肝癌Bel7402细胞HLA-E基因上调对NK细胞体外杀伤作用的影响

目的: 探讨肝癌Bel7402细胞人类白细胞抗原-E(HLA-E)基因上调对NK细胞体外杀伤作用的影响。方法: 体外构建Lentivirus/CMV/GFP-HLA-E慢病毒表达载体并转导肝癌Bel7402细胞,采用RT-PCR和Western blotting方法检测肝癌Bel7402细胞HLA-E基因mRNA水平和蛋白水平的表达,分析肝癌Bel7402细胞HLA-E基因上调及HLA-ABC抗体封闭靶细胞相应位点对NK细胞体外杀伤作用的影响。结果: Lentivirus/CMV/GFP-HLA-E重组慢病毒载体感染Bel7402细胞后不同时段(24 h、48 h、72 h、96 h)的HLA-E mRNA的表达明显上调,除在感染后24 h(P＜0.05)外,慢病毒过表达组在感染后48 h、72 h、96 h与Bel7402组比较,显著差异 (P＜0.01)。蛋白水平的表达亦明显上调:24 h、48 h、72 h、96 h外源性/内源性HLA-E吸光度比值与12 h比较显著差异 (P＜0.01)。未封闭的Bel7402组和Bel7402 Lenti HLA-E组比较,NK细胞的杀瘤活性差异显著(P＜0.05或P<0.01);封闭组和未封闭组比较,NK细胞的杀瘤活性差异显著(P＜0.05);Bel7402(封闭组) 和Bel7402 Lenti HLA-E(封闭组)比较,NK细胞的杀瘤活性有显著差异(P＜0.01)。结论: Lentivirus/CMV/GFP-HLA-E慢病毒表达载体能有效增加HLA-E的表达。NK细胞对HLA-E基因表达上调的Bel7402细胞的靶杀伤作用降低;抗HLA-ABC单抗封闭靶细胞相应位点后,NK细胞对靶细胞的杀伤能力普遍有所提高。     

携带人MAGE-1基因重组腺病毒的构建及其在肝癌细胞中的表达

目的 构建人黑色素瘤相关抗原-1(MAGE-1)基因的腺病毒,并研究其感染肝癌细胞系HepG2细胞的效率.方法 采用基因克隆技术,将人MAGE-1基因克隆到腺病毒穿梭质粒pShuttle-IRES-hrGFP中,构建pShuttle-MAGE-1-IRES-hrGFP质粒,利用细菌BJ5183将穿梭质粒与pAdEasy-1进行重组,获得重组的腺病毒质粒.线性化的腺病毒质粒经脂质体转染AD293细胞,进行重组腺病毒的包装和扩增.采用CsCl密度梯度离心进行病毒浓缩和纯化.获得的腺病毒感染肝癌细胞HepG2,观察其感染效率和MAGE-1表达水平.结果 通过酶切鉴定证明腺病毒载体构建成功,包装出携带人MAGE-1基因的腺病毒,滴度达2.1×1010pfu/mL,获得的腺病毒对HepG2细胞的感染效率高于98%以上.结论 成功地利用细菌内同源重组方法构建了携带人MAGE-1基因的腺病毒,并能够在肝癌细胞中高效地表达,为利用MAGE-1进行免疫治疗提供了材料.     

HLA-G+调节性T细胞在胎膜早破患者的表达差异

目的通过观察人类白细胞抗原G(HLA-G)在胎膜早破患者与正常妊娠胎盘组织及外周血中的表达差异,探讨HLA-G在母胎耐受中的意义。方法分组:选取本院2010年5月～10月行剖宫产分娩产妇40例,其中胎膜早破组20例(实验组),正常产妇组20例(对照组)。采用流式细胞术检测两组产妇外周血及胎盘组织的HLA-G+的表达情况,比较两组表达差异。结果胎膜早破患者胎盘组织中HLA-G+表达显著低于正常妊娠组,差异有统计学意义(P<0.05)。结论 HLA-G+在胎盘中的低表达可能与胎膜早破的发生有关,对于母胎耐受的维持有意义。     

人肝癌细胞系HepG2和Huh7中的基因组拷贝数变异分析

目的 利用人肝细胞癌拷贝数变异和转录组实验,结合临床公共数据,探索肝细胞癌的拷贝数变异对肝癌发生发展的影响。方法 用光学基因组图谱技术识别肝癌细胞系HepG2和Huh7中的拷贝数变异,随后分析两种细胞系拷贝数变异基因的功能,并根据富集通路绘制蛋白质相互作用网络。选取两个细胞系核心网络中的关键基因,分析肝细胞癌拷贝数变异与基因表达之间的关系。GEPIA数据库分析基因表达与患者临床生存的关系。用RNA-seq实验和公共数据验证基因的表达水平。结果 HepG2细胞主要存在拷贝数增加,相关基因富集在雌激素信号通路、金黄色葡萄球菌感染等通路;Huh7细胞系中既有拷贝数增加又有减少,相关基因主要富集嗅觉传导、细胞因子-细胞因子受体相互作用等通路。关键基因SRC、MAPK3和MAP3K7拷贝数与基因表达成正比,并且高表达这些基因的患者生存期显著降低(P<0.05)。相比于HEK293T细胞系,SRC、MAP3K7基因在两个肝癌细胞系的表达显著升高(P<0.001),提示了肝细胞癌的特异性变异,MAPK3无差异。结论 肝细胞癌拷贝数变异基因SRC、MAP3K7的表达与患者的预后显著相关,可...     

人类白细胞抗原G和人类白细胞抗原E在宫颈癌中的表达及临床意义

目的 评估人类白细胞抗原G(HLA-G)和人类白细胞抗原E(HLA-E)的表达在宫颈癌进展中的意义及对患者预后的影响.方法 采用聚合酶链反应和流式细胞术检测正常组、CIN Ⅰ、CINⅡ、CINⅢ(宫颈上皮内瘤变Ⅰ-Ⅲ级)及浸润癌组HLA-G和HLA-E基因及蛋白的表达,比较两者在各组表达的差异及其对病人长期生存的影响.结果 CINⅢ及浸润癌组HLA-G表达与CIN Ⅰ、CINⅡ和正常组比较U值分别为564.0、536.0和565.5,P均＜0.05,HLA-E前两组的表达与后者比较U值分别为550.0、542.0和578.5,P均＜0.05;并且HLA-G和HLA-E阳性的患者生存率明显低于阴性者(P＜0.05).结论 HLA-G和HLA-E的异常表达促使宫颈癌的发生和发展,并且影响患者的术后长期生存.     

Gas2基因在肝细胞癌中的表达及意义

已知Ｇａｓ2在细胞周期阻滞时特异性表达 ,并因其被证实参与细胞凋亡而引起人们的关注[1] 。我们对肝细胞肝癌(ＨＣＣ)和癌旁及正常肝组织中Ｇａｓ2的原位表达作对比观察 ,结合ＨＣＣ中细胞增殖及凋亡状况 ,分析Ｇａｓ2在ＨＣＣ发生、发展中的可能作用。一、材料与方法1.标本处理 :36例ＨＣＣ及相应癌旁组织取自 1999年复旦大学附属中山医院首次住院手术患者 ,另有 8例正常组织 ,取自同期因良性肿瘤而部分切除的肝手术标本。2 .原位杂交检测Ｇａｓ2ｍＲＮＡ :探针由上海肿瘤研究所提供 (与ＧｅｎｅＢａｎｋ的Ｇａｓ2ｍＲＮＡ序列有 98.6 %同…     

Expression of apolipoprotein M in human hepatocellular carcinoma tissues

The present study examined mRNA levels and protein mass of apolipoprotein M (apoM) in human hepatocellular carcinoma (HCC) tissues and in the adjacent tissues. Plasma apoM levels in these HCC patients were also determined and compared to the normal subjects. The mean level of plasma apoM in the HCC patients was 0.61±0.30 OD mm⁻², which was significantly higher than that in the normal subjects 0.37±0.07 OD mm⁻² (P<0.01). However, both apoM mRNA levels and apoM protein mass in the HCC tissues were significantly lower than in the adjacent tissues (P<0.05). It is concluded that human hepatocellular carcinoma tissues had a reduced capacity to produce apoM than the adjacent non-tumor tissues. However, the plasma apoM levels were higher in the HCC patients than in normal subjects, which suggested that tissues adjacent to the tumors or extra-hepatic apoM production in the HCC patients may contribute to the higher plasma apoM levels in these patients. The clinical significance of apoM in relation to HCC still needs further investigation.     

HLA-DR抗原在慢性乙型肝炎和肝细胞癌中的表达及其意义

目的　探讨HLA DR抗原在慢性乙型肝炎和肝细胞癌 (HCC)中的表达及其意义。方法采用免疫组化技术对 2 0例正常肝组织、36例慢性乙型肝炎和 44例HCC中HLA DR抗原的表达进行检测。结果　正常肝组织中肝细胞未见HLA DR抗原表达。慢性乙型肝炎肝细胞HLA DR抗原表达阳性率为 2 7.8% ,其中 ,中度和重度肝炎HLA DR抗原阳性率明显高于轻度肝炎 (阳性率分别为 37.5 %和 2 0 % ,χ2 =13.6 ,P <0 .0 1)。HCC中肿瘤细胞HLA DR抗原表达阳性率为 43.2 %。HLA DR抗原表达与癌周淋巴细胞浸润 (χ2 =0 .5 1,P >0 .0 5 )和转移 (χ2 =2 .9,P >0 .0 5 )无关 ,但与癌组织分化程度有关 (χ2 =4.9,P <0 .0 5 )。结论　HLA DR抗原的异常表达在慢性乙型肝炎免疫损伤、免疫保护和HCC发生、发展中起重要作用     

肺癌组织的HLA-I类分子表达与临床病理因素关系

目的 研究肺癌组织的人类白细胞抗原I类分子HLA-A,B,C,G的表达情况以及临床病理因素关系.方法 抗HLA-A,B,C单克隆抗体(mAb)W6 /32 ,抗HLA-G (mAb)GG11,以免疫组化S-P法检测49例肺癌中的HLA-A,B,C,G抗原表达情况, 同时收集肺癌患者临床特征.结果 小细胞肺癌、鳞癌和腺癌的HLA-A,B, C抗原表达有不同程度的下降或缺失,小细胞肺癌HLA-A,B,C抗原下降和缺失率最高(P ＜0.05);而不同病理分期、分级的肺癌HLA-A,B,C抗原表达并无差异(P＞0.05).H LA-G在49例原发肺癌癌细胞中均无阳性表达,但4例肿瘤组织旁的部分免疫细胞上有阳性表达.结论 肺癌HLA-A,B,C抗原表达下降与肺癌组织类型相关,而与临床病理因素无关;HLA-G在肺癌细胞无表达,而部分免疫细胞表达HLA-G.肺癌可通过HLA-I 类分子诱导免疫耐受逃避宿主的免疫监控     

Hepatitis B surface antigen and hepatocellular carcinoma in Southern Africa

Summary Most series in Africa show a high percentage of hepatitis B surface Hepatitis antigen in hepatocellular carcinoma. Two groups of cases were investigated in this study. The one was derived from the autopsy material at Baragwanath hospital from subjects who had lived in Soweto, a large Black urban town. The second group consisted of male Black mineworkers generally originating from rural areas. A combination of the aldehydefuchsin stain and immunoperoxidase technique was used. The two groups showed totally different results. The Baragwanath series consisted of 24 hepatocellular carcinomas of which only 4 (17%) were HBsAg positive. Of the 24 cases, 14 had cirrhosis of which 9 were macronodular and 5 micronodular. Ten of these cases showed heavy iron overload. The series of male Black mineworkers comprised 22 cases of which 16 (72%) were HBsAg positive. Twelve of the 22 cases showed a macronodular cirrhosis and there were no micronodular cirrhoses. Only one case showed severe iron overload. These findings delineate two different populations of hepatocellular carcinoma in Southern Africa.     

Non-classical MHC-I human leukocyte antigen (HLA-G) in hepatotropic viral infections and in hepatocellular carcinoma

The human leukocyte antigen (HLA)-G is a “nonclassical” major histocompatibility complex (MHC) class Ib gene, located at chromosome 6, in the 6p21.3 region.     

建立猪MHCⅠ类抗原特异性人T细胞系的探讨

目的　建立间接识别中国版纳猪SLAⅠ类P1分子的特异性人T细胞系的方法 ,以进一步研究SLAⅠ类分子的间接识别在猪→人异种细胞性排斥中的作用。方法　以E·coli中表达并纯化的SLAⅠ类分子P1为抗原 ,体外反复刺激健康人PBMC ,3H TdR掺入法筛选特异性增殖的T细胞 ,FACS作表型初步分析。结果　初步测定健康人外周血版纳猪SLAⅠ类P1分子特异性T细胞反应频率约 6 .67× 1 0 - 7,所建 4个T细胞系表型均为TCRαβ+ ,其中 3株以CD4+ 为主 ,1株以CD8+ 为主。结论　利用E .coli表达的纯蛋白抗原可建立间接识别版纳猪SLAⅠ类P1分子的特异性人T细胞系。     

肝癌Hep3B细胞HLA-G过表达对NK细胞体外杀伤作用的影响

目的: 探讨肝癌Hep3B细胞白细胞抗原-G(HLA-G)过表达对NK细胞体外杀伤作用的影响。方法: 实验分为正常对照组(无干预措施, Hep3B组)、空载体对照组(转染空质粒, Hep3B-GFP组)和实验组(转染pEGFP-C1-HLA-G质粒,Hep3B-HLA-G组)。构建HLA-G真核表达质粒,转染至Hep3B细胞,经G418筛选,建立稳定过表达HLA-G的肝癌Hep3B细胞。Real-time PCR及Western blotting检测肝癌Hep3B细胞HLA-G mRNA及蛋白质水平的表达。分析肝癌Hep3B细胞HLA-G过表达及HLA-ABC抗体封闭靶细胞相应位点对NK细胞体外杀伤作用的影响。结果: 经酶切及测序鉴定证实, 插入序列与设计的序列相符。Real-time PCR和Western blotting结果均表明,转染重组质粒的Hep3B细胞HLA-G过表达(P<0.01)。NK细胞对转染pEGFP-C1-HLA-G的Hep3B细胞杀伤作用明显降低(P<0.01),HLA-ABC单抗封闭靶细胞相应位点后,NK细胞对Hep3B、Hep3B-GFP和Hep3B HLA-G细胞的杀伤均增加(P<0.01)。结论: pEGFP-C1-HLA-G表达载体可有效增加HLA-G表达,HLA-G过表达可以削弱NK细胞对肝癌细胞的杀伤效应。HLA-ABC单抗封闭靶细胞相应位点后,NK细胞对靶细胞的杀伤能力普遍有所提高。     

Carcinoembryonic antigen and lectin binding in the bile canalicular structures of hepatocellular carcinoma

Summary Bile canalicular structures of 32 hepatocellular carcinomas and 36 cirrhotic livers were investigated by light microscopy using immunohistochemistry for carcinoembryonic antigen (CEA) and lectin histochemistry. CEA positive bile canalicular structures were found in 17 out of 32(53%) hepatocellular carcinomas and in 33 of 36(92%) cirrhotic livers. Among the 10 lectins examined, about 40% of the CEA positive bile canalicular structures of hepatocellular carcinomas showed positive binding of MPA, DBA, WGA, RCA-I or UEA-I, whereas only MPA or RCA-I bound to the CEA positive bile canalicular structures of cirrhotic liver, in about 20% of cases. It has been shown that the CEA positive bile canalicular structures of hepatocellular carcinomas are heterogeneous and differ from those of cirrhotic liver in lectin histochemistry.Presented at the XVI International Congreess of International Academy of Pathology (Rikuo Machinami 1986)