、甲胎蛋白（AFP）、细胞角蛋白-18（CK-18）、细胞角蛋白-19（CK-19）等肝细胞特征性表型的表达情况。（2）直接共培养：将荧光染料羧基荧光素二醋酸盐琥珀酰亚胺酯（CFSE,绿色）标记的ZHJ-MAPC与L02细胞（均为1×10^4个／ml）按照各50％比例混合行直接共培养。5d后采用荧光免疫细胞化学SABC-Cy3法（红色）双标后在激光共聚焦显微镜下分别观察ZHJ-MAPC表达白蛋白、AFP、CK-18的情况。结果（1）间接共培养结果：AFP在ZHJ-MAPC间接共培养第1天即表现为强阳性,随后表达逐渐减弱;白蛋白在第5天达到高峰;CK-18在第5天开始出现阳性,第7天出现较强表达。CK-19在各时间点均为阴性。（2）直接共培养结果：共培养第5天在检测白蛋白和CK-18表达情况时呈黄色荧光的阳性细胞即向肝细胞分化的ZHJ-MAPC出现较多,而AFP阳性表达则较少。结论人骨髓来源的ZHJ-MAPC与人肝细胞系L02行间接或直接共培养均能够诱导其向成熟肝样细胞定向分化。     

The comparative study on ultrastructure and immunohistochemistry in AFP negative and positive hepatocellular carcinoma

AFP,asasensitiveandspecificbiochemicalmarkerintheprimaryhepatocellularcarcinoma,playsanimportantroleingeneralsurvey,earlierdiagnosisanddeterminationofthetreatingeffectofhepatocellularcarcinoma(HCC).SerumAFPlevelofmostHCCincreased,bywhichthediagnosisrateof…     

鼻咽癌细胞株CNE-2及其亚克隆S-18裸鼠皮下移植生长速度比较

目的 研究鼻咽癌细胞株CNE-2及其亚克隆S-18在体内的生长速度差异及其可能机制.方法 体外培养CNE-2和S-18细胞,以6×105细胞/只移植到裸鼠背部皮下,移植后3、7、10、14 d观测肿瘤大小;移植后第14天处死小鼠,称取肿瘤重量并检测肿瘤组织内的HSP27和NF-ΚB转录水平.结果 (1)裸鼠皮下移植后7 d,S-18已明显成瘤,CNE-2尚未成瘤;移植后10 d和14 d,2种细胞均明显成瘤,但S-18肿瘤体积[(223.13±21.32)mm3,10 d;(420.25±24.52)mm3,14 d]明显大于CNE-2肿瘤体积[(113.70±11.70)mm3,10 d;(279.86±25.78)mm3,14 d],P<0.05;移植后14 d S-18所形成的肿瘤的重量(0.521±0.137)g明显高于CNE-2所形成的肿瘤重量(0.449±0.125)g,P<0.05;(2)S-18肿瘤内的HSP27、NF-ΚB的转录水平明显高于CNE-2肿瘤,P<0.05.结论 鼻咽癌细胞株S-18在体内的生长速度明显快于CNE-2细胞,HSP27和NF-ΚB可能参与了鼻咽癌细胞生长速度的调节.     

The comparative study on ultrastructure and immunohistochemistry in AFP negative and positive hepatocellular carcinoma.

Summary: To comparatively investigate ultrastructural characteristics and expressions of AFP (alpha-fetoprotein) and Tn (Thomsen-Friedenreich-related antigen) protein in AFP negative (AFP-) and AFP positive (AFP+) primary hepatocellular carcinoma. Fourty-three cases of AFP- and AFP+ hepatocellular carcinoma (HCC) tissues and five cases of normal liver tissues were divided into three groups: control group (normal liver tissue, n=5); AFP+ HCC group (the serum AFP level was higher than 10 ng/ml, n = 22); AFP- HCC group (the serum AFP level was lower than 10 ng/ml, n=21). The ultrastructural morphology was studied by transmission electron microscopy, the expressions of AFP and Tn protein were detected by immunohistochemistry and cell image analysis. 1. The immunohistochemical study showed that (1) the expression intensity and positive rate of Tn protein in AFP- HCC group were markedly higher than that in AFP+ HCC group (P<0.01); (2) The expression intensity of AFP in AFP- HCC group was lower than that in AFP+ HCC group (P<0.01). 2. The transmission electron microscopy demonstrated that some AFP- HCC cells linked closely with each other, others dispersed loosely just as cultured cells, the remarkable morphologic features in AFP- HCC cells were simple organelles, but they were abundant in the free polyribosomes. In AFP+ HCC group, all the HCC cells linked closely together and were rich organelles in their cytoplasm, especially the rough endoplasmic reticula. In addition, mitochondria and Golgi complex were obviously observed. (1) The AFP and Tn protein had discrepancy distribution in AFP- and AFP+ HCC tissues, Tn protein may be one of the early diagnostic indicators in AFP- HCC; (2) The synthetic locations of the AFP and Tn protein were different in hepatocarcinoma cells by ultrastructural observation.     

Hepatic differentiation from embryonic stem cells in vitro

Objective To investigate an method for hepatic differentiation from embryonic stem cells (ES cells) in vitro and the resulting differentiation ratio, in order to develop a procedure for producing a new type of hepatocyte for hepatocyte replacement therapy in the treament of liver failure.Methods ES cells from Balb/C mice were cultured and maintained in an undifferentiated state in gelatin-coated dishes using Dulbecco’s modified Eagle’s medium (DMEM) containing 1000 U/ml leukemia inhibitory factor (LIF). Then, LIF was withdrawn from the DMEM to allow the ES cells to develop into embryonic bodies (EBs). EBs were plated onto tissue culture dishes, and growth factors such as acidicfibroblast growth factor (aFGF) and hepatocyte growth factor (HGF) were added to the medium to promote directional differentiation. The course of development and differentiation was observed dynamically using an inversion microscope. The expression of hepatic proteins, such as α-fetoprotein (AFP), albumin (ALB), cytokeratin 8 (CK8), cytokeratin 18 (CK18), in cytoplasm was analyzed by immunocytochemistry (ICC). The concentration of ALB in the medium was determined dynamically by radioimmunoassay (RIA). Results ES cells replicated as clones, without differentiating, in DMEM containing LIF. They developed into EBs in medium without LIF. Our ICC assay showed that differentiating cells did not express hepatic proteins, such as AFP, ALB, CK8, and CK18 until day 7, day 9, day 11, and day 11, respectively (up to 2 days later when growth factors are not present). The concentration of AFP in the medium was first detected on day 8, at a concentration of 3.4 ng/ml, and increased to 22.8 ng/ml by day 15. The concentration of ALB in the medium was 0.2 μg/ml on day 11, and increased to 2.2 μg/ml by day 15. ALB-positive cells under ICC manifest morphological structures were consistent with normal mouse hepatocytes. The differentiation ratio of hepatocytes in the ES cell differentiation system was 30％ on day 15 (significantly lower without the presence of growth factors).Conclusions ES cells can differentiate into mature hepatocytes. Growth factors, such as aFGF and HGF, can enhance this differentiation and produce sufficient numbers of functional hepatocytes. This method may be a reliable new way of differentiating ES cells into hepatocytes for use in replacement therapy in the treament of liver failure.