罗丹明123分离的肝癌细胞系MHCC97亚群AFP的表达

目的:应用罗丹明123(Rho123)结合流式细胞术(FCM)对人肝癌细胞MHCC97进行分选,检测甲胎蛋白(AFP)在不同肝癌细胞亚群中的表达差异.方法:应用罗丹明123(Rho123)结合FCM将人肝癌细胞株MHCC97分为Rholow和Rhohigh组,采用免疫细胞化学方法观察AFP表达和含量.结果:Rholow组和Rhohigh组比较,AFP表达率有显著性差异(P<0.01).结论:AFP在肝癌细胞亚群中表达有差异,Rho123结合FCM可以进一步证实肿瘤的异质性.     

α-胎儿蛋白对人A和B型红细胞抗原与其特异性抗血清间凝集反应的抑制作用

α-胎儿蛋白(AFP)是来自胎儿肝脏的糖蛋白,在胎儿血清和羊水中浓度很高.母亲血清中AFP浓度在妊娠中期增高,分娩后迅速降低到正常成人水平.已证实AFP在体内、外对体液和细胞免疫有抑制作用,并参与妊娠时的免疫调节,在保护胎儿免受排斥中起作用.作者证实人AFP可抑制实验性重症肌无力(EAMG)和实验性变应性脑脊髓炎(EAE)的发展.还观察到AFP既能抑制重症肌无力又能抑制     

携带AFP基因慢病毒载体制备并感染树突状细胞的实验研究

目的:制备携带AFP基因的慢病毒, 并体外感染树突状细胞(DC) , 制备AFP-DC疫苗.方法:以人HepG2肝癌细胞cDNA为模板PCR 扩增出AFP基因片段, 将该片段克隆入慢病毒载体, 构建成Lenti-AFP慢病毒载体.其后, 用慢病毒载体转染293T细胞, 包装慢病毒表达载体.通过酶切、 PCR对Lenti-AFP慢病毒载体进行鉴定.包装好的慢病毒体外感染DC, 制备AFP-DC疫苗, 流式细胞仪检测感染率, 免疫荧光、 RT-PCR 法及电化学发光法检测AFP表达.结果:酶切、 PCR鉴定证实, 慢病毒载体插入片段为AFP基因.包装的慢病毒具有良好的感染性, 可以在293T细胞中形成病毒颗粒, 携带AFP基因的慢病毒感染DC, 经免疫荧光、 RT-PCR法及电化学发光法证实DC能够表达转染基因AFP, 并且不会影响DC表型, 感染率高达78.91%.结论:成功构建携带AFP基因的慢病毒载体, 感染树突状细胞后表达AFP, 为DC疫苗在肝癌中的临床应用提供了实验基础.     

肝硬化患者血清AFP、TNF-α、IL-6、IL-10检测的临床意义

目的:探讨了肝硬化患者血清中AFP、TNF-α、IL-6、IL-10水平及意义.方法:分别应用RIA和ELISA对63例肝硬化患者进行了血清AFP、TNF-α、IL-6、IL-10水平测定,并与35例正常健康人做比较.结果:肝硬化患者血清AFP、TNF-α、IL-6、IL-10水平均非常显著的高于正常人组(P＜0.01),且AFP水平与TNF-α、IL-6、IL-10水平呈明显的正相关(r=0.5988、0.6138、0.6284,P＜0.01).结论:AFP、TNF-α、IL-6、IL-10在肝硬化的防病机理中有一定的临床价值.     

一种敏感的人甲胎蛋白测定新方法——单克隆抗体三点夹心放射免疫测定法

人甲脂蛋白(AFP)是一种分子量约为70,000dalt的简单多肽,其表面有多种抗原决定簇。本文通过抗人AFP单克抗体(mAb)检测,发现人AFP至少有a、b和c三种抗原决定簇。a、b和c决定簇在AFP分子上有一定的排列格局,其中任何一种被相应特异mAb结合封闭后,并不影响另二种决定簇的活性。利用这一原理,本文作者创建了三点夹心固相放射免疫分析法(3—site sandwichtype solid phase radioimmunoassay)来检测人AFP。     

抗人AFP单抗及多抗与丝裂霉素联接物对肝癌细胞的杀伤作用

用本室制备的分泌IgG AFP McAb的杂交瘤株G2及G10,接种于BALB/c小鼠腹腔,取其腹水经硫酸铵及Sepharose 4B柱亲和层析纯化,获得抗人AFP单抗。将马抗人AFP血清经硫酸铵及Sepharose 4B柱亲和层析提取AFP抗体IgG,得到AFP多抗。将纯化     

MspI核酸内切酶消化α胎蛋白基因的DNA限制性多态性

最近已分离出人类α胎蛋白(AFP)cDNA探针.AFP基因定位在4号染色体q11-22,它与血清清蛋白基因连锁.本文作者利用限制性酶MspⅠ消化DNA,并利用5′中部及3′cDNA-AFP探针与人的DNA杂交,发现了AFP多态性的证据.作者研究了法国群体中大约60个巴黎人的DNA.当cDNA探针与受几种酶(EcoRⅠ,HindⅢ,HaeⅢ,PvuⅡ,PstⅠ,TaqⅠ)限制的人体DNA杂交时,没有观察到DNA的变异型.     

原发性肝癌患者介入治疗前后血清IGF-Ⅱ、CA19-9和AFP联检的临床意义

目的:探讨了原发性肝癌患者介入治疗前后血清IGF-Ⅱ、CA19-9和AFP水平的变化及临床意义.方法:应用放射免疫分析对35例原发性肝癌患者进行了血清IGF-Ⅱ、CA19-9和AFP含量检测,并与30名正常健康人作比较.结果:介入治疗前血清IGF-Ⅱ、CA19-9和AFP含量非常显著地高于正常人组(P＜0.01),介入治疗后一个月皆已接近正常.介入治疗6个月再测,未复发者仍然属正常水平,而复发者又恢复至介入前水平.结论:测定原发性肝癌患者血清中IGF-Ⅱ、CA19-9和AFP水平的变化对患者的治疗和预后均具有重要的临床价值.     

肝硬化患者血清Hcy水平与AFP、HA、PCⅢ的关系

目的:探讨肝硬化患者血清Hcy水平与AFP、HA、PCⅢ的关系.方法:分别应用放射免疫分析和酶免法对68例肝硬化患者进行了血清Hcy、AFP、HA和PCⅢ含量检测,并与35名正常人作比较.结果:肝硬化患者血清Hcy水平非常显著地高于正常人组(P＜0.01),尤以肝硬化腹水组(P＜0.001)更为显著,且与AFP、HA、PCⅢ呈明显的正相关.结论:检测肝硬化患者血清Hcy水平对观察病情和判断预后均具有重要的临床价值.     

AFP增强子驱动的HSV-TK/GCV自杀基因系统对肝癌细胞杀伤的实验研究

目的探讨人AFP增强子驱动的单纯疱疹病毒胸苷激酶/丙氧鸟苷（HSV-TK/GCV）自杀基因系统体外靶向杀伤肝癌细胞效应。方法构建人AFP增强子驱动的pAFP-CDNA3.1-TK自杀基因真核表达质粒,脂质体转染肝癌细胞,检测TK mRNA和蛋白表达,MTT法检测GCV对肝癌细胞的杀伤作用。结果成功构建pAFP-CDNA3.1-TK自杀基因真核表达质粒,在AFP阳性HepG2细胞中检测到TK mRNA和蛋白表达,添加GCV可特异性地杀伤HepG2细胞,而AFP阴性的SMMC7721细胞生长不受影响。结论 AFP增强子驱动的TK/GCV自杀基因系统可以靶向杀伤AFP阳性肝癌细胞。     

Characterization of Homologous Anti-Alpha-Feto-protein Antibodies Produced in Rabbits

The natural tolerance to alpha-fetoprotein (AFP) was broken in rabbits by immunizations with purified AFP from different species and homologous AFP chemically modified by haptenation. Some of the rabbits were boostered with rabbit AFP. The highest titers were obtained with human AFP, which shows a strong cross-reaction with rabbit AFP. The homologous antibodies were of lower avidity than heterologous (sheep) antisera. Injections with rabbit AFP did not alter the avidity or titer. All antibodies produced by injections with human AFP could be absorbed with the original immunogen, and no reactivity against determinants unique to rabbit AFP could be found. These results indicate that the antibody activity against autologous AFP is based on cross-reactivity and that the immunizations did not make the autologous AFP immunogenic. These findings may be important in view of the possible use of AFP as a target antigen in tumor immunotherapy.     

A Population of Human Cord Blood Mononuclear Cells with Surface Alpha Fetopfotein

Suspensions of mononuclear cells from adult peripheral blood (PBL) and mononuclear cells from cord blood (CBL) were examined for the presence of surface alpha-fetoprotein (AFP) using a fluoresceinated F(ab')₂ fragment of rabbit IgG anti-human AFP. The mean proportion of CBL with AFP was increased (10%) when compared with PBL (1%) although some CBL specimens did not demonstrate such an increase (range 0-15%). The presence of AFP on CBL could be either due to cytophilic AFP attached to a unique surface receptor or intrinsic AFP synthesis. The following observations could not distinguish between these two possibilities: (1) After treatment with trypsin, only minor reappearance of surface AFP could be observed in AFP - free medium in contrast to the larger numbers observed in medium containing AFP. Such selective reappearance depending on the media could be related to either cytophilic attachment of heterologous or homologous AFP or preferential stimulation of intrinsic AFP synthesis. (2) The reappearance of AFP positive CBL following trypsin treatment and incubation in media with or without AFP containing sera was inhibited by cyclohexamide. Such inhibition could be due to inhibition of synthesis of an AFP surface recpetor or intrinsic AFP. (3) The shedding of surface AFP observed at 2-4°C could be due to release of exogenous cytophilic AFP or the continued “turnover” of intrinsic AFP without concomitant AFP synthesis due to the cold temperature. Finally, the removal of AFP positive cells via selective depletion of B cells using bead columns coated with IgG-anti-IgG and the absence of depletion of AFP positive cells after successive gradient centrifugation of E-rosettes and cells with IgG-Fc receptors are consistent with the identity of AFP positive CBL as cells without IgG-Fc receptors or lymphocytes without conventional T-cell markers as defined by E-rosettes.     

A population of human cord blood mononuclear cells with surface alpha fetoprotein.

Suspensions of mononuclear cells from adult peripheral blood (PBL) and mononuclear cells from cord blood (CBL) were examined for the presence of surface alpha-fetoprotein (AFP) using a fluoresceinated F(ab')2 fragment of rabbit IgG anti-human AFP. The mean proportion of CBL with AFP was increased (10%) when compared with PBL (1%) although some CBL specimens did not demonstrate such an increase (range 0--15%). The presence of AFP on CBL could be either due to cytophilic AFP attached to a unique surface receptor or intrinsic AFP synthesis. The following observations could not distinguish between these two possibilities: (1) After treatment with trypsin, only minor reappearance of surface AFP could be observed in AFP-free medium in contrast to the larger numbers observed in medium containing AFP. Such selective reappearance depending on the media could be related to either cytophilic attachment of heterologous or homologous AFP or preferential stimulation of intrinsic AFP synthesis. (2) The reappearance of AFP positive CBL following trypsin treatment and incubation in media with or without AFP containing sera was inhibited by cyclohexamide. Such inhibition could be due to inhibition of synthesis of an AFP surface receptor or intrinsic AFP. (3) The shedding of surface AFP observed at 2--4 degrees C could be due to release of exogenous cytophilic AFP or the continued "turnover" of intrinsic AFP without concomitant AFP synthesis due to the cold temperature. Finally, the removal of AFP positive cells via selective depletion of B cells using bead columns coated with IgG-anti-IgG and the absence of depletion of AFP positive cells after successive gradient centrifugation of E-rosettes and cells with IgG-Fc receptors are consistent with the identity of AFP positive CBL as cells without IgG-Fc receptors or lymphocytes without conventional T-cell markers as defined by E-rosettes.     

Immunohistochemical Differentiation of Yolk Sac-type Alpha-Fetoprotein from Hepatic-type Alpha-Fetoprotein

In order to differentiate yolk sac type alpha fetoprotein (AFP) from hepatic-type AFP, 5 yolk sac tumors (YSTs) and 6 hepatocellular carcinomas (HCCs) were examined immunohistochemically by the peroxidase antiperoxidase (PAP) method for AFP, and paradoxical concanavalin A (P Con A) staining, which has been reported to detect glycoprotein including AFP. In all 5 YSTs, AFP was negative for P Con A staining. On the other hand, AFP was strongly positive for the same staining in all 6 HCCs. A similar staining pattern for AFP was observed in human yolk sac endodermal cells and embryonal hepatocytes. Thus, it was clarified that yolk sac type AFP was unable to bind with Con A, in contrast with hepatic-type AFP, on tissue sections. It was concluded that the PAP method for AFP and P Con A staining might facilitate the immunohistochemical differentiation of these two types of AFP, and that it would be us et ul for clarifying the histogenesis of various AFP secreting tumors.     

Immunohistochemical differentiation of yolk sac-type alpha-fetoprotein from hepatic-type alpha-fetoprotein

In order to differentiate yolk sac-type alpha-fetoprotein (AFP) from hepatic-type AFP, 5 yolk sac tumors (YSTs) and 6 hepatocellular carcinomas (HCCs) were examined immunohistochemically by the peroxidase-antiperoxidase (PAP) method for AFP, and paradoxical concanavalin A (P-Con A) staining, which has been reported to detect glycoprotein including AFP. In all 5 YSTs, AFP was negative for P-Con A staining. On the other hand, AFP was strongly positive for the same staining in all 6 HCCs. A similar staining pattern for AFP was observed in human yolk sac endodermal cells and embryonal hepatocytes. Thus, it was clarified that yolk sac-type AFP was unable to bind with Con A, in contrast with hepatic-type AFP, on tissue sections. It was concluded that the PAP method for AFP and P-Con A staining might facilitate the immunohistochemical differentiation of these two types of AFP, and that it would be useful for clarifying the histogenesis of various AFP-secreting tumors.     

Rabbit alpha-fetoprotein: normal levels and breakage tolerance with haptenated homologous alpha-fetoprotein

Nanogram quantities of alpha-fetoprotein (AFP), a tumor associated fetal protein, were found in the serum of normal adult rabbits by radioimmunoassay. This AFP was isolated and shown to be immunologically identical to fetal AFP by immunodiffusion. Immunizations of rabbits with unmodified or desialylated AFP in complete Freund's adjuvant did not cause antibody formation, indicating the existance of tolerance against homologous AFP. The tolerance could be terminated by immunizing with hapten-coupled AFP, which resulted in production of rabbit antibodies reacting with unmodified rabbit AFP.     

Differences between human and mouse alpha‐fetoprotein expression during early development

Alpha‐fetoprotein (AFP) is the major serum protein during development. AFP is one of the earliest proteins to be synthesised by the embryonic liver. The synthesis of AFP decreases dramatically after birth and only trace amounts are expressed in the adult liver. The tissue distribution of AFP in early human embryogenesis has not been defined. We have studied the expression pattern of AFP mRNA in human and mouse embryos by in situ hybridisation. In humans, AFP is expressed in the hepatic diverticulum at 26 d postovulation as it differentiates from the foregut endoderm (i.e. in the most primitive hepatocytes). It is also expressed in the endoderm of the gastrointestinal tract and in the yolk sac at this age. AFP is subsequently expressed in the mesonephros and transiently in the developing pancreas. In the mouse, no expression of AFP was observed in the mesonephros but other sites of expression were similar. Thus AFP has a distinct temporospatial expression pattern during the embryonic period and this differs between human and mouse species. It is interesting that AFP is expressed by tumours such as primitive gastrointestinal, renal cell and pancreatic tumours as well as those of hepatocyte origin. This distribution reflects the sites of AFP expression during development.     

Differences between human and mouse alpha-fetoprotein expression during early development

Alpha-fetoprotein (AFP) is the major serum protein during development. AFP is one of the earliest proteins to be synthesised by the embryonic liver. The synthesis of AFP decreases dramatically after birth and only trace amounts are expressed in the adult liver. The tissue distribution of AFP in early human embryogenesis has not been defined. We have studied the expression pattern of AFP mRNA in human and mouse embryos by in situ hybridisation. In humans, AFP is expressed in the hepatic diverticulum at 26 d postovulation as it differentiates from the foregut endoderm (i.e. in the most primitive hepatocytes). It is also expressed in the endoderm of the gastrointestinal tract and in the yolk sac at this age. AFP is subsequently expressed in the mesonephros and transiently in the developing pancreas. In the mouse, no expression of AFP was observed in the mesonephros but other sites of expression were similar. Thus AFP has a distinct temporospatial expression pattern during the embryonic period and this differs between human and mouse species. It is interesting that AFP is expressed by tumours such as primitive gastrointestinal, renal cell and pancreatic tumours as well as those of hepatocyte origin. This distribution reflects the sites of AFP expression during development.     

Tyrosinemia type I treated by NTBC: how does AFP predict liver cancer?

BACKGROUND: Tyrosinemia type I is associated with an increased risk of liver cancer development. The formation of the pathogenic fumarylacetoacetate is prevented by 2-(2-nitro-4-3 trifluoro-methylbenzoyl)-1,3-cyclohexanedione (NTBC). Still, some patients with NTBC treatment develop liver cancer. A rise of alpha-fetoprotein (AFP) is an indicator of liver cancer. AIM: To study the predictive value of AFP in tyrosinemia type I patients for the discrimination between patients at high and low risk of liver cancer development. METHODS: We examined the course of AFP values of 11 Dutch patients with tyrosinemia type I treated by NTBC, of whom four were diagnosed with liver cancer. RESULTS: The four patients with liver cancer had a course of AFP different from the other patients in either velocity of the decrease of AFP, achieving normal AFP and/or having a rise of AFP concentrations. CONCLUSION: Apart from a rise of AFP, a slow AFP decrease, and never normalizing levels of AFP are important predictors of liver cancer development in further life.     

α-Fetoprotein Synthesis in Yolk Sac Tumor: Sex-dependent Production of α-Fetoprotein by Transplantable Rat Yolk Sac Tumor

Transplantable rat yolk sac tumor cells produce α-fetoprotein (AFP) and their ability to synthesize AFP is maintained for generations in female rats. However, when they are transplanted in male rats, the AFP production decreases markedly.
To examine this sex dependency in AFP production, experiments at cellular and subcellular levels were carried out. In cell incubation studies, yolk sac tumor cells maintained in female rat (YST-F cells) synthesized more AFP than yolk sac tumor cells maintained in male rat (YST-M cells). Using cytosol RNAs prepared from YST-F and YST-M cells, AFP production was studied in cell-free protein synthesizing system derived from wheat germ.
In this system, cytosol RNA from both YST-F and YST-M cells directed AFP synthesis. But the amount of AFP synthesized was smaller in the presence of RNA from YST-M cells. From these results, it was suggested that the reduced AFP synthesis by YST-M cells is, at least partly, due to a quantitative decrease in their cytosol messenger RNA coded for AFP.