转移及非转移肿瘤移植后615小鼠血液流变学变化的研究

血道高转移瘤株FC、淋巴合并血道高转移瘤株U14、淋巴道高转移瘤株H22、非转移瘤株P615分别接种于336只纯系近交615小鼠.不同时间取血并处死动物,进行组织学及血液流变学检查.将转移瘤发展过程分为潜伏期、侵袭期、转移早、中、晚期,非转移瘤发展过程分为潜优期、增殖期、囊腔形成期及中心坏死期.本实验结果显示,不同转移能力及途径肿瘤发展的不同时期血液流变学变化规律不同,因而表明肿瘤侵袭、转移与血液流变学变化之间存在互为因果的紧密关系.其临床诊断及治疗意义被讨论.     

可移植性小鼠组织细胞肉瘤侵袭与转移模型的建立

将小鼠可移植性组织细胞肉瘤ＬⅡ分别接种在近交系６１５小鼠的胁部皮下、后肢肌肉和爪垫皮下组织内,取全肺和各部位淋巴结进行组织学检查,观察肿瘤自发转移率及转移程度。胁部皮下与后肢肌肉移植组待荷瘤小鼠自然死亡,平均存活时间分别为３９．３、２７．９ｄ,淋巴结转移率分别为９５．７％和１００％,肺转移率均为１００％。爪垫皮下移植组在肿瘤接种后１、３、５、１０、２０、３０和４０ｄ处死荷瘤小鼠,早期淋巴结转移出现在肿瘤接种后第１０天,至第３０天时形成肺转移瘤。局部侵袭达Ⅲ～Ⅳ级时才发生肿瘤转移,肺转移出现时间晚于淋巴转移。结果表明,ＬⅡ瘤株具有淋巴道合并血道转移的特性,是研究肿瘤侵袭和转移的理想实验肿瘤模型。     

具不同淋巴道转移潜能小鼠肝癌瘤株细胞膜鞘糖脂比较研究

采用高效薄层层析（ＨＰＴＬＣ）对两株具有不同淋巴道转移潜能的小鼠腹水型肝癌瘤株细胞膜鞘糖脂组分进行了比较分析。低转移的ＣＬ－Ａ２瘤株神经节苷脂以ＧＭ３为主,高转移的ＣＬ－１６Ａ３瘤株则以ＧＭ２为主。两细胞株中性鞘糖脂各组分相对百分含量无较显著差异。脂结合唾液酸含量测定表明,ＣＬ－１６Ａ３瘤株脂结合唾液酸含量约为ＣＬ－Ａ２瘤株的三倍。提示,具有不同淋巴道转移潜能的瘤细胞,其质膜鞘糖脂的组成也不同。     

具不同淋巴道转移潜能小鼠肝癌瘤株细胞膜鞘糖脂比较研究

采用高效薄层层析（ＨＰＴＬＣ）对两株具有不同淋巴道转移潜能的小鼠腹水型肝癌瘤株细胞膜鞘糖脂组分进行了比较分析．低转移的ＣＬ－Ａ_２瘤株神经节苷脂以ＧＭ_３为主,高转移的ＣＬ－１６Ａ_３瘤株则以ＧＭ_２为主．两细胞株中性鞘糖脂各组分相对百分含量无较显著差异．脂结合唾液酸含量测定表明,ＣＬ－１６Ａ_３瘤株脂结合唾液酸含量约为ＣＬ－Ａ_２瘤株的三倍．提示,具有不同淋巴道转移潜能的瘤细胞,其质膜鞘糖脂的组成也不同．     

淋巴道转移相关标志物与癌

肿瘤早期的淋巴道转移是恶性肿瘤患者高死亡率的主要原因,其机制复杂,一直是肿瘤研究的难点. 肿瘤淋巴道转移特异性蛋白标记物的发现有助于揭示恶性肿瘤早期发病机制、早期临床诊断和治疗. 本文综述了近年来肝癌、胃癌、食管癌、结肠癌、乳腺癌、肺癌、鼻咽癌和前列腺癌淋巴道转移研究所取得的蛋白质组学成果,并对发现的与以上几种肿瘤淋巴道转移相关的蛋白标记物及其在肿瘤的发生、浸润、转移及病人预后中作用及机制进行了探讨.     

软骨多糖抑制Hca-F肝癌细胞转移的初步研究

目的:研究软骨多糖抑制小鼠淋巴结高转移肝癌细胞Hca-F侵袭转移的作用。方法:以615近交系小鼠右脚垫皮下注射肝癌细胞Hca-F建立转移模型,设模型组和给药组。检测软骨多糖抑瘤率、淋巴道转移抑制率、淋巴结石蜡切片HE染色观察肿瘤细胞侵袭情况和采用ELISA试剂盒测定血清中TNF-α的含量。结果:软骨多糖具有明显的抑瘤作用,抑瘤率达61.9%;淋巴结转移抑制率为22.2%;通过比较模型组和给药组瘤细胞转移部位淋巴结的HE染色,发现给药组转移情况明显低于模型组;ELISA检测表明给药组血清中TNF-α的含量明显低于模型组(P<0.05)。结论:软骨多糖一定程度上能够抑制肝癌细胞Hca=F向周围淋巴结转移,为开发治疗恶性肿瘤的药物提供了选择。     

肿瘤转移研究的现状与趋势

肿瘤转移是恶性肿瘤的主要特征,是引起癌症患者死亡的首要因素．肿瘤转移的发生涉及到肿瘤细胞及其所处的微环境中复杂的信号通路,这些信号通路的激活及相互作用介导了肿瘤的转移、侵袭和在血液/淋巴循环系统中存活,以及在转移靶部位的生长过程．肿瘤转移是一个复杂的、多因素调控的动态过程,对于肿瘤转移机制的研究将有助于深入了解转移过程,并可以鉴定到有意义的治疗靶标,为临床诊断和治疗奠定基础．     

A549人肺癌细胞系/615-SCID小鼠转移瘤的生物学特征

目的通过建立A549人肺腺癌细胞/615-SCID小鼠模型,评价重度联合免疫缺陷615-SCID小鼠在建立人类肺癌转移模型方面的应用价值.方法将1×107A549细胞接种到615-SCID及SCID小鼠右上肢背部皮下,观察成瘤时间、成瘤率、肿瘤生长速度及转移发生.结果两品系小鼠接种后的成瘤率均为100%,615-SCID小鼠移植瘤潜伏期较长、生长较缓慢,更容易发生转移.结论 615-SCID小鼠比SCID小鼠更易于构建人类肺腺癌转移模型,对于肺癌转移特性研究具有较大的意义.     

Ad-GFP-nm23-H1体内外抑制人高转移肺巨细胞癌株95D生长和转移作用的研究

目的 观察Ad-GFP-nm23-Hl抑制人高转移肺巨细胞癌株95D细胞生长和转移的作用,为后期nm23-Hl基因和腺病毒载体用于95D及其他肿瘤的基因治疗提供一定的理论和方法.方法 应用MTT法及Transwell小室法分别检测95D细胞经Ad-GFP-nm23-Hl作用后的细胞体外增殖活性、黏附能力以及侵袭力的变化.同时应用人高转移肺巨细胞癌株95D裸鼠移植瘤模型,研究Ad-GFP-nm23-Hl对此移植瘤生长的抑制作用.结果 10~8PFU/ml、10~9PFU/ml、10~(10)PFU/ml处理组,可以明显抑制95D细胞的增殖、黏附和侵袭能力,其抑制作用呈剂量-效应关系.10~9PFU/ml 的Ad-GFP-nm23-Hl对移植瘤的抑瘤率为38.23%,较对照组差异显著.结论 Ad-GFP-nm23-Hl对人高转移肺巨细胞癌株95D细胞的生长和转移以及95D实体瘤具有抑制作用.     

GFP标记的肺癌细胞生长和转移的在体荧光成像

目的:利用稳定表达GFP的人高转移肺巨细胞癌细胞株95D,在体观察肿瘤的生长和淋巴结转移情况.方法:利用逆转录病毒感染人高转移肺巨细胞癌细胞株95D,经过G418筛选获得稳定表达GFP的细胞株.接种裸鼠皮下,成瘤后使用Nikon公司SMZ1000 P-FLA型荧光体视显微镜观察皮下原发肿瘤病灶及转移淋巴结.结果:使用逆转录病毒感染,可以获得稳定表达高强度GFP的肺癌细胞株,且生物学行为无明显改变.接种至裸鼠皮下后,2周左右成瘤,皮下肿瘤原发病灶在荧光体视镜下可清晰观察到肿瘤侵犯范围及周边血管生长情况.切开皮肤,可以观察到表达的GFP的转移淋巴结.结论:GFP标记人高转移肺巨细胞癌细胞株95D,建立裸鼠移植瘤模型,能更方便的观察肿瘤的生长、侵润和转移,为研究肿瘤的生长转移机制打下基础.     

肿瘤淋巴管入侵与无淋巴结转移膀胱癌复发及预后的相关性分析

目的:分析肿瘤淋巴管入侵与无淋巴结转移膀胱癌复发和预后之间的关系。方法:选取临床资料完整的膀胱癌病例72例,分为淋巴结转移组(32例)和无淋巴结转移组(40例)。采用Spearman相关分析探讨淋巴管入侵与膀胱癌复发和预后的相关性,应用Kaplan-Meier法描绘生存曲线,Cox比例危险度模型筛选影响膀胱癌患者预后的因素。结果:在72例膀胱癌组织中,淋巴管入侵的阳性率是48.6%(35/72),淋巴管入侵的阳性率随肿瘤分期和分级增加而显著升高(P0.05);淋巴结转移组的淋巴管入侵阳性率为68.8%(22/32),显著高于无淋巴结转移的32.5%(13/40)。淋巴管入侵与膀胱癌的临床分期、分级、淋巴结转移以及无淋巴结转移膀胱癌复发均显著相关(P0.05)。淋巴管入侵阴性的患者的五年总体生存率显著高于淋巴管入侵阳性者,淋巴管入侵是无淋巴结转移膀胱癌复发和预后不良的危险因素。结论:肿瘤淋巴管入侵与膀胱癌临床分期和淋巴结转移密切相关,并影响膀胱癌患者的总体生存率,可作为无淋巴结转移膀胱癌复发和预后的预测因素。     

两种不同淋巴转移能力小鼠肝癌细胞亚系的建立和生物学特性

通过单细胞分离(克隆)培养和局部淋巴结转移灶筛选的方法,建立两种不同淋巴转移能力HepA肝癌细胞亚系,分别命名为HepA-H和HepA-L,并比较其生长、游走和贴壁能力等生物学特性。瘤细胞接种于小鼠足垫后,同侧腘窝淋巴结转移率,HepA-H为83.3%,HepA-L为16.7%,两者具有显著差异(p<0.01)。两种细胞亚系均未见肺、肝、脾、肾等脏器转移灶。体外实验显示,HepA-H的生长、游走和贴壁能力均强于HepA-L。HepA肝癌不同淋巴转移能力亚系的建立,为研究肿瘤经淋巴管转移机理提供了良好的肿瘤淋巴转移动物模型。     

Peritumoral lymphangiogenesis induced by vascular endothelial growth factor C and D promotes lymph node metastasis in breast cancer patients

ABSTRACT: BACKGROUND: Mounting clinical and experimental data suggest that the migration of tumor cells into lymph nodes is greatly facilitated by lymphangiogenesis. Vascular endothelial growth factor (VEGF)-C and D have been identified as lymphangiogenic growth factors and play an important role in tumor lymphangiogenesis. The purpose of this study was to investigate the location of lymphangiogenesis driven by tumor-derived VEGF-C/D in breast cancer, and to determine the role of intratumoral and peritumoral lymphatic vessel density (LVD) in lymphangiogenesis in breast cancer. METHODS: The expression levels of VEGF-C/D were determined by immunohistochemistry, and intratumoral LVD and peritumoral LVD were assessed using immunohistochemistry and the D2-40 antibody in 73 patients with primary breast cancer. The associations of intratumoral LVD and peritumoral LVD with VEGF-C/D expression, clinicopathological features and prognosis were assessed. RESULTS: VEGF-C and D expression were significantly higher in breast cancer than benign disease (P < 0.01). VEGF-C (P < 0.001) and VEGF-D (P = 0.005) expression were significantly associated with peritumoral LVD, but not intratumoral LVD. Intratumoral LVD was associated with tumor size (P = 0.01). Peritumoral LVD was significantly associated with lymph node metastasis (LNM; P = 0.005), lymphatic vessel invasion (LVI; P = 0.017) and late tumor,node,metastasis(TNM) stage (P = 0.011). Moreover, peritumoral LVD was an independent risk factor for axillary lymph node metastasis, overall survival and disease-free survival in multivariate analysis. CONCLUSIONS: This study suggests that tumor-derived VEGF-C/D induce peritumoral lymphangiogenesis, which may be one mechanism that leads to lymphatic invasion and metastatic spread. Peritumoral LVD has potential as an independent prognostic factor in breast cancer patients.     

乳腺癌组织中VEGF—C和p38MAPK的表达及与伴淋巴结转移的关系

目的探讨乳腺浸润性导管癌组织中血管内皮生长因子C（VEGF—C）和丝裂原激活蛋白激酶p38（p38MAPK）的表达关系,以及与乳腺浸润性导管癌淋巴结转移等生物学行为的关系。方法采用免疫组织化学sP法检测70例乳腺浸润性导管癌组织及15例癌旁正常组织中VEGF-C和p38MAPK蛋白的表达,并采用Westernblot法检测13例伴有淋巴结转移的乳腺癌及12例无淋巴结转移的乳腺癌的新鲜组织中VEGF—C和p38MAPK蛋白表达。结果VEGF—C和p38MAPK在乳腺浸润性导管癌组织中的表达（阳性率分别为67．0％和61．4％）明显高于癌旁正常组织;VEGF-C和p38MAPK蛋白在伴有淋巴结转移组的乳腺癌组织中的表达均高于无淋巴结转移组（P=0．005,P=0．005）;在乳腺浸润性导管癌组织中VEGF-C和p38MAPK表达存在显著正相关（r=0．383,P=0．001）,并与乳腺浸润性导管癌的TNM分期（P=0．019,P=0．010）有关;VEGF-C和p38MAPK蛋白表达与乳腺浸润性导管癌肿块的大小（P=0．203,P=0．086）和患者的年龄（P=0．0．266,P=0．087）无明显关系。Western blot也证实,VEGF-C和p38MAPK蛋白在有淋巴结转移组中表达高于无淋巴结转移组。结论VEGF-C和p38MAPK的蛋白表达与乳腺浸润性导管癌的淋巴结转移密切相关,其有望成为乳腺癌治疗的新靶点。     

Divergent expression and roles for caveolin-1 in mouse hepatocarcinoma cell lines with varying invasive ability

Caveolin-1 is the major component protein of caveolae and associated with a lot of cellular events such as endocytosis, cholesterol homeostasis, signal transduction, and tumorigenesis. The majority of results suggest that caveolin-1 might not only act as a tumor suppressor gene but also a promoting metastasis gene. In this study, the divergent expression and roles of caveolin-1 were investigated in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have high, low, and no metastatic potential in the lymph nodes, as compared with normal mouse liver cell line IAR-20. The results showed that expression of caveolin-1 mRNA and protein along with the amount of caveolae number in Hca-F cells was higher than that in Hca-P cells, but was not detectable in Hepa1-6 cells. When caveolin-1 expression in Hca-F cells was down-regulated by RNAi approach, Hca-F cells proliferation rate in vitro declined and the expression of lymphangiogenic factor VEGFA in Hca-F decreased as well. Furthermore, in vivo implantation assay indicated that reduction of caveolin-1 expression in Hca-F prevented the lymphatic metastasis tumor burden of Hca-F cells in 615 mice. These results suggest that caveolin-1 facilities the lymphatic metastasis ability of mouse hepatocarcinoma cells via regulation tumor cell growth and VEGFA expression.     

胰腺癌原位种植瘤中VEGF-C的器官差异表达和作用

目的 研究胰腺癌裸鼠原位种植瘤自发性淋巴结转移模型中VEGF-C表达的器官差异,以及VEGF-C反义寡核苷酸对不同生长部位胰腺癌细胞生长、凋亡能力的影响。方法 建立人胰腺癌细胞株PANC-1裸鼠原位种植瘤自发性淋巴结转移模型,分离、原代培养原发灶和自发性淋巴结转移灶中的胰腺癌细胞,并应用荧光定量PCR、MTT、流式细胞术检测VEGF-C反义寡核苷酸转染对原发胰腺癌细胞和淋巴结转移的胰腺癌细胞各自生长特性、凋亡能力的影响。结果 淋巴结转移胰腺癌细胞VEGF-C的mRNA表达水平显著高于原发灶胰腺癌细胞（P〈0.05）。VEGF-C反义核苷酸抑制胰腺癌细胞VEGF-C的表达后,淋巴结转移灶中胰腺癌细胞生长抑制率、凋亡率均显著提高（P〈0.01）,而原发灶中胰腺癌细胞无明显影响（P〉0.05）。结论 VEGF-C反义寡核苷酸能显著抑制淋巴结转移灶中胰腺癌细胞生长、促进凋亡,但对原发灶胰腺癌细胞无影响;VEGF-C基因的表达和作用存在器官差异性。     

Exosomal miRNAs as circulating biomarkers for prediction of development of haematogenous metastasis after surgery for stage II/III gastric cancer

Exosomes secreted by living cancer cells can regulate metastasis. Exosomal miRNAs can reflect pathological conditions of the original cancer cells. Therefore, we aim to identify exosomal miRNAs as circulating biomarkers for haematogenous metastasis of gastric cancer. Pre-treatment serum samples of eighty-nine patients with stage II/III gastric cancer were collected. Thirty-four of them developed haematogenous metastasis after surgery and the other fifty-five did not. Extraction of exosomes was validated by western blot, transmission electron microscopy and nanoparticle tracking analysis. MiRNA qPCR array was performed in three matched pairs of samples. Internal control was selected from PCR array and validated in the remaining samples. Expressions of exosomal miRNAs were evaluated in the remaining samples by RT-qPCR, as well as in gastric cancer tissue samples and cell culture medium. Expression levels of exosomal miRNAs were analysed with clinical characteristics. The results indicated thirteen up-regulated and six down-regulated miRNAs were found after normalization. MiR-379-5p and miR-410-3p were significantly up-regulated in metastatic patients (P < .01). Higher expression of exosomal miR-379-5p or miR-410-3p showed shorter progression-free survival of the patients (P < .05). It was also found that miR-379-5p and miR-410-3p were down-regulated in gastric cancer tissue samples, while they were significantly up-regulated in gastric cancer cell culture medium compared with cancer cells. In conclusion, exosomal miRNAs are promising circulating biomarkers for prediction of development of haematogenous metastasis after surgery for stage II/III gastric cancer.     

Cell Budding from Normal Appearing Epithelia: A Predictor of Colorectal Cancer Metastasis?

Background: Colorectal carcinogenesis is believed to be a multi-stage process that originates with a localized adenoma, which linearly progresses to an intra-mucosal carcinoma, to an invasive lesion, and finally to metastatic cancer. This progression model is supported by tissue culture and animal model studies, but it is difficult to reconcile with several well-established observations, principally among these are that up to 25% of early stage (Stage I/II), node-negative colorectal cancer (CRC) develop distant metastasis, and that circulating CRC cells are undetectable in peripheral blood samples of up to 50% of patients with confirmed metastasis, but more than 30% of patients with no detectable metastasis exhibit such cells. The mechanism responsible for this diverse behavior is unknown, and there are no effective means to identify patients with pending, or who are at high risk for, developing metastatic CRC.Novel findings: Our previous studies of human breast and prostate cancer have shown that cancer invasion arises from the convergence of a tissue injury, the innate immune response to that injury, and the presence of tumor stem cells within tumor capsules at the site of the injury. Focal degeneration of a capsule due to age or disease attracts lymphocyte infiltration that degrades the degenerating capsules resulting in the formation of a focal disruption in the capsule, which selectively favors proliferating or “budding” of the underlying tumor stem cells. Our recent studies suggest that lymphocyte infiltration also triggers metastasis by disrupting the intercellular junctions and surface adhesion molecules within the proliferating cell buds causing their dissociation. Then, lymphocytes and tumor cells are conjoined through membrane fusion to form tumor-lymphocyte chimeras (TLCs) that allows the tumor stem cell to avail itself of the lymphocyte''s natural ability to migrate and breach cell barriers in order to intravasate and to travel to distant organs. Our most recent studies of human CRC have detected nearly identical focal capsule disruptions, lymphocyte infiltration, budding cells, and the formation of TLCs. Our studies have further shown that age- and type-matched node-positive and -negative CRC have a significantly different morphological and immunohistochemical profile and that the majority of lymphatic ducts with disseminated cells are located within the mucosa adjacent to morphologically normal appearing epithelial structures that express a stem cell-related marker.New hypothesis: Based on these findings and the growth patterns of budding cells revealed by double immunohistochemistry, we further hypothesize that metastatic spread is an early event of carcinogenesis and that budding cells overlying focal capsule disruptions represent invasion- and metastasis-initiating cells that follow one of four pathways to progress: (1) to undergo extensive in situ proliferation leading to the formation of tumor nests that subsequently invade the submucosa, (2) to migrate with associated lymphocytes functioning as “seeds” to grow in new sites, (3) to migrate and intravasate into pre-existing vascular structures by forming TLCs, or (4) to intravasate into vascular structures that are generated by the budding cells themselves. We also propose that only node-positive cases harbor stem cells with the potential for multi-lineage differentiation and unique surface markers that permit intravasation.