原发性肝癌患者肿瘤浸润T淋巴细胞亚群分析

目的:了解原发性肝癌患者肿瘤浸润T淋巴细胞中CD4+及CD8+的表达特点。方法:收集30例原发性肝癌患者(均有乙型肝炎病毒感染背景)的癌组织(乙肝肝癌组)和癌旁组织(乙肝癌旁组)以及30例因良性病变而行肝切除的患者的新鲜肝组织(对照组),分离组织浸润淋巴细胞,用抗CD3、CD4和CD8单克隆抗体同时荧光染色,用流式细胞仪检测各表面标志的表达情况。结果:1)乙肝肝癌组、乙肝癌旁组及对照组CD3+CD4+T细胞占浸润淋巴细胞的比例分别为(22.31±3.68)%、(10.69±2.47)%及(4.21±4.26)%。乙肝肝癌组显著高于乙肝癌旁组及对照组,差异有统计学意义(P=0.000,P=0.001),乙肝癌旁组高于对照组,差异亦有统计学意义(P=0.019)。2)乙肝肝癌组、乙肝癌旁组及对照组CD3+CD8+T细胞占浸润淋巴细胞的比例分别为(26.10±5.82)%、(21.82±2.70)%及(41.31±14.01)%,乙肝肝癌组及乙肝癌旁组显著低于对照组,差异有统计学意义(P=0.014,P=0.004),而乙肝肝癌组与乙肝癌旁组之间差异无统计学意义(P=0.651)。3)乙肝肝癌组组织浸润淋巴细胞中CD3+CD4+T细胞与CD3+CD8+T细胞的比值为0.91±0.30,显著高于乙肝癌旁组(0.47±0.11,P=0.003)及对照组(0.11±0.13,P=0.000),CD3+CD4+与CD3+CD8+T细胞的比值出现失衡。结论:原发性肝癌患者肿瘤浸润淋巴细胞中T细胞亚群失调,表现为CD3+CD4+T细胞所占比例升高,CD3+CD8+T细胞所占的比例下降,CD3+CD4+/CD3+CD8+明显升高。     

22 CD4+CD25+调节性T细胞在肝癌微环境中的分布状况与局部免疫状态的关系

笔者对52例肝癌组织和癌旁组织用CD4和CD25双重酶标免疫组化染色标记Treg和CD4+T细胞，对20例肝癌组织和癌旁组织用CD8EnVision法染色标记CD8+T细胞，光镜下计数阳性细胞，对癌组织中Treg细胞和CD4+T,CD8+T及CD4+T/CD8+T值进行相关性分析，并用正常肝组织作对照。结果示8例正常肝组织中未发现Treg细胞。52例肝癌、癌旁组织中Treg细胞单个高倍视野平均数分别为7.6308±2.8368和5.1654±1.6718（P＝0.000）;肝癌中Treg细胞数目与肿瘤大小、有无子灶、有无癌栓及肿瘤临床病理分期有关，组间差异有显著性（P<0.05），而与患者年龄、性别、有无肝硬化、术前AFP浓度、肿瘤有无包膜及肿瘤Edmondson分级无明显关系（均P>0.05）;肝癌中Treg细胞数量与其浸润性CD4+T淋巴细胞的数量以及CD4+T/CD8+T值呈显著负相关（r=-0.539，P＝0.014;r=-0.545，P＝0.000），而与浸润性CD8+T淋巴细胞的数量分布无关（r=-0.403 P＝0.078）。提示肝癌微环境中的Treg细胞可能通过抑制肿瘤局部免疫，使肿瘤细胞逃避免疫监视，促进肿瘤的进展以及侵袭或转移;肝癌组织中的Treg细胞数量可能作为判断肝细胞癌预后的指标之一。     

免疫磁珠及无血清悬浮培养法对肝癌干细胞的分离与富集

目的评估免疫磁珠分选技术分选肝癌干细胞的效率。方法 (1)CD90的表达检测:采用免疫组织化学染色SP法检测8例正常肝组织、58例肝癌及其癌旁组织中CD90的表达。(2)细胞系筛选:将Huh-7、MHCC97-H、SMMC-7721及Bel7402细胞系分为空白对照组和实验组(细胞数量均为5.5×10~5个,均为1孔),实验组细胞中加入5μL CD90~–PE抗体,空白对照组加入5μL CD90~–无荧光抗体。采用流式细胞术检测4种细胞系空白对照组和实验组的CD90~+细胞比例,以筛选细胞。(3)免疫磁珠分选:将Huh-7和MHCC97-H细胞系分别进行磁珠标记后进行磁珠分选。收集流经磁珠分选柱(MS)后的细胞悬液1 m L,作为CD90~–组;将MS移出磁场区域,加入1 m L PBS缓冲液快速冲洗至离心管中,标记为CD90~+组;以未分选细胞作为空白对照组(加入CD90~–无荧光抗体)和未分选实验组(加入CD90~–PE抗体)。对Huh-7细胞系进行二次分选。采用流式细胞仪检测4组细胞中的CD90~+细胞比例。(4)无血清培养和含血清培养:将Huh-7细胞接种到无血清培养基专用6孔板中进行培养(无血清和含血清悬浮培养均为1孔),1周后采用流式细胞仪检测无血清培养和含血清培养后细胞中的CD90~+细胞比例。结果 (1)正常肝组织中CD90的表达阳性率为0(0/8),肝癌组织为65.5%(38/58),癌旁组织为20.7%(12/58)。肝癌组织中CD90的表达阳性率较正常肝组织(χ2=6.78,P0.05)和癌旁组织高(χ~2=20.83,P0.05)。(2)Huh-7、MHCC97-H、SMMC-7721及Bel7402细胞系中实验组的CD90~+细胞比例分别为0.851%、1.090%、2.710%及4.050%,空白对照组分别为0.241%、0.688%、1.890%及2.080%,故选择Huh-7和MHCC97-H细胞系进行下一步的免疫磁珠分选。(3)Huh-7细胞经一次分选后:未分选空白对照组的CD90~+细胞比例为0.241%,未分选实验组为0.851%,CD90~–组为0.574%,CD90~+组为1.100%;二次分选后:未分选空白对照组的CD90~+细胞比例为0.032%,未分选实验组为0.961%,CD90~–组为0.426%,CD90~+组为9.700%。结论正常肝组织实质细胞不表达CD90,肝癌组织高表达CD90。肝癌细胞系Huh-7和MHCC97-H中存在少量的CD90~+细胞,免疫磁珠分选法能明显提高CD90~+细胞比例,但作用十分有限。无血清悬浮培养法对富集CD90~+细胞无明显作用。     

原发性肝癌患者微环境中CD8+CD28-调节性T细胞表达变化及意义

目的：探讨原发性肝癌患者微环境中CD8+CD28-调节性T细胞(CD8+CD28-Treg)的表达变化及其意义。 方法：采用流式细胞检测技术对60例原发性肝癌患者及其50例健康患者的外周血中CD8+CD28-Treg进行检测,对于顺利手术切除的46例患者的肝癌组织及癌旁组织进行CD8+CD28-Treg的检测,再选取30例肝癌石蜡标本,检测其肝癌组织和癌旁组织中的CD8+CD28-Treg的表达。 结果：通过对肝癌组与健康对照组检测结果比较发现：CD8+CD28-Treg在原发性肝癌患者的外周血中测定值明显高于健康对照组[（15.55±3.14）% vs.（10.12±3.16）%],肝癌组织的CD8+CD28-Treg表达较癌周组织中的CD8+CD28-Treg表达增高[（19.34±3.12）% vs.（13.59±2.47）%],石蜡标本中肝癌组织CD8+CD28-Treg表达高于癌旁组织中CD8+CD28-Treg的表达[（13.58±2.46）% vs. （9.47±3.04）%],差异均有统计学意义（均P<0.05）。 结论：CD8+CD28-调节性T细胞在原发性肝癌患者的微环境中表达明显增加,其可能在机体对原发性肝癌的免疫应答中有重要的意义。     

肝癌患者CD8+CD28-Foxp3+调节性T细胞的变化及意义

目的:探讨肝癌患者外周血和癌组织中CD8~+CD28~-Foxp3~+调节性T细胞(CD8~+CD28-Foxp3~+Tregs)的改变及意义。方法:采用流式细胞仪检测72例肝癌患者和22例健康对照人群外周血CD8~+CD28~-Foxp3~+T细与CD8~+T细胞比值(CD8~+CD28-Foxp3~+Tregs/CD8~+T),分析CD8~+CD28~-Foxp3~+Tregs/CD8~+T与肝癌患者临床病理因素的关系;分别用免疫组化和Western blot检测肝癌患者癌组织及癌旁组织中Foxp3阳性细胞与Foxp3蛋白表达水平。结果:与健康对照人群比较,肝癌患者外周血CD8~+CD28~-Foxp3~+Tregs/CD8~+T明显升高(P0.05);外周血CD8~+CD28~-Foxp3~+Tregs/CD8~+T与患者TNM分期、淋巴结转移、分化程度有关(均P0.05);肝癌组织中平均Foxp3阳性细胞数与Foxp3蛋白表达量均明显高于癌旁组织(均P0.05);外周血CD8~+CD28~-Foxp3~+Tregs/CD8~+T,肝癌组织Foxp3阳性细胞数与Foxp3蛋白表达量在高、中、低分化的肝癌中均呈依次升高趋势,但差异均无统计学意义(均P0.05)。结论:CD8~+CD28~-Foxp3~+Tregs在肝癌患者外周血及癌组织中增多,可能与肿瘤免疫抑制作用有关,其检测对肝癌患者病情有一定评估价值。     

聚糖蛋白抗体的制备及其在肝癌组织及细胞系蛋白表达检测中的应用

目的制备聚糖蛋白3(glypican-3,GPC3)抗体并应用其对肝癌组织及细胞系中GPC3蛋白表达进行研究,探讨GPC3蛋白作为肝癌潜在的新的肿瘤标志物的可能性。方法利用原核表达系统诱导表达了GPC3抗原并制备了特异性好的兔抗GPC3多克隆抗体,应用该纯化的抗体对40例组织(正常肝7例,肝细胞型肝癌26例,胆管细胞型肝癌7例)及4个细胞系(肝癌细胞系HepG2、HuH-7、Hep3B,非肝癌细胞系Hela)进行了蛋白印记杂交试验。结果Western blot结果显示,肝癌组织及肝癌细胞系HepG2、HuH-7、Hep3B细胞系中有相对相对分子质量约70000的蛋白主带,与GPC3的核心蛋白带相对分子质量大小相一致;正常肝组织不表达GPC3蛋白,肝细胞型肝癌中癌组织(HCC)及癌旁组织表达GPC3蛋白的比例分别为84.6%(22/26)及30.8%(8/26),7例胆管细胞型肝癌(ICC)中有2例癌组织及其旁组织表达GPC3。结论GPC3蛋白广泛存在于肝癌组织中,很可能成为一种新的肝癌肿瘤标志物。     

长链非编码RNA Lnc-DQ在肝癌组织中的表达及对其对SMMC7721细胞干性特征的影响

目的 检测长链非编码RNALnc-DQ在肝癌组织中的表达,探讨其对肝癌SMMC7721细胞干性特征的影响。方法 采用实时定量反转录聚合酶链反应（RT-qPCR）检测Lnc-DQ在30例肝癌组织和癌旁组织中的表达;shRNA干扰下调Lnc-DQ在SMMC7721细胞中的表达,克隆形成和成球培养实验观察其对SMMC7721肿瘤干细胞特征的影响;流式细胞仪检测CD133+细胞亚群的比例。结果 Lnc-DQ在肝癌组织中的相对表达显著高于癌旁组织（3.24±0.34 vs 1.81±0.17）,差异具有统计学意义（P<0.05）。shRNA转染可显著下调Lnc-DQ在SMMC7721细胞中的表达（P<0.05）。实验组细胞（sh-Lnc-DQ）克隆形成数为43.62±5.35,较对照组（sh-NC）显著减少（94.31±5.07）,差异具有统计学意义（P<0.05）。成球培养实验显示sh-Lnc-DQ可显著抑制SMMC7721细胞的成球能力（P<0.05）。实验组细胞CD133+细胞比例显著低于对照组织（2.32%±0.25% vs 9.24%±0.69%）,差异具有统计学意义（P<0.05）。结论 Lnc-DQ在肝癌中高表达,下调Lnc-DQ的表达能够抑制肝癌SMMC7721细胞的干性功能。     

人肝癌组织原代巨噬细胞的提取与鉴定

目的研究人肝癌组织原代肿瘤相关性巨噬细胞（TAMs）的提取与鉴定方法。 方法收集原发性肝癌患者术中切取的肝癌组织标本15份,免疫组织化学染色方法检测肝癌组织中肿瘤相关巨噬细胞表面标志物CD68及CD206的表达。利用机械法和酶消化法结合分离TAMs,贴壁培养24 h后在倒置显微镜下进行细胞形态学观察,利用CD68和CD206作为标志物,流式细胞术鉴定细胞纯度。 结果癌巢和癌旁组织中均可见CD68、CD206阳性表达,阳性细胞表现为棕褐色;获得一定纯度的原代巨噬细胞,具备巨噬细胞的形态特征,其中CD68单阳性比例为36.29%,CD68和CD206双阳性比例为15.96%。 结论本研究采用的方法可有效提取和培养人肝癌组织来源的原代TAMs,对进一步研究人类肝癌组织TAMs特性及对肿瘤生物学行为的影响具有重要价值。     

原发性肝细胞癌、癌旁及肝硬化组织中CD3+细胞的研究

摘要：为探讨CD3在原发性肝细胞癌、癌旁、肝硬化及正常肝组织中的表达，并探讨CD3+细胞与肝癌、肝硬化的关系。笔者对60例原发性肝细胞癌组织、62例单纯性肝硬化及21例正常肝组织，用SP免疫组化法进行染色，定量分析其阳性细胞数。结果示： (1)CD3+细胞平均数从高到低依次为：肝癌癌旁组织、癌组织、肝硬化及正常肝组织（P<0.05）；(2)肝癌组织中CD3+细胞平均数与组织学分级和临床TNM分期无关；(3)肝癌15个月内有转移组的癌组织中CD3+细胞数少于无转移组（P<0.01）。提示CD3+细胞可作为反映机体抗肿瘤免疫状态及判断患者预后的重要指标。     

CD4+CD25+FOXP3+T细胞在肝癌肝移植肿瘤复发中的作用

目的 探讨肝癌肝移植病人移植前后外周血和肿瘤组织中CD4+CD25+FOXP3+T细胞比例变化及其临床意义.方法 用流式细胞仪检测肝癌肝移植病人和其他肝移植病人术后外周血中CD4+CD25+FOXP3+T细胞的比例,并采用正常人作对照.用免疫组化法检测肝癌病人和非肝癌病人肿瘤组织中FoxP3的表达及CD8+T细胞浸润的比例.观察肝癌肝移植病人术后及肿瘤复发后调节性T细胞的变化及其对肿瘤复发的影响.结果 流式细胞检测显示肝癌肝移植、非肝癌肝移植的病人术后外周血中CD4+CD25+FOXP3+T细胞占CD4+T细胞的比例较正常人明显升高,分别为(10.15±1.00)%、(5.30±1.64)%和(3.20±1.18)%,P<0.05.肝癌肝移植肿瘤复发病人较未复发病人外周血CD4+CD25+FOXP3+T比例明显升高,分别为(15.15±1.50)%和(6.80±1.50)%,P<0.01.免疫组化检测显示肿瘤组织中FOXP3+T细胞增多,CD8+T细胞浸润明显减少.结论 肝癌肝移植肿瘤复发的病人外周血中CD4+CD25+FOXP3+T细胞比例升高.调节性T细胞可能通过减少CD8+T细胞浸润,加速肿瘤复发.     

Effect of lentinan on lymphocyte subsets of peripheral blood,lymph nodes,and tumor tissues in patients with gastric cancer

The effect of lentinan on lymphocyte subsets of peripheral blood, lymph nodes, and tumor tissues in gastric cancer patients was investigated. A 2-mg dose of lentinan was administered to 12 patients intravenously twice, the first at 3–9 days before and the second the day before surgery. The results were then compared with a control group without lentinan administration comprising 12 patients. Regarding peripheral blood and lymph nodes without metastasis, lymphocyte subsets defined with anti-CD3, anti-CD4, anti-CD8, anti-Leu7, and anti-Leu11 were analyzed by flowcytometry. As for tumor tissues, lymphocyte subsets defined with anti-CD3, anti-CD4, anti-CD8, anti-Leu11, and anti-M3 were analyzed after immunohistochemical staining. There were no significant changes in the lymphocyte subsets of peripheral blood between the two groups. In the lymph nodes, the CD4 cell ratios increased; otherwise in regard to tumor-infiltrating lymphocytes (TILs), the number of CD4, Leu11 and LeuM3 cells showed a prominent increase. Therefore, lentinan was observed to produce different effects in accordance with the subjective organs.     

Failure of B7.1-modified tumor to evoke full activation of CD8+ tumor-infiltrating lymphocytes in the central nervous system: prevention of parental tumor growth in the subcutaneous environment

OBJECT: It is well known that the central nervous system (CNS) is an immunologically privileged site. To characterize CD8+ tumor-infiltrating lymphocytes (TILs) recovered from the CNS, the authors compared these cells with TILs recovered from subcutaneous tissue by using a B7.1 gene-modified tumor implantation model. METHODS: The authors established a B7.1 gene-modified EL4 murine lymphoma cell line (EL4-B7.1) and implanted the cells into the CNS to observe the duration of tumor-free survival. Although EL4-B7.1 cells were completely rejected in a subcutaneous implantation model, 40% of animals died after the CNS implantation (all animals in which the parent tumor was implanted died within 16 days). Therefore, the authors isolated TILs from each implantation site and analyzed the expressions of activation antigens CD25 and CD69 by performing the anti-CD8 magnetic beads separation method and flow cytometric analysis. After implantation of the parent tumor, there was no difference in the number of TILs from each site (CD25 1.7-3.2%, CD69 21.9-34.3%). After implantation of the B7.1-modified tumor, the CD25-expressing TIL population from the subcutaneous site was 4.68 times higher than that from the CNS site (17.8% compared with 3.8%). Based on these findings, the authors used a mitomycin C-treated EL4-B7.1 subcutaneous vaccination with various protocols. Vaccination before tumor challenge was sufficient to prevent the development of the tumor. For animals with established tumor, the vaccination protocol was able to prolong host survival (p = 0.0053). CONCLUSIONS: The data clearly demonstrate that the CNS environment fails to activate CD8+ TILs fully. These are the first data indicating in detail a difference between CD8+ TILs from the CNS and those from other sites based on a B7.1-modified tumor model.     

The mechanism of anti-CD3 monoclonal antibodies. Mediation of cytolysis by inter-T cell bridging

OKT3, an anti-CD3 MAB, depletes T cells in vivo and is among the most potent inhibitors of acute allograft rejection. The mechanism of this inhibition is unknown. The present studies investigate whether anti-CD3 antibodies have the ability to crosslink CD3 on two different cells and induce TCR-dependent antibody-bridged cell-mediated cytolysis (TCR-ABCMC) between T cells. Two different anti-CD3 antibodies (OKT3 and CD3,3) and OKT3 F(ab')2 were all highly effective in inducing cytolysis of CD8+ and CD4+ T cells by CD8+ T cells, and CD8+ T cells by CD4+ T cells. Monovalent OKT3 Fab was 25-125-fold less potent than OKT3 F(ab')2. Monovalent CD3,X was totally ineffective. The necessity for intercellular bridging was evidenced by the observation that an anti-CD3:anti-CD4 (CD3,4) bispecific MAB (BSMAB) was effective in mediating lysis of CD4+ but not CD8+ T cells by CD8+ T cells. These studies indicate that neither FcR-mediated ADCC nor complement fixation is necessary for bivalent anti-CD3 MAB to lyse T cells. Inter-T cell TCR-ABCMC may be particularly effective in inflammatory tissues, such as rejecting allografts and autoimmune diseases, in which numerous cytolytic T lymphocytes are present in close association with other T cells.     

Improved in vivo efficacy of tumor-infiltrating lymphocytes after restimulation with irradiated tumor cells in vitro

Background: We investigated different culture conditions for tumor-infiltrating lymphocytes (TILs) with regard to proliferation, phenotypic changes, in vitro cytotoxicity, and in vivo therapeutic efficacy. Methods: After enzymatic digestion of the murine fibrosarcoma, MCA-105, TIL cultures were initiated as pure lymphocyte (groups 1 and 2) or mixed lymphocyte/tumor suspensions (groups 3 and 4). Group 1 TILs were grown in culture medium containing 100 IU/ml recombinant interleukin-2 (rIL-2). Group 2 TILs were stimulated with solid-phase anti-CD3 monoclonal antibody (mAb) for 48 h and cultured in rIL-2 (100 IU/ml)-containing medium. Group 3, which consisted initially of a surplus of tumor cells, received the same treatment as group 2. Group 4 was also activated with anti-CD3 mAb and rIL-2 but was additionally restimulated weekly with irradiated tumor cells (TILs to tumor, 20:1). Results: Groups 1 and 2 showed up to twofold higher increases in TIL numbers compared with groups 3 and 4 by the end of culture week 5. Although the original lymphocyte/tumor cell suspension consisted of 12.0 ± 3.8% CD4⁺ T cells and 5.3 ± 3.3% CD8⁺ T cells, all four TIL cultures showed 80% CD8⁺ TILs and no CD4⁺ TILs by the end of culture week 4. In vitro cytotoxicity did not correlate with in vivo efficacy of the examined TIL cultures. By using the MCA-105 pulmonary metastases model in C57BL/6 mice, only suboptimal doses of TILs (2 × 10⁶) from group 4, which had been restimulated weekly with irradiated tumor, showed significant tumor eradication compared with all other treatment groups (p<0.01). Conclusions: We conclude that in vitro tumor restimulation of TILs improves in vivo efficacy, most likely through the education of tumor-reactive T cells.Presented at the 48th Annual Meeting of The Society of Surgical Oncology, Boston, Massachusetts, March 23–26, 1995.     

Characterization of T lymphocytes infiltrating human pancreas allograft affected by isletitis and recurrent diabetes.

We studied a human leukocyte antigen-identical pancreas graft transplanted into an insulin-dependent (type I) diabetic patient shortly after onset of recurrent diabetes to characterize the putative autoreactive T lymphocytes mediating the lesion. The immunohistopathological analysis revealed the presence of isletitis and a selective loss of beta-cells. The isletitis was mostly constituted by CD8+/T-lymphocyte receptor alpha,beta (TCR alpha,beta +) T lymphocytes surrounding and infiltrating the affected islets. CD4-/CD8-/TCR gamma, delta + T lymphocytes were observed within the islets. Incubation of the tissue in 15% interleukin 2 induced the migration and initial expansion of the infiltrating cells (66% CD3+ lymphocytes) for up to 2 wk; most T lymphocytes in this initial isolate were CD4+ (92% CD4+ and 7% CD8+). Long-term anti-CD3 stimulation of this T-lymphocyte population induced the selective growth of CD8+/TCR alpha,beta + (75%) and CD4-/CD8-/TCR gamma,delta + (all V1 delta +) (17%) T lymphocytes. Therefore, this strategy selectively expanded the T lymphocytes, found to be the predominantly islet-infiltrating cells, rather than the lymphocytes predominating in the initial isolate. Anti-CD3 did not stimulate growth of T lymphocytes in cultures of three isletitis-free pancreas graft biopsies. In a control experiment with a CD4(+)-rich T-lymphocyte population, long-term anti-CD3 stimulation and cloning of cytomegalovirus (CMV)-primed peripheral blood mononuclear cells from a CMV+ subject selectively induced the growth of CD4+ T-lymphocyte clones, all CMV specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clinical significance of tumor-infiltrating lymphocytes in neoplastic progression and lymph node metastasis of human breast cancer

To investigate the clinical significance of tumor-infiltrating lymphocytes (TILs) within the tumor milieu, we quantitatively measured and compared the subpopulations of TILs in 24 patients with stage I–III breast carcinoma. Peripheral blood mononuclear cells (PBMCs), normal breast parenchyma-infiltrating lymphocytes (NILs), and TILs were isolated from tissue specimens and quantified by flow cytometry. The results showed that increased proportion of CD8⁺ T cells, with decreased proportion of CD4⁺ T cells, was significant in gated CD3⁺ TILs as compared to autologous NILs or PBMCs (P < 0.001). The tumor-infiltrating CD8⁺ T cells significantly increased with stage progression, reflected in a more strongly decreased CD4/CD8 percentage (P = 0.003). The CD4/CD8 percentage of TILs was strongly correlated with lymphovascular permeation and subsequent lymph node metastasis (P < 0.001). Increased percentages of tumor-infiltrating CD8⁺ T cells with decreased CD4/CD8 percentages are of prognostic importance for cancer progression in human breast cancer.     

人肝癌血管内皮细胞分离培养、鉴定及比较

目的 建立人肝癌血管内皮细胞(TEC)分离培养方法并鉴定比较.方法 采用CD105、CD31抗体交联免疫磁珠方法优化分选TEC并传代培养;通过形态观察、功能实验、流式检测及实时聚合酶链反应(real-time PCR)等验证比较.结果 CD105+TEC呈细长"纤维条索状",CD31+TEC则呈"梭形";流式检测TEC表达CD68和α-平滑肌肌动蛋白(α-SMA)低于5%,排除巨噬细胞和成纤维细胞污染,PCR检测CD105+TEC、CD31+TEC甲胎蛋白mRNA显著低于肿瘤细胞HepG2(P＜0.01),表达水平低于HepG2的1/8,排除肿瘤细胞污染;95%以上阳性分选细胞呈乙酰化低密度脂蛋白摄取实验阳性;增殖实验检测CD105+TEC和CD31+TEC生长48 h的吸光度值分别为0.45±0.04和0.35±0.02(P＜0.05),CD105+TEC增殖能力强于CD31+TEC;TEC在Matrigel胶中可形成毛细管网状结构,CD31+TEC强于CD105+TEC.结论 CD105、CD31抗体交联免疫磁珠分选法均可分离得到高纯度人肝癌TEC.

Abstract：

Objective To isolate, identify and compare vascular endothelial cells derived from human hepatocellular carcinoma (HCC). Methods Modified immunomagnefic methods using magnetic beads conjugated with anti-CD105 and anti-CD31 antibody were used to isolate tumor-derived endothelial cells (TEC), namely CD31 +TEC and CD105 +TEC. CD31 +TEC and CD105 +TEC were cultured in vitro,identified and compared by morphological appearance, functional characterization, flow cytometry and realtime polymerase chain reaction (PCR) analyses. Results The isolated CD105 + TEC presented "fibrous bands" appearance while CD31 + TEC presented "fusiformis" appearance. Macrophages, fibrocytes, and tumor cells contamination was excluded. Surface CD68 and α-SMA expression was less than 5%. Alpha fetoprotein expression was significantly lower in CD31 + TEC and CD105 + TEC than that in HepG2 cells ( P ＜0. 01 ), and the expression level was less than 1/8 folds of HepG2 cells. Internalization of acetylated lowdensity lipoprotein was positive in more than 95% of isolated cells. The absorbance (A) values of CD105 +TEC and CD31 + TEC grown for 48 h were 0. 45 ± 0. 04 and 0. 35 ± 0. 02 respectively ( P ＜ 0. 05 ). Proliferation of CD105 + TEC was significantly more active than CD31 + TEC when cultured in the serum supplemented with medium for 24, 48, and 72 h. The isolated cells could form capillary-like tubes on Matrigel matrix, stronger capability of CD31 + TEC than CD105 + TEC. Conclusion CD31 + TEC and CD105 + TEC can be conveniently purified by modified immunomagnetic methods.     

长链非编码RNA ST8SIA6-AS1通过调节微小RNA-142-3p促进肝细胞癌的增殖和侵袭

目的探讨ST8SIA6-AS1靶向结合微小RNA(microRNA,miR)-142-3p,影响肝细胞癌的增殖和侵袭能力。方法用基因表达谱数据动态分析(GEPIA)验证ST8SIA6-AS1在肝细胞肝癌(HCC)和邻近正常组织中的差异表达。采用实时定量反转录聚合酶链反应(RT-qPCR)检测ST8SIA6-AS1和miR-142-3p在组织水平和细胞水平的表达量。采用细胞计数试剂盒(CCK-8)和集落形成实验检测人肝癌细胞Hep3B的增殖情况;采用迁移侵袭实验(Transwell法)检测肝癌Hep3B细胞迁移和侵袭情况。生物信息学、双荧光素酶报告实验验证ST8SIA6-AS1与miR-142-3P的靶向关系。组间采用t检验,多组间采用单因素方差分析进行比较,相关性分析采用Spearman秩检验。结果GEPIA结果显示ST8SIA6-AS1在肝癌组织中的表达明显高于正常组织。RT-qPCR结果显示ST8SIA6-AS1在HCC中的表达明显高于癌旁组织(0.778±0.363比1.951±0.575,t=11.370,P<0.05),差异有统计学意义。miR-142-3p在肝癌组织的表达量显著低于癌旁组织(0.881±0.374比0.371±0.164,t=9.395,P<0.05),差异有统计学意义。ST8SIA6-AS1在4种人HCC细胞株(HepG2、SMMC-7721、HCCLM3和Hep3B)的表达(3.84±0.27、5.81±0.60、4.47±0.55、5.94±0.69)明显高于正常肝细胞株L02(1.00±0.09,t=17.280,t=13.730,t=10.780,t=12.300,P<0.05),差异有统计学意义,ST8SIA6-AS1在敲低ST8SIA6-AS1的Hep3B细胞中的相对表达量低于对照组(0.26±0.04比1.00±0.01,t=9.423,P<0.05),差异有统计学意义。集落形成实验结果显示,敲低ST8SIA6-AS1的Hep3B细胞集落个数显著低于对照组[(32.60±6.70)个比(100.00±10.00)个,t=10.040,P<0.05],差异有统计学意义;CCK-8实验中敲低ST8SIA6-AS1的Hep3B细胞72 h吸光度(A)值低于对照组[(0.71±0.06)%比(1.14±0.09)%,t=6.886,P<0.05],差异有统计学意义。Transwell实验结果显示敲低ST8SIA6-AS1的Hep3B细胞垂直迁移率明显低于对照组[(53.38±6.49)%比(100.00±13.00)%,t=5.641,P<0.05],差异有统计学意义。敲低ST8SIA6-AS1的Hep3B细胞的侵袭率低于对照组[(44.98±8.42)%比(100.00±10.00)%,t=7.485,P<0.05],差异有统计学意义。双荧光素酶报告实验证实miR-142-3p过表达组明显低于对照组ST8SIA6-AS1-野生型(WT)细胞的荧光活性,ST8SIA6-AS1负向调控miR-142-3p的表达(0.42±0.05比1.00±0.09,t=9.757,P<0.05)。干扰ST8SIA6-AS1的表达可对肝癌细胞增殖、迁移、侵袭产生抑制作用,这种作用可通过沉默miR-142-3p发生逆转。结论ST8SIA6-AS1可通过竞争性结合miR-142-3p在肝癌中发挥致癌作用。     

T CELLS ACTIVATED IN VITRO AS IMMUNOTHERAPY FOR RENAL CELL CARCINOMA: CHARACTERIZATION OF 2 EFFECTOR T-CELL POPULATIONS

PURPOSE: Effector T cell populations generated using 2 methods of in vitro activation are currently being tested in separate clinical trials as immunotherapy for patients with advanced cancer, including renal cell carcinoma. To determine the most appropriate method of activation for cancer immunotherapy in vitro antitumor activity of the 2 effector T-cell populations were compared. METHODS AND METHODS: The effector T-cell populations were generated concurrently by activation of peripheral blood mononuclear cells from patients with advanced renal cell carcinoma or other cancer using soluble anti-CD3 monoclonal antibody (3T cells) or anti-CD3 and anti-CD28 monoclonal antibodies immobilized on beads (3/28T cells). After 14-day culture the phenotype and functional activity of the cells were tested. RESULTS: Fold expansion of CD4(+) cells for 3T cultures was lower than for 3/28T cultures but expansion of CD8(+) cells was similar for both cultures. Expression of CD69 was higher on 3T cells. 3T and 3/28T cells exhibited similar ability to kill various human tumor cell lines. Although both effector T-cell populations produced Th1-type cytokines upon re-stimulation, 3T cells secreted a higher level of interferon-gamma and tumor necrosis factor-alpha compared with 3/28T cells. Intracellular cytokine analysis demonstrated that the percent of cells producing interferon-gamma was higher in CD4(+), CD8(+), CD25(+), CD69(+) and CD45RO(+) 3T cells compared with 3/28T cells. CONCLUSIONS: These data suggest that 3T cells may have increased efficacy as immunotherapy for patients with cancer due to higher levels of tumoricidal cytokine production than 3/28T cells.