子宫腺肌病组织中PTTG、bFGF和VEGF的表达及意义

采用免疫组化法检测26例子宫腺肌病及30例正常子宫平滑肌组织中的垂体转化基因蛋白(PTTG)、碱性成纤维细胞生长因子(bFGF)及血管内皮生长因子(VFGF).结果显示,在子宫腺肌病组织中PTTG、bFGF及VEGF的阳性表达率分别为84.6%、73%及57.7%,正常子宫组织三个指标的阳性表达率均为20%,两者三指标相比,P均<0.01;三指标在子宫腺肌病组织中的表达呈正相关(r分别为0.621、0.518、0.576,P均<0.01).认为PTTG、bFGF和VEGF共同参与了子宫腺肌病的发生和发展.     

沙利度胺联合三氧化二砷腹腔注射对肝癌细胞SMMC7721移植瘤生长的影响及机制

目的观察沙利度胺(Tha)联合三氧化二砷(AsT)对肝癌细胞SMMC7721裸鼠移植瘤生长的影响,并探讨其可能作用机制。方法将35只雄性BALB/c裸鼠随机分为7组各5只,均于颈部皮下接种人肝癌细胞株SMMC7721细胞悬液(密度为5×107/mL)0.2 mL,其后按如下方法处理:高、低剂量Tha组分别腹腔注射Tha 25、2.5 mg/kg,AsT组腹腔注射AsT 2.5 mg/kg,高、低剂量Tha联合AsT组分别同前联合给予Tha、AsT,阳性对照组腹腔注射环磷酰胺(CTX)20 mg/kg,阴性对照组腹腔注射生理盐水20 mL/kg,均连续给药4周。观察各组裸鼠皮下移植瘤体积变化及裸鼠行动、对外界刺激的反应;用药结束后处死裸鼠,取部分瘤组织采用RT-PCR法检测血管内皮生长因子(VEGF)mRNA表达,免疫组化法检测Bax、Caspase-3表达。结果裸鼠移植瘤成瘤率100%,瘤体积在高剂量Tha联合AsT组<低剂量Tha联合AsT组<高剂量Tha组<低剂量Tha组低剂量Tha联合AsT组>AsT单药组>高剂量Tha组>低剂量Tha组>阳性对照组>阴性对照组,差异均有统计学意义(P均<0.05)。结论 Tha联合AsT对SMMC 7721移植瘤的生长具有显著抑制作用,效果优于两单药;机制分别与通过下调VEGF mRNA表达抑制肿瘤血管生成、上调Bax及Caspase-3蛋白表达促进肿瘤细胞凋亡相关。     

人参皂甙对食管癌细胞VEGF表达的影响

目的 探讨人参皂甙(Rg3)抑制人食管癌鳞癌细胞血管生成的机制.方法 将75 μmol/L的二氧化钴单独或联合30、60 μg/ml的Rg3作用于食管癌EC9706细胞24 h和48 h,分别收取细胞培养上清液,用 ELISA法检测其中血管内皮生长因子(VEGF)水平,以含10%胎牛血清RPMI1640培养基做空白对照.结果 与空白组对比,二氯化钴作用24、48 h后,VEGF表达明显升高;二氯化钴与Rg3联合作用后VEGF表达明显下降,并与Rg3的剂量和作用时间密切相关.结论 Rg3可抑制二氯化钴诱导的EC9706细胞血管生成;其可能机制是抑制VEGF表达.     

PTTG和bFGF在原发性胆囊癌中表达及意义

探讨垂体肿瘤转化基因 (PituitaryTumorTransformingGene ,PTTG)和碱性成纤维细胞生长因子 (basicFi broblastGrowthFactor,bFGF)在原发性胆囊癌中的表达及相互关系 ,研究它们的表达与肿瘤临床病理指标及预后的联系。应用免疫组织化学 ,检测 4 1例原发性胆囊癌及 2 0例慢性结石性胆囊炎中PTTG和bFGF的表达水平。在原发性胆囊癌中PTTG和bFGF阳性表达率分别为 85 4 %、5 6 1% ,其阳性率及表达等级均明显高于慢性胆囊炎 (P <0 0 1)。PTTG和bFGF表达等级均与胆囊癌的Nevin分期和淋巴结转移密切相关 (P <0 0 5或P <0 0 1) ;PTTG表达与bFGF表达成等级正相关 (r =0 5 2 6 ,P <0 0 1)。PTTG或bFGF阳性患者的累计生存期明显低于阴性者 (P <0 0 5 )。PTTG或bFGF与胆囊癌生物学行为及预后有密切关系 ,二者的联合检测 ,有助于原发性胆囊癌恶性程度和预后的判断     

PTTG在垂体瘤组织中的表达及意义

采用免疫组化SP法检测垂体瘤转化基因(PTTG)在18份侵袭性垂体瘤组织、32份非侵袭性垂体瘤组织及10份正常垂体瘤组织中的表达水平.结果 PTTG蛋白在袭性垂体瘤组织中的表达水平明显高于非侵袭性垂体瘤组织及正常垂体瘤组织,提示PTTG基因高表达可促进侵袭性垂体瘤的发生.     

抗人VEGF发卡状核酶基因的构建及其对肝癌细胞VEGF蛋白表达的影响

利用计算机辅助设计合成针对人VEGF的发卡状核酶（RZ）,定向亚克隆于真核表达载体pcDNA3．1^＋中,转染肝癌细胞SMMC-7721,RT—PCR鉴定。ELISA法和MTT法检测SMMC-7721细胞对照组、SMMC-7721／pcDNA3．1^＋空载体对照组和SMMC-7721／RZ基因转染组细胞VEGF表达和增殖情况,流式细胞术检测各组细胞周期和凋亡情况。结果为SMMC-7721／RZ基因转染组细胞VEGF表达水平和细胞增殖速率显著低于SMMC-7721细胞对照组和SMMC-7721／pcDNA3．1’空载体对照组。MC-7721／RZ基因转染组细胞出现凋亡峰,其凋亡率达11．O％,而细胞对照和空载体细胞对照均末出现。认为SMMC-7721／RZ转基因细胞株的成功构建和生物学特性的初步研究为进-步建立裸鼠人肝癌模型及评价RZ对体内肝癌生长的抑制作用奠定了-定的基础。     

三氧化二砷治疗肝肿瘤的机制研究

As2O3对肝细胞肝癌(HCC)的抗肿瘤机制尚不明确。一般认为其主要作用途径包括：(1)诱导肿瘤细胞凋亡；(2)抑制肿瘤转移；(3)影响肝癌细胞免疫功能；(4)通过抑制VEGF合成间接抗肿瘤血管形成；(5)与化疗药联用的增效作用。本实验以原发性肝细胞肝癌模型为基础，观察砷剂的抗肿瘤作用。     

大肠癌组织PTTG、VEGF-C表达及其与微淋巴管生成的关系

目的探讨垂体肿瘤转化基因(PTTG)、血管内皮生长因子-C(VEGF-C)在大肠癌发生、发展中的作用。方法采用RT-PCR技术及免疫组织化学法检测20份大肠正常黏膜(正常组)、80份大肠癌组织(大肠癌组)中PTTG mRNA及PTTG蛋白的表达水平;用D2-40标记微淋巴管,采用免疫组化法检测VEGF-C、D2-40表达,计算微淋巴管密度(LMVD)。结果大肠癌组PTTG、VEGF-C表达量及LMVD均明显高于正常组,且与大肠癌浸润深度、淋巴结转移、Duke’s分期呈正相关。结论 PTTG与VEGF-C在大肠癌的发生发展中起重要作用,促淋巴管生成为其主要作用机制;PTTG与VEGF-C可作为判断大肠癌转移及预后的预测指标。     

颌面部血管瘤VEGF及其受体的表达及意义

为探讨血管内皮生长因子（VEGF）及其受体（VEGFR／KDR）在颌面部血管瘤发生、发展中的作用，采用二步En Vision免疫组织化学方法，分别检测11例血管瘤（HA）和8例血管畸形（VM）组织中VEGF和VEGFR／KDR的表达；同时采用原位杂交Gene Point法检测VEGF mRNA的表达。结果显示，VEGF和VEGFR／KDR在HA中高表达，而在VM中低表达，两者差异有显著性（P＜0.01）；VEGF和VEGF mRNA在血管瘤细胞及周围间质细胞明显表达，VEGF／KDR主要表达于HA中内皮细胞膜上。提示VEGF可通过自分泌和旁分泌的形式影响HA内皮细胞的增殖，可能与HA发生、发展和退化密切相关。     

三氧化二砷对肝癌细胞侵袭转移的影响及机制

目的 探讨三氧化二砷(As2O3)抗肝癌侵袭转移的机制.方法 体外培养人肝癌细胞株SMMC-7721,将0、1.0、2.0 μmol/L浓度的As2O3作用于SMMC-7721细胞,采用过河实验检测肿瘤细胞体外运动;MTT法观察细胞黏附能力;Transwell体外侵袭转移模型检测细胞迁徙、侵袭能力;并用PCR法检测乙酰肝素酶(HPA)mRNA表达.结果 0、1.0、2.0 μmol/L As2O3作用SMMC-7721细胞后,表现为过河时间延长,细胞黏附抑制率明显上升,过膜细胞数减少(P＜0.01),HPA基因表达量降低(P均＜0.01);且呈剂量依赖性.结论 As2O3可明显抑制人肝癌细胞SMMC-7721细胞黏附、迁徙和侵袭能力,下调HPA mRNA表达,此可能为其抗肿瘤侵袭转移作用的机制之一.     

三氧化二砷对肺腺癌细胞凋亡及肺耐药蛋白基因多药耐药蛋白基因表达的影响

目的　研究三氧化二砷 (As2 O3 )对人肺腺癌的抑制和诱导凋亡作用及对LRP、MRP基因表达的影响及可能机制。方法　哈尔滨医科大学第一临床医院呼吸内科于 2 0 0 2 - 0 5～ 2 0 0 4 - 0 6选用人肺腺癌A5 4 9细胞株 ,运用体外细胞培养法 ,MTT法 ,流式细胞术检测As2 O3 对人肺腺癌的抑制和诱导凋亡作用 ;用逆转录 -聚合酶链反应(RT -PCR)方法检测LRP、MRPmRNA的表达。结果　As2 O3 对人肺腺癌A5 94细胞具有抑制作用 ,其抑制率呈时间 -剂量依赖关系。不同浓度的As2 O3 均可诱导凋亡。 1 0 μmol/L、2 0 μmol/L的As2 O3 可下调LRP、MRPmRNA的表达。结论　As2 O3 具有抗肿瘤作用 ,主要是通过诱导细胞凋亡实现的 ,其机制与下调LRP、MRPmRNA表达有密切关系。     

Anti-hepatoma effect of arsenic trioxide on experimental liver cancer induced by 2-acetamidofiuorene in rats

AIM: To study the anti-hepatoma efficiency of arsenic trioxide (As2O3) in the treatment of experimental rat hepatocellular carcinoma (HCC) induced by 2-acetamidofluorene (2-FAA)and to elucidate the possible mechanisms.METHODS: SD rats (2 mo old) had been fed with 2-FAA for 8 wk to induce HCC, and then they were treated with As2O3 or matrine. On d 29, the rats were killed and the liver was weighed and liver tumors were counted. The histological changes of liver tissue were observed under microscope, and the cellular dynamic parameters were studied by flow cytometry. Immunohistochemistry (two-step method) was used to observe the expression of vascular endothelial growth factor (VEGF) and micro-vessel density (MVD) on consecutive sections. The pathological parameters were also analyzed, the levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT),total bilirubin (TBi), and direct bilirubin (DBi).RESULTS: The number of liver tumors decreasedsignificantly in groups treated with As2O3, especially in medium-dose (1 mg/kg) group (t = 2.80, P＜0.01). As2O3 caused HCC cell death via apoptosis; necrosis was seen and apoptosis was common when the dose was 1 mg/kg.Proliferation index decreased sharply in medium-dose (1 mg/kg) group (7.87±4.11 vs 24.46±6.49, t = 2087,P＜0.01), but not in 0.2 mg/kg group. However, S-phase fraction decreased dramatically in both groups, it reached the bottom level only when the dose was 1 mg/kg compared with control (0.40±0.13 vs 3.01±0.51, t = 2.97, P＜0.01),and it was obviously accompanied with accumulation of cells in G0/G± (G0/G1 restriction). The expressions of VEGF and MVD in medium-dose (1 mg/kg) group were significantly lower than normal saline group (0.63±0.74 vs 2.44±0.88, P＜0.05; 15.75±3.99 vs47.44±13.41, t= 2.80,P＜0.01). Compared with normal saline group, mediumand low-dose groups As2O3 and matrine lowered the levels of ALT in serum (61.46±9.46, 63.75±20.40, 61.18±13.00 vs 108.98±29.86, t= 2.14, P＜0.05), but had no effect on the level of serum AST, TBi, and DBi.CONCLUSION: As2O3 had inhibitory effect on growth of experimental HCC in rats induced by 2-FAA, but had no obvious effect on normal hepatic cells. The mechanisms may involve decrease of cell division, accumulation of cells in G0/G1 phase, apoptosis of tumor cells, and inhibitory effect on angiogenesis through blocking VEGF.     

Anti-hepatoma effect of arsenic trioxide on experimental liver cancer induced by 2-acetamidofluorene in rats

AIM: To study the anti-hepatoma efficiency of arsenic trioxide (As2O3) in the treatment of experimental rat hepatocellular carcinoma (HCC) induced by 2-acetamidofluorene (2-FAA) and to elucidate the possible mechanisms. METHODS: SD rats (2 mo old) had been fed with 2-FAA for 8 wk to induce HCC, and then they were treated with As2O3 or matrine. On d 29, the rats were killed and the liver was weighed and liver tumors were counted. The histological changes of liver tissue were observed under microscope, and the cellular dynamic parameters were studied by flow cytometry. Immunohistochemistry (two-step method) was used to observe the expression of vascular endothelial growth factor (VEGF) and micro-vessel density (MVD) on consecutive sections. The pathological parameters were also analyzed, the levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBi), and direct bilirubin (DBi). RESULTS: The number of liver tumors decreased significantly in groups treated with As2O3, especially in medium-dose (1 mg/kg) group (t = 2.80, P><0.01). As203 caused HCC cell death via apoptosis; necrosis was seen and apoptosis was common when the dose was 1 mg/kg. Proliferation index decreased sharply in medium-dose (1 mg/kg) group (7.87±4.11 vs 24.46±6.49, t= 2087, P<0.01), but not in 0.2 mg/kg group. However, S-phase fraction decreased dramatically in both groups, it reached the bottom level only when the dose was 1 mg/kg compared with control (0.40±0.13 vs .01±0.51, t= 2.97, P<0.01), and it was obviously accompanied with accumulation of cells in G0/G1 (G0/G1 restriction). The expressions of VEGF and MVD in medium-dose (1 mg/kg) group were significantly lower than normal saline group (0.63±0.74 vs 2.44±0.88,P<0.05; 15.75±3.99 vs 47.44±13.41, t = 2.80, P<0.01). Compared with normal saline group, mediumand low-dose groups As2O3 and matrine lowered the levels of ALT in serum (61.46±9.46, 63.75±20.40, 61.18±13.00 vs 108.98?9.86, t = 2.14, P<0.05), but had no effect on the level of serum AST, TBi, and DBi. CONCLUSION: As2O3 had inhibitory effect on growth of experimental HCC in rats induced by 2-FAA, but had no obvious effect on normal hepatic cells. The mechanisms may involve decrease of cell division, accumulation of cells in G0/G1 phase, apoptosis of tumor cells, and inhibitory effect on angiogenesis through blocking VEGF.     

三氧化二砷治疗晚期原发性肝癌的临床观察

目的探讨三氧化二砷(As2O3)治疗晚期原发性肝癌(PLC)的临床价值。方法将48例患者随机分为治疗组(26例)和对照组(22例)。两组患者在保肝、抗病毒、支持对症等治疗基础上,治疗组加用As2O3 10 mg,10%葡萄糖注射液500 ml,每日1次静脉输注,每2周为1个疗程,间隔2周进行下一个疗程,共2个疗程,观察结束复查并进行疗效评估。结果两组患者观察结束的客观有效率、获益率分别为0.0%、19.2%(P=0.0376)和22.8%、53.8%(P=0.0083),两组患者生活质量改善和稳定率分别为31.8%和65.4%(P=0.0291)。结论 As2O3治疗晚期PLC具有一定的临床价值,对于肝功能差且无法耐受其他治疗的PLC患者,As2O3仍不失是一种较有效的治疗手段。     

三氧化二砷对人体胃肠癌细胞凋亡及p53、survivin表达的影响

目的研究三氧化二砷（arsenic trioxide,As2O3）在人体内诱导胃肠癌细胞凋亡作用及对凋亡相关基因p53、survivin表达的影响,探讨其抗肿瘤作用及发生机制。方法采用原位末端转移酶标记技术（TUNEL）和免疫组化法分别检测As2O3用药前后胃肠癌细胞凋亡指数和凋亡相关基因蛋白P53、Survivin的表达变化情况。结果 As2O3用药后胃肠癌细胞凋亡指数为（17.04±3.67）%,与用药前（6.07±2.21）%相比差异有统计学意义（P〈0.01）;用药后胃肠癌细胞P53蛋白阳性表达率为（35.05±19.64）%,与用药前（34.80±16.48）%相比差异无统计学意义（P〉0.05）;用药后胃肠癌细胞Survivin蛋白阳性表达率为（15.59±3.94）%,与用药前（36.74±20.5）%相比明显下调,差异有统计学意义（P〈0.01）;As2O3作用后胃肠癌细胞Survivin蛋白表达下调时凋亡率升高,二者之间具有相关性（r=-0.47,P=0.04）。结论 As2O3在人体内可诱导胃肠癌细胞凋亡而发挥抗肿瘤作用,其机制可能与下调survivin基因的表达有关。     

Synergistic effect of all-trans-retinoic acid and arsenic trioxide on growth inhibition and apoptosis in human hepatoma, breast cancer, and lung cancer cells in vitro

AIM: To investigate the effect of all-trans-retinoic acid (ATRA) on arsenic trioxide (As_2O_3)-induced apoptosis of human hepatoma, breast cancer, and lung cancer cells in an attempt to find a better combination therapy for solid tumors. METHODS: Human hepatoma cell lines HepG2, Hep3B, human breast cancer cell line MCF-7, and human lung adenocarcinoma cell line AGZY-83-a were treated with As_2O_3 together with ATRA. Cell survival fraction was determined by MTT assay, cell viability and apoptosis were measured by annexin V-fluorescein isothiocyanate (FITC) and PI staining, and intracellular glutathione (GSH) and glutathione-S-transferase (GST) activities were determined using commercial kits. RESULTS: Cytotoxicity of ATRA was low. ATRA (0.1, 1, and 10 umol/L) could synergistically potentiate As_2O_3 to exert a dose-dependent inhibition of growth and to induce apoptosis in each of the cell lines. HepG2 and Hep3B with low intracellular GSH or GST activities were remarkably sensitive to As_2O_3 or As_2O_3+ATRA, while AGZY-83-a with higher GSH or GST activities was less sensitive to As_2O_3 or As_2O_ 3+ATRA. Treatment with 2 umol/L As_2O_3 for 72 h significantly decreased intracellular GSH and GST levels in each of the cell lines, and 1 umol/L ATRA alone reduced minimal intracellular GSH and GST levels. ATRA potentiated the effect of As_2O_3 on intracellular GSH levels, but intracellular GST levels were not significantly affected by the combination of As_2O_3 and ATRA for 72 h as compared to As_2O_3 alone. CONCLUSION: ATRA can strongly potentiate As_2O_3-induced growth-inhibition and apoptosis in each of the cell lines, and two drugs can produce a significant synergic effect. The sensitivity to As_2O3_ or As_2O_3+ATRA is inversely proportional to intracellular GSH or GST levels in each of the cell lines. The GSH redox system may be the possible mechanism by which ATRA synergisticaily potentiates As_2O_3 to exert a dose-dependent inhibition of growth and to induce apoptosis.     

氧化砷诱导胃癌细胞凋亡的实验研究

目的 研究三氧二化砷（氧化砷）对胃癌细胞的诱导凋亡作用。方法 应用ＴＵＮＥＬ染色、流式细胞仪技术研究氧化砷对胃癌细胞ＭＫＮ－４５、ＭＫＮ－２８的诱导凋亡作用。结果 氧化砷作用于不同分化程度的胃癌细胞后,可看到较为典型的细胞凋亡的形态学变化：细胞核固缩,染色质凝集,呈新月型紧贴于核膜周边,核碎裂,染色质片断化,凋 亡小体形成等。流式细胞仪ＤＮＡ直方图上出现典型的亚二倍体的“凋亡峰”。ＴＵＮＥＬ染色法     

三氧化二砷诱导肝癌HepG2细胞凋亡作用

目的 探讨三氧化二砷（As2O3）诱导肝癌HepG2细胞凋亡作用。方法 应用普通光学显微镜、荧光显向镜和流式细胞仪观察As2O3对HepG2细胞株的形态学改变和诱发凋亡率。结果 As2O3作用于细胞后,可看到较典型的细胞凋亡形态学改变,细胞体积缩小,染色质固综合成斑块状,呈半月型紧贴于核膜周边,核碎裂,凋亡小体形成等,AO/EB荧光染色法显示。细胞凋亡率为4.3%-9.1%.As2O3诱导HepG2细胞凋亡作用呈时间依赖性,最佳浓度为2umol/L。流式细胞仪DNA直方图上呈现典型的亚二倍体凋亡峰。As2O3主要作用于细胞周期的G2/M期。结论 As2O3具有诱导HepG2细胞凋亡作用,存在治疗肝癌的潜在价值。     

Effect of arsenic trioxide on vascular endothelial cell proliferation and expression of vascular endothelial growth factor receptors Flt-1 and KDR in gastric cancer in nude mice

AIM: To investigate the effect of arsenic trioxide (As2O3) on expression of vascular endothelial growth factor receptor-1 (VEGFR-1, Flt-1) and VEGFR-2 (KDR) in human gastric tumor cells and proliferation of vascular endothelial cells. METHODS: The solid tumor model was formed in nude mice with the gastric cancer cell line SGC-7901. The animals were treated with As2O3. Microvessel density (MVD) and expression of Flt-1 and KDR were detected by immunofluorescence laser confocal microscopy. SGC-7901 cells were treated respectively by exogenous recombinant human VEGF165 or VEGF165 As2O3. Cell viability was measured by MTT assay. Cell viability of ECV304 cells was measured by MTT assay, and cell cycle and apoptosis were analyzed using ? ow cytometry. RESULTS: The tumor growth inhibition was 30.33% and 50.85%, respectively, in mice treated with As2O3 2.5 and 5 mg/kg. MVD was signifi cantly lower in arsenic-treated mice than in the control group. The ? uorescence intensity levels of Flt-1 and KDR were significantly less in the arsenic-treated mice than in the control group. VEGF165 may accelerate growth of SGC7901 cells, but As2O3 may disturb the stimulating effect of VEGF165. ECV304 cell growth was suppressed by 76.51%, 71.09% and 61.49% after 48 h treatment with As2O3 at 0.5, 2.5 and 5 μmol/L, respectively. Early apoptosis in the As2O3- treated mice was 2.88-5.1 times higher than that in the controls, and late apoptosis was 1.17-1.67 times higherthan that in the controls. CONCLUSION: Our results showed that As2O3 delays tumor growth, inhibits MVD, down-regulates Flt-1 and KDR expression, and disturbs the stimulating effect of VEGF165 on the growth of SGC7901 cells. These results suggest that As2O3 might delay growth of gastric tumors through inhibiting the paracrine and autocrine pathways of VEGF/VEGFRs.     

三氧化二砷对裸鼠膀胱癌细胞凋亡的影响

目的探讨三氧化二砷(As2O3)抑制膀胱癌细胞生长的作用。方法体外培养膀胱癌T24细胞,取指数生长期T24细胞裸鼠皮下接种,成瘤后连续7 d尾静脉分别注射As2O3(5、10 mg·kg-1·d-1)、丝裂霉素(MMC)及生理盐水。应用电镜、流式细胞术和免疫组化等方法检测了癌细胞的超微结构变化、细胞的凋亡和Caspase3蛋白的表达。结果电镜下As2O3组可见典型癌细胞凋亡的形态学改变;流式细胞分析,As2O3组可见癌细胞凋亡峰,凋亡率分别为10．67％和23．42％,明显高于对照组(0．83％);免疫组化检测As2O2组Caspase 3蛋白表达(33．54％和56．22％)也明显高于对照组(6．25％)。结论As2O3可以通过诱导膀胱癌细胞凋亡,抑制膀胱癌细胞生长。