不同分化肝癌细胞株中上皮细胞黏附分子、CD90及CD24的表达分析

目的 验证不同分化肝癌细胞株MHCC97L、MHCC97H和HCCLM3的恶性表型,并探索其中肿瘤干细胞表面标志分子CD90、上皮细胞黏附分子(EpCAM)及CD24的表达情况.方法 Transwell侵袭小室检测三种不同分化细胞株MHCC97L、MHCC97H和HCCLM3的侵袭能力,细胞计数试剂盒8检测三种不同分化细胞株对奥沙利铂的敏感性,流式细胞仪检测肿瘤干细胞表面标志分子CD90、EpCAM、CD24在三种细胞株中的表达情况.多个样本均数比较采用方差分析. 结果 MHCC97L、MHCC97H和HCCLM3三种细胞穿膜细胞数存在明显差异,HCCLM3(30.57±8.95)个＞MHCC97H (21.33±4.17)个＞HCC97L (9.33±3.85)个,F=38.484,P＜ 0.01.对奥沙利铂的半数抑制浓度IC50 HCCLM3为(36.57±6.95)μmol/L,MHCC97H (26.35±3.88) μmol/L,MHCC97L (17.68±3.25) μmol/L,F=40.181,P＜0.01. CD90在MHCC97L、MHCC97H和HCCLM3细胞中的表达比例分别为0.92％±0.21％、1.98％±0.23％和2.55％±0.34％,差异有统计学意义(F=60.481,P＜0.01);EpCAM在三种细胞中的表达比例分别为2.11％±0.32％、3.23％±0.18％和4.38％±0.49％,表达差异也有统计学意义(F=84.800,P＜0.01);CD24在三种细胞中的表达比例分别为0.68％±0.37％、1.22％±0.26％和1.36％±0.24％,表达差异有统计学意义(F=15.677,P＜0.01).结论 分化越低的肝癌细胞株侵袭能力越强,对化疗敏感性越差,其中的肝癌干细胞比例则越高.     

不同转移潜能人肝癌细胞株趋化因子受体谱差异表达

目的对比观察不同转移潜能人肝癌细胞株趋化因子受体谱差异性表达。方法Pre- mier软件设计18对趋化因子受体引物,RT-PCR分析SMMC-7721、MHCC97-L、MHCC97-H和HCCLM6细胞侵袭转移潜能逐渐增强的人肝癌细胞株趋化因子受体谱。结果4组不同转移潜能细胞株趋化因子受体表达谱存在明显差异（P＜0．01）,其中CCR10、CXCR4、CXCR6表达随转移潜能增加逐渐降低。HCCLM6表达谱中CCR3、CCR4、CCR10、CCR12及XCR1比SMMC-7721表达明显降低甚至缺失（P＜0．01）,而CXCR1（P=0．006）、CXCR5（P=0．003）表达高于低转移潜能组SMMC-7721。MHCC97-H和MHCC97-L比较,除CXCR2、CXCR6、XCR1外差异均有统计学意义,其中CCR1（P=0．002）、CCR2（P=0．004）、CCR5（P=0．046）表达高于MHCC97- L。CXCR4在模板减量时只能在SMMC-7721组检测到。结论高低转移潜能肝癌细胞株趋化因子受体表达在mRNA水平存在差异性表达,与肝癌细胞株差异性转移潜能相关。     

不同转移潜能肝癌细胞株血管内皮生长因子及其受体的表达和意义

目的 探讨血管内皮生长因子受体-1(VEGFR-1)在不同转移潜能肝癌细胞株中的表达及其意义.方法 应用半定量RT-PCR、酶联免疫吸附试验和(或)Western blot检测4株不同转移潜能的肝癌细胞株及一株正常肝细胞中VEGFR-1、VEGF-A、VEGF-B及VEGFR-2的mRNA和(或)蛋白质表达.结果 MHCC97-H、MHCC97-L和SMMC-7721细胞均有VEGFR-1 mRNA和蛋白质表达,且VEGFR-1 mRNA和蛋白质在MHCC97-H中的表达高于MHCC97-L、SMMC-7721的表达,两者比较,差异有统计学意义(P＜0.05),而其配体VEG-A和VEGF-B在检测的4种肝癌细胞株和正常肝细胞株L-02中均有表达.同时在检测的四种肝癌细胞株和正常肝细胞株L-02中均能检测到VEGFR-2 mRNA和蛋白质表达,但各组间表达差异无统计学意义(P＞0.05).结论 具有转移潜能的肝癌细胞株有VEGFR-1表达,而且其表达强弱与肝癌细胞株的转移潜能呈正相关,VEGFR-1的表达可能促进了肝细胞癌的侵袭转移.     

CD24在肝癌组织中的表达

目的探讨CD24在肝细胞癌组织中的表达情况及作为肝癌早期诊断指标及分子靶向治疗的可能性。方法应用寡核苷酸基因芯片、RT-PCR和免疫组织化学技术检测CD24在人正常肝组织、肝硬化组织及肝癌组织中的表达,分析CD24与临床病理学特征的关系及其发生的分子生物学机制。结果寡核苷酸基因芯片、RT-PCR及免疫组织化学方法证明CD24在肝癌组织中mRNA及蛋白水平均呈高表达,与病理学分级无显著相关性。结论 CD24在肝癌组织中显著增高,但与肿瘤分化程度无密切相关性。     

黏附分子CD44的表达及其糖基化与肝癌转移的相关性

目的 探讨CD44S及其变异体CD44v分子的表达和其糖基化与肝癌细胞侵袭转移的关系. 方法 用免疫组织化学法、量子点、RT-PCR、Western blot、细胞免疫荧光染色、甲基化特异性聚合酶链反应等技术检测转移与非转移性肝癌组织、不同转移潜能人肝癌细胞株中CD44S及其变异体CD44v的定位与表达;并应用多重凝集素印迹法检测这些细胞株中CD44v6的糖基化差异.组间均数比较应用方差分析及t检验,多组间等级资料的比较采用Wilcoxon秩和检验,各组间率的比较采用x2检验.结果 免疫组织化学结果显示,CD44S蛋白定位以细胞质为主; CD44v3、CD44v4/5蛋白定位于细胞膜与细胞质;而CD44v6主要定位于细胞膜.组织芯片结果显示,相对于CD44S及其他CD44v,CD44v6在转移组的表达水平高于非转移组(阳性率为75％与46％),差异具有统计学意义(x2=8.828,P＜0.05);量子点检测(t=2.392,P＜0.05)与血清学检测(t=2.56,P＜0.05)也证实这一结果.Western blot结果显示,CD44v6的表达与肝癌细胞转移潜能呈正相关.此外,在MHCC97L、MHCC97H肝癌细胞中CD44v6基因启动子发生不完全甲基化,而在Hep3B细胞中则发生完全甲基化.而且,相对于Hep3B细胞,MHCC97L及MHCC97H细胞中CD44v6蛋白对朝鲜槐凝集素、黑接骨木凝集素及麦胚凝激素的亲和力较高. 结论 在CD44S及其变异体CD44v中,CD44v6蛋白的高表达与肝癌转移潜能的增强呈正相关;其高表达可能与基因启动子低甲基化有关.此外,CD44v6蛋白唾液酸寡糖链的增加可能与肝癌细胞转移潜能增高有关.     

CD34与CD117在人肝癌组织中的表达及其临床意义

目的 研究肝细胞肝癌组织中CD34与CD117的表达情况,探讨其与临床病理的关系及其对肝细胞肝癌(HCC)患者预后的评价.方法 应用免疫组织化学PV-9000二步法检测55例HCC组织标本中CD34和CD117的表达,并与临床病理学指标和术后无瘤生存期进行比较分析,对照组为肝硬化组织10例,正常肝组织6例.采用SPSS16.0统计分析软件对CD34和CD117表达结果及与临床病理参数的关系进行Fisher精确检验,Pearson χ2检验,Kaplan-Meier生存分析,Log-Rank检验,Cox回归模型分析等检验.结果 CD34在HCC组、肝硬化组和正常肝组织组表达阳性率分别为65.4%、20.0%和16.7%,HCC组织中的阳性率大于肝硬化组织(P=0.012)及正常肝组织组(P=0.031),差异有统计学意义.但正常肝组织组与肝硬化组比较,差异无统计学意义.CD34表达在HCC组中与脉管瘤栓、中瘤分化程度有密切相关性,χ2值分别为4.000和11.008,P值分别为0.046和0.001.CD117在HCC组、肝硬化组和正常肝组织组表达阳性率分别为47.3%、10%和0,HCC组阳性率大于肝硬化组(P=0.037)及正常肝组织(P=0.033),差异无统计学意义.但正常肝组织与肝硬化组比较,差异无统计学意义.CD117表达在HCC组中与肿瘤分化程度、肿瘤分期有相关性,χ2值分别为5.115和15.459,P值分别为0.024和0.000.HCC组CD34阳性组及CD34阴性组的中位无瘤生存时间分别为17个月和19个月,CD34阳性组较CD34阴性组无瘤生存时间缩短,χ2=4.105,P=0.043,差异有统计学意义.CD117阳性组及CD117阴性组的中位无瘤生存时间分别为12个月和19个月,CD117阳性组的无瘤生存时间较CD117阴性组也明显缩短,χ2=28.023,P=0.000,差异有统计学意义.COX多因素分析显示CD117表达、血清甲胎蛋白水平及肿瘤大小是HCC患者术后2年无瘤生存时间的独立预后因素.结论 CD34与CD117可能在HCC发生、发展过程中具有重要作用,有望成为判断预后的指标.     

不同转移潜能肝癌细胞株的差异蛋白质组的二维液相色谱分析

目的:对不同转移潜能肝癌细胞株MHCC97- H(高转移)和MHCC97-L(低转移)差异表达的蛋白质进行二维液相色谱分离和MALDI-TOF质谱鉴定.方法:将肝癌细胞株MHCC97-H和MHCC97- L细胞裂解样品按蛋白质PI进行一维的色谱聚焦分离,然后每个PI组分再按疏水性经二维反相无孔硅胶HPLC分离,利用ProteoVue软件将UV光吸收图谱转换成PI对疏水性的胶图,再利用DeltaVue软件比较升高或降低的差异蛋白.收集差异蛋白峰进行胰酶酶解,然后进行MALDI-TOF质谱鉴定.结果:2D图谱显示共有72个差异蛋白条带,共鉴定出了9个差异蛋白.分别为M2型丙酮酸激酶、ATP合成酶α亚单位、热休克蛋白60、Toll样受体9、含黄素单加氧酶、钙网硬蛋白前体、锰超氧化物岐化酶、nm23-H1、G-蛋白偶连受体激酶5;其中4个蛋白在高转移细胞株MHCC97-H中表达升高,5个蛋白在低转移细胞株MHCC97-L表达升高.结论:这些差异蛋白可能在肝癌的转移中起关键作用.     

CD34和Ki67的表达预测肝癌术后复发的风险

目的探讨免疫组化蛋白表达与肝细胞癌术后复发风险的关系。方法回顾性分析我院肝细胞癌行根治性手术治疗患者的免疫组化结果,共50例纳入分析,统计分析免疫组化结果和复发时间的关系。结果患者以是否在术后5年内复发为界定为复发组和治愈组,肝癌组织中免疫组化结果显示,CD34的表达在复发组高于治愈组,有统计学差异(P<0.05),Ki-67表达的百分比复发组高于治愈组,有统计学差异(P<0.05),CD10、CK19、AFP、Hep、Arg和GPC-3两组比较无统计学差异。结论CD34和Ki67的高表达预示着术后复发风险较高。     

原代口腔鳞癌细胞的分离培养及CD133 CD44的表达

目的探讨从人新鲜口腔鳞癌组织中培养原代鳞癌细胞的方法以及干细胞表面标志物CD133、CD44表达量的检测情况。方法选取该院2016~2017年行口腔鳞状细胞癌切除术的新鲜肿瘤标本6例,应用机械法获得原代口腔鳞癌细胞并用酶消化法传代。应用细胞形态学及角蛋白免疫组化方法对原代鳞癌细胞进行鉴定。流式细胞仪技术检测鳞癌细胞中干细胞表面标志物CD133、CD44的表达情况。结果(1)成功培养出纯化的口腔鳞癌细胞,并能成功传代,至目前为止已传至27代。(2)培养出的OSCCs呈多边形铺路石状,角蛋白染色其胞浆中可见阳性棕色染色。(3)流式细胞检测鳞癌细胞中干细胞表面标志物CD133的平均表达率为0.41%、CD44的平均表达率为33.76%。结论从人新鲜口腔鳞癌标本中利用机械法可成功获得大量原代口腔鳞癌细胞并能顺利传代,口腔鳞癌细胞中干细胞表面标志物CD44表达偏高,CD133的表达量明显偏低。     

CD133和CD90在肝细胞癌中的表达及其意义

存在于肿瘤组织中的少数具有干细胞性质的细胞群体被称为肿瘤干细胞(CSCs),可促进肿瘤的发生和发展,也是肿瘤耐药性、复发及转移的根源.有报道CD133和CD90可能为肿瘤干细胞表面标志物,但CD133和CD90在肝癌中的表达及其意义报道尚少.本研究采用免疫组织化学方法检测不同肝组织中CD133及CD90蛋白的表达水平,探讨其在肝癌中的表达情况及其与肝癌生物学特性及预后的关系.     

Cancer stem cell markers correlate with early recurrence and survival in hepatocellular carcinoma

AIM:To investigate whether expression of cancer stem cell(CSC)markers is associated with recurrence and survival in hepatocellular carcinoma(HCC)patients.METHODS:A consecutive series of 90 HCC patients who underwent curative hepatectomy between April2007 and April 2009 were analyzed.Of the 90 patients,38(42%)experienced recurrence within two years of surgery.To adjust for baseline differences between this early recurrence group and the other patients,propensity-score matching was used to generate 25 pairs of patients.Immunohistochemistry was used to compare expression of CD133,CD90,and epithelial cell adhesion molecule(EpCAM)in liver tissues from propensity score-matched patients and from 10 healthy adults.Associations of the three markers with HCC,clinicopathological characteristics,early recurrence,and survival time were explored.RESULTS:The expression of all three CSC markers was significantly higher in HCC tissue than in healthy liver tissue(P<0.001 for all).Among the HCC clinicopathology characteristics examined,the absence of tumor capsule was associated with CD133 expression(P=0.005);higher histopathology grade and larger tumor size were associated with CD90 expression(P=0.010 and 0.034,respectively);and elevated serum alpha-fetoprotein levels were associated with EpCAM expression(P=0.021).Expression of CD90 and EpCAM was significantly higher in the early recurrence group than in other patients(P=0.001 and 0.045,respectively),whereas CD133 expression was not significantly different between the two groups(P=0.440).Multivariate analysis identified only CD90 expression as significantly associated with early recurrence.Log-rank analysis identified expression of both CD90 and EpCAM as significantly associated with survival time of HCC patients.Cox regression identified EpCAM expression as an independent predictor of survival time.CONCLUSION:Expression of CD133,CD90,and EpCAM CSC markers may be linked to HCC tumor onset and/or progression.In addition,EpCAM expression is associated with shorter survival time,while CD90 expression is associated with early HCC recurrence.     

CD133和CD90在肝细胞癌中的表达及其意义

存在于肿瘤组织中的少数具有干细胞性质的细胞群体被称为肿瘤干细胞(CSCs),可促进肿瘤的发生和发展,也是肿瘤耐药性、复发及转移的根源.有报道CD133和CD90可能为肿瘤干细胞表面标志物,但CD133和CD90在肝癌中的表达及其意义报道尚少.本研究采用免疫组织化学方法检测不同肝组织中CD133及CD90蛋白的表达水平,探讨其在肝癌中的表达情况及其与肝癌生物学特性及预后的关系.     

Cancer stem cell markers CD133 and CD24 correlate with invasiveness and differentiation in colorectal adenocarcinoma

AIM： To verify that CD markers are available for detecting cancer stem cell populations and to evaluate their clinical significance in colon cancer.METHODS： Immunohistochemistry for CD133, CD24 and CD44 was performed on the tissue microarray of 523 colorectal adenocarcinomas. Medical records were reviewed and clinicopathological analysis was performed.RESULTS： In colorectal adenocarcinoma, 128 of 523 cases （24.5%） were positive and 395 cases （75.5%） were negative for CD133 expression. Two hundred and sixty-four of 523 cases （50.5%） were positive and 259 cases （49.5%） were negative for CD24 expression. Five hundred and two of 523 cases （96%） were negative and 21 cases （4%） were positive for CD44 expression. Upon clinicopathological analysis, CD133 expression was present more in male patients （P = 0.002） and in advanced T stage cancer （P = 0.024）. Correlation between CD24 expression and clinicopathological factors was seen in the degree of differentiation （P= 0.006）. Correlation between CD44 expression and clinicopathological factors was seen in the tumor size （P = 0.001）. Survival was not significantly related to CD133, CD24 and CD44 expression.CONCLUSION： CD markers were related to invasiveness and differentiation of colorectal adenocarcinoma. However, CD expression was not closely related to survival.     

肝细胞肝癌组织中CD90表达变化及意义

目的观察肝细胞肝癌(HCC)组织中肿瘤干细胞标记物CD90的表达变化,并探讨其临床意义。方法采用免疫组化法检测60例HCC组织(HCC组)、60例癌旁组织(癌旁组)及60例正常肝脏组织(对照组)中的CD90,分析其表达与HCC临床病理参数的关系。结果 CD90阳性表达率HCC组为76.7%(46/60)、癌旁组为48.3%(29/60)、对照组为0,3组组间两两比较,P均<0.01。CD90表达与HCC患者的性别、民族、有无门静脉瘤栓、包膜是否完整、TMN分期、病理分级有关(P均≤0.05),与HCC患者的年龄、肿瘤大小、甲胎蛋白高低、乙肝病毒是否阳性无关(P均>0.05)。结论 CD90在HCC组织及癌旁组织中高表达,HCC组织中表达更高;癌组织中CD90表达有助于HCC患者预后的判断,可为患者进行个体化诊断治疗提供参考。     

Clinical significance of CD44v mRNA detection by PCR in peripheral blood of patients with hepatocellular carcinoma

AIM: To study the clinical significance of detecting the expression of CD44v mRNA in the blood of patients with hepatocellular carcinoma (HCC).METHODS: The expression of CD44v mRNA was detected in blood with RT and diploid PCR, and the clinical significance was discussed based on the result of pathological examination and follow-up.RESULTS: CD44v mRNA was detected in the blood of 10/15 patients, giving a positive rate of 66.67%. In the 13 patients who showed response in the follow up period, the CD44v mRNA expression was positive in 9 and negative in 4. Recurrence rate was higher in the patients with positive CD44v mRNA expression than in those with negative CD44v mRNA expression, and the clinical pathological indices were also higher in the former than in the latter.CONCLUSION: Detection of the expression level of CD44v mRNA in blood of the patients with HCC can be used as an adjuvant means for differential diagnosis, prediction and monitoring of HCC recurrence.     

Alpha-fetoprotein expression is a potential prognostic marker in hepatocellular carcinoma

AIM: To characterize the alpha-fetoprotein (AFP) positive and negative hepatocellular carcinoma (HCC) samples. METHODS: Thirty-seven paraffin-embedded human HCC samples were analyzed by immunohistochemistry for the following antigens: AFP, β-catenin, p53, CD44, MSH-2, MLH-1, and HNF-4. The tumors were divided into two groups based on the AFP expression. The immunophenotypic data and important clinical parameters were studied between the two groups. RESULTS: Twenty-one of the thirty-seven examined HCCs were AFP positive. Seven with nuclear p53 staining were AFP positive, while seven tumors with nuclear β-catenin staining were AFP negative. CD44 staining and high histological tumor grade were more frequent among the AFP-positive HCCs. The other immunophenotypical and dinical parameters did not show statistically significant difference in their distribution between the AFP positive and negative samples. CONCLUSION: AFP expression in HCC correlates with unfavorable prognostic factors, while nuclear β-catenin positivity is more common among the AFP-negative liver tumors. This observation supports the microarray data on in vivo human tumors.