L-选择蛋白可能介导小鼠肝癌HCa-F细胞经淋巴道转移的研究

目的观察L-选择蛋白是否介导小鼠肝癌HCa-F细胞与淋巴结黏附，探讨HCa-F细胞特异性向周围淋巴结转移的分子机制。方法应用免疫细胞化学方法验证小鼠肝癌HCa-F细胞为非淋巴细胞来源；用免疫印迹分析、RT-PCR及流式细胞术检测L-选择蛋白在HCa-F细胞的表达；并用L-选择蛋白的抗体抑制HCa-F细胞与冰冻淋巴结切片间的黏附实验(Stamper-Woodruff method)检测L-选择蛋白的功能。结果HCa-F细胞为非淋巴细胞来源的肝癌细胞，在其表面有L-选择蛋白的表达，且HCa-F细胞与淋巴结之间的黏附可被L-选择蛋白的抗体抑制。结论非淋巴细胞来源的HCa-F细胞表面表达功能性L-选择蛋白分子，并可协助其与淋巴结黏附，从而可能介导HCa-F细胞向淋巴道转移。     

CXCR4对小鼠腹水型肝癌细胞淋巴转移潜能的影响

目的探讨趋化因子受体CXCR4表达水平对小鼠腹水型肝癌细胞淋巴转移潜能的影响。方法采用RT-PCR,流式细胞仪检测趋化因子受体CXCR4在小鼠腹水性肝癌细胞株Hca-F和Hca-P细胞的表达。通过归巢实验测定用抗体封闭CXCR4表达前后细胞特异性的向淋巴管迁移能力。结果CXCR4及其mRNA在高转移潜能小鼠肝癌细胞系Hca-F的表达高于低转移潜能小鼠肝癌细胞系Hca-P的表达。CX-CR4中和抗体能够抑制Hca-F细胞在体内向淋巴结转移。结论CXCR4在Hca-F、Hca-P细胞表面表达水平不同,可能是导致它们向淋巴管转移的潜能不同的影响因子之一,CXCR4表达水平与Hca-F、Hca-P细胞的特异性淋巴管转移潜能有关。     

Sorcin在小鼠腹水型肝癌高、低淋巴道转移株中的表达及定位

目的：研究抗药蛋白sorcin（又称可溶性抗药相关蛋白）在小鼠腹水型肝癌高、低淋巴道转移潜能不同的细胞株Hca-F/Hca-cP中的定位及表达差异,进一步探讨其在肿瘤淋巴道转移中的作用。方法：以小鼠腹水肝癌高淋巴道转移株Hca-F和小鼠腹水肝癌低淋巴道转移株Hca-P为研究对象,采用实时定量PCR技术检测sorcin在Hca-F/Hca-P细胞中基因水平的表达差异;采用Western免疫印迹技术检测sorcin在Hca-F/Hca-P细胞中蛋白水平的表达差异;采用免疫荧光技术检测sorcin在细胞中的定位情况;采用细胞计数试剂盒-8检测肿瘤细胞的增殖能力;应用Transwell小室检测肿瘤细胞的迁移和侵袭能力。结果：Hca-F细胞的体外增殖、迁移和侵袭能力都明显高于Hca-P细胞;sorcin在Hca-F细胞中的基因水平和蛋白水平表达分别为Hca-P细胞的1.5和1.8倍;sorcin在两种小鼠腹水型肝癌细胞株中的定位主要位于细胞质。结论： sorcin在Hca-F中呈高表达,主要定位于细胞质,可能在其增殖、迁移和侵袭过程中发挥重要作用,为进一步探讨其作用机制和功能奠定了基础。     

小鼠肝癌细胞特异性淋巴道转移与其基质金属蛋白酶分泌的关系

目的　探讨肿瘤细胞基质金属蛋白酶 (MMPs)分泌与特异性淋巴结转移的关系。方法　将自建高、低转移小鼠肝癌细胞系HCa F(高转移 )和HCa P(低转移 )在培养时 ,分别加入淋巴结、肝脏或脾脏匀浆 ,离心收集培养上清 ,用明胶酶谱法检测两株癌细胞在不同培养环境下分泌的MMPs酶谱 ,分析两者的差异。结果　在单纯RPMI16 40培养基中HCa F和HCa P细胞仅能分泌少量MMP 9(12 5 6± 15 7,2 6 42± 385 ) ,加入淋巴结匀浆后HCa F细胞分泌MMP 9显著增强 (12 40 3± 894) ,且分泌MMP 9的活性型和MMP 2 ;HCa P细胞也分泌MMP 9(90 6 8± 6 86 )及其活性型 (3 914± 12 5 3)和MMP 2(2 997± 1990 ) ,但量均明显低于HCa F细胞 ;加入肝脏或脾脏匀浆后 ,HCa F和HCa P细胞均不分泌MMPs。结论　HCa F细胞强于HCa P细胞的转移能力可能与HCa F细胞分泌MMPs的能力强于HCa P细胞相关 ,而HCa F和HCa P细胞仅在淋巴结成分诱导下才能分泌较多的MMPs,这可能与它们具有特异性地向淋巴结转移的功能密切相关。     

榄香烯处理瘤苗抗原致敏的树突状细胞的抗肿瘤效应研究

本文采用 β 榄香烯处理小鼠H2 2肝癌亚系HCa F ,提取榄香烯瘤苗可溶性上清作为粗制肿瘤抗原 ,制备抗原冲击(pulsed)的树突状细胞 ,研究其抗瘤作用及机制。结果表明榄香烯瘤苗抗原冲击的树突状细胞可诱导对HCa F瘤株的MHC非限制性免疫应答 ,继承性转输后对HCa F瘤株攻击有免疫保护效应 ;体外可激发脾不粘附细胞活化增殖和加强杀瘤细胞活性。     

细胞内氯离子通道蛋白1在肝癌高、低淋巴道转移细胞株中的定位及表达差异

目的 研究细胞内氯离子通道蛋白1(CLIC1)在小鼠腹水型肝癌高、低淋巴道转移细胞株中的定位及表达差异.方法 以小鼠腹水型肝癌高淋巴道转移细胞株Hca-F和低淋巴道转移细胞株Hca-P为研究对象,采用双向凝胶电泳和质谱鉴定、细胞免疫荧光、细胞免疫化学染色方法和Western blot方法检测CLIC1在高、低淋巴道转移细胞株中的定位及蛋白表达情况.结果 双向凝胶电泳和质谱鉴定CLIC1在Hca-F细胞中高表达,是Hca-P细胞的1.6倍;细胞免疫荧光和免疫化学结果显示CLIC1定位于Hca-F和Hca-P细胞的细胞质和细胞膜,功能形式的细胞膜定位更多存在于Hca-F细胞中,并且在Hca-F细胞中的表达强于Hca-P细胞.Western blot结果显示CLIC1在Hca-F细胞中的表达是Hca-P细胞的1.6倍.结论 CLIC1在Hca-F和Hca-P细胞中定位于细胞质和细胞膜,在Hca-F细胞中高表达,且在Hca-F中更多定位于细胞膜,可能在肝癌淋巴道转移过程中发挥作用.     

小鼠肝癌淋巴道转移细胞系中NF-κB基因的表达及意义

目的 探讨NF-κB基因表达在小鼠肝癌淋巴道转移细胞系中的表达及意义.方法 采用RQ-PCR方法检测NF-κB基因在肝癌淋巴道低转移潜能的小鼠肝癌细胞系Hca-P和高转移潜能的小鼠肝癌细胞系Hca-F中的表达水平.结果 NF-κB基因在Hca-P和Hca-F细胞系基因表达分别为(1.41±0.48)×10~(-3)、(2.95±0.22)×10~(-3),Hca-P和 Hca-F之间,NF-κB基因表达差异有统计学意义 (P<0.01),其随转移潜能的增高,表达水平增加.结论 NF-κB基因表达水平增高与肝癌淋巴道侵袭转移相关.     

不同淋巴道转移潜能小鼠肝癌细胞系中Toll样受体2基因的表达

目的 探讨TLR-2基因表达水平与小鼠肝癌淋巴道转移中的关系.方法 采用RQ-PCR方法检测TLR-2基因在肝癌细胞系H22、淋巴道低转移潜能的小鼠肝癌细胞系Hca-P和高转移潜能的小鼠肝癌细胞系Hca-F中的表达水平.结果 TLR-2基因在H22、Hca-P和HcaF细胞系基因表达分别为(1.08±0.25)×10-3、(2.14±0.42)×10-3、(4.31±0.62)×10-3,差异有统计学意义(P<0.01),其随转移潜能的增高,表达水平增加.结论 TLR-2基因表达水平增高提示其在肝癌的淋巴道侵袭转移中起作用,有可能成为预测肝癌预后新的靶点.     

应用表达芯片筛选小鼠肝癌淋巴道转移相关基因

目的采用表达芯片比较高、低淋巴道转移力小鼠肝癌腹水型细胞株Hca—F和Hca—P的基因表达谱,以筛选出与肿瘤淋巴道转移相关的基因。方法分别提取Hca—F和Hca—P细胞的总RNA,反转录合成生物素标记的cRNA探针,并与Affy—metrix GeneChip MOE430A（包括22690个转录本,对应于约14500个小鼠已知基因和4371个EST）杂交,检测结果利用生物信息学进行分析。结果Hca—F和Hca—P相比,在14500个已知基因中,有901个（6．2％）表达上调幅度≥2倍;在4371个EST中,有129个（3％）上调幅度≥2倍。公布了差异表达≥8倍的37个基因并根据Gene Ontology（GO）分类和TreeView分析,按照其参与的生物学过程和具有的分子功能进行了功能分类。其中有19个基因参与了组织发生、细胞黏附、信号转导、细胞生长、分化及代谢等的生物学过程,29个基因分别具有转运、移动、蛋白激酶、蛋白结合及受体活性等分子功能,7个基因的生物学功能尚不清楚。结论表达芯片检测与肿瘤淋巴道转移模型相结合,为肿瘤转移研究提供新方法、新思路,一些过量表达的基因将为肿瘤转移机制的研究提供新线索。     

两株肝癌细胞淋巴结转移机理的初步探讨

Hca－F（F）和Hca－P（P）是具有不向淋巴道转移能力的小鼠腹水型肝癌,F的转移率大于SO％,P的转移率小于20％,本实验观察了在体内外条件下它们对淋巴结内成分的粘附,以期找出其器官特异性的结构基础。将F和P细胞分别加到淋巴结冰冻切片上,于37－C,5％C02条件下孵育,分别于第24’48、72、96h取出,冲洗。将此对瘤株分别接种于SCID小鼠皮下,2周后取增大的淋巴结,部分制成石蜡切片,嗜银染色,部分制成透射电镜标本,观察瘤细胞与细胞外基质（ECM）的关系。酶谱法测定瘤细胞在有无淋巴结成分时的IV型胶原酶的分泌。F细胞粘…     

Galectin-3 Expression in Tumor Progression and Metastasis of Papillary Thyroid Carcinoma

Galectin-3 plays important roles in cell adhesion, cell proliferation, apoptosis, neoplastic transformation, and metastasis. Galectin-3 expression has been evaluated in various malignant neoplasms to determine its effectiveness in differential diagnosis from benign lesions and its effects on carcinogenesis. There are few and somewhat controversial results regarding its changes through cancer progression in thyroid malignancies. We studied the presence of galectin-3 expression immunohistochemically and its relation with tumor invasiveness and lymph node metastasis in 89 cases of papillary carcinoma of the thyroid. Galectin overexpression was less frequent in cases with lymph node metastases compared with cases without lymph node metastasis (P = 0.001). Metastatic foci in lymph nodes showed a lower degree of galectin-3 overexpression than their primary lesions (P = 0.001). Degree of galectin-3 overexpression was also lower in larger tumors (P = 0.009). Additionally, a decreased level of galectin-3 overexpression was observed at the invasive edges of the tumors (P = 0.001). Galectin-3 overexpression is more profound in early stages of papillary carcinoma, and its expression intensity decreases during tumor progression. This finding is consistent with roles for galectin-3 in cell adhesion to other tumor cells and the matrix.     

The involvement of the sLe-a selectin ligand in the extravasation of human colorectal carcinoma cells

The extravasation of tumor cells is a pivotal stage in the formation of hematogenous metastasis. An interaction of selectins expressed on endothelial cells and selectin ligands expressed by tumor cells has been implicated to play a role in extravasation. In the present study we used a human-mouse model to prove the hypothesis that the selectin ligand sialyl Lewis-a (sLe-a) is indeed involved in the in vivo extravasation of colorectal carcinoma (CRC) cells. The results indicated that highly metastatic CRC cells expressing high levels of sLe-a extravasate more efficiently than non-metastatic CRC cells expressing low levels of sLe-a. It was also demonstrated that down regulating the expression levels of sLe-a in CRC cells by genetic manipulations, significantly reduced CRC extravasation. Non-specific effects of these manipulations were ruled out. The results of this study indicate that the arrest and adhesion of CRC cells, and possibly of other types of cancer cells as well, to endothelium depend on the expression of the selectin ligand sLe-a by the tumor cells.     

不同转移特性瘤细胞系的筛选及其生物学特性

目的：探讨与肿瘤转移相关的某些生物学特性。方法：将小鼠乳腺癌Ｃａ７６１－ＦＰ８／Ｌ和Ｃａ７６１－ＦＬ１０／Ｌ经体内筛选得到细胞系Ｃａ７６１－Ｐ５Ｂ和Ｃａ７６１＝Ｌ６Ｂ，并观察其瘤细胞与凝集反应、靶器官组织条件培养基对瘤细胞真挚化作用等，结果：Ｃａ７６１－Ｐ５Ｂ具有高肺转移、低淋巴结转移特性，Ｃａ７６１－Ｌ５Ｂ具有低肺、低淋巴结转移特性。两个瘤细胞系的细胞表面的糖基表达，对条件培养的趋化反应不同。结     

间质细胞衍生因子1及其受体CXCR4在浸润性乳腺癌中的表达及其临床意义

目的 研究间质细胞衍生因子1(SDF-1)及其受体CXCR4在浸润性乳腺癌中的表达,并分析其与浸润性乳腺癌相关临床病理指标及淋巴结转移之间的关系.方法 采用免疫组织化学LSAB方法检测SDF-1/CXCR4在120例浸润性乳腺癌中的表达情况;采用地高辛标记的寡核苷酸探针进行原位杂交以检测趋化因子SDF-1在肿瘤环境中表达的部位及来源.结果 (1)SDF-1主要表达于肿瘤细胞的胞质和胞膜;SDF-1的胞质表达在淋巴结阳性组高于阴性组(P=0.033),且其表达程度与淋巴结受累数目、病理学分期、组织学分级、肿瘤大小及ER表达等指标呈正相关(P<0.05);(2)罕见SDF-1 mRNA表达的脉管内皮却可见SDF-1蛋白的表达,其表达程度与肿瘤胞质SDF-1着色正相关(P<0.01);且淋巴管内皮SDF-1的着色与淋巴结转移程度为正相关(P=0.005);血管内皮SDF-1的着色与肿瘤环境中的淋巴细胞浸润正相关(P<0.01),且同时伴有较多淋巴细胞浸润及SDF-1血管内皮着色阳性的病例,其淋巴结的转移程度分别高于仅有上述条件之一或二者均不具备的各组病例(P<0.05);(3)CXCR4也主要表达于肿瘤细胞的胞质和胞核;CXCR4的胞质表达在淋巴结阳性组高于阴性组(P<0.05),且其表达程度与淋巴结受累数目、病理学分期、组织学分级、肿瘤大小及HER2表达等呈正相关(P=0.005),而胞核的表达仅与PR的表达情况呈正相关(P<0.01);(4)瘤细胞质CXCR4与SDF-1的表达呈正相关(P=0.001).结论 浸润性乳腺癌肿瘤细胞SDF-1和CXCR4的表达与多项临床病理指标,尤其是淋巴结转移率及转移程度有关,可作为预测乳腺癌淋巴结转移及预后的免疫病理学指标,同时应注意肿瘤微环境中SDF-1的多个来源及定位的不同意义.     

树突状细胞在榄香烯复合瘤苗主动免疫效应中的作用

本实验以HCa F或L6 1 5榄香烯复合瘤苗 (H TCV或L TCV )、H TCV溶解物 (TH )、短小棒状菌 (CP )免疫小鼠 ,分离制备其脾脏DC ,在体外分别用相应瘤细胞溶解物 (H或L )和瘤苗溶解物 (TH或TL )冲激后 ,以MTT法检测其诱导同系小鼠脾不粘附细胞增殖的能力。结果表明用榄香烯复合瘤苗或其溶解物免疫DC供鼠和体外冲激DC ,可增强其诱导同系小鼠脾不粘附细胞增殖的作用 ,给DC供鼠注射CP可进一步增强DC的这一作用     

Establishment of lymph node metastatic model for human gastric cancer in nude mice and analysis of factors associated with metastasis

The actual mechanisms responsible for lymph node metastasis in gastric cancer are still unclear. To investigate the mechanisms of lymph node metastasis in gastric cancer, we established a lymph node metastatic model for human scirrhous gastric carcinoma. Lymph node metastasis had frequently developed after orthotopic implantation of OCUM-2M LN derived from a scirrhous gastric cancer cell line, OCUM-2M, which had low capacity for lymph node metastasis. We elucidated the different characteristics including binding ability, migratory capacity and immunoresponses induced by the cell surface molecules of these two cell lines. The binding ability to Matrigel and migratory capacity of OCUM-2M LN cells were significantly greater than those of OCUM-2M cells. On flow cytometric analysis, both OCUM-2M and OCUM-2M LN cells strongly expressed HLA-I (99.5 and 97.1%) and LFA-3 (76.6 and 99.2%) in level of expression between the two cell lines, but neither cell line expressed HLA-II (0.0 and 0 .0%), B7-1 (0.0 and 0.0%) or B7-2 (0.4 and 0.3%). ICAM-1 expression in OCUM-2M LN cells was weaker (0.7%) than that in OCUM-2M cells (36.8%). Strong adhesiveness and cytotoxicity of mononuclear lymphocytes for OCUM-2M cells were observed in adhesion and cytotoxic assays, both of which were significantly decreased by the addition of anti-ICAM-1 antibodies. On the other hand, the adhesiveness and cytotoxicity of OCUM-2M LN cells were significantly less than those of OCUM-2M cells, and were not affected by the addition of anti-ICAM-1 antibodies. These findings suggest that decreased ICAM-1 expression in a new gastric cancer cell line with a high rate of lymph node metasta-sis may in turn decrease immune responses mediated through LFA-1-dependent effector cell adhesion, and that this escape from the immunosurveillance system may be one of the factors inducing lymph node metas-tasis. In conclusion, we established a gastric cancer cell line, OCUM-2M LN, with a high rate of lymph node metasta sis. An in vivo lymph node-metastatic model with this cell line should be useful for analysing the mech-anism and therapeutic approach of lymph node metastasis. © Rapid Science Ltd.     

CD44v6与MMP—9在口腔鳞癌中的表达意义

目的：探讨细胞粘附分子CD44v6和基质金属蛋白酶MMP－9相互关系及其在评估口腔鳞癌的组织学分级,肿瘤浸润以及转移等生物学特性中的意义。方法：运用免疫组化S－P法测定22例口腔鳞癌和6例正常口腔黏膜组织中CD44v6和MMP－9的表达,结果：CD44v6在正常口腔黏膜组织呈强阳性表达;癌组织中CDv6的表达明显弱于正常组织,高表达CD44v6的口腔鳞癌不仅分化程度较差而且易发生淋巴结转移,MMP－9在正常口腔黏膜组织中呈阴性或弱阳性表达,癌组织中MMP－9的阳性表达高达68．2％（15／22）。MMP－9的表达与病理分级和颈淋巴结转移呈正相关（P＜0．05）,口腔鳞癌中MMP－9与CD44v6的表达呈正相关（P＜0．01）,多因素分析示两者之间的交互作用是影响口腔鳞癌病理分化和颈淋巴结转移的最重要因素。结论：口腔鳞癌中CD44v6与MMP－9的表达密切相关。MMP－9和CD44v6可作为临床上评估口腔鳞癌浸润,转移以及预后的指标。     

Correlation between E-cadherin and CD44 adhesion molecules expression and cervical lymph node metastasis in oral tongue SCC: Predictive significance or not

The aim of this article was to evaluate the expression of E-cadherin and CD44 adhesion molecule in oral tongue squamous cell carcinoma (SCC) since inappropriate expression of adhesion molecules raises the metastatic ability of the tumor cells.Biopsy specimens from 92 patients with tongue SCC were examined for the expression of E-cadherin and CD44 by immunohistochemistry. The relationship of immunoreactivity with tumor stage and cervical lymph node metastasis was then analyzed.Sixty-one patients (66.3%) had reduced or negative staining for CD44. Weak or absent staining for E-cadherin was seen in 14 patients (15.21%). Cervical lymph node metastasis is associated with decreased or negative staining for CD44, but no association was found between E-cadherin immunoreactivity and nodal metastasis.Our study reveals that reduced expression of CD44 could be an indicator of high invasiveness of tumor by increasing cervical lymph node metastasis.     

小细胞肺癌中CD24的表达及与微血管密度关系

目的检测CD24在小细胞肺癌(small cell lung cancer,SCLC)原发灶、淋巴结转移灶中的表达,探讨其与SCLC临床参数、侵袭转移及微血管密度(micro-vascular density,MVD)关系。方法应用免疫组化检测45例SCLC原发灶、33例区域淋巴结转移灶CD24表达及CD34-MVD。结果 SCLC原发灶、淋巴结转移灶CD24阳性率分别为53.3%(24/45)和60.6%(20/33),CD24阳性癌细胞常在癌巢周围密集分布,多见于侵袭边缘。CD34主要表达于血管内皮细胞胞膜,呈条索状甚至形成小管腔,主要位于癌巢边缘或癌细胞密集区;CD24、MVD与患者性别、年龄、肿瘤直径无关,而与淋巴结转移及胸膜侵袭有关(P<0.05);SCLC原发灶和淋巴结转移灶CD24阳性组MVD(33.44±8.51、47.65±14.31)均高于CD24阴性组(20.40±6.44、30.64±10.20)(P<0.05)。结论 CD24表达与SCLC侵袭转移行为和MVD有关。     

乳腺浸润性微乳头状癌上皮性钙黏附素的表达及意义

目的 研究细胞黏附分子,在乳腺浸润性微乳头状癌肿瘤细胞的集团性浸润、转移中的表达和作用。方法 复习2002年1月～2003年5月所有手术切除乳腺癌组织切片,按WHO乳腺癌分类分组,浸润性微乳头状癌(IMPC)64例、浸润性导管癌(IDC)57例。采用免疫组织化学标记的链霉素抗生物素蛋白-生物素(LSAB)法检测64例IMPC中E-钙黏附素的表达,并同IDC加以比较。结果 E-钙黏附素主要表达于IMPC细胞膜;IMPCE-钙黏附素表达率(85．9％,55／64)明显高于IDC(43．9％,25／57),并且在微乳头状肿瘤细胞集团内的细胞间连接面表达正常,而在细胞集团面向间质侧的表达明显减弱或不表达;IMPC组的淋巴结转移率(85．9％,55／64)明显高于IDC(52．6％,30／57)．．其淋巴结阳性、E-钙黏附素阳性病例的d-连接素、B-连接素共同表达率(45．1％,26／51)也明显高于IDC(15．4％,2／13)。结论 IMPC的微乳头状肿瘤细胞集团内细胞间黏附性强、而与间质间的黏附性减弱或消失的特性可能是IMPC具有高转移潜能的原因之一。