RhoA在胃癌细胞中的表达及其作用

目的 探讨RhoA在胃癌细胞系中的表达及其作用。方法 Westem blot方法检测RhoA在多株人消化道肿瘤细胞系中的表达;构建RhoA反义核酸真核表达载体,脂质体法转染至高表达RhoA的人胃癌细胞系AGS,MTT比色法观察细胞生长,流式细胞仪检测细胞周期分布。结果 10株肿瘤细胞系中RhoA蛋白表达均显著高于永生化正常人肠黏膜细胞系HIEC。转染RhoA反义核酸真核载体后,AGS细胞中RhoA蛋白表达降低,细胞生长受到抑制,细胞周期分析显示聚集在S期的细胞明显增多。结论 RhoA蛋白在消化道肿瘤细胞系中表达明显增高,降低RhoA表达能部分逆转胃癌细胞的恶性表型。     

T细胞表面细胞表型的变化与胃癌分期及术式的关系

背景与目的:肿瘤患者的免疫功能,尤其是细胞免疫与肿瘤的发生、发展密切相关。关于T细胞与胃癌分期及术式的关系尚未明了。本研究目的是研究不同分期、不同术式胃癌患者围手术期的免疫状态。方法:应用流式细胞仪检测33例胃癌患者手术前后T细胞表面6种细胞表型,并与良性病变患者的检测结果进行对比分析。结果:随着胃癌分期的增高,术前CD3、CD4、CD4/CD8、CD16、CD69逐渐下降,CD8逐渐增高(P<0.05),Ⅰ、Ⅱ、Ⅲ期胃癌患者术前活化T细胞CD3+/HLA-DR+无显著性差异(P>0.05);根治术后,CD8明显降低,CD3、CD4、CD4/CD8、CD16、CD69、CD3+/HLA-DR+显著性增高(P<0.01);姑息切除术后CD3、CD4无变化;肿瘤未切组术后CD16、CD4/CD8进一步下降(P<0.05)。结论:胃癌患者术前免疫状态与疾病程度呈负相关,切除肿瘤有利于改善患者细胞免疫功能。     

VEGFs家族及其与胃癌关系的研究进展

目前发现血管内皮生长因子（vascular endothelial growth factors, VEGFs）家族包括7个成员：VEGFA，胎盘生长因子（placenta growth factor,PlGF)，VEGFB，VEGFC，VEGFD，VEGFE 和蛇毒VEGF(snake venom VEGF,svVEGF )；其同源受体有酪氨酸激酶受体VEGFR 1(Flt1)、VEGFR2(KDR)、VEGFR3(Flt4)等多种类型。其中，VEGFA与VEGFR1 和VEGF     

幽门螺杆菌基因表型与胃癌的关系

幽门螺杆菌 (helicobacterpylori ,Hp)是 1种革兰氏阴性、尿素酶阳性的人胃病原体。流行病学统计 ,人群的Hp感染率为15 %～ 90 % [1] 。二十多年来 ,Hp与慢性胃炎、消化性溃疡和胃癌等疾病的关系得到了广泛而深入的研究。已证实Hp是B型慢性胃炎和消化性溃疡发生的病因 ,但是 ,H     

RhoA小干扰RNA对胃癌AGS细胞基因表达谱的影响

目的：应用RhoA小干扰RNA（small interfering RNA，siRNA）转染胃癌细胞系AGS细胞，采用高通量基因芯片技术，筛选基因表达谱的变化，为进一步研究RhoA信号转导通路奠定基础。方法：用pSilencer3．1载体构建RhoA／siRNA重组质粒。Effectene转染试剂转染AGS细胞，空载体pSilencer3．1转染对照组。应用G418筛选转染细胞系，获得稳定转染细胞；美国Agilent基因芯片检测细胞基因表达谱变化。结果：基因芯片检测全基因组，发现表达上调的有1151个基因，表达下调的有1079个基因，涉及信号转导、细胞周期、细胞凋亡等相关信号分子。生物信息学分析显示，与细胞凋亡有关的caspase家族，以及细胞周期调控基因存在明显的差异表达。结论：基因芯片结果提示干扰RhoA表达后，与凋亡和增殖有关的分子在RhoA介导的肿瘤细胞异常增殖信号转导中起着重要的调控作用，为下一步研究其信号调控网络机制提供重要的信息。     

胃癌SP表型细胞侵袭转移相关性研究

目的：观察人胃癌SNU5肿瘤细胞系及其高侵袭细胞亚系中是否含有SP细胞,并验证该表型细胞与侵袭转移的关系。方法：采用Transwell小室,体外筛选出高侵袭细胞;经Hoechst33342染色,应用流式检测并分选出SP表型以及非SP表型细胞;应用无血清培养方法检测SNU5亲本细胞、SP表型以及非SP表型细胞在无血清培养中的成球能力。将分选出的SP表型以及非SP表型细胞进行侵袭实验、划痕迁移实验。裸鼠皮下接种,检测SP表型以及非SP表型细胞的自发性肺转移能力。基因芯片的方法分析SP表型以及非SP表型细胞的基因表达谱差异。结果：经过三轮筛选建立了稳定的高侵袭亚细胞群SNU5-P3。SP分选检测发现,SNU5-P3的SP细胞比例为1.6%;无血清培养发现,SNU5-P3-SP细胞成球数为104±19,显著强于SNU5以及SNU5-P3-non-SP细胞。侵袭实验检测发现,SNU5-P3-SP细胞的侵袭能力比SNU5、SNU5-P3-non-SP细胞增强近2.5倍。划痕迁移检测发现,SNU5为-P3-SP运动能力显著增强,划痕24h愈合。体内移植瘤转移实验发现SNU5-P3-SP细胞自发性肺转移率为100%,SNU5为50%,而SNU5-P3-non-SP仅有16.7%。基因芯片分析,获得733个差异基因,其中上调基因450个,36%的基因与侵袭转移有关。结论：胃癌SP表型细胞具有干细胞的特征,在胃癌侵袭、转移中起到关键作用。     

胃癌SP表型细胞侵袭转移相关性研究

目的:观察人胃癌SNU5肿瘤细胞系及其高侵袭细胞亚系中是否含有SP细胞,并验证该表型细胞与侵袭转移的关系。方法:采用Transwell小室,体外筛选出高侵袭细胞;经Hoechst33342染色,应用流式检测并分选出SP表型以及非SP表型细胞;应用无血清培养方法检测SNU5亲本细胞、SP表型以及非SP表型细胞在无血清培养中的成球能力。将分选出的SP表型以及非SP表型细胞进行侵袭实验、划痕迁移实验。裸鼠皮下接种,检测SP表型以及非SP表型细胞的自发性肺转移能力。基因芯片的方法分析SP表型以及非SP表型细胞的基因表达谱差异。结果:经过三轮筛选建立了稳定的高侵袭亚细胞群SNU5-P3。SP分选检测发现,SNU5-P3的SP细胞比例为1.6%;无血清培养发现,SNU5-P3-SP细胞成球数为104±19,显著强于SNU5以及SNU5-P3-non-SP细胞。侵袭实验检测发现,SNU5-P3-SP细胞的侵袭能力比SNU5、SNU5-P3-non-SP细胞增强近2.5倍。划痕迁移检测发现,SNU5为-P3-SP运动能力显著增强,划痕24h愈合。体内移植瘤转移实验发现SNU5-P3-SP细胞自发性肺转移率为100%,SNU5为50%,而SNU5-P3-non-SP仅有16.7%。基因芯片分析,获得733个差异基因,其中上调基因450个,36%的基因与侵袭转移有关。结论:胃癌SP表型细胞具有干细胞的特征,在胃癌侵袭、转移中起到关键作用。     

维甲酸对小鼠前胃癌细胞恶性表型及抑癌基因表达的调控作用

采用全反式维甲酸（ＲＡ）对小鼠前胃癌细胞系进行了体外加药处理及将处理的细胞接种于近交系６１５小鼠皮下的实验观察。结果表明：经维甲酸处理的瘤细胞基本丧失在软琼脂中生长形成集落的能力，将该处理细胞接种于小鼠皮下，可见其自发性转移能力明显降低。ＲＮＡ狭线杂交显示经ＲＡ处理的瘤细胞其抑癌基因ｐ５３和转移抑制基因ｎｍ２３的表达明显增强，提示ＲＡ可能通过调控抑癌基因的表达，降低肿瘤细胞的转移能力。     

人NHE1反义基因转染对SGC-7901胃癌细胞恶性表型的逆转作用

目的:探讨NHE1反义基因转染对SGC-7901胃癌细胞恶性表型的逆转作用及其在肿瘤基因治疗中的价值。方法:将构建好的人NHE1反义基因真核表达载体,采用脂质体法将其转染至SGC-7901胃癌细胞中,观察NHE1反义基因转染后SGC-7901胃癌细胞的细胞形态学、NHE1蛋白表达、体外生长增殖特性、双层软琼脂集落形成能力的变化。结果:NHE1反义基因转染的SGC-7901胃癌细胞形态恶性程度降低,NHE1蛋白表达明显减少,双层软琼脂集落形成能力、体外生长增殖能力降低,接触抑制恢复,细胞凋亡率增加。结论:NHE1反义基因转染能使SGC-7901胃癌细胞的NHE1蛋白表达明显下调并逆转其恶性表型,提示NHE1基因可成为肿瘤治疗的新靶点,具有一定的临床应用前景。     

胃癌的家族聚集性研究

本文采用遗传流行病学病例对照研究方法,探讨遗传因素在胃癌病因中所起的作用,并研究胃癌的家族聚集性.结果病例组有胃癌家族史的比例(22.53%)显著高于对照组(10.79%),病例组一级亲属患胃癌的比例(3.21%)高于对照组(1.44%,0R=2.27,95%CI1.38～3.73).用(p+q)n模型拟合结果家族中实际病例数的分布高于二项分布的概率范围,经频数分布拟合优度的检验,P＜0.01,说明胃癌在家族中并非按机会均等的概率分布,而是呈现明显的家族聚集现象,胃癌家族史是胃癌发生的危险因素.     

RhoA在胃癌细胞核中的表达及预后意义

[目的]探讨RhoA蛋白在胃癌细胞核中的表达及预后意义。[方法]选取有详细临床资料的胃癌组织179例及正常胃黏膜组织80例,应用免疫组织化学改良二步法检测RhoA蛋白的胞核表达。[结果]RhoA蛋白在胃癌及正常胃黏膜组织细胞核中的表达阳性率分别为69.8%(125/179)和25.0%(20/80),差异有显著性(P<0.05)。RhoA蛋白胞核表达率与胃癌肿瘤分化、TNM分期、淋巴结转移有关(P<0.05)。RhoA胞核阳性患者的术后生存期短于阴性患者(P=0.022)。Cox风险比例模型表明,RhoA胞核阳性是胃癌术后预后的独立指标(OR=3.09,P=0.005)。[结论]RhoA蛋白在胃癌细胞核中过表达,是判断胃癌患者术后生存期的独立预后指标。     

Tanshinone IIA Reverses the Malignant Phenotype of SGC7901 Gastric Cancer Cells

Backgrounds: Tanshinone IIA (TIIA), a phenanthrenequinone derivative extracted from Salvia miltiorrhizaBUNGE, has been reported to be a natural anti-cancer agent in a variety of tumor cells. However, the effect ofTIIA on gastric cancer cells remains unknown. In the present study, we investigated the influence of TIIA on themalignant phenotype of SGC7901 gastric cancer cells. Methods: Cells cultured in vitro were treated with TIIA (0,1, 5, 10 μg/ml) and after incubation for different periods, cell proliferation was measured by MTT method andcell apoptosis and cell cycling were assessed by flow cytometry (FCM). The sensitivity of SGC7901 gastric cancercells to anticancer chemotherapy was investigated with the MTT method, while cell migration and invasion wereexamined by wound-healing and transwell assays, respectively. Results: TIIA (1, 5, 10 μg/ml) exerted powerfulinhibitory effects on cell proliferation (P < 0.05, and P < 0.01), and this effect was time- and dose-dependent.FCM results showed that TIIA induced apoptosis of SGC7901 cells, reduced the number of cells in S phase andincreased those in G0/G1 phase. TIIA also significantly increased the sensitivity of SGC7901 gastric cancer cellsto ADR and Fu. Moreover, wound-healing and transwell assays showed that TIIA markedly decreased migratoryand invasive abilities of SGC7901 cells. Conclusions: TIIA can reverse the malignant phenotype of SGC7901gastric cancer cells, indicating that it may be a promising therapeutic agent.     

Farnesyl transferase inhibitor treatment of breast cancer cells leads to altered RhoA and RhoC GTPase activity and induces a dormant phenotype

Farnesyl transferase inhibitors (FTIs) were shown to be effective in modulating tumor growth in Ras‐transformed tumor cells. Recent studies have focused on Rho GTPases as putative targets of FTI action. Previously, we demonstrated that FTIs were effective in inhibiting the growth and invasiveness of RhoC GTPase‐overexpressing inflammatory breast cancer (IBC) cells however, RhoC activity was increased. In this study, we examine the mechanisms of FTI action on breast cancer cells in culture through modulation of RhoC and RhoA GTPases. We found that FTI inhibition of breast cancer cell growth was reversible and resembled what has been described for an in vitro model of tumor cell dormancy. On FTI treatment, levels of active RhoA decreased significantly, whereas levels of active RhoC increased 3.8‐fold. We studied the role of these two GTPases in a fibronectin and basic FGF‐induced model of breast cancer cell dormancy. Hypoactivation of RhoA and hyperactivation of RhoC were seen to induce morphology and growth changes consistent with tumor cell dormancy in culture. In addition, the JNK/SAPK pathway was induced on FTI treatment. A pharmacologic inhibitor of the JNK/SAPK pathway significantly reduced the number of dormant cells. This study has implications for the use of FTIs as therapeutic agents as well as potential mechanisms for breast cancer cell dormancy.     

DN-hTERT对MCF7细胞端粒酶活性及细胞生长的抑制作用

目的：观察端粒酶逆转录酶负突变体（DN-hTERT）对乳腺癌细胞MCF7端粒酶活性和恶性表型的抑制作用.方法：将带DN-hTERT基因的真核表达载体（DN-hTERT-IRES2-EGFP，DN）、IRES2-EGFP空载体（Ⅰ）通过脂质体2000（lipofectamine2000）介导转入MCF7细胞，以G418进行稳定筛选，得到阳性克隆 倒置荧光显微镜观察转染细胞的生长情况 逆转录聚合酶链反应（RT-PCR）检测转染细胞hTERTmRNA的表达 TRAP-ELISA方法检测转染细胞端粒酶活性 流式荧光原位杂交法（FLOW-FISH）检测转染细胞端粒长度的变化 裸鼠肿瘤细胞移植观察转染细胞动物致瘤率.结果：MCF7细胞转染DN-hTERT后生长受到抑制 转染DN-hTERT的MCF7细胞（MCF7/DN）hTERT mRNA表达升高 TRAP-ELISA检测MCF7/DN细胞端粒酶活性（2.36±0.12）与转染空载体MCF7细胞（MCF7/I）（3.32±0.14）比较显著降低（P＜0.05） 反应端粒长度的荧光强度差比较中，MCF7/DN细胞（13.89±0.23）比MCF7/I（19.87±0.36）低，转染DN端粒长度出现缩短 裸鼠肿瘤细胞移植，2周动物致瘤率MCF7/DN为0，与MCF7/I的35%比较有极显著意义（P＜0.01）.结论：DN-hTERT能特异性抑制MCF7细胞生长和端粒酶活性.     

RhoA和核因子κB在胃癌中的表达及其临床意义

目的 探讨RhoA和核因子κB(NF-κB)在胃癌组织中的表达,分析其与胃癌患者临床病理特征及预后的关系.方法 利用免疫组化、组织芯片技术检测189例胃癌、54例相应癌旁及32例正常胃黏膜组织中RhoA蛋白和NF-κB蛋白的表达.用Kaplan-Meier法行单因素生存分析,应用Cox回归模型进行多因素生存分析.结果 RhoA蛋白在胃癌、癌旁和正常胃黏膜组织中的表达阳性率分别是84.7%、68.5%和65.6%,胃癌与癌旁及正常胃黏膜组织的阳性率差异有统计学意义(P＜0.05).NF-κB蛋白在胃癌、癌旁和正常胃黏膜组织中的表达阳性率分别是75.1%、42.6%和15.6%,且三者的阳性率相互比较,差异均有统计学意义(P＜0.05).RhoA和NF-κB在胃癌组织中的蛋白表达呈正相关(r=0.203,P=0.005).RhoA的蛋白表达与胃癌的浸润深度有关(P＜0.05).NF-κB的蛋白表达与胃癌的浸润深度和有无淋巴结转移有关(P＜0.05).单因素分析结果显示,肿瘤大小、有无淋巴结转移、浸润深度和NF-κB的蛋白表达影响胃癌患者术后的生存(P＜0.05);多因素回归分析结果显示,NF-κB蛋白表达、有无淋巴结转移和浸润深度为影响胃癌患者预后的独立因素(均P＜0.05).结论 RhoA和NF-κB参与胃癌的发生、发展,并在胃癌的浸润和转移中起重要作用;NF-κB的蛋白表达水平、有无淋巴结转移和浸润深度是影响胃癌患者预后的独立因素.

Abstract：

Objective To investigate the expression of RhoA and NF-κB in gastric carcinoma and their correlation with clinicopathological fearures.To determine the effective prognostic factors of long-term suivival of gastric carcinoma patients.Methods The role of RhoA and NF-κB in gastric carcinoma was assessed by tissue array technology and the levels of RhoA and NF-κB expression in paraffin-embedded tissues was quantified by immunohistochemistry from 189 cases of gastric carcinoma, 54 cases of their adjacent tissues, and 32 cases of normal gastric mucosa.The prognosis of gastric carcinoma was evaluated by Kaplan-Meier survival analysis and Cox multivariate regression analysis.Results The positive rates of RhoA expression were 84.7%, 68.5% and 65.6% in gastric carcinoma, adjacent tissues and normal mucosa, respectively.The expression of RhoA in gasric carcinoma was significantly higher than that in adjacent tissues and normal mucosa ( P ＜ 0.05 ).The positive rates of NF-κB expression were 75.1%,42.6% and 15.6%% in gastric carcinoma, adjacent tissues and normal mucosa, respectively.The expression of NF-κB in gasric carcinoma was significantly higher than that in adjacent tissues and normal mucosa (P ＜ 0.05 ).RhoA was positively linked with NF-κB ( r = 0.203, P = 0.005 ).In gastric carcinoma, the expression of RhoA was related with depth of invasion (P ＜ 0.05), and the expression of NF-κB was related with depth of invasion and lymph node metastasis ( P ＜ 0.05 ).The Kaplan-Meier survival analysis showed that the tumor size, lymph node metastasis, depth of invasion, expression of RhoA and NF-κB can shorten the cumulative survival rate.With these paramaters entering the Cox multivariate regression analysis mode, it was revealed that expression of NF-κB, lymph node metastasis and depth of invasion are independent prognostic factors.Conclusions The overexpression of RhoA and NF-κB is involved in the occurrence and development of gastric carcinoma.RhoA is positively linked with NF-κB.They are correlated with the invasion and metastasis of gastric carcinoma.The expression of NF-κB, lymph node metastasis, depth of invasion are independent prognostic factors playing an important role in prediction of the clinical outcome after radical resection of gastric carcinoma.     

幽门螺杆菌与胃癌相关性研究

［目的］研究幽门螺杆菌（Ｈｐ）与胃癌发生之间的关系。［方法］用美蓝染色对７４１例胃炎性病变和１３１例胃癌病人之胃粘膜活检组织的Ｈｐ感染情况进行检测。［结果］胃癌组和慢性萎缩性胃炎组（ＣＡＧ）Ｈｐ检出率分别为６２．５９％和６１．６０％,明显高于慢性浅表性胃炎组（ＣＳＧ）的３８．６１％（Ｐ＜０．０１）;胃癌组中,伴有癌周粘膜肠化生者Ｈｐ检出率明显高于不伴肠化生者（Ｐ＜０．０５）;在活动性ＣＳＧ和ＣＡＧ中,Ｈｐ检出率分别为８９．４１％和９０．５３％,而在非活动性ＣＳＧ和ＣＡＧ中仅分别为１．７１％和１．２３％,两者差异非常显著（Ｐ＜０．０１）。［结论］提示Ｈｐ感染与胃癌的发生有显著的相关性;Ｈｐ有可能通过引发活动性胃炎、腺体萎缩、上皮肠化生等方式参与胃癌的发生。     

非小细胞肺癌中PRL-3与RhoC的表达及相关性意义

背景与目的 PRL-3是新近发现的酪氨酸蛋白磷酸酶,尚属于肝再生磷酸酶家族,具有促进肿瘤转移作用;RhoC属于小分子G蛋白超家族中的Rho亚家族,二者作用机制不清楚.本研究通过检测非小细胞肺癌(nonsmall cell lung cancer,NSCLC)中PRL-3和RhoC的表达,分析二者表达的相关性及在不同分组之间的差异,为进一步研究PRL-3在肿瘤发生发展中的作用机理提供实验依据.方法 采用免疫组化SP法检测PRL-3和RhoC在92例NSCLC中的表达,用统计学方法检验分析二者在不同分组间的表达差异性及二者的相关性.结果 在NSCLC中PRL-3和RhoC的表达阳性率分别为69.6%(64/92)、73.9%(68/92),二者的表达在不同的TNM分期、淋巴结及胸膜是否转移的分组之间差异有统计学意义(P＜0.01),同时二者的表达具有相关性(r=0.754,P＜0.001).结论 在NSCLC中PRL-3和RhoC在TNM分期较高以及合并淋巴结、胸膜转移的分组中表达较高,同时二者的表达具有相关性,提示PRL-3可能通过RhoC及其下游因子促进癌细胞的远处转移.     

H2O2 Inhibits Proliferation and Mediates Suppression of Migration via DLC1/RhoA Signaling in Cancer Cells

Background: RhoGTPase-activating proteins (RhoGAPs) regulate RhoGTPases in cells, but whetherindividual reactive oxygen species (ROS) regulate RhoGAPs is unknown. Our previous published papers haveshown that deleted in liver cancer 1 (DLC1) inhibits cancer cell migration by its RhoGAP activity. The presentstudy was designed to explore the role of H2O2 in regulation of DLC1. Materials and Methods: We treated cells withH2O2 for 24h and phenotypic changes were analyzed by MTT, RT-PCR, Western blotting, immunofluorescencestaining and wound healing assays. Results: H2O2 downregulated cyclin D1 and cyclin E to inhibit proliferation,and upregulated BAX to induce apoptosis in MCF-7 cells. Compared with non-tumorigenic cells, H2O2 increasedexpression of DLC1 and reduced activity of RhoA in cancer cells. Stress fiber production and migration werealso suppressed by H2O2 in MDA-MB-231 cells. Conclusions: Our study suggests that H2O2 inhibits proliferationthrough modulation of cell cycle and apoptosis-related genes, and inhibits migration by decreasing stress fibersvia DLC1/RhoA signaling.