小干扰RNA沉默基质金属蛋白酶-2基因对卵巢癌细胞恶性行为的影响

 目的　探讨靶向基质全属蛋白酶-2（MMP-2）基因的RNA干扰（RNAi）技术对卵巢癌OVCAR-3细胞MMP-2的沉默作用,以及沉默MMP-2基因对OVCAR-3细胞生长、黏附、侵袭和迁移能力的影响。方法　合成特异性靶向MMP-2基因的小干扰RNA（siRNA）并转染卵巢癌OVCAR-3细胞,以非特异性序列转染细胞作为阴性对照组,以培养液代替siRNA转染细胞作为空白对照组,采用实时荧光定量PCR和Western blot分别检测转染后24 、48 和72 h MMP-2 mRNA和蛋白的表达水平,通过四甲基偶氮唑蓝（MTT）法绘制细胞生长曲线,利用Transwell、划痕实验检测细胞侵袭和迁移能力。结果　与阴性对照组相比,OVCAR-3细胞在转染siRNA-MMP-2后24 、48 和72 h MMP-2 mRNA表达量分别下降了73.8 %、78.8 %和78.4 %（均P＜0.05）,蛋白表达量分别下降了72.6 %、81.2 %和76.4 %（均P＜0.05）,以48 h作用最强;细胞生长曲线显示细胞生长无明显变化（P＞0.05）;60 min和90 min时的黏附抑制率分别为55.0 %和44.8 %（均P＜0.05）;细胞的侵袭和迁移能力分别下降29.7 %和35.8 %（均P＜0.05）。结论　siRNA介导的MMP-2基因沉默能明显抑制OVCAR-3细胞的黏附、侵袭和迁移能力,而对其生长无明显影响,MMP-2基因有可能成为卵巢癌基因治疗的重要靶点。     

siRNA下调LSD1基因对卵巢癌OVCAR-3细胞增殖和周期的影响

目的应用小分子干扰RNA（small interfering RNA,siRNA）抑制赖氨酸特异性组蛋白去甲基化酶1（lysine specific demethylase 1,LSD1）基因的表达,探讨其对卵巢癌OVCAR-3细胞增殖和生长周期的影响。方法采用脂质体介导法将LSD1-siRNA转染卵巢癌OVCAR-3细胞;实时荧光定量PCR（RFQ-PCR）和Western blot法检测基因的表达;MTT法检测细胞增殖能力;FCM分析细胞周期。结果成功转染LSD1-siRNA后,OVCAR-3细胞中LSD1 mRNA和蛋白表达均明显下降;细胞增殖明显抑制;G₂/M期细胞比例增加。结论 LSD1-siRNA可显著下调LSD1基因在卵巢癌OVCAR-3细胞中的表达水平,在一定程度上抑制卵巢癌细胞的增殖,导致细胞周期阻滞。     

siRNA沉默结肠癌转移相关基因1表达对人类卵巢癌细胞株侵袭转移的影响

目的 检测结肠癌转移相关基因1（MACC1）在卵巢癌细胞株中的表达,并探讨用siRNA技术抑制其表达对卵巢癌细胞生物学行为的影响.方法 应用实时荧光定量PCR （RT-qPCR）及Western blot法检测OVCAR3、ES-2、SKOV3、HO-8910卵巢癌细胞株中MACC1的表达,合成MACC1特异性siRNA并转染OVCAR3细胞,利用RT-qPCR筛选并鉴定MACC 1基因有效沉默后,应用体外黏附实验、Transwell迁移及侵袭实验、体外血管拟态实验检测MACC1基因沉默后OVCAR3细胞的体外黏附、迁移、侵袭及血管生成能力的变化.结果 OVCAR3细胞较其他卵巢癌细胞株高表达MACC1.MACC1基因沉默后,OVCAR3细胞的体外黏附能力受到不同程度的抑制;Transwell迁移实验中,MACC1 siRNA干扰的OVCAR3细胞（干扰组）转入底层膜的细胞数为（245.5±12.8）个,低于阴性对照组和空白对照组[分别为（500.3±16.5）、（496.3±13.1）个,均P＜0.05]; Transwell侵袭实验中,干扰组转入底层膜的细胞数为（185.3±14.1）个,低于阴性对照组和空白对照组[分别为（405.7±9.1）、（416.3±11.5）个,均P＜0.05];体外血管拟态显示干扰组细胞多呈散在分布,连接减少,形成的完整结构少.结论 利用siRNA技术抑制MACC1基因表达可有效抑制卵巢癌OVCAR3细胞的体外转移和侵袭能力,MACC1有望成为卵巢癌治疗的靶基因.     

HMGA1基因小分子RNAi对卵巢癌转移抑制的实验研究

目的:探讨高迁移率蛋白家族A1(high mobility group A1,HMGA1)基因小分子干扰RNA对卵巢癌转移抑制的机理.方法:RT-PCR一步法检测3种不同的卵巢癌细胞株中HMGA1、E钙黏蛋白mRNA的表达水平;设计并合成HMGA1 siRNA,转染HO8910PM细胞:半定量RT-PCR分析法观察HMGA1 siRNA转染对E钙黏蛋白mRNA表达的逆转作用,及对卵巢癌细胞株体外侵袭、运动的抑制作用.结果:RT-PCR半定量分析结果显示:OVCAR-3细胞中HMGA1 mRNA表达量相对较低(0.64±0.13),而E-钙黏蛋白mRNA仅在OVCAR-3细胞中有表达;HO8910PM细胞稳定转染HMGA1 siRNA后,转染组、对照组HMGA1 mRNA表达水平分别为0.16±0.08、0.47±0.11(P＜0.01),E 钙黏蛋白mRNA表达水平分别 为0.38±0.07、0.09±0.05(P＜0.01);体外运动实验显示,跨膜细胞数转染组明显低于对照组(P＜0.05);重组细胞基底膜侵袭实验显示,穿透基底膜细胞数转染组明显低于对照组(P＜0.01).结论:HMGA1基因与卵巢癌转移密切相关,HMGA1 基因siRNA 可上调卵巢癌细胞中E钙黏蛋白的表达,抑制卵巢癌细胞的运动和侵袭,为卵巢癌转移的基因治疗提供新的靶点.     

HMGA1基因小分子RNAi对卵巢癌转移抑制的实验研究

目的：探讨高迁移率蛋白家族A1（high mobility group A1,HMGA1）基因小分子干扰RNA对卵巢癌转移抑制的机理。方法：RT—PCR一步法检测3种不同的卵巢癌细胞株中HMGA1、E钙黏蛋白mRNA的表达水平;设计并合成HMGA1 siRNA,转染H08910PM细胞：半定量RT—PCR分析法观察HMGA1 siRNA转染对E钙黏蛋白mRNA表达的逆转作用,及对卵巢癌细胞株体外侵袭、运动的抑制作用。结果：RT—PCR半定量分析结果显示：OVCAR-3细胞中HMGA1 mRNA表达量相对较低（0．64±0．13）,而E-钙黏蛋白mRNA仅在OVCAR-3细胞中有表达;H08910PM细胞稳定转染HMGA1 siRNA后,转染组、对照组HMGA1 mRNA表达水平分别为0．16±0．08、0．47±0．11（P〈0．01）,E钙黏蛋白mRNA表达水平分别为0．38±0．07、0．09±0.05（P〈0．01）;体外运动实验显示,跨膜细胞数转染组明显低于对照组（P〈0．05）;重组细胞基底膜侵袭实验显示,穿透基底膜细胞数转染组明显低于对照组（P〈0．01）。结论：HMGA1基因与卵巢癌转移密切相关,HMGA1基因siRNA可上调卵巢癌细胞中E钙黏蛋白的表达,抑制卵巢癌细胞的运动和侵袭,为卵巢癌转移的基因治疗提供新的靶点。     

SPAG6促进卵巢癌细胞的增殖、迁移和侵袭

目的:探讨抑制精子相关抗原6（sperm-associated antigen 6,SPAG6）对卵巢癌细胞增殖、侵袭和迁移的影响。方法:体外培养正常卵巢上皮细胞（NOE-71）和卵巢癌细胞（SKOV-3、OVCAR-3）,采用实时荧光定量PCR（RT-qPCR）实验检测细胞中SPAG6基因的表达。将4种潜在抑制SPAG6基因表达的“SPAG6-shRNA”质粒分别转染到SKOV-3细胞,RT-qPCR和Western blot实验筛选有效抑制SPAG6基因表达的细胞株;采用细胞计数法和平板克隆实验检测抑制SPAG6基因表达对卵巢癌细胞增殖的影响;运用细胞划痕实验和Transwell实验检测抑制SPAG6基因表达对细胞迁移和侵袭能力的影响;流式细胞实验检测抑制SPAG6基因表达对细胞周期的影响;免疫荧光实验和Western blot检测在SKOV-3细胞中抑制SPAG6表达对p27kip1分布的影响。结果:SPAG6基因在卵巢癌细胞SKOV-3和OVCAR-3中的表达均显著高于正常卵巢上皮细胞NOE-71,且在SKOV-3细胞中表达最强。RT-qPCR和Western blot实验显示2种“SPAG6-shRNA”质粒能够有效抑制SKOV-3细胞中SPAG6基因的表达;与对照组细胞相比,抑制SPAG6基因表达能够降低SKOV-3细胞的增殖,差异有统计学意义（P＜0.01）;平板克隆实验显示抑制SPAG6基因表达能降低SKOV-3细胞的克隆形成率,差异有统计学意义（P＜0.01）;细胞划痕实验和Transwell实验显示抑制SPAG6基因表达能够降低卵巢癌细胞的迁移和侵袭能力（P＜0.01）;流式细胞实验显示抑制SPAG6基因表达能使细胞G0/G1期显著上调,S期显著下调(P＜0.01)。SKOV-3细胞中抑制SPAG6表达可降低细胞质中p27kip1的水平而增加细胞核中p27kip1的水平。结论:SPAG6促进卵巢癌细胞的增殖、迁移和侵袭。     

Survivin基因沉默下调血管形成相关因子表达抑制视网膜母细胞瘤侵袭性及其分子机制研究

目的:研究基因沉默Survivin对视网膜母细胞瘤HXO-RB44细胞凋亡、侵袭以及血管内皮生长因子（VEGF）、基质金属蛋白酶 2（MMP-2）、基质金属蛋白酶9（MMP-9）活性的影响。方法:培养视网膜母细胞瘤HXO-RB44细胞,构建Survivin-shRNA载体。按照处理不同分为Survivin-shRNA组、GFP组和CON组。分别检测三组HXO-RB44细胞凋亡指数、细胞侵袭能力以及VEGF、MMP-2、MMP-9、CAS-3蛋白的表达水平。结果:流式细胞术结果表明,与CON组和GFP组相比,Survivin-shRNA组细胞凋亡率增加,差异有统计学意义（P＜0.05）;Transwell侵袭小室实验显示Survivin-shRNA能明显降低HXO-RB44细胞的侵袭能力;Western blot结果发现,Survivin-shRNA可降低VEGF、MMP-2、MMP-9蛋白的表达水平,并提高CAS-3的表达水平。结论:Survivin-shRNA可诱导HXO-RB44细胞凋亡,抑制细胞侵袭能力,下调血管形成相关因子VEGF、MMP-2、MMP-9的表达,进而抑制肿瘤新生血管的形成。     

乳腺癌转移抑制基因1对卵巢癌细胞血管生成的影响及其机制探讨

目的探讨乳腺癌转移抑制基因1(breast cancer metastasis suppressor 1,BRMS1)对人卵巢癌细胞OVCAR3诱导血管内皮细胞形成管腔能力的影响及可能的机制。方法应用脂质体介导的方法将BRMS1 shRNA重组质粒转染人卵巢癌细胞OVCAR3,经G418筛选获得稳定转染株。荧光定量PCR和Western blot法检测BRMS1 mRNA和蛋白的表达水平;体外血管形成实验检测OVCAR3诱导人脐静脉内皮细胞(HUVECs)的血管形成能力;Western blot法检测生长抑制因子4(ING4)和白细胞介素-6(IL-6)的蛋白表达情况。结果稳定转染BRMS1 shRNA后,OVCAR3细胞BRMS1的表达在 mRNA和蛋白水平均受到明显抑制。体外血管形成实验提示,BRMS1表达下调后能显著促进卵巢癌细胞诱导的HUVECs形成管腔样结构的能力;Western blot法显示干扰组中ING4蛋白表达量较对照组下降30%,而IL-6蛋白表达上调,是空白对照组的1.5倍,差异有统计学意义(P<0.05)。结论干扰BRMS1基因的表达可促进卵巢癌细胞诱导血管形成能力,其机制可能与调节下游基因ING4和IL-6的表达有关。     

沉默MTA1抑制肝癌细胞黏附、迁移和侵袭能力的实验研究

目的:研究沉默转移相关基因1(MTA1)对肝癌细胞黏附、迁移和侵袭能力的影响。方法:在肝癌细胞中转染MTA1 siRNA、siRNA control,同时以不做转染的细胞作为对照,qRT-PCR和Western blot分别检测细胞中MTA1的表达水平,MTT检测细胞增殖,细胞黏附试验检测细胞黏附能力,Transwell小室检测细胞侵袭和迁移能力,Western blot检测细胞中基质金属蛋白酶-2（MMP-2）、上皮钙黏附素(E-cadherin)、基质金属蛋白酶-9（MMP-9）、神经型钙黏素(N-cadherin)蛋白水平,明胶酶谱实验检测MMP-2和MMP-9活性。结果:细胞中转染MTA1 siRNA能够下调肝癌细胞中MTA1 mRNA和蛋白水平,转染siRNA control对细胞中MTA1 mRNA和蛋白水平没有影响。沉默MTA1后的肝癌细胞存活率和黏附率降低,侵袭细胞数目减少,与没有转染的细胞比较,差异有统计学意义（P＜0.05）。沉默MTA1后的肝癌细胞中MMP-2、MMP-9蛋白水平及活性降低,细胞中E-cadherin蛋白水平升高,N-cadherin蛋白水平降低,与没有转染的细胞比较,差异有统计学意义（P＜0.05）。结论:沉默MTA1抑制肝癌细胞黏附、迁移和侵袭能力,作用机制与抑制细胞分泌MMP-2、MMP-9及抑制细胞EMT有关。     

lncRNA PVT1沉默对卵巢癌SKOV3细胞增殖、迁移及STAT3通路的影响

目的:研究沉默变异性浆细胞瘤异位1（plasmacytoma heterotopic 1,PVT1）基因对卵巢癌SKOV3细胞增殖、迁移、信号转导及转录活化因子（STATs）3通路的影响。方法:体外培养卵巢癌SKOV3细胞株,设计PVT1 siRNA序列,转染细胞,分为siRNA组、阴性对照组（NC）、空白对照组（BC）,利用qRT-PCR检测各组细胞PVT1相对表达量,利用MTT法检测细胞增殖情况,利用划痕实验检测细胞迁移能力,利用Transwell实验检测细胞侵袭能力,利用WB法检测STAT3通路相关蛋白表达情况。结果:转染后,siRNA3组细胞中PVT1表达水平较BC组与NC组显著降低（P＜0.05）;转染后,与BC组与NC组比较,siRNA3组细胞增殖率较BC组与NC组显著降低（P＜0.05）,迁移、侵袭细胞数显著降低（P＜0.05）,p-STAT3蛋白,MMP2、MMP9、CD44、PCNA蛋白表达水平显著降低（P＜0.05）。结论:沉默PVT1可能抑制卵巢癌SKOV3细胞增殖、迁移与侵袭,其具体机制可能与STAT3信号通路有关。     

RNA干扰抑制血管内皮生长因子影响舌癌耐药细胞移植瘤增殖及血管生成

目的研究RNA干扰抑制血管内皮生长因子（VEGF）的表达对舌癌耐药细胞移植瘤增殖及血管生成的影响。方法用顺铂体外连续诱导法诱导舌癌Tca8113细胞系获得耐药细胞,将耐药细胞接种裸鼠皮下建立移植瘤模型,并随机分为空白对照组、空载体组、无关片段组、干扰质粒组。以脂质体法对空载体组、无关片段组、干扰质粒组进行瘤内及瘤周注射转染,RNA干扰抑制VEGF的表达,测量瘤体重量及体积,免疫组化法检测移植瘤PCNA、CD34,比较肿瘤增殖性及微血管参数,原位杂交法检测VEGFmRNA,免疫印迹法检测VEGF蛋白。结果干扰质粒组的瘤体体积、重量、PCNA平均光密度值、微血管参数均小于其他各组（P〈0．05）。干扰质粒组中VEGFmRNA及蛋白表达水平较其他各组明显下降（P〈0．05）。结论通过靶向VEGF的RNA干扰下调VEGF的表达,能抑制人舌癌耐药细胞移植瘤的增殖及血管生成,提示RNA干扰抑制VEGF可能为临床治疗耐药舌痛柽供新途径。     

Anti-angiogenic effects of somatostatin receptor subtype 2 on human pancreatic cancer xenografts.

Somatostatin receptor subtypes, especially subtype 2 (SSTR2), exert their antitumor (cytostatic and/or cytotoxic) and anti-angiogenic effects. Here we aimed to investigate the anti-angiogenic effect of SSTR2 gene transfer into pancreatic cancer cell line PC-3, and the mechanisms involved in this effect. The full-length human SSTR2 complementary DNA was introduced into pancreatic cancer cell line PC-3 by lipofectamine-mediated transfection, and stable expression of SSTR2 was detected by immunohistochemistry and RT-PCR. Athymic mice were separately xenografted with SSTR2-expressing cells (experimental group), vector control and mock control cells. Intratumoral microvessel density (MVD) was assessed by immunohistochemistry. Immunohistochemistry and RT-PCR were used to determine the expression of angiogenic factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase (MMP)-2 in xenograft tumors. MVD was significantly lower in the experimental group (5.16 +/- 1.34) than that in the vector control (16.52 +/- 2.25) and mock control (15.32 +/- 2.53) (P < 0.05). The immunohistochemical assay showed a significant decrease in the expression of VEGF, bFGF and MMP-2 protein in the experimental group compared with the vector control and mock control, considering both the integral optical density and area of staining (P < 0.05). RT-PCR showed a significant reduction of VEGF, bFGF and MMP-2 mRNA expression in the experimental group compared with the vector control and mock control (P < 0.05). Thus, introduction of the SSTR2 gene, the expression of which is frequently lost in human pancreatic adenocarcinoma, exerts its anti-angiogenic effects by down-regulating the expression of the factors, which are involved in tumor angiogenesis and metastasis, suggesting SSTR2 gene transfer as a promising strategy of gene therapy for pancreatic cancer.     

Enhanced in vitro invasiveness of ovarian cancer cells through up-regulation of VEGF and induction of MMP-2

Vascular endothelial growth factor (VEGF) has been identified to be important in tumor angiogenesis, which is essential for the growth, invasion, and metastasis of solid tumors. The aim of this study was to determine the effect of VEGF overexpression on the invasion of human epithelial ovarian cancer cells in vitro and the possible mechanism involved. The VEGF165 cDNA was transfected into ovarian tumor cell lines CAOV3 and COC1 to promote the expression of VEGF. The VEGF expression and matrix metalloproteinase (MMP)-2 activity were examined by RT-PCR, Western blot analysis and gelatin zymography. A modified Boyden chamber assay was used to test tumor cell invasion in vitro. All cells overexpressing VEGF displayed an enhanced in vitro invasiveness through Matrigel-coated filters with Boyden chamber invasion assay. MMP-2 mRNA and protein were significantly increased during VEGF165 cDNA transfection; MMP-2 activity was also increased. The invasion property of ovarian cancer cells was abrogated with VEGF neutralizing antibody. Our data indicated that the expression of VEGF gave impetus to the in vitro invasion of ovarian cancer cells by stimulating the production and functional activities of MMP-2, which may be a key component of VEGF in promoting ovarian cancer cell invasion. VEGF may constitute a novel therapeutic target for antiangiogenic cancer therapy.     

基质金属蛋白酶-9及血管内皮生长因子在卵巢癌组织中的表达及临床意义

目的:观察卵巢癌患者基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)和血管内皮生长因子(vascular endothelial cell growth factor,VEGF)的表达水平,分析卵巢癌MMP-9和VEGF表达的临床意义.方法:将60份卵巢癌组织作为恶性组,60例卵巢良性肿瘤组织作为良性组,将获取的60份健康卵巢组织作为健康组,应用免疫组化法检测各组卵巢组织中MMP-9、VEGF阳性表达情况,应用RT-PCR检测不同卵巢组织中MMP-9 mRNA、VEGF mRNA相对表达量,应用Western blot法检测MMP-9和VEGF的蛋白水平,同时对比不同临床病理特征的卵巢癌组织中MMP-9、VEGF表达水平.结果:恶性组MMP-9阳性率(53.33%)、强阳性率(25.00%)和VEGF阳性率(55.00%)、强阳性率(26.67%)显著高于其他组(P<0.05),良性组MMP-9阳性率(23.33%)、VEGF阳性率(26.67%)显著高于健康组(P<0.05);恶性组MMP-9 mRNA、MMP-9蛋白、VEGF mRNA、VEGF蛋白显著高于其他组,其次为良性组,三组间MMP-9和VEGF表达水平差异显著(P<0.05);卵巢癌组织不同临床分期、不同淋巴结转移者的MMP-9、VEGF表达水平具有显著差异(P<0.05),且MMP-9表达和VEGF表达具有显著正相关性(P<0.05).结论:MMP-9、VEGF在卵巢癌中均为高表达,且二者表达强度与卵巢癌临床分期、淋巴结转移情况密切相关.     

Vector-based RNA interference against vascular endothelial growth factor-A significantly limits vascularization and growth of prostate cancer in vivo

RNA interference technology is emerging as a very potent tool to obtain a cellular knockdown of a desired gene. In this work we used vector-based RNA interference to inhibit vascular endothelial growth factor (VEGF) expression in prostate cancer in vitro and in vivo. We demonstrated that transduction with a plasmid carrying a small interfering RNA targeting all isoforms of VEGF, dramatically impairs the expression of this growth factor in the human prostate cancer cell line PC3. As a consequence, PC3 cells loose their ability to induce one of the fundamental steps of angiogenesis, namely the formation of a tube-like network in vitro. Most importantly, our "therapeutic" vector is able to impair tumor growth rate and vascularization in vivo. We show that a single injection of naked plasmid in developing neoplastic mass significantly decreases microvessel density in an androgen-refractory prostate xenograft and is able to sustain a long-term slowing down of tumor growth. In conclusion, our results confirm the basic role of VEGF in the angiogenic development of prostate carcinoma, and suggest that the use of our vector-based RNA interference approach to inhibit angiogenesis could be an effective tool in view of future gene therapy applications for prostate cancer.     

钙释放激活钙通道调节分子1促进结肠癌细胞系SW480的迁移和侵袭

  目的  探讨钙释放激活钙通道调节分子1（calcium release-activated calcium channel modulator 1,ORAI1）对SW480迁移和侵袭的影响及其机制。  方法  以ORAI1干扰慢病毒感染SW480细胞,用RT-qPCR和Western blot检测细胞中ORAI1mRNA和蛋白的表达,Transwell小室、黏附实验、血管形成及拟态分别检测细胞侵袭、迁移能力,同、异种细胞间黏附及血管生成能力,激光共聚焦显微镜检测细胞钙内流（store-operated Ca2+ entry,SOCE）,Western blot法检测细胞中ERK1/2、p-ERK1/2、MMP-2、VEGF和E-cadherin蛋白的表达。  结果  SW480转染ORAI1干扰慢病毒72h后,可见明显的荧光表达;较空病毒组和（或）对照组,干扰组ORAI1的表达降低（P < 0.01）;侵袭和迁移能力减弱（P < 0.01）;同种黏附能力增强（P < 0.05）;异种黏附能力减弱（P < 0.05）;血管生成能力减弱（P < 0.01）;SOCE内流峰值降低（P < 0.05）;p-ERK1/2、MMP-2和VEGF的表达降低（P < 0.01）、E-cadherin表达增加（P < 0.01）。  结论  ORAI1可以促进SW480的迁移和侵袭,其机制可能与SOCE增加有关。      

卵巢癌细胞体外侵袭与血管内皮生长因子和明胶酶-A表达的关系

背景与目的:血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)和明胶酶-A(matrixmetalloproteinase-2,MMP-2)均为介导肿瘤转移的重要因子,但两者在卵巢癌转移中的相关性尚未见相关报道。本研究通过比较卵巢癌细胞的体外侵袭力及VEGF和MMP-2的表达水平,探讨VEGF、MMP-2分别与卵巢癌转移的相关性及VEGF与MMP-2的关系。方法:用Boyden小室体外侵袭实验比较两株卵巢癌细胞CaOV-3、COC1的体外侵袭力强弱;采用半定量逆转录聚合酶链反应(RT-PCR)和Western免疫印迹检测CaOV-3、COC1细胞VEGF与MMP-2的mRNA及蛋白表达情况;用明胶酶谱法比较分析两株癌细胞MMP-2酶活性。结果:Boyden小室体外侵袭实验表明卵巢癌细胞系CaOV-3体外侵袭力较强,其穿膜细胞相对百分率为(21.9±1.5)%,而COC1细胞体外侵袭力相对较弱,其穿膜细胞相对百分率为(8.8±0.9)%,两者差异具有显著性意义(P<0.05);RT-PCR、Westernblot提示CaOV-3细胞VEGF、MMP-2mRNA及蛋白表达水平均较COC1细胞高(P<0.05);明胶酶谱法结果表明CaOV-3细胞MMP-2酶活性约为COC1细胞的2倍。结论:卵巢癌细胞体外侵袭力与VEGF、MMP-2表达正相关,在卵巢癌侵袭转移中,VEGF与MMP-2可能具有某种内在联系。     

Thymidine phosphorylase-mediated angiogenesis regulated by thymidine phosphorylase inhibitor in human ovarian cancer cells in vivo

Metastasis or progression of ovarian cancer cells is known to be due to the action of various angiogenic factors. We determined the expression of thymidine phosphorylase/platelet-derived endothelial cell growth factor (TP/PD-ECGF) and vascular endothelial growth factor (VEGF) in cell lines established from 3 serous adenocarcinomas, 3 clear cell carcinomas and 2 mucinous carcinomas of the human ovary. TP activity and the TP mRNA level were much higher in the serous adenocarcinoma cells than in the clear cells and mucinous carcinoma cells, and TP expression was extremely low in the clear cell carcinoma cells. Expression of VEGF mRNA was variable, but not significantly different between the 3 histological types of ovarian cancer. In vivo angiogenesis in the ovarian cancer cells was evaluated by the dorsal air sac assay and revealed that SHIN-3 and HRA serous adenocarcinoma cells, which have high levels of TP expression, induced angiogenesis, while KK clear cell carcinoma cells with low TP expression, did not. The degree of ovarian-cancer-induced angiogenesis seemed to be independent of expression of VEGF in the cells. To confirm that the serous adenocarcinoma-induced angiogenesis is dependent on TP levels, a potent and specific inhibitor of TP was administered orally to mice implanted with a chamber containing SHIN-3 or HRA cells. The TP inhibitor significantly inhibited the angiogenesis induced by the serous adenocarcinoma cells. These results suggest that the angiogenic potency of ovarian cancer cells differs with the histological type and is controlled by expression of TP/PD-ECGF, not by VEGF, and that TP-mediated angiogenesis may be the main factor responsible for progression or metastasis of ovarian serous adenocarcinomas.     

多效蛋白基因沉默对小细胞肺癌H446细胞中部分肿瘤生长相关基因表达的影响

目的 观察多效蛋白(PTN)基因沉默后,人小细胞肺癌H446细胞中与血管新生、肿瘤生长、侵袭过程相关的血管内皮细胞生长因子(VEGF)、angiomotin(Amot)、schlafen5(Slfn5)和金属蛋白酶9(MMP-9)4种基因的表达变化.方法 利用逆转录聚合酶链反应(RT-PCR)和Western blot方法 检测PTN在H446细胞中的表达.构建干扰PTN表达的慢病毒载体,在293T细胞中包装成病毒后感染H446细胞.RT-PCR检测未干扰细胞组、阴性对照组、PTN抑制组和PTN抑制加化疗组细胞中Amot、Slfn5、MMP-9和VEGF的表达.结果 RT-PCR和Western blot检测结果 均表明,PTN基因在H446细胞中高表达.所构建的shRNA序列(GCAGCTGTGGATACTGCTGAA)对H446细胞PTN mRNA表达的抑制效率达72.1%,对PTN蛋白表达的抑制效率为59.2%.FIN基因沉默后,H446细胞中Slfn5和MMP-9的表达分别较阴性对照组上调了165.1%和47.3%,而Amot和VEGF的表达分别较阴性对照组下调了33.1%和26.6%.协同化疗后,上述趋势更为明显.结论 通过构建PTN小干扰RNA(siRNA)慢病毒表达载体沉默PTN基因,可以影响H446细胞增殖和转移相关基因的表达变化.