肿瘤血管生长抑制因子与肝细胞癌关系研究进展

血管生成抑制因子对于调控肿瘤的生物学行为和肿瘤的治疗方面具有重要的作用.血管生成受正、负调控因子的双重作用,目前发现的与肝细胞癌（hepatocellular carcinoma,HCC）有关的血管生成抑制因子有血管生成抑制素（AS）、内皮抑素（ES）、干扰素（IFN）、血小板反应素（TSP）、组织金属蛋白酶抑制剂（TIMP）、转化生长因子β1（TGFβ1）、白细胞介素12（IL 12）、肿瘤坏死因子（TNF）、血小板因子4（PE4）及纤溶酶原抑制因子（PAI）等.其作用机制为：抑制内皮细胞的分裂和迁移,降低内皮细胞的活性,促进内皮细胞的凋亡或直接杀伤内皮细胞,干扰基底膜与内皮细胞的相互作用,抑制蛋白水解反应和内皮细胞的黏附运动,下调各种促血管生成因子的表达等.这些因子具有抗HCC血管生成治疗药物的潜在意义.     

抗肿瘤新生血管靶向治疗进展

肿瘤的生长和存活依赖于生成的血管为它所提供的氧气和营养物质，没有血管的生成，肿瘤最大也只能长至1～2mm^3。1971年，Folkman等提出可通过阻断肿瘤血管的生成来抑制肿瘤的生长，防止肿瘤的转移。基于肿瘤血管生成机理，抑制肿瘤血管生成的策略包括：（1）利用小分子药物和内源性血管生成抑制因子直接抑制血管内皮细胞增殖、迁移，抑制细胞外基质形成，诱导血管内皮细胞凋亡；（2）利用中和抗体、可溶性受体、受体拮抗剂阻断血管生成因子传递；（3）利用反义核酸或生物因子抑制血管生成促进因子表达。     

内抑素(endostatin)对移植大肠癌新生血管密度影响的动物实验研究

目的：探讨内皮细胞生长抑素（内抑素endostatin）对裸鼠大肠血管生成的影响及其作用机理，为endostatin的临床应用提供理论依据。方法：采用免疫组化方法检测肿瘤新生血管密度，对用药过程中瘤 体积进行测量，对endostatin抑制肿瘤的效果进行分析。结果：endostatin能够抑制裸鼠大肠癌肿瘤血管生成，减轻肿瘤的血管密度。结论：endostatin通过抑制血管形成抑制肿瘤的生长。     

缺氧共培养胶质瘤细胞对内皮细胞生长及凋亡的影响

目的：通过体外模拟胶质瘤缺氧微环境,研究在缺氧条件下C6胶质瘤细胞对人脐静脉内皮细胞（HUVECs）生长及凋亡的影响。方法：利用Transwell共培养装置及二氯化钴（CoCl2）模拟缺氧法,建立模拟胶质瘤缺氧微环境的体外共培养模型。通过绘制生长曲线明确内皮细胞生长状况,流式细胞仪检测内皮细胞凋亡及细胞周期。结果：缺氧能够显著抑制内皮细胞生长,促进内皮细胞凋亡。缺氧培养24小时后,内皮细胞中G1期细胞增加,S期细胞减少。相反,共培养胶质瘤细胞能够减少缺氧诱导的内皮细胞凋亡。同时,细胞周期中G1期细胞比例下降,S期细胞比例升高。结论：缺氧能够诱导内皮细胞凋亡,抑制内皮细胞增殖。而共培养胶质瘤细胞能够有效减少这种缺氧诱导的内皮细胞凋亡,促进内皮细胞有丝分裂。胶质瘤细胞可能正是通过这一机制,维持肿瘤血管发生。     

血管生成抑制素在实体瘤治疗中的研究进展

血管生成抑制素是肿瘤衍生的血管生成抑制因子，能有效地抑制血管内皮细胞的增生和迁移，在实体瘤的治疗中有重要的应用价值。综述了血管生成抑制素的研究现状，分析了它与实体瘤血管生成的关系，及其在实体瘤抗血管生成治疗中的应用前景。     

和厚朴酚抗血管生成作用的实验研究

目的：本研究通过检测和厚朴酚在体内对鸡胚尿囊膜（chicken chorilallantoic membranes，CAM）血管生成的抑制作用及体外对肿瘤血管生成的影响，探讨和厚朴酚的抗血管生成作用及其初步机制。方法：（1）用四甲基偶氮唑盐（MTT）法检测和厚朴酚对人脐静脉内皮细胞（human umbilical vein endothelial cell，HUVEC）、人成纤维细胞及人结肠癌RKO细胞增殖的抑制作用；（2）采用鸡胚绒毛尿囊膜模型，观测和厚朴酚对CAM新生血管生成的抑制作用；（3）逆转录聚合酶链反应（RT-RCR）方法检测和厚朴酚对RKO细胞血管内皮生长因子A（VEGF-A）mRNA表达及细胞培养上清液VEGF蛋白分泌的影响。结果：和厚朴酚对血管内皮细胞具有优先抑制作用，对HUVEC的半数抑制剂量（inhibitory concentration 50％，IC50）为14．5μmol/L，而对原代培养的人成纤维细胞的IC50高达150μmol/L，对人结肠癌RKO细胞的IC50为42μmol/L；和厚朴酚0．1μg／只和0．2μg／只剂量时显著抑制CAM新生血管形成，抑制率分别为58％和86％；和厚朴酚在10μmol/L和20μmol/L剂量时显著抑制RKO细胞的VEGF-A mRNA表达及细胞培养上清液中VEGF蛋白的分泌，具有显著性差异。结论：和厚朴酚在非凋亡剂量时具有抗血管形成作用，其机制与抑制血管内皮细胞增殖以及抑制肿瘤细胞表达VEGF有关。     

血管生成抑制素在实体瘤治疗中的研究进展

血管生成抑制素是肿瘤衍生的血管生成抑制因子 ,能有效地抑制血管内皮细胞的增生和迁移 ,在实体瘤的治疗中有重要的应用价值。综述了血管生成抑制素的研究现状 ,分析了它与实体瘤血管生成的关系 ,及其在实体瘤抗血管生成治疗中的应用前景     

caveolin-1与内皮细胞的细胞周期关系研究

目的：研究caveolin-1与内皮细胞增殖和细胞周期的关系。方法：以腺病毒为载体，在内皮细胞内高表达caveolin-1蛋白，以H^3-胸腺嘧啶掺入法检测caveolin-1与内皮细胞增殖的关系，应用流式细胞仪检测caveolin-1对内皮细胞周期的影响。结果：高表达caveolin-1导致内皮细胞H^3-胸腺嘧啶掺入率降低了67％（P〈0．05），处于G0／G1期的内皮细胞百分比增加（P〈0．05）。结论：caveolin-1通过抑制内皮细胞于G0／G1期来抑制内皮细胞增殖。     

TSP-1抗血管生成机制研究进展

肿瘤的生长和转移依赖于的血管的生成,血管生成是促血管生成因子与抑制血管生成因子共同调控的结果。凝血酶敏感蛋白-1（TSP-1）是一个有力的抗血管生成因子,它能通过阻止内皮细胞对各种血管生成因素的刺激反应而抑制血管生长。本文就TSP-1抗血管生成机制研究进展做一综述。     

肿瘤相关巨噬细胞对膀胱癌细胞增殖和血管生成的影响

目的:探讨肿瘤相关巨噬细胞（TAMs）对膀胱癌细胞增殖和血管生成的影响。方法:采用巨噬细胞U937的上清液培养膀胱癌细胞系T24,CCK-8法检测膀胱癌细胞的增殖能力;流式细胞术检测其对细胞周期的影响;通过实时荧光定量PCR和Western blot检测膀胱癌细胞中血管内皮生长因子（VEGF）的mRNA和蛋白表达以及AKT磷酸化水平。结果:CCK-8结果表明TAMs促进膀胱癌细胞的增殖;流式结果显示TAMs能促进细胞周期G1-S期转换;实时荧光定量PCR和Western blot 结果显示TAMs促进膀胱癌细胞内VEGF的mRNA和蛋白表达,并促进AKT磷酸化。结论:TAMs促进细胞周期G1-S期转换,促进AKT磷酸化和VEGF的表达,且TAMs有望成为膀胱癌治疗和预后判断新的靶点。     

GBX2过表达促进人宫颈癌SiHa细胞增殖、迁移及侵袭

[摘要] 目的：探讨GBX2 基因在人宫颈癌SiHa 细胞增殖和侵袭转移中的作用及其机制。方法：应用质粒转染技术,分别将过表达GBX2 基因重组质粒pCMV6-entry-GBX2（实验组）及空载体质粒pCMV6-entry（阴性对照组）转染到宫颈癌SiHa 细胞中,用WST-1 法、集落形成实验、流式细胞术分别检测转染细胞的增殖、集落形成和细胞周期,用划痕愈合实验、Transwell 实验检测细胞的迁移、侵袭能力,用ELISA 法检测细胞培养上清中IL-6 的表达水平,用WB检测EMT相关蛋白的表达变化并探讨其可能的作用机制。结果：与SiHa/pCMV6 组相比,上调GBX2 表达后：（1）SiHa/GBX2 组细胞的增殖、集落形成、迁移和侵袭能力明显增强（均P<0.01）,G0/G1 期的细胞比例减少、S期与G2/M期的细胞比例增加（均P<0.01）;（2）SiHa/GBX2 组细胞EMT相关蛋白上皮钙黏蛋白表达水平下降,神经钙黏蛋白、波形蛋白和snail 表达水平上调（均P<0.01）;（3）SiHa/GBX2 组细胞培养上清中IL-6 的表达水平明显增高（P<0.01）;（4）SiHa/GBX2 组细胞STAT3 磷酸化水平增强,并能被STAT3 抑制剂S31-201 抑制（P<0.01）。结论：GBX2可能通过IL-6/STAT3 通路诱导宫颈癌SiHa 细胞EMT,从而促进宫颈癌细胞的增殖、迁移和侵袭能力。     

软骨细胞培养液对鸡胚绒毛尿囊膜血管抑制效应的研究

目的检测软骨细胞培养液中是否存在抑制血管生长的因子。方法用酶消化、分离鸡胚关节软骨细胞,体外单层培养,收集细胞培养液。采用鸡胚绒毛尿囊膜实验检测该培养液对血管的抑制效应。结果加软骨细胞培养液的实验组血管数为2.75±0.99且血管较细、分支少;不加样的对照组血管数为4.5±1.08且血管较粗、分支多。两组有显著差异(P＜0.05)结论软骨细胞培养液具有抑制血管生长的作用。     

Effect of direct cell-to-cell interaction between the KM-102 clonal human marrow stromal cell line and the HL-60 myeloid leukemic cell line on the differentiation and proliferation of the HL-60 line

Hematopoietic cellular interaction was investigated in a coculture of the human clonal marrow stromal line, KM-102, and the myeloid leukemia cell line, HL-60. In the coculture, a large number of HL-60 cells remained in the supernatant but some of them became firmly attached to KM-102 cells. The attached HL-60 cells showed little positive reaction in the NBT test when the culture was supplemented with 10(-9) to 10(-7) M 1 alpha, 25-dihydroxyvitamin D3. In contrast, differentiation in the supernatant HL-60 cells was strikingly responsive to the agent in a dose-dependent way. Furthermore, the complete inhibition was observed in the incorporation of [3H]thymidine into the attached HL-60 cells with autoradiography, but 23.6% of the supernatant cells moderately incorporated [3H] thymidine into their nuclei. There was no attachment between HL-60 cells and stromal cells from human thymus and lymph node, or between lymphocytic leukemia cells and marrow stromal cells. These results indicate that there is direct cellular interaction between myeloid leukemic cells and marrow stromal cells which modulates the proliferation and differentiation of the myeloid leukemic cells. This modulation by marrow stromal cells is more strongly affected by this interaction than by exogenously added differentiation-inducing agents. Apparently marrow stromal cells produce a definitive milieu for the proliferation and differentiation of myeloid leukemic cells.     

Stromal cells promote angiogenesis and growth of human prostate tumors in a differential reactive stroma (DRS) xenograft model

Reactive stroma has been reported in many cancers, including breast, colon,and prostate. Although changes in stromal cell phenotype and extracellular matrix have been reported, specific mechanisms of how reactive stroma affects tumor progression are not understood. To address the role of stromal cells in differential regulation of tumor incidence, growth rate, and angiogenesis, LNCaP xenograft tumors were constructed in nude mice with five different human prostate stromal cell lines as well as GeneSwitch-3T3 cells engineered to express lacZ under mifepristone regulation. Alone, LNCaP prostate carcinoma cells were essentially nontumorigenic, whereas combinations of LNCaP cells with three different human prostate stromal cell lines (L/S tumors) resulted in a tumor incidence (50-63%) similar to that of control LNCaP plus Matrigel (L/M) tumors over a 9-week period. In contrast, LNCaP combinations with two other human prostate stromal cell lines were nontumorigenic, illustrating that stromal cell effects are differential. L/S tumors exhibited well-developed blood vessels at 2 weeks, whereas control L/M tumors were avascular at 2 weeks and exhibited blood lakes in lieu of extensive vessels at later time points. Xenografts constructed under three-way conditions (LNCaP, Matrigel, and stromal cells; L/M/S tumors) exhibited a 100% tumor incidence and showed rapid blood vessel formation as early as day 7 with mature vessels formed by day 10. L/M/S tumors exhibited a 10.3-fold increase in microvessel density, and the corresponding hemoglobin:tumor weight ratio was increased 2-fold relative to L/M control tumors at day 10. L/M/S tumor wet weight and volume increased by 1.6- and 2.4-fold, respectively, by day 21, compared with control L/M tumors. L/M/S tumors made with LNCaP cells plus GeneSwitch-3T3-pGene/lacZ stromal cells showed similar results. Mifepristone-regulated gene expression was observed in stromal cells immediately adjacent to clusters of carcinoma cells and in vessel walls in a mural cell (pericyte) position. This study shows that regulation of angiogenesis is one mechanism through which stromal cells affect LNCaP tumor incidence and growth rate. This regulation may be mediated through direct recruitment and interactions of stromal cells with endothelial cells. Furthermore, this study describes for the first time a model system with regulated transgene expression in the stromal compartment of an experimental carcinoma. These findings point to the stromal compartment as a potential source of new prognostic markers and therapeutic targets and show the utility of the carcinoma-stromal xenograft model system in dissecting specific mechanisms of reactive stroma.     

Tumor-stroma interactions reduce the efficacy of adenoviral therapy through the HGF-MET pathway

Many preclinical studies have shown the potential of adenovirus-based cancer gene therapy. However, successful translation of these promising results into the clinic has not yet been achieved. Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant desmoplastic stroma, and tumor-stromal cell interactions play a critical role in tumor progression. Therefore, we hypothesized that tumor-stroma interactions reduce the efficacy of adenoviral therapy. We investigated the effect of fibroblasts on adenovirus-based gene therapy using SUIT-2 and PANC-1 pancreatic cancer cells cultured with or without fibroblast-conditioned culture supernatant then infected with Ad-LacZ. After 48 h, the cells were stained for β-galactosidase. The results showed that the number of β-galactosidase-positive cells was significantly reduced after culture with fibroblast-conditioned supernatant (P < 0.05). Because the hepatocyte growth factor (HGF)/MET pathway plays an important role in tumor-stroma interactions we next investigated the involvement of this pathway in tumor-stroma interactions leading to the decreased efficacy of adenoviral therapy. SUIT-2 cells were cultured with or without SU11274 (a MET inhibitor) and/or fibroblast-conditioned culture supernatant, then infected with Ad-GFP. After 48 h, GFP-positive cells were counted. The number of GFP-positive cells in cultures containing fibroblast-conditioned supernatant plus SU11274 was significantly greater than in cultures without SU11274. In conclusion, our results suggest that stromal cells in PDAC reduce the efficacy of adenoviral therapy through a mechanism involving the HGF/MET pathway. Control of such tumor-stroma interactions may lead to improvements in adenoviral gene therapy for PDAC.     

Antitumor effects of an antiangiogenic polysaccharide from an Arthrobacter species with or without a steroid

A sulfated polysaccharide-peptidoglycan complex, DS-4152, isolated from the culture supernatant of an Arthrobacter species inhibited angiogenesis and tumor growth and enhanced the antiangiogenic activity of 11 steroid hormones by 2 to 100 times. In the presence of cortisone acetate or tetrahydro S, DS-4152 suppressed chick chorioallantoic membrane angiogenesis and murine tumor M5076 cell-induced s.c. angiogenesis. The antitumor effects of DS-4152 administered in combination with a steroid whose dose level did not affect tumor growth were examined. DS-4152 significantly inhibited the growth of s.c.-implanted B16 melanoma in combination with cortisone acetate. DS-4152 plus tetrahydro S inhibited the growth of s.c. solid tumors and prolonged the survival time of mice bearing highly metastasizing M5076. The body weight increase was not affected by any administration. On the other hand, the survival of mice with ascitic M5076 tumors was not affected by the combination of DS-4152 plus tetrahydro S. The antiangiogenic activity of DS-4152 was more potent than that of heparin. Furthermore, DS-4152 is an angiogenesis inhibitor by itself, without steroid hormones. Successive s.c. treatment with heparin caused hemorrhagic death, but with DS-4152, suppressed tumor growth without reducing body weight.