大肠癌细胞增殖中HGF/SF作用的观察

目的研究肝细胞生长因子/离散因子(HGF/SF)在诱导大肠癌细胞增殖中的作用。方法采用W estern B lot方法,检测HGF的受体c-met在受检大肠癌细胞株Caco-2,Colo3 2 0中的表达;观察Caco-2,Colo3 2 0中HGF/SF活化p 4 2/p 4 4MAPK和p 3 8MAPK的动态变化;应用[3H]-TdR,MTT方法观察p 4 2/p 4 4MAPK和p 3 8MAPK传导通路阻滞剂PD 9 8 0 5 9和SB 2 0 3 5 8 0对HGF/SF诱导的大肠癌细胞增殖的抑制作用。结果(1)c-met在Caco-2和Colo3 2 0中有表达。(2)HGF/SF激活p 4 2/p 4 4MAPK,p 3 8MAPK:2 0 ng/mL的HGF/SF处理细胞,p 4 2/p 4 4MAPK磷酸化在1 0m in达高峰(2.2 8±0.0 1);p 3 8MAPK变化与之相似(2.2 5±0.0 1)。(3)HGF/SF诱导大肠癌细胞的DNA合成增加依赖于p 4 2/p 4 4MAPK的激活,在2 4 h时点分别以2 0 ng/m lHGF/SF,不同浓度(1μmol/L,5μmol/L,1 0μmol/L)的PD 9 8 0 5 9和SB 2 0 3 5 8 0处理细胞,HGF/SF使胸腺啶吸收增加(P<0.0 1);PD 9 8 0 5 9以浓度依赖性抑制胸腺啶的吸收(P<0.0 1)。(4)HGF促进Caco-2细胞的增殖,而PD 9 8 0 5 9对这种增殖有抑制作用。结论HGF激活大肠癌细胞Caco-2和Colo3 2 0中p 4 2/p 4 4MAPK和p 3 8MAPK;p 4 2/p 4 4MAPK参与HGF/SF诱导的大肠癌细胞Caco-2有丝分裂;HGF促进大肠癌细胞Caco-2增殖;HGF/SF和p 4 2/p 4 4MAPK在大肠癌细胞中发挥作用可能有细胞选择性。     

姜黄素对雄激素依赖性前列腺癌细胞的诱导凋亡作用

目的:探讨姜黄素对雄激素依赖性前列腺癌细胞株(LNCaP)的诱导凋亡作用。方法:分别用10、25、50、75、100μmol/L浓度的姜黄素作用于LNCaP细胞,5、12、24 h后MTT法检测细胞生长活性;24 h后流式细胞仪测定细胞周期及细胞凋亡的变化,透射电镜观察细胞超微结构变化;5 h后W estern印迹法检测细胞内IκBα的表达。结果:姜黄素能显著抑制LNCaP细胞的生长,呈剂量与时间依赖性,不同浓度姜黄素组之间与不同作用时间组之间的差异均有显著性意义(P均<0.05)。姜黄素诱导LNCaP细胞出现剂量依赖性G2/M期阻滞(P<0.01),各浓度组凋亡细胞比例均显著高于空白对照组(P均<0.05),差异有显著性意义;姜黄素作用24 h后LNCaP细胞出现凋亡的形态学改变;不同浓度姜黄素作用后,LNCaP细胞内IκBα的表达无变化。结论:姜黄素能显著抑制LNCaP细胞的体外生长,并促进其凋亡。     

姜黄素诱导急性单核细胞白血病细胞凋亡的实验研究

目的观察姜黄素对人白血病细胞株K562的致凋亡作用,及其对凋亡相关基因NF-κB与Caspase-3表达的影响。方法将姜黄素加入白血病细胞株THP-1中,用流式细胞仪检测其凋亡率,免疫组化方法检测NF-κB与Caspase-3的表达,比色法检测Caspase-3活性。结果流式细胞仪检测15μmol/L姜黄素作用24h后,凋亡率为（26.504±0.617）%,与对照组（1.232±0.120）%相比明显增高,差异有显著性（P〈0.01）,姜黄素诱导THP-1细胞凋亡具有量-效关系;免疫组化法检测发现姜黄素可降低NF-κB的表达,增加Caspase-3蛋白的表达,且表达量与姜黄素的作用呈剂量相关。结论姜黄素能诱导人白血病细胞株THP-1凋亡,其机制可能与姜黄素抑制NF-κB信号转导通路,下调NF-κB表达继而上调Caspase-3表达有关。     

奥曲肽对人胃癌细胞的凋亡诱导作用

目的 探讨生长抑素对人胃癌细胞的抑制作用及其作用机理。方法 以生长抑素类似物奥曲肽作用于人胃癌细胞株SGC-7901细胞，以人成纤维细胞株HF作为正常细胞对照，以5-FU作为阳性药物对照，通过MTT实验、荧光显微镜观察及流式细胞术分析，得出结论。结果 一定浓度范围内的奥曲肽对SGC-7901细胞产生抑制作用，其抑制作用具有量效关系和饱和性。奥曲肽对SGC-7901细胞的抑制程度接近于5-FU对他的抑制，而对HF细胞无影响。奥曲肽可诱导SGC-7901细胞凋亡。结论 奥曲肽在体外对SGC-7901细胞具有抑制作用，诱导肿瘤细胞凋亡可能是其作用机理之一。     

L-精氨酸诱导人肝癌细胞凋亡的作用

目的 探讨L-精氨酸诱导人肝癌细胞凋亡的作用及机制。方法 采用倒置显微镜、透射电镜和流式细胞仪，观察和分析不同浓度L-精氨酸作用下体外培养人肝癌细胞株QGY-7703的增殖和凋亡情况。结果（1）低浓度L-精氨酸对肝癌细胞生长和细胞大体形态无明显影响。高浓度的L-精氨酸引起肝癌细胞形态变圆、体积变小，细胞皱缩，细胞核固缩，核碎裂，染色质边聚，凋亡小体形成。（2）低浓度L-精氨酸对肝癌细胞生长周期和增殖指数无影响；而高浓度的L-精氨酸引起G0／G1期较对照组显著升高，S期显著降低，细胞增殖指数显著下降（P〈0．05），而G2／M无明显变化。（3）低浓度L-精氨酸（1mmol／L）和对照组细胞未见凋亡峰（亚G1峰），凋亡率分别为2．1％和2．5％；而16mmol／L和64mmol／L浓度组则出现典型的凋亡峰，凋亡率分别为12．4％和30．8％，较对照组显著增高（P〈0．05）。结论 高浓度的L-精氨酸通过合成NO，诱导肝癌细胞凋亡。     

肝细胞生长因子在肝星状细胞调控卵圆细胞凋亡中的作用

目的 探讨肝细胞生长因子(hepatocyte growth factor,HGF)在肝星状细胞(hepatic stellate cells,HSCs)调控卵圆细胞凋亡中的作用.方法 将健康雄性SD大鼠随机分为实验组、条件对照组和假手术组.三组大鼠均接受2-AAF灌胃,实验组和条件对照组进行2/3肝切除,而假手术组仅进行开关腹.于术后第0、1、3、6、9、12和15天提取HSCs,收集该细胞的条件培养液,将HGF受体抑制剂PHA665752(5 μmol/L)加入条件对照组的条件培养液中,用各时间点的条件培养液作用于LE6卵圆细胞系24 h,经PI/Annexin-Ⅴ染色后采用流式细胞术检测LE6细胞的凋亡情况.提取第9天条件培养液处理后的LE6细胞蛋白,采用Western Blotting检测Bim和cleaved-Casepase 3蛋白的表达.结果 经第6、9、12天条件培养液处理后,实验组LE6细胞的凋亡率显著低于假手术组和条件对照组(P＜0.05),其中第9天差异最显著,而且第9天实验组的Bim和cleaved-Casepase 3蛋白的表达量均低于假手术组和条件对照组(P＜0.05).结论 HGF可能通过降低Bim蛋白表达和抑制Caspase 3活性参与HSCs对卵圆细胞凋亡的负调控.     

肝细胞生长因子对实验性急性胰腺炎细胞凋亡作用的研究

目的:观察外源性肝细胞生长因子(HGF)对小鼠轻症实验性急性胰腺炎的炎症反应及胰腺细胞凋亡的作用。方法:以雨蛙肽腹腔注射诱发小鼠轻症实验性急性胰腺炎。第1组以盐水腹腔注射作为对照,第2、3及4组每小时腹腔注射雨蛙肽诱发急性胰腺炎;第3组在造模前及造模中两次皮下注射重组人肝细胞生长因子(rhHGF);第4组同时皮下注射rhHGF及其单抗。观察HGF对炎症反应的干预作用及对腺泡细胞凋亡的影响。结果:炎症小鼠予每公斤体重4、20μgrhHGF时,与单纯炎症组相比,血清淀粉酶、脂肪酶下降,胰腺组织学炎症评分减少,胰腺腺泡细胞TUNEL凋亡指数增加,caspase3活性增强。予每公斤体重4或20μgrhHGF时,其效应无显著性差异。同时予rhHGF单抗的小鼠,上述指标与单纯炎症组相比差异无显著性。结论:HGF能减轻雨蛙肽诱导的小鼠轻症实验性急性胰腺炎。HGF单抗能消除HGF的生物学作用。HGF可以促进急性胰腺炎时腺泡细胞的凋亡,这可能是其保护胰腺的作用机制之一。     

TdT法分析羟基喜树碱对人大肠癌细胞凋亡的影响

目的：喜树碱（Ｃａｍｐｔｏｔｈｅｃｉｎ，ＣＰＴ）是从植物喜树中分离得到的一种生物碱，在６０年代就作为一种抗肿瘤药物用于临床。近年来，又从喜树中分离到比其更具抗癌活性的羟基喜树碱（ＨＣＰＴ），且无论从基础实验研究，抑或从临床应用上均发现ＨＣＰＴ是一种有效的抗癌药物。随着分子生物学理论和技术的不断发展，目前得知许多抗肿瘤药物是通过诱导肿瘤细胞凋亡来发挥其治疗作用的。为了阐明ＨＣＰＴ对大肠肿瘤细胞的杀伤     

mda-7/IL-24对人前列腺癌细胞的凋亡诱导效应

黑色素瘤分化相关基因-7(mda-7)是从人黑色素瘤细胞中分离出的一种肿瘤抑制基因，基于其蛋白结构、细胞因子样特性以及生物学作用模式，mda-7蛋白最近被正式归类为IL-24。携带mda-7的无复制能力的腺病毒(Ad．mda-7)可以诱导多种癌细胞凋亡．同时对正常纤维细胞和上皮细胞不产生毒作用。最近,Lebedeva等人证实Ad．mda-7通过诱导雄激素敏感性前     

姜黄素对HepG-2细胞增殖与凋亡的影响

目的：检测姜黄素对肝癌HepG-2细胞增殖及细胞凋亡的作用,以及对核转录因子κB（NF-κB）表达的影响。方法：不同浓度（10,25,50,100 μmol/L）姜黄素处理肝癌HepG-2细胞不同时间后（16,24,48 h）,采用CCK-8法检测细胞增殖抑制率;50 μmol/L的姜黄素处理HepG-2细胞24 h后,用流式细胞仪检测细胞凋亡情况;不同浓度（10,25,50,100 μmol/L）姜黄素处理肝癌HepG-2细胞24 h后,用Western blot方法检测NF-κB p65蛋白的表达。结果：姜黄素能明显抑制HepG-2细胞的增殖,并呈时间和浓度依赖性（均P<0.05）;姜黄素作用后,HepG-2细胞凋亡率较对照组明显增高（P<0.05）;姜黄素能浓度依赖性抑制地HepG-2细胞 NF-κB p65蛋白的表达。结论：姜黄素能抑制HepG-2细胞增殖并促进其凋亡,其机制可能系通过阻断NF-κB信号通路有关。

     

肝细胞生长因子基因修饰间充质干细胞治疗股骨头坏死的探索性研究

目的探讨肝细胞生长因子基因修饰自体骨髓间充质干细胞治疗股骨头坏死的可行性,并评价其疗效。方法对1例ARCOⅢB股骨头坏死患者,手术过程中局部注射肝细胞生长因子基因修饰的自体骨髓间充质干细胞1×10^7个,随访观察评价其临床疗效和影像学改变。结果治疗过程顺利,术后无感染及过敏等严重并发症发生。术后随访9个月,患者髋关节疼痛指数较治疗前明显降低,髋关节临床症状Harris评分分值由治疗前的63分提高到治疗后的76分,髋关节活动度Harris评分由治疗前分值2.5分提高至4.2分。CT扫描显示损伤局部出现重建性骨修复,低密度区范围缩小,密度增高。ARCO分期由ⅢB改善至ⅢA。结论肝细胞生长因子基因修饰的自体骨髓间充质干细胞治疗股骨头坏死,安全有效,本次探索为进一步临床研究奠定了基础。     

The Hypoxic Environment in Tumor-Stromal Cells Accelerates Pancreatic Cancer Progression via the Activation of Paracrine Hepatocyte Growth Factor/c-Met Signaling

Background

Pancreatic cancer is one of the representative solid tumors, in which the hypoxic microenvironment plays a crucial role in malignant progression. We previously demonstrated that tumor-stromal interaction under hypoxia enhances the invasiveness of pancreatic cancer cells through hepatocyte growth factor (HGF)/c-Met signaling.

Methods

We investigated the immunohistochemical expression of hypoxia inducible factor-1α (HIF-1α) c-Met, and HGF in both cancer and stromal cells using 41 pancreatic cancer tissue specimens, and tried to identify any correlations with the clinical features and survival.

Results

Positive staining for HIF-1α was observed in both pancreatic cancer and the surrounding stromal cells in more than 30% of the cases, and it significantly correlated with lymph node metastasis (P < .05). A significant correlation was observed between the expression of HIF-1α and HGF in stromal cells (P < .05). In addition, the c-Met expression in cancer cells was found to significantly correlate with the HGF expression in not only cancer but also stromal cells. The disease-free survival rates of the patients with HIF-1α in cancer, stromal, c-Met in cancer, and an HGF expression in stromal cells was significantly worse than those without such expressions (P < .05).

Conclusions

These data suggest that the HGF/c-Met signaling via HIF-1α ?may therefore negatively affect the prognosis in patients with pancreatic cancer, and targeting tumor stroma under hypoxia might thus be potentially useful as a novel therapy for this cancer.

     

血管内皮细胞生长因子与增殖细胞核抗原联合检测用于大肠癌临床预后判定

目的　探讨大肠癌血管内皮细胞生长因子 (VEGF)及增殖细胞核抗原 (PCNA)与大肠癌术后有无潜在性转移及复发的关系。方法　对 5 9例已获确诊的大肠癌石蜡标本作免疫组化SP法染色 ,检测其VEGF及PCNA的表达 ,并与 12例正常大肠组织和 16例大肠腺瘤进行比较。结果　大肠癌VEGF的表达明显高于大肠腺瘤 (P<0 .0 5 ) ;DukesA +B期大肠癌VEGF的表达与DukesC +D期比较 ,差异有显著性 (P<0 .0 5 ) ;术后复发组VEGF的表达明显高于无复发组 (P<0 .0 5 )。增殖活性表达提示 ,大肠癌分化程度越低 ,有淋巴结或肝转移时 ,其PCNA指数增高 ;术后有、无复发者之间 ,其PCNA指数差异有显著性 (P<0 .0 5 )。结论　大肠癌VEGF与PCNA的表达与肿瘤的浸润、转移密切相关。手术时虽然无明显转移灶 ,VEGF阳性及PCNA活性增强时仍可能有潜在的转移存在。     

Regulation of lnvasive Potential of Human Prostate Cancer Cell Lines by Hepatocyte Growth Factor

Background: The growth and progression of prostate cancer depends on the stromal-epithelial interaction which is under paracrine control. Hepatocyte growth factor (HGF), produced by mesenchymal cells, is a multifunctional growth factor stimulating the movement and growth of epithelial cells including cancer cells. We therefore assessed the relationship between the invasive potential of prostate cancer and HGF in vitro.
Methods: Three human prostate cancer cell lines were used including PC-3 and DU145 (androgenindependent), and LNCaP (androgen-dependent). We studied the expression of the HCF receptor c-met proto-oncogene (c-met) by Western blotanalysis, and alsodetermined theeffectsof HGF on cell scattering, and the mechanisms of invasion and proliferation, by microscopic observation, the matrigel invasion chamber assay, and the MTT assay.
Results: c-met was detected in PC-3 and DU145 cells, but not in the LNCaP cells. There was increased cell motility in the scatter assay and an increased cell invasive potential in the matrigel invasion chamber assay by stimulation with HGF only with DU145 cells.
Conclusion: HGF plays an important role in the invasion and metastasis of the DU145 cell line through a paracrine mechanism mediated by the c-metreceptor. In the PC-3 cell line, the lack of downstream signal transduction after the c-met receptor is suggested.     

大肠癌围手术期血清血管内皮生长因子表达与预后的关系

为探讨大肠癌围手术期患者血清血管内皮生长因子（VEGF）表达与预后的关系,采用酶链免疫吸附测定试剂盒检测8名健康体检者和40例大肠癌患者术前与术后1周血清VEGF的含量,并分析大肠癌患者术后VEGF水平下降程度对持续无瘤生存期的影响。结果显示,大肠癌患者术前VEGF水平为（497．68±128．36）pg／ml,术后1周为（368．56±98．72）Pg／ml,均明显高于健康体检者（224．54±68．23）pg／ml,q术前=8．951,q术后=4．720,P均〈0．01。大肠癌患者术后1周VEGF水平明显下降,q=7．329,P〈0．01。术后VEGF水平下降〈50％组持续无瘤生存期低于VEGF水平下降≥50％组,x2=8．903,P〈0．05。结果表明,VEGF在大肠癌患者血清中高表达,与患者预后显著相关,可作为判断大肠癌预后的有效指标。     

血清肝细胞生长因子与结直肠癌肝转移的关系

目的研究结直肠癌患者血清中肝细胞生长因子(hepatocyte growth factor,HGF)的表达水平及其与结直肠癌肝转移的关系。方法收集我院2006年9月至2009年1月期间手术治疗并经病理证实为结直肠癌患者35例,应用夹心ELISA法检测血清中HGF表达水平。结果血清中HGF表达在结直肠癌有远处转移者中为(0.37±0.11)ng/ml,明显高于无转移者的(0.20±0.06)ng/ml(P0.05);T3、T4期者为(0.32±0.29)ng/ml,亦明显高于T1、T2期者的(0.20±0.14)ng/ml(P0.05);肝转移者为(0.43±0.10)ng/ml,明显高于其他部位转移者的(0.31±0.09)ng/ml(P0.05)。结论血清HGF水平与结直肠癌发展及肝转移发生有密切关系,血清HGF水平有望作为监测结直肠癌转移的临床指标。     

Comparison of Angiogenic Factor Levels in Tumor Drainage and Peripheral Venous Blood From Colorectal Cancer Patients

Background The main objective of this study was to determine whether there was a correlation in the levels of various angiogenesis-related factors between the tumor drainage and peripheral venous blood and whether appraisal of angiogenic factor levels in the tumor drainage venous blood could provide better prognostic information for patients with colorectal cancer than assessment of the peripheral venous blood.Methods Plasma levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor, and endostatin were measured and compared in both tumor drainage and peripheral venous blood from 52 patients with colorectal cancer. Plasma levels of angiogenesis-related factors were also correlated with tumor stage and clinical outcomes.Results The plasma endostatin level was significantly higher in peripheral blood than in tumor drainage venous blood (P < .001). The plasma VEGF level was significantly correlated with plasma endostatin levels (P = .028 in tumor drainage venous blood and P = .002 in peripheral venous blood). In both tumor drainage and peripheral venous blood, the VEGF level (but not the basic fibroblast growth factor or endostatin level) was significantly correlated with tumor stage and disease recurrence. However, in multivariate analysis, only plasma VEGF level in tumor drainage venous blood remained an independent predictor of disease recurrence.Conclusions The plasma VEGF level in tumor drainage venous blood provided better prognostic information than that in peripheral venous blood. The plasma endostatin level was paradoxically significantly higher in peripheral than in tumor drainage blood, and this strongly suggests additional sources of endostatin in peripheral blood.     

酸性成纤维细胞生长因子及表皮生长因子对兔韧带细胞增殖的影响

目的：观察酸性成纤维细胞生长因子(aFGF)和表皮生长因子(EGF)对内侧副韧带(MCL)和前十字韧带(ACL)细胞增殖行为的影响。方法：培养10周龄新西兰白兔内侧副韧带和前十字韧带细胞，在培养液中分别加入aFGF和EGF，以XTT方法测定细胞的增殖行为。结果：aFGF在1ng／ml时即对两种细胞具有显著的促进增殖作用，其浓度达50ng／ml时，对MCL细胞的促进作用最大，达100ng／ml时对ACL细胞的促进作用最大。EGF在0．78ng／ml时即对MCL细胞有显著的促增殖作用，在1．56ng／ml时始对ACL细胞有显著的促增殖作用，其浓度达3．125ng／ml时对2种细胞的促进作用最大。aFGF和EGF在超过其最佳浓度后，随浓度升高促进作用均下降。结论：aFGF和EGF可以促进韧带成纤维细胞增殖。     

肝细胞生长因子基因修饰的人骨髓间充质干细胞促进大鼠肢体血管新生

目的评价肝细胞生长因子(Hepatocyte growth factor,HGF)修饰的人骨髓间充质干细胞(Human bone marrow mesenchymal stem cells,hMSC)体内促血管新生效应,探讨其作用机制。方法结扎Wistar大鼠右侧股动脉,建立局部肢体缺血模型。将hMSC、hMSC/Ad-HGF或PBS,通过肌肉注射接种于缺血区,21d后以对侧肢体为正常对照,进行激光多普勒灌注成像测定、HE染色和免疫组化检查,评价骨骼肌微血管密度。以MTT、Transwell及内皮细胞体外管状结构形成实验,评价hMSC/Ad-HGF对血管内皮细胞的体外增殖、迁移及成管状能力的影响。结果 21d后,hMSC/Ad-HGF和hMSC组血流灌注水平明显高于对照组(P0.01),前者接近PBS组,显著高于hMSC组(P0.05)。组织学分析表明,每高倍视野肌肉组织中,hMSC/Ad-HGF组微血管数量最高,PBS组和hMSC组次之,对照组最低(P0.01)。hMSC/Ad-HGF培养上清可明显促进ECV304细胞的增殖,刺激作用高于表皮生长因子(20ng/mL,P0.001);促进细胞跨膜迁移和管状样结构形成,强于表皮生长因子。结论 hMSC/Ad-HGF可促进血管内皮细胞的增殖、迁移及血管结构重建,从而引发血管新生效应。     

Expression of Hepatocyte Growth Factor in Chronic Hepatitis: Effect of Hemodialysis

Background and aims. Elevated serum levels of hepatocyte growth factor (HGF) have been documented in patients on chronic hemodialysis (HD) and with chronic hepatitis (CH). The aim of this study was to determine the presence and density of HGF in liver tissue and the source of this factor in HD patient with CH.?Material and methods. Seventy-five hepatic needle-biopsy specimens were investigated, 11 from CH patients (Group 1) and 44 from CH patients on HD (Group 2), plus 20 liver biopsies obtained from healthy individuals as controls (Group 3). All specimens were analyzed immunohistochemically with an HGF antibody. Results. Groups 1 (CH only) and 2 (CH?+?HD) had significantly (p?<?0.05) more HGF-positive nonparenchymal cells than the control group. Also, specimens from CH-only patients (Group 1) had significantly (p?<?0.05) more positive staining than those from CH patients on HD (Group 2). Groups 1 and 2 had similar results for grading of CH and fibrosis score. No association was found between the number of HGF-positive cells and necroinflammatory or fibrosis score. Conclusions. The expression of HGF in liver tissue in CH patients on HD was much lower than in CH-only patients. The extrahepatic tissue might be the source of increased HGF serum levels in HD patients.