CD44s、CD44v6在颅内转移瘤与胶质母细胞瘤中的表达研究

目的:探讨粘附分子CD44基因蛋白在颅内转移瘤及胶质母细胞瘤中的表达及其与这些肿瘤侵袭、转移之间的关系。方法:应用标准型CD44(CD44s)和变异型CD44v6基因蛋白单克隆抗体,微波-LSAB免疫组化染色检测20例正常脑组织、35例脑胶质母细胞瘤和30例颅内转移瘤中CD44基因蛋白的表达情况。结果:20例正常脑组织中CD44s和CD44v6表达均为阴性:35例脑胶质母细胞瘤CD44s阳性表达率为100％(35／35),CD44v6表达为阴性:30例颅内转移瘤中CD44s阳性表达率为86.7％(26／30),CD44v6阳性表达率为66.7％(20／30)。CD44v6在颅内转移瘤及脑胶质母细胞瘤的表达差异有显著性(P<0.01)。结论:脑胶质母细胞瘤中CD44S的表达可能与其脑内侵袭过程有关,无CD44v6表达可能与其很少发生颅外转移有关,并有可能成为颅内转移瘤诊治的有用指标之一。     

骨桥蛋白和CD44v6在垂体腺瘤中的表达及意义

目的 检测骨桥蛋白(OPN)、CD44v6在垂体腺瘤中表达并探讨其与垂体腺瘤临床病理特征的关系.方法 应用免疫组化SP法检测80例垂体腺瘤组织中OPN和CD44v6蛋白的表达,并对二者表达与垂体腺瘤临床病理特征之间的关系进行分析.结果 OPN、CD44v6在垂体腺瘤中阳性表达率分别为66.3%、65.0%,OPN、CD44v6的表达均与患者的年龄、性别、肿瘤组织学类型无关,但在侵袭性垂体腺瘤组与非侵袭性垂体腺瘤组、复发组及非复发组之间的差异均有统计学意义(P＜0.01),而且二者在垂体腺瘤中表达呈正相关(r=0.585,P＜0.01).结论 OPN和CD44v6蛋白在垂体腺瘤中高表达与肿瘤侵袭性生长及复发相关,且两者在肿瘤侵袭性改变中可能起协同作用,二者联合检测有望更客观评价垂体腺瘤的侵袭性.

Abstract：

Objective To explore the expressions of osteopontin (OPN) and CD44v6 in pituitary adenomas and to evaluate the relationship between the expressions and invasiveness of the tumor.Methods Immunohistochemistry was used to detect the expressions of OPN and CD44v6 in 80 pituitary adenomas,and the relationships between the expressions of OPN and CD44v6 and the clinicopathologic characteristics were also analyzed.Methods The positive expression rates of OPN and CD44v6 were 66.3% and 65.0% respectively in the pituitary adenomas.Both OPN and CD44v6 expressions were different statistically in invasive group and recurrent group(P<0.01 ),but not correlated with patient' s age,sex and histological types.There was also a significantly positive relation between OPN and CD44v6 expression in pituitary adenomas (P<0.01 ).Conclusion The increased expressions of OPN and CD44v6 are associated with the invasiveness and recurrence of pituitary adenomas.They might have a synergetic effect in the invasive growth of the tumor,which can be taken as leading candidates for evaluating the biological behavior.     

EGFR基因突变非小细胞肺癌脑转移对预后的影响

非小细胞肺癌（NSCLC ）占肺癌的80％～85％,诊断时约40％已出现转移［1］。肺癌转移是导致肺癌预后差的一个原因,脑转移的预后更差,肺癌脑转移患者的中位生存期为73 d［2］,全脑颅脑照射后的中位生存期延长至4～6个月［3‐4］。存在EGFR突变的非小细胞肺癌脑转移患者接受靶向药物治疗（EGFR‐TKI）及颅脑照射的预后优于野生型非小细胞肺癌脑转移患者（12.9个月vs.3个月）［5］。虽EGFR突变的非小细胞肺癌脑转移患者的预后较好,但EGFR突变的肺癌患者较易发生脑转移。EGFR突变患者约占非小细胞肺癌患者的10％,却占脑转移患者的44％～63％［6］。随着 EG‐FR‐TKI药物等治疗的应用,EGFR突变的非小细胞肺癌脑转移患者的预后与颅外转移的患者是否存在差异,我们对此进行研究,报告如下。     

非小细胞肺癌脑转移的靶向药物治疗研究进展

中枢神经系统转移肿瘤绝大多数起源于肺癌,其中非小细胞肺癌发病率占80%左右。非小细胞肺癌早期患者即有10%进展为颅内转移,手术治疗及放疗是脑转移的经典治疗方法,但治疗后易复发。由于血脑屏障的存在,常规化疗药物难以在转移灶内发挥疗效,总体预后不佳。近年来,靶向药物如表皮生长因子受体酪氨酸激酶抑制剂、间变性淋巴瘤激酶抑制剂在脑转移患者中表现出了较好的疗效,已成为非小细胞肺癌脑转移患者的可选治疗方案。文中就非小细胞肺癌脑转移的靶向药物治疗的进展进行综述。     

Oct-4在激素非依赖性前列腺癌干细胞中的表达及意义

背景：干细胞特异性基因Oct-4在维持干细胞功能和调节干细胞的分化过程中起着关键性作用,了解前列腺癌干细胞中Oct-4的表达,对前列腺癌的预后、复发和耐药的评估具有重要意义。 目的：观察Oct-4在激素非依赖性前列腺癌干细胞中的表达。 设计、时间及地点：观察性实验,于2008-02/10在南昌大学第一附属医院中心实验室完成。 材料：取12例前列腺癌患者手术后组织标本,其中6例在手术前没有经过化疗和激素治疗,另外6例患者经过激素治疗出现抵抗而手术。 方法：制备前列腺癌组织单细胞悬液,流式细胞仪分选未经治疗前列腺癌和治疗后出现耐药的前列腺癌干细胞,RT-PCR和Western blot检测激素非依赖性前列腺癌Oct-4基因和蛋白的表达。 主要观察指标：激素依赖和非依赖性前列腺癌CD133+/CD44+以及CD133-/CD44-细胞的比例;Oct-4基因和蛋白在激素非依赖性前列腺癌CD133+/CD44+、CD133+/CD44-、CD133-/CD44+、CD133-/CD44-细胞的表达。 结果：流式细胞仪检测显示未经过治疗的前列腺癌CD133+/CD44+细胞占(0.83±0.21)%,而CD133-/CD44-细胞多达(95.62±1.35)%;治疗出现激素抵抗的前列腺癌CD133+/CD44+细胞为(54.62±0.86)%,而CD133-/CD44-细胞为(9.56±0.47)%;与其他亚群细胞相比,激素非依赖性前列腺癌CD133-/CD44-细胞Oct-4基因和蛋白显著降低(P < 0.01);CD133+/CD44+细胞Oct-4基因和蛋白表达显著升高(P < 0.01);而CD133+/CD44-和CD133-/CD44+细胞之间Oct-4基因和蛋白表达无显著差异(P > 0.05)。 结论：Oct-4在激素非依赖性前列腺癌干细胞中高表达,可能与前列腺癌的预后和耐药有关。     

肺及肺癌组织芯片PBK/TOPK蛋白表达：检测的可靠性

背景：组织芯片技术可高通量检测组织中的生物分子。但是组织芯片所用的组织很小,直径仅0.6 mm,且肿瘤细胞具有异质性,所以组织芯片检测结果是否可靠尚不清楚。 目的：比较石蜡组织芯片和对应组织常规石蜡切片PBK/TOPK 蛋白在正常成人肺泡Ⅱ型上皮细胞、人胚胎肺泡上皮细胞、肺癌原发灶和相应的淋巴结转移灶的表达差异,探讨组织芯片检测PBK/TOPK蛋白表达的可靠性。 方法：采用组织微阵列技术按照正常成人肺组织,胚胎肺组织,肺鳞癌原发灶及其相应淋巴结转移癌,肺腺癌原发灶及其相应淋巴结转移癌,肺小细胞癌原发灶及其相应淋巴结转移癌,肺大细胞癌原发灶及其相应淋巴结转移癌的顺序依次构建包含760点阵的石蜡组织芯片。应用免疫组化SP法,检测石蜡组织芯片和对应组织常规石蜡切片PBK/TOPK蛋白的表达。应用Leica Q500MC图像分析系统分别对石蜡组织芯片和常规石蜡切片上PBK/TOPK蛋白表达的阳性单位和阳性表达率进行定量测试。 结果与结论：组织芯片和对应常规切片相应细胞中PBK/TOPK 的阳性强度及阳性表达率基本相同,相对偏差不足0.6%,两者比较差异无显著性意义。结果说明石蜡组织芯片和常规组织切片检测PBK/TOPK蛋白表达结果高度一致;应用组织芯片检测肺癌、胚胎肺及正常肺组织中PBK/TOPK蛋白的表达结果可靠。 关键词：肺癌;组织芯片;PBK/TOPK;免疫组化;可靠性     

透明质酸偶联壳聚糖微球对非小细胞肺癌的靶向性作用★

背景：部分肿瘤细胞表面的CD44受体表达上调，如大肠癌、非小细胞肺癌，提示透明质酸与CD44受体在肿瘤的生长与扩散有着一定的关系。 目的：观察透明质酸偶联壳聚糖纳米微球对非小细胞肺癌的靶向性。 方法：采用离子交联法制备负载多西紫杉醇的透明质酸偶联壳聚糖微球(DTX-HACTNPs)，电镜观察其形态学特征，激光粒度分析仪测定粒径大小及分布。FITC标记微球，作用于CD44+人非小细胞肺癌细胞株(A549)，荧光显微镜观察。MTT法检测载药微球的体外细胞毒性。 结果与结论：DTX-HACTNPs形态规则，粒径分布较为均匀，平均粒径为(228.0±2.6) nm。DTX-HACTNPs对A549细胞的杀伤力高于非透明质酸偶联载药微球，但仍低于普通注射用DTX，3者的半数抑制浓度(IC50)分别为(15.06±0.94)，(25.73±3.37)，(5.35±0.61) mg/L(F=73.871，P=0.000)。提示HACTNPs通过受体途径主动靶向性结合于非小细胞肺癌细胞，可提高化疗药对肿瘤细胞的选择性杀伤力。     

脂质体介导CD基因转染SHG-44胶质瘤细胞凋亡的实验研究

目的以含胞嘧啶脱氨酶（CD）基因的pCMVCD重组表达质粒转染SHG-44胶质瘤细胞，体外观察5-氟胞嘧啶（5-FC）对转染CD基因的胶质瘤细胞的凋亡诱导作用。方法体外扩增、酶切鉴定pCMVCD质粒并采用DNA序列测定pCMVCD质粒中的CD基因；脂质体Lipofectamine 2000介导pCMVCD质粒转染SHG-44细胞，G418筛选培养获取抗性细胞克隆（即SHG-44／CD细胞）；免疫细胞化学检测SHG-44／CD细胞的CD基因蛋白水平表达；流式细胞仪、TUNEL实验及透射电子显微镜观察5-FC对表达CD基因的SHG-44／CD细胞凋亡的诱导作用。结果含CD基因的pCMVCD质粒成功转染进入SHG-44细胞，获取了含CD基因的SHG-44／CD细胞，免疫细胞化学染色显示SHG-44／CD细胞成功地表达了CD。在含5-FC的培养液中培养，SHG-44／CD细胞出现典型的凋亡形态，TUNEL显示凋亡细胞比例极高；透射电镜可见凋亡改变；流式细胞术检测凋亡率达18．6％，凋亡率呈剂量依赖性。结论建立了SHG-44恶性人脑胶质瘤细胞的CD／5-FC自杀基因系统。诱导SHG-44／CD胶质瘤细胞产生凋亡可能是脑胶质瘤CD基因疗法的重要机制。     

神经胶质瘤E-cd、MMP-2和CD44v6的表达及临床意义

目的 探讨钙粘素(E-cadherin,E-cd)、基质金属蛋白酶-2(matrix metalloproteinases-2,MMP-2)和粘附分子(CD44v6)在脑神经胶质瘤中的表达及临床意义。方法 采用免疫组织化学方法对50例神经胶质瘤患者和9例对照者脑组织中E-cd、MMP-2和CD44v6蛋白表达进行检测。结果 (1)对照组脑组织E-cd的阳性表达率为100％;神经胶质瘤Ⅰ～Ⅱ级和Ⅲ～Ⅳ级组阳性表达率分别为40％(10/25)和24％(6/25),两组差异无显著性意义(P>0.05);对照组与神经胶质瘤组之间差异有显著性意义(P<0.05)。(2)对照组MMP-2阳性表达率为11％;在经胶质瘤Ⅰ～Ⅱ级组为32％(8/25),Ⅲ～Ⅳ级组为64％(16/25),两组比较差异有显著性意义(P<0.05)。对照组与神经胶质瘤组相比,MMP-2的表达差异亦有显著性意义(P<0.05)。(3)CD44v6在对照组及神经胶质瘤组均无表达。结论 E-cd和MMP-2在神经胶质瘤的牛物学行为中起重要作用,且可能与其侵袭性有关,而CD44v6则与神经胶质瘤的生物学行为无关。     

结直肠癌组织中CD44+肿瘤细胞与S期标志物增殖细胞核抗原的关系*◆

背景：目前尚未找到结直肠癌干细胞理想的标志物,结合周期蛋白能更精确的找到GO/G1期癌干细胞。 目的：了解结直肠癌组织中CD44和增殖细胞核抗原PCNA的表达及CD44+肿瘤细胞与S期标志物增殖细胞核抗原PCNA的关系。 方法：采用组织芯片检测、免疫组织化学和双重免疫组织化学染色技术检测61例结直肠癌组织、10例正常肠黏膜组织中增殖细胞核抗原PCNA和CD44表达情况。 结果与结论：在结直肠癌组织中,PCNA+细胞数远多于CD44+细胞数,CD44+癌细胞形态有两种,一种核较大,染色质边集,呈空泡状,核膜核仁清晰,这些细胞大多数呈PCNA阳性;另一种核较小,染色质致密,呈小圆形,部分表达PCNA。在大多数病例中,CD44+细胞中90%以上的瘤细胞同时为PCNA阳性,只有(3.38±2.08)%细胞为单纯PCNA阳性。说明结直肠癌组织中大多数CD44+细胞呈现S期标志物PCNA阳性,处于S期;只有极少数CD44+/PCNA-处于G0/G1期的细胞可能为肿瘤干细胞。     

CD44v6在脑转移癌中的表达

目的:研究ＣＤ４４ｖ６在脑转移癌中的表达情况及其相关性。方法:采用逆转录－聚合酶链反应(ＲＴ－ＰＣＲ)法和Ｗｅｓｔｅｒｎ Ｂｌｏｔ法检测９例脑转移癌标本中ＣＤ４４ｖ６基因和蛋白的表达。结果:８例转移癌标本中有ＣＤ４４ｖ６ｍＲＮＡ和ＣＤ４４ｖ６蛋白的异常表达,对照组中全为阴性。结论:ＣＤ４４ｖ６在脑转移癌中有异常表达,可能在肿瘤粘附、突破血脑屏障,脑内定居、繁殖中有重要作用。     

Her-2与CD44在恶性脑肿瘤中的表达及意义

目的 探讨人类表皮因子受体2型(Her-2)及CD44在常见恶性脑肿瘤中的表达情况及与肿瘤预后的关系.方法 采用免疫组织化学和间接免疫荧光标记法检测胶质母细胞瘤、胶质肉瘤、髓母细胞瘤、间变性少突胶质细胞瘤共120例及10例正常脑组织标本中Her-2、CD44的表达,对数据进行统计学分析.结果 Her-2的表达阳性率在胶质母细胞瘤、胶质肉瘤、髓母细胞瘤和间变性少突胶质细胞瘤中分别是 80.0%、73.3%、66.7%和33.3%.CD44的表达阳性率在上述四种肿瘤中分别为86.7%、80.0%、90.0%和73.3%.Her-2在正常脑组织中无表达,CD44在正常脑组织中表达阳性率为20.0%.Her-2与CD44在恶性脑肿瘤中的表达无相关性(P>0.05),但两者均与肿瘤预后关系密切.免疫荧光标记显示两者存在共表达现象.结论 Her-2在胶质母细胞瘤、胶质肉瘤、髓母细胞瘤中高表达,而在间变性少突胶质细胞瘤中相对低表达,CD44则普遍高表达.未发现两者在恶性脑肿瘤中表达具有相关性,但二者均与肿瘤的预后有密切关系,具有协同作用.因此Her-2与CD44有望成为脑肿瘤联合免疫治疗的联合靶位,在脑肿瘤诊治中发挥重要作用.     

Estimation of prognostic value of CD44 expression in neuroblastic tumours in children

Adversities observed in treatment of children with neoplastic disease based on new diagnostic markers and new prognostic factors. Both of them allow prognosis to be established for a single patient. The aim of our study was to examine the expression of CD44 adhesion molecule in different histologic types in a neuroblastoma group of tumours (35 cases of neuroblastoma from current files and archives) and to estimate the possible prognostic value of CD44 expression by comparison with widely accepted prognostic markers and chosen histoclinical parameters (9 cases of neuroblastoma with follow-up data). We did not find a statistically significant correlation between CD44 expression and histologic type of the tumour. However, we found that all relapses appeared among patients with tumours with the strongest CD44 expression, and that in none of the investigated tumours without relapses was strong CD44 expression ever observed. We noticed CD44 expression in 88.88% of examined tissue samples which underwent statistical analysis and we found the strongest CD44 expression in tumours situated in the retroperitoneal space. Results of log-rank test and Kaplan-Meier estimation showed that a correlation between CD44 expression and survival time was close to a statistically significant value (p=0.065). We conclude that lack of a clear statistically significant correlation between CD44 expression and histoclinical parameters and currently known prognostic factors in our study is due to the presence of many CD44 isoforms, which cannot be distinguished with commercially used antibodies, but they may play a different role in pathogenesis and spread of neuroblastoma.     

T cell transformation with Herpesvirus saimiri: a tool for neuroimmunological research

Splice variants of CD44 molecule-harboring exon 10 (v6), often called v6 variants (v6v), are shown to confer tumor progressive, metastatic or invasive capacities. Furthermore, CD44 molecule on activated T-cells are shown to be required for infiltration of these cells into the inflammatory site and for accelerated immune response. Human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is caused by HTLV-I infection and characterized by spastic paraparesis and urinary disturbance with perivascular HTLV-I-infected and activated CD4+ T-cell infiltration. In order to explore the underlying mechanism causing the disease after HTLV-I infection, we analyzed CD44 variant expression on peripheral blood mononuclear cells (PBMC) and in the spinal cord specimens from patients with HAM/TSP, and compared them with those from other HTLV-I-infected individuals and controls. We found that v6v expression with special direct link of exons 10 (v6) and 14(v10) was highly expressed in PBMC from patients with HAM/TSP and that v6v and CD4 double positive T-cell infiltration into the spinal cord lesion of HAM/TSP. This combination of CD44 splice variant has not been previously reported in the study of chronic inflammatory disorders and may be a marker molecule for T-cells infiltrating into the central nervous system (CNS), especially the spinal cord.     

CD44 expression in primary and recurrent oligodendrogliomas and in adjacent gliotic brain tissue

CD44 expression was evaluated in 114 primary and recurrent oligodendrogliomas (46 primary oligodendrogliomas grade II and 15 recurrences grade II; 17 primary anaplastic oligodendrogliomas and nine recurrent oligodendrogliomas grade III; 14 glioblastomas with oligodendroglial growth pattern and 13 tumour recurrences grade IV). CD44 expression was found to correlate with tumour grading (P<0.001) and with survival (Kaplan-Meier Log Rank P<0.01, median survival 28 months in oligodendrogliomas with CD44 expression vs. 108 months in CD44-negative tumours). However, multivariate Cox regression analysis of grading and CD44 expression revealed that CD44 expression had no prognostic relevance independent of histological grading. Characterization of CD44 positive cells by double labelling with GFAP revealed that in addition to oligodendroglioma cells, reactive astrocytes within the tumour, at the invasive margin and along the pathways of oligodendroglioma invasion in the subpial matrix, and in the vicinity of vessels, frequently expressed CD44. It is suggested that in analogy to carcinoma invasion where a tumour-induced production of hyaluronan was found in fibroblasts at the invasive margin of the tumour, in the brain reactive astrocytes may produce hyaluronan which would facilitate the adhesion of new CD44-positive astrocytic processes but which would also promote tumour invasion.     

Human endosialin (tumor endothelial marker 1) is abundantly expressed in highly malignant and invasive brain tumors

Endosialin (tumor endothelial marker 1) is expressed preferentially by tumor endothelial cells but not by normal endothelium. Its protein domain architecture is homologous to that of CD93 and thrombomodulin (CD141), suggesting a similar function in mediating cell-cell interactions. The aim of this study was to investigate the expression pattern of endosialin in human brain tumors in a bid to decipher its contribution to tumor angiogenesis. We generated an antibody specifically recognizing human endosialin and used it to study endosialin expression in 30 human brain tumor specimens by immunoblotting and immunohistochemistry. Twenty of 30 tumors expressed endosialin in a heterogeneous manner. The largest proportion of endosialin-expressing tumors was found in highly invasive glioblastoma multiforme, anaplastic astrocytomas, and metastatic carcinomas. Endosialin was localized to the endothelium of small and large vessels strongly stained for CD31 and was also expressed by Thy-1-positive fibroblast-like cells close to the meninges and alpha-smooth muscle actin-positive cells in some vessels. Endosialin colocalized with thrombomodulin, suggesting the proteins may have complementary functions in tumor progression.     

Expression of leucocyte adhesion molecules at the human blood-brain barrier (BBB).

The expression of leucocyte adhesion molecules was studied on cerebral endothelia by immunocytochemistry. In peritumoral "normal" brain tissue we found low endothelial expression of ICAM1, LFA3, CD44, and CD9, whereas VLA1 was present on vessels in high incidence and density. LFA1, CD2, and CR3 were found on intraluminal and parenchymal leucocytes, but were absent on brain vessels. In brain tumors and inflammatory brain lesions, we observed an up-regulation of endothelial ICAM1 and LFA3 expression, whereas other adhesion molecules on endothelial cells remained unchanged. Within the brain parenchyma, ICAM1 and LFA3 were found on astrocytes and tumor cells; on the contrary, LFA1 was expressed on microglial cells similar to CR3. CD44 and CD9 showed a diffuse neuropil expression in normal and tumoral tissue, whereas VLA1 was not expressed on any parenchymal cells. Our data show that multiple different adhesion molecules are present on blood-brain barrier endothelium (BBB) under normal conditions and some adhesion molecules are up-regulated in brain tumors and under inflammatory conditions. The presence of adhesion molecules in the vessel walls as well as on parenchymal cells like astrocytes and microglia may guide inflammatory cells into and through the brain in the course of immune surveillance and inflammation.