大黄素、芹菜素抑制人卵巢癌细胞侵袭的体外实验研究

背景与目的:大黄素抑制酪氨酸蛋白激酶、酪蛋白激酶2的活性,抑制I-κB降解;芹菜素抑制丝裂原活化蛋白激酶、PI3K的活性。大黄素、芹菜素是否能抑制高恶性度肿瘤侵袭与转移的研究还未见报道,本研究选用大黄素、芹菜素,观察其对人卵巢癌细胞体外侵袭的作用。方法:台盼蓝活细胞拒染法观察药物对人卵巢癌细胞生长、增殖的影响;以人工重组基底膜(Matrigel)体外侵袭实验观察药物对细胞体外侵袭、粘附、运动能力的影响;SDS-聚丙烯酰胺凝胶电泳法观察对Ⅳ型胶原酶分泌及活性的影响。结果:大黄素及芹菜素均抑制HO-8910PM细胞的生长、增殖,其48h的IC50分别为(35.30±3.50)μmol/L和(28.92±2.60)μmol/L。大黄素有效抑制HO-8910PM细胞体外侵袭、粘附、运动,在40μmol/L时,抑制率分别为(45.31±3.10)%、(25.42±1.70)%和(41.59±1.90)%;大黄素抑制基质金属蛋白酶-9(MMP-9)分泌,但不能直接抑制其活性。芹菜素能抑制细胞粘附、运动,在40μmol/L时,抑制率分别为(30.80±3.00)%和(29.04±1.70)%,但抑制细胞体外侵袭作用不显著,仅为(12.08±0.50)%,且不能抑制MMP-9分泌也不能直接抑制其活性。结论:大黄素、芹菜素对人卵巢癌HO-8910PM细胞均有一定的毒性,而大黄素更具有成为抗肿瘤侵袭药物的潜力。     

抗Ⅳ型胶原酶单链抗体的表达及其对肿瘤细胞侵袭的抑制作用

目的：制备抑制肿瘤细胞侵袭转移的单链抗体片段,用于构建小型化的免疫偶联物。方法：应用噬菌体呈现技术,以产生抗Ⅳ型胶原酶单抗的杂交瘤细胞为基础克隆构建单链抗体（scFv)基因。以表达质粒pET-30a（＋）为载体在大肠杆菌中进行诱导表达。ELISA法测定表达产物的免疫反应性,明胶酶谱法测定表达产物对靶酶活性的影响。Boyden Chamber测定表达产物对肿瘤细胞侵袭的影响。结果：表达的单链抗体以包涵体形式存在,经变性和复性后,检测表明表达产物保留了对抗原的免疫反应性,对高转移性人肺癌PG细胞分泌的Ⅳ型胶原酶有抑制作用。同时在体外能抑制肿瘤细胞的侵袭,抑制率与表达产物的剂量浓度相关,1mg/ml浓度的片段具有68％的抑制率。结论：运用基因工程手段获得的单链抗体片段保留了亲本抗体的若干特性,对Ⅳ型胶原酶有抑制作用,并能抑制肿瘤细胞的侵袭。     

胶原酶A与大肠癌侵袭转移性的关系研究

目的 揭示胶原酶Ａ（ＭＭＰ－２）与大肠癌分期、临床表现的关系，探讨以胶原酶Ａ在肿瘤组织中的含量及活性比值来判定大肠癌侵袭转移的可能性及方法。方法 采用明胶酶谱法检测３２例大肠癌组织和１７例癌旁组织及７例外伤或尸检的正常大肠组织中ＭＭＰ－２的酶原和酶含量，并计算其活性比值。结果 ＭＭＰ－２含量及活性在癌、癌旁及正常大肠组织中存在显著性差异（Ｐ〈０．０１），且与大肠癌Ｄｕｋｅ’ｓ分期（Ｐ〈０．０１）、     

抗Ⅳ型胶原酶胞内抗体对人巨细胞肺癌PG细胞侵袭的抑制作用

目的探讨内质网滞留型胞内抗体对Ⅳ型胶原酶分泌及其对人巨细胞肺癌PG细胞侵袭的抑制作用。方法构建了编码胞浆和内质网滞留型抗Ⅳ型胶原酶抗体的表达载体pcDNA3．1-CP．scFv和pcDNA3．1-ER．scFv。对人巨细胞肺癌PG细胞系进行基因转染。以Western blot检测pcDNA3．1-CP．scFv和pcDNA3．1-ER．scFv的表达,明胶酶谱检测PG细胞Ⅳ型胶原酶分泌情况,Matrigel实验检测细胞侵袭。结果CP．scFv和ER．scFv胞内抗体在PG细胞内表达,ER．scFv对Ⅳ型胶原酶分泌具有显著的抑制作用,对基质金属蛋白酶-9和基质金属蛋白酶-2的抑制率分别为85．7％和51．2％;而CP．scFv对Ⅳ型胶原酶分泌无抑制作用。ER．scFv编码基因转染的PG细胞与野生型和空白质粒组比较,对体外侵袭有明显的抑制,抑制率为76．3％;而CP．scFv编码基因转染的PG细胞未显示出有抑制作用。结论内质网滞留型胞内抗体技术可以在蛋白加工、分泌通路中抑制Ⅳ型胶原酶的活性,进而抑制肿瘤侵袭,可能在肿瘤基因治疗中具有重要的应用前景。     

恶性黑色素瘤的黑色素含量与其进展和转移相关性探讨

  目的  探讨黑色素含量与细胞周期蛋白D1（cyclin D1）表达在恶性黑色素瘤中的临床病理意义。  方法  采用组织芯片和免疫组织化学（immunohistochemistry,IHC）方法,检测购于西安艾丽娜生物有限公司189例恶性黑色素瘤患者组织中的黑色素含量和cyclin D1表达。  结果  189例恶性黑色素瘤组织中的黑色素高含量者为76例（40.2%）,cyclin D1高表达者为80例（45.7%）。黑色素含量与患者的年龄、性别、肿瘤组织来源和有无淋巴结转移无关,与肿瘤的侵袭深度（P=0.001）和临床分期（P=0.038）有关。T3、T4期黑色素瘤中的黑色素含量明显低于T1、T2期。同时,临床分期Ⅲ、Ⅳ期皮肤恶性黑色素瘤中的黑色素含量也明显低于Ⅰ、Ⅱ期。cyclin D1表达与年龄、性别、侵袭深度、临床分期、有无淋巴结转移和肿瘤组织来源无关。黑色素含量和cyclin D1表达在58例淋巴结转移性恶性黑色素瘤中呈负相关（r=-0.271,P=0.039）。  结论  黑色素含量下调可能与恶性黑色素瘤的侵袭、进展和转移有关。为恶性黑色素瘤的预后因素分析和病理诊断提供新的依据。      

内质网滞留型抗Ⅳ型胶原酶胞内抗体对肿瘤细胞侵袭和增殖的抑制作用

背景与目的:侵袭转移是恶性肿瘤细胞的重要特性,Ⅳ型胶原酶(包括基质金属蛋白酶2和基质金属蛋白酶9)在恶性肿瘤转移扩散中发挥着极其重要的作用。我们通过胞内抗体技术,阻断Ⅳ型胶原酶的分泌,以期达到抑制恶性肿瘤细胞侵袭转移的效果。方法:构建编码可在内质网滞留的抗Ⅳ型胶原酶单链抗体的表达载体pcDNA3.1-ER.scFv,并转染入人巨细胞肺癌PG细胞系内。Westernblot检测pcDNA3.1-ER.scFv的表达,免疫共沉淀实验分析胞内抗体ER.scFv在PG细胞内与靶蛋白相互作用情况,明胶酶谱检测Ⅳ型胶原酶的分泌,以及体外侵袭和增殖实验。结果:ER.scFv在PG细胞内表达,而且能够识别和结合靶蛋白基质金属蛋白酶9。通过胞内抗体的基因转染,显著抑制了Ⅳ型胶原酶的功能和活性。ER.scFv转染的PG细胞较野生型和空白质粒组,侵袭能力明显降低,抑制率为76.3%(P<0.05),在Matrigel上进行细胞培养,ER.scFv对PG细胞有明显的抗增殖效果。结论:内质网滞留型胞内抗体技术可以在蛋白加工、分泌这一关键通路中抑制肿瘤细胞Ⅳ型胶原酶的活性,进而抑制了肿瘤侵袭和增殖,在肿瘤基因治疗中具有应用前景。     

三种腺苷类似物抑制人高转移卵巢癌细胞侵袭的实验研究

背景与目的:腺苷作用于细胞信号转导系统,可抑制磷脂酰肌醇4-激酶活性、升高腺苷酸环化酶活性和细胞内cAMP水平、抑制血小板肌动蛋白聚合等。腺苷类似物有上述腺苷的生物活性,且不易为腺苷脱氨酶所降解,具有相对较长的生物半衰期。本研究拟观察2-氯腺苷、2-氯脱氧腺苷、2'-脱氧腺苷对人高转移卵巢癌细胞HO-8910PM体外侵袭的抑制作用。方法:以3种腺苷类似物处理HO-8910PM朱峰,等.三种腺苷类似物抑制人细胞、以5-氟尿嘧啶为细胞敏感对照,用台盼蓝拒染法观察药物对细胞的毒性效应;以人工重组基底膜观察细胞的侵袭、粘附和运动能力;以明胶酶显影法观察细胞基质金属蛋白酶-9(matrixmetalloproteinase-9,MMP-9)的分泌及活性;以RT-PCR检测细胞mta1mRNA、nm23H1mRNA表达。结果:2-氯腺苷和2-氯脱氧腺苷处理高转移卵巢癌细胞72h的IC50值分别为5.80μmol/L和2.61μmol/L,2'-脱氧腺苷对该细胞无明显抑制(对细胞的IC50值大于100μmol/L);6.0μmol/L的2-氯腺苷和2-氯脱氧腺苷对细胞侵袭的抑制率分别为(42.5±1.5)%(与对照组比较,P<0.01)和(9.9±0.5)%(P<0.05);对细胞运动的抑制率分别为(36.9±2.1)%(P<0.01)和(17.1±0.4)%(P<0.05);对细胞粘附的抑制率分别为(32.2±2.3)%(P<0.01)和(19.5±3.1)%(P<0.05)。而2'-脱氧腺     

Adinbitor对B16黑色素瘤细胞侵袭能力的影响

目的观察Adinbitor对黑色素瘤细胞侵袭迁移能力的影响。方法应用细胞培养技术,培养B16黑色素瘤细胞,经不同浓度的Adinbitor处理后,采用结晶紫染色法,检测其对基膜黏附的影响;Transwell小室模型观察其侵袭和迁移能力的变化。结果Adinbitor能显著抑制黑色素瘤细胞与基膜的黏附、抑制其迁移和侵袭（P〈0．01）,随Adinbitor浓度的增高其抑制作用增强。结论Adinbitor具有抑制B16黑色素瘤细胞侵袭迁移的作用。     

二种体外肿瘤侵袭模型的比较研究

本文采用双层培养板法和双套管是浮培养法，分别以人羊膜和ＳＤ胚鼠心肌组织作为靶组织，以小鼠Ｂ１６细胞，人ＳＧｃ－７９０１和ＨＲ８３４８细胞作为侵袭细胞，研究观察了上述三种肿瘤细胞体外侵袭行为。实验表明Ｂ１６和ＳＧｃ－７９０１细胞对人羊膜以及ＨＲ８３４８和ＳＧｃ－７９０１细胞对ＳＤ胚鼠心肌都有不同程度的侵袭作用。本文还对二种不同的体外侵袭模型的特点作了分析比较。     

小鼠黑色素瘤B16F10-Red细胞株的建立与生物学特性分析

目的:构建表达香菇珊瑚红色荧光蛋白(discosomasp red fluorescent protein,DsRed)的小鼠黑色素瘤B16F10-Red细胞株,并检测其生物学特性.方法:用GenEscortTMⅡ转染试剂将pDsRed质粒导入小鼠黑色素瘤B16F10细胞,G418加压培养联合极限稀释法建立稳定、高水平表达DsRed的单克隆细胞系.FCM检测B16F10和B16F10-Red细胞的细胞周期.比较B16F10-Red和B16F10细胞的克隆球形成能力和小鼠体内致瘤能力.结果:稳定表达DsRed的小鼠黑色素瘤B16F10-Red细胞株基本保持了其亲代细胞的特征,能在C57BL/6小鼠腹部皮下形成肿瘤并继续生长和转移.结论:B16F10-Red细胞株构建成功,其移植瘤模型成瘤率和转移情况同B16F10肿瘤相比无明显差别.     

Correlation between cell deformability and metastatic potential in B16-F1 melanoma cell variants

Four B16 melanoma cell variants were investigated to determine if there exists a correlation between their deformability and their metastatic potential. Cell deformability was measured as the percentage of cells traversing 10-mum diameter Nuclepore filter membranes at constant pressure as a function of time. A method was devised to circumvent common problems encountered in cell filtration experiments, i.e., cell aggregation and adhesion to the filter and failure to recover the input. F1a cells with the lowest spontaneous metastatic rate required 44 s for 50% of the cell input to traverse the filter, whereas No. 4 cells, featuring the highest metastatic rate, needed 12 s despite the fact that the cells had identical dimensions. Other variants tested showed intermediate filterability which also correlated with their metastatic potential. Cells, when pretreated with cytochalasin B at a final concentration of 21 microM exhibited increased filterability (75% and 42% greater than control for F1a and No. 4 cells, respectively). Somewhat smaller increases were observed after colchicine treatment. The findings imply major involvement of the cytoskeleton in the filterability and thus deformability of these B16 variants. Such physiochemical factors may play an important role in the metastasis of this and possibly other tumor types.     

Comparative study of the relationship between in vitro motility and invasion potentials of three mouse forestomach cancer (MFC) cell lines]

The relationship between motility of cancer cells and their invasiveness is important in understanding the invasion mechanisms of malignant tumors. The in vitro motility of three MFC cell lines was measured by the Boyden chamber technique. Statistical analysis of the results showed significant differences in vitro motility among these three cell lines. The motility correlated with in vitro invasion potentials.     

Generation of drug-resistant variants in metastatic B16 mouse melanoma cell lines

Genetic instability is recognized as an important aspect of the development of tumor heterogeneity and malignancy. In a previous study [Hill et al. Science (Wash. DC), 244:998-1001, 1984], we demonstrated that metastatic variants are generated at a more rapid rate in the highly metastatic B16F10 mouse melanoma cell line than in the less metastatic B16F1 cell line. The metastatic variants were phenotypically unstable, being generated and lost at high rates; consequently, we proposed a dynamic heterogeneity model of tumor metastasis which describes these properties quantitatively. As an extension of this work, we have examined the ability of these two melanoma cell lines to generate variants resistant to the drugs methotrexate and N-(phosphonacetyl)-L-aspartate. We observed that the highly metastatic B16F10 cell line generated variants resistant to a given concentration of methotrexate or N-(phosphonacetyl)-L-aspartate at higher rates than the B16F1 cell line. We conclude that B16F10 cells are genetically less stable than B16F1 cells and since resistance to methotrexate and N-(phosphonacetyl)-L-asparate usually results from gene amplification that B16F10 cells possess increased ability to amplify DNA. This higher rate of generation of drug-resistant variants corresponds to the higher rate of generation of metastatic variants we observed previously and suggests that a gene amplification mechanism may be involved in the generation of a metastic phenotype in B16 melanoma cells.     

Discrimination between human melanoma cell lines by fluorescence anisotropy

The fluorescence polarization of dipheylhexatriene (DPH) and trimethylammonium diphenylhexatriene (TMA-DPH) was measured when these markers were imbedded in cells of the human melanoma cell lines IGR37, IGR39, IGR3 and IGRA4, as well as in cells of the mouse melanoma cell lines B16 F1 and B16 F10. These measurements were performed on cell cultures which were grown on quartz plates as well as on cell suspensions. Considerable differences are found between the polarization values of the human cell lines that are related to their different origins. Differences for the plated cells are considerably greater than those for the suspensions. No differences in the polarization values were found for the two mouse melanoma lines. It is concluded that differences in lipid structural order can be found between cell types endowed with different metastasizing capabilities.     

Correlation between metastatic potency and the down-regulation of E-cadherin in the mouse hepatoma cell lines G-1 and G-5

Cell-cell adhesiveness, involving the adherens junction system including homophilic adhesion of cadherin and intracellular catenins, is a critical factor for tumor cell invasion and metastasis. We evaluated the levels of E-cadherin and beta-catenin in hepatoma cell sublines with high and low metastatic capacities. Stimulation of these cells with serum growth factors for more than 3 h after 24 h of starvation caused decreases in levels of E-cadherin and beta-catenin in the subline with high metastatic capacity, G-5. In contrast, no significant changes were observed in the subline with low metastatic capacity, G-1. Concomitantly with the decreases in E-cadherin and beta-catenin levels, G-5 cells were dissociated and detached from the culture dish, although G-1 cells again showed no morphological alterations. These in vitro results reflected the in vivo metastatic potencies of these hepatoma sublines, and further suggested the importance of the adherens junction system in determining metastatic potency of these parenchymal tumor cell lines as in epithelial/endothelial tumors.     

Correlation of frequency of induced mutation and metastatic potential in tumor cell lines from a single mouse mammary tumor

Spontaneous mutation rates were determined in mouse mammary tumor subpopulation lines that differ in metastatic phenotype. Although there was almost a 9-fold difference in spontaneous rates to ouabain resistance among the three lines tested, the difference did not correlate with ability to metastasize. Similarly a 10-fold difference in spontaneous rates to 6-thioguanine resistance did not correlate with metastatic ability. In contrast, the frequency of ethyl methanesulfonate-induced mutations was associated with metastatic potential. Thus, ethyl methanesulfonate only induced significant numbers of 6-thioguanine resistant colonies in 66 and 410.4 cells, the only 2 of 5 lines tested that spontaneously metastasize at high frequency, and of ouabain resistant colonies in 66, 410.4, and 168 cells, the only lines tested that produce experimental lung metastases after i.v. injection. Differential sensitivity to induced mutation was not correlated with differences in plating efficiency, wild type sensitivity to ethyl methanesulfonate, 6-thioguanine, or ouabain toxicity, ploidy, cell shape, cell size, or ability to engage in metabolic cooperation.     

Adhesion characteristics of human melanoma cell lines of varying metastatic potential

The adhesive behaviour of a series of human melanoma cell lines, of varying metastatic potential, to basement membrane and stromal components was investigated in vitro. Experimental metastatic propensity was assessed from the number of pulmonary nodules formed after i.v. injection of cells into BALB/c nude mice. All cell lines showed similar kinetics of attachment when tested on plastic, type-I collagen films, type-I collagen hydrated gels, fibronectin, laminin type-IV collagen substrates and bovine aortic endothelial monolayers. Fibronectin-coated plastic compared to plastic alone produced increased cell attachment and spreading to the same extent in all the cell lines. The melanoma lines attached preferentially to cryostat sections of lung compared to other organs reflecting the pattern of organ involvement of metastasis in vivo. However, no significant quantitative differences in attachment to lung sections were seen between melanoma variants of differing metastatic capacities. Cells labelled with [125I]iododeoxyuridine to determine their initial organ distribution following i.v. injection showed that tumour-cell arrest was not significantly changed enough to explain the differing metastatic capacities. Thus it appears that adhesive properties of these melanoma cells are not correlated with their capacity to form metastases in vivo.     

血管内皮生长因子-C的表达与宫颈癌生物学行为的关系

目的研究VEGF-C表达与宫颈癌临床病理生物学行为的关系。方法免疫组化S-P法检测59例宫颈癌手术标本中VEGF-C蛋白表达情况。结果宫颈癌中VEGF-C蛋白表达率为66.1%(39/59),与盆腔淋巴结转移显著相关(P=0.005)。但与年龄、国际妇产科联盟(FI-GO)分期、组织学类型、组织学分级和肿瘤直径无关。VEGF-C表达阳性组5年生存率显著低于阴性组(P=0.006)。结论宫颈癌组织中VEGF-C蛋白表达与宫颈癌侵袭转移和预后有关,检测VEGF-C对了解宫颈癌生物学行为和评估预后具有一定的临床价值。