15-羟基前列腺素脱氢酶基因转染对人胃癌细胞生长和迁移的影响

目的:探讨含有NAD+依赖性l5-羟基前列腺素脱氢酶(NAD+-dependent 15-hydroxyprostaglandin dehydrogenase, 15-PGDH)的真核表达质粒(pcDNA3/15-PGDH)转染对人胃癌细胞生长和迁移的影响. 方法:Real time PCR检测多种胃癌细胞株中15-PGDH的表达情况.双酶切方法鉴定15-PGDH真核表达质粒pcDNA3/15-PGDH后,应用Lipofectamine 2000将其转染入胃癌细胞SGC7901中.MTT法检测质粒转染对胃癌细胞增殖的影响,划痕实验观察质粒转染对胃癌细胞迁移的影响,软琼脂克隆形成实验观察质粒转染对胃癌细胞克隆形成的影响.结果:SGC7901细胞中15-PGDH表达显著偏低.pcDNA3/15-PGDH质粒转染入SGC7901细胞的效率较高,转染后24 h及48 h时pcDNA3/15-PGDH组的15-PGDH mRNA表达量分别是pcDNA3空质粒组的188倍和271倍.pcDNA3/15-PGDH质粒转染后SGC7901细胞增殖、迁移和克隆形成能力均显著低于空质粒组和未转染组(P<0.05). 结论:15-PGDH基因转染可显著抑制人胃癌SGC7901细胞的增殖、迁移和克隆形成能力,推测15-PGDH可能是抑制胃癌发生、发展的重要因素之一.     

15-羟基前列腺素脱氢酶抗肿瘤作用的分子机制研究

15-羟基曲列腺素脱氢酶是前列腺素生物降解的重要限速酶,多项研究显示其与体内多种肿瘤发生发展关系非常密切,其表达量减低促进肿瘤的发生发展、浸润、转移及肿瘤血管形成。文章就15-羟基前列腺素脱氯酶抗肿瘤的分子机制作一综述。     

肝细胞生长因子基因转染对阿霉素诱导胃癌细胞调亡的影响

目的研究肝细胞生长因子（HGF）基因转染对阿霉素诱导的胃癌细胞凋亡的影响。方法先构建HGF基因的真核表达质粒pIRES2-EGFP-HGF,应用pIRES2-EGFP-HGF重组质粒和pIRES2-EGFP空质粒转染人胃癌MKN-45细胞,以未转染组为对照。用转染细胞的培养液培养MDCK细胞后,以细胞形态学改变来分析目的基因mRNA、蛋白的表达,分别用RT-PCR、Western blot测定其功能。MTT法测定阿霉素对细胞生长的抑制作用,DNA凋亡条带法和PI染色法检测细胞凋亡。结果稳定转染HGF基因的MKN-45细胞株可表达HGF mRNA,其分泌的HGF蛋白具有正常功能。MTT检测表明,HGF质粒转染组活细胞数高于空质粒转染和未转染组。0．1μg／ml阿霉素作用细胞后DNA凋亡条带分析发现,空质粒转染及未转染组的MKN-45细胞出现典型阶梯状条带, HGF转染组细胞凋亡条带不显著。流式细胞术结果显示,HGF质粒转染组细胞凋亡率显著低于未转染及空质粒转染组。结论HGF基因稳定转染可显著抑制阿霉素诱导的胃癌细胞凋亡。     

VEGF-C基因转染对人胃癌OCUM-2MD3细胞VEGF-C表达的影响

目的:探讨脂质体介导VEGF-C基因转染对人胃癌OCUM-2MD3细胞VEGF-C表达的影响。方法:双酶切pCRⅡ-VEGF-C重组质粒获得人VEGF-CcDNA片段,与真核表达载体pcDNA3.1连接构建pcDNA3.1-VEGF-C重组质粒,脂质体介导转染人胃癌OCUM-2MD3细胞并筛选,RT-PCR检测转染后VEGF-CmRNA表达水平,免疫组化法、流式细胞术检测VEGF-C蛋白表达变化。结果:经酶切鉴定,pcDNA3.1-VEGF-C重组质粒含人VEGF-CcDNA。转染组VEGF-CmRNA相对吸光度值为(0.73±0.16)%,较空载组(0.54±0.13)%表达显著上调(P＜0.01)。免疫组化和流式细胞术检测转染组VEGF-C蛋白阳性表达率分别为(52.16±6.72)%、(41.83±3.65)%,较空载组(30.33±5.04)%、(30.16±3.23)%显著增加(P＜0.01或0.05)。结论:脂质体介导VEGF-C基因转染人胃癌OCUM-2MD3细胞可显著增加VEGF-C表达水平,对探索胃癌淋巴转移机制及其治疗具有重要意义。     

人yrdc基因表达促进了胃癌细胞的增殖

Objective  

The aim of the research was to study the function of human yrdC gene in the gastric carcinoma cells.     

Cx43基因转染胶质瘤细胞抑制细胞增殖与下调若干重要生长因子

目的：研究连接蛋白43(Cx43)基因对胶质瘤细胞增殖的抑制作用及其可能的机制。方法：以脂质体介导,将Cx43cDNA转染于内源性Cx43表达缺失的鼠C6胶质瘤细胞和人TJ905胶质母细胞瘤细胞。采用MTT法及AgNORs(核仁组成区嗜银蛋白)平均数检测细胞增殖、。TUNEL法检测细胞凋亡、划痕标记染料示踪技术检测细胞间隙连接通讯(GJIC)。应用Western蛋白印迹及免疫组化法检测CX43cDNA转染细胞前后若干与胶质瘤发生密切相关的重要生长因子及受体表达,其中包括IGF1、bFGF、PDGF、IGFBP3、EGFR。结果：胶质瘤细胞转染Cx43cDNA后细胞增殖被抑制,细胞凋亡并未增加;GJIC明显恢复。BFGF、PDGF,IGF1、IGFBP3表达显著下调,但EGFR表达无变化。结论：Cx43基因对胶质瘤细胞增殖的抑制作用与GJIC恢复以及bFGF,PDGF,IGF1,IGFBP3表达下调相关,CX43可望成为胶质瘤基因治疗的靶基因。     

蛇毒cystatin基因转染抗人胃腺癌细胞体外侵袭作用的研究

目的:探讨蛇毒半胱氨酸蛋白酶抑制剂(sv-cystatin)对人胃腺癌细胞SGC7901侵袭转移的抑制作用.方法:采用人工拼接方法合成蛇毒cystatin cDNA,构建pcDNA3.1/sv-cystatin真核表达质粒,经脂质体转染将pcDNA3.1/sv-cystatin质粒和pcDNA3.1质粒分别导入胃腺癌细胞系SGC7901:利用RT-PCR和Western blot检测SGC7901细胞中sv-cystatin基因的表达;应用细胞-基质粘附实验、细胞运动实验及重建基底膜侵袭实验分析svcystatin表达对SGC7901细胞粘附、运动和侵袭能力的影响.结果:转染sv-cystatin基因后,SGC/sv-cystatin细胞克隆中可检测到sv-cystatin的明显表达,SGC/sv-cystatin细胞的运动能力和体外穿越重建基底膜的能力明显低于转染空载体细胞和未转染的SGC7901细胞,但其体外粘附能力未见明显变化.结论:sv-cystatin基因的表达可使胃癌SGC7901细胞体外运动能力及侵袭能力明显减弱,提示sv-cystatin具有抑制胃癌细胞侵袭转移的作用.     

B7-1基因抑制小鼠前胃癌细胞增殖活性的实验研究

目的 :研究B7 1基因对小鼠前胃癌细胞 (MFC)增殖活性的抑制作用 ,探讨其在肿瘤免疫中的作用。方法 :将B7 1基因转染MFC细胞后 ,用MTT法和流式细胞仪 (FCM )分别测定转染细胞增殖活性的变化 ,并将已转染B7 1的MFC细胞种植到裸鼠皮下 ,研究其对体内成瘤的影响。结果 :转染B7 1基因的MFC细胞增殖活性受到抑制 ,P <0 0 5。结论 :B7 1基因对MFC细胞增殖活性有抑制作用。     

薏苡仁提取物诱导胃癌细胞SGC-7901凋亡和抑制增殖的体内实验

细胞凋亡 ( Apoptosis)是由基因调控的主动的细胞死亡过程 ,与细胞增殖互补 ,共同维持机体的器官组织细胞数量的稳定。恶性肿瘤的发生和发展的过程 ,是细胞的过度增殖和细胞凋亡受到抑制的过程 [1] 。化疗诱导肿瘤细胞凋亡已成为肿瘤综合治疗中的重要手段。胃癌是我国最常见的恶性肿瘤 ,但目前有关化疗诱导胃癌细胞凋亡的研究极少。本研究用小鼠 SGC- 790 1胃癌模型 ,研究体内薏苡仁提取物 (康莱特 ,KLT)对 SGC- 790 1胃癌细胞凋亡和增殖的影响 ,以评价 KLT化疗的临床价值。材料与方法一、动物模型 　昆明种小鼠 2 0只 ,2 5g左右 ,由…     

15-hydroxyprostaglandin dehydrogenase is downregulated and exhibits tumor suppressor activity in gastric cancer

We investigated the tumor suppressor activity and regulatory mechanism of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in gastric cancer; 15-PGDH expression was lost in 70.1% of malignant human gastric tissues, but was preserved in normal and metaplastic gastritis. KATO III and SNU-719 cells were transfected with pcDNA3.1-empty vector or an expression vector encoding wild-type 15-PGDH. In TUNEL assays apoptotic cell numbers were increased in KATO-PGDH-WT cells compared with control. We found that EGFR and ERK1/2 inhibitors clearly increased the expression of 15-PGDH in KATO III cells. Our findings demonstrate both downregulation and a tumor suppressor activity of 15-PGDH in gastric cancer.     

siRNA靶向沉默hyrdC基因对胃癌细胞SGC-7901生长作用的研究

目的:观察RNA干扰靶向沉默hyrdC基因对胃癌SGC-7901细胞生长的影响.方法:将已成功构建的携带hyrdC基因以及沉默hyrdC基因的重组腺病毒Ad-hyrdC、Ad-shyrdC分别转染胃癌SGC-7901细胞,台盼蓝细胞计数和MTT法观察各组胃癌细胞生长情况,将胃癌细胞接种裸鼠皮下构建裸鼠荷瘤模型观察各组胃癌瘤体的生长情况.结果:Ad-shyrdC组细胞生长明显慢于Ad-hyrdC、Ad-null和正常对照组,差异有统计学意义,P=0.02;Ad-shyrdC组细胞的MTT值明显低于Ad-hyrdC、Ad-null和正常对照组,P=0.03;Ad-shyrdC组胃癌瘤体生长率较Ad-hyrdC、Ad-null组以及正常对照组明显减慢,P=0.02.结论:hyrdC基因具有促进胃癌细胞生长的功能,而沉默hyrdC基因可抑制胃癌细胞的生长,hyrdC基因有望成为胃癌基因治疗领域的新靶点.     

NRP-1mAb对胃癌细胞BGC 823增殖的影响

目的 观察自主研发的抗NRP-1 b1／b2 IgG单克隆抗体（NRP 1mAb）对胃癌BGC 823细胞生长的影响,并初步探讨可能的作用机制。方法 实验室制备NRP-1mAb,采用SDS-PAGE检测纯度。采用0、25、100、200、400 μg／ml NRP-1 mAb培养胃癌BGC-823细胞,光学显微镜下观察细胞形态学变化;采用MTT法、集落形成实验、流式细胞术观察胃癌细胞BGC-823的增殖、集落形成和凋亡的情况;Western blotting法检测NRP 1mAb作用后Akt、p38、ERK、JNK信号蛋白磷酸化情况。结果 SDS-PAGE检测显示,NRP-1mAb的纯度在95％以上。光学显微镜观察显示,NRP-1mAb作用后,BGC-823细胞形态呈凋亡改变。MTT实验显示,NRP-1mAb抑制BGC-823细胞的增殖作用呈浓度和时间依赖性（P＜0.01）。集落形成实验显示,不同浓度NRP-1mAb均能显著抑制BGC-823细胞的集落形成,其抑制率呈浓度依赖性（P＜0.01）。流式细胞术检测显示,不同浓度NRP-1mAb均明显促进BGC-823细胞凋亡,且大部分集中在早期凋亡。Western blotting检测显示,BGC-823细胞的Akt磷酸化受到抑制, ERK、p38、JNK的磷酸化水平无明显变化。结论NRP-1mAb能抑制胃癌细胞BGC-823的生长、促进凋亡,可能与抑制Akt磷酸化有关。     

15-hydroxyprostaglandin dehydrogenase is a tumor suppressor of human breast cancer

Prostaglandin E(2) plays a growth-stimulatory role in breast cancer, and the rate-limiting enzyme in its synthesis, cyclooxygenase-2, is often overexpressed in these cancers. Little is known about the role of the key prostaglandin catabolic enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in breast cancer pathogenesis. Using a pharmacologically based screen for epigenetically silenced genes, we found low levels of 15-PGDH in MDA-MB-231 cells [estrogen receptor (ER) negative] but high levels in MCF-7 cells (ER positive) and observed its up-regulation following demethylation treatment. Further analysis revealed methylation of the 15-PGDH promoter in one breast cancer cell line and 30% of primary tumors. Analysis of 15-PGDH expression revealed low levels in 40% of primary breast tumors and identified a correlation between 15-PGDH and ER expression. Transfection assays showed that transient up-regulation of 15-PGDH levels in MDA-MB-231 cells resulted in a decreased clonal growth, and stable up-regulation significantly decreased the ability of these cells to form tumors in athymic mice. In contrast, transient silencing of 15-PGDH in MCF-7 cells resulted in their enhanced proliferation, and a stable silencing in these cells enhanced cell cycle entry in vitro and tumorigenicity in vivo. Forced expression of 15-PGDH inhibited the ER pathway and silencing of 15-PGDH up-regulated expression of aromatase. In addition, 15-PGDH levels were down-regulated by estrogen but up-regulated by the tumor suppressor gene CAAT/enhancer binding protein alpha. Our results indicate for the first time that 15-PGDH may be a novel tumor suppressor gene in breast cancer, and suggest that this enzyme can modulate the ER pathway.     

KAI1基因对乳腺癌细胞体外增殖的抑制作用

目的研究KAI1基因对乳腺癌细胞体外增殖的抑制作用。方法应用脂质体法将pCMV-KAI1质粒转染人高转移性人乳腺癌细胞株MDA-MB-231中,免疫印迹方法检测蛋白表达;利用四甲基偶氮唑盐(MTT)法、平板克隆形成实验、体外黏附及侵袭力实验判断细胞增殖能力及黏附、侵袭力的变化。流式细胞术检测细胞生长周期的变化。结果获得了稳定表达KAI1基因的MDA- MB-231乳腺癌细胞克隆。MTT法显示,转染KAI1基因组的集落形成率(25.33%±2.36%)较转染前(43.17%±2.75%)明显降低(P<0.05)。体外黏附及侵袭力实验表明,转染组的细胞黏附侵袭能力低于未转染组和转染空载体组(P<0.05)。流式细胞术显示,转染KAI1后,G_0/G_1期细胞数量增高,从36.78%±0.61%升高至64.00%±7.56%;G_2/M期数量降低,由17.88%±0.76%降至7.63%±0.60%,差异有统计学意义。结论KAI1基因可以抑制乳腺癌细胞的增殖黏附和侵袭能力,并可能通过调节细胞周期来影响细胞的增殖。     

生长抑素类似物抑制人胃癌裸鼠种植瘤的生长增殖的研究

目的:观察生长抑素类似物奥曲肽对SGC-7901胃癌种植瘤生长增殖的影响。方法:建立人SGC-7901胃癌裸鼠移植瘤动物模型48只,随机分为4组:奥曲肽组、5-氟尿嘧啶组、奥曲肽 5-氟尿嘧啶组、对照组,相应药物干预后,处死动物,测量各组移植瘤体积、瘤质量、坏死体积,计算抑瘤率,Elivisionplus免疫组化法检测移植瘤Ki67、Bcl-2和VEGF表达情况。结果:奥曲肽组、5-FU组、联合治疗组和对照组的平均瘤体体积(cm3)分别为2·17±0·78、2·19±0·79、1·36±0·75和3·23±0·74(F=9·317,P=0·0001);平均瘤质量(g)分别为:6·88±2·06、7·22±2·47、4·47±2·28和10·30±2·80(F=5·452,P=0·0001);平均坏死体积(cm3)分别为:0·55±0·38、0·52±0·53、1·14±0·83和0·32±0·27(F=13·987,P=0·0001)。奥曲肽组、5-FU组、联合治疗组抑瘤率分别为33·20%、29·92%和56·60%(Chi-square=6·461,P=0·04)。Ki67:奥曲肽组、5-FU组、联合治疗组和对照组弱阳性百分率分别为50·0%、33·3%、25·0%和0;强阳性百分率分别为41·7%、50·0%、0和100·0%;VEGF:奥曲肽组、5-FU组、联合治疗组和对照组弱阳性百分率分别为41·7%、33·3%、8·3%和16·7%;强阳性百分率分别为33·3%、33·3%、8·3%和83·3%;Bcl-2:奥曲肽组、5-FU组、联合治疗组和对照组弱阳性百分率分别为50·0%、41·7%、16·7%和8·3%;强阳性百分率分别为:16·7%、25·0%、0和91·7%。结论:奥曲肽可以抑制SGC-7901裸鼠移植瘤的生长,且与5-FU可能有协同作用。     

Inhibitory effect of adenovirus-uteroglobin transduction on the growth of lung cancer cell lines

Uteroglobin is a secretory protein synthesized by most epithelia, including the respiratory tract. It has strong anti-inflammatory properties that appear to be related to the inhibition of phospholipase A2. Recent experimental evidence indicates that uteroglobin has an inhibitory effect on the proliferation and invasion of cancer cells. We investigated the effects of the adenovirus-uteroglobin (ad-UG) transduction on the growth of lung cancer cell lines, which did not express the uteroglobin gene. Upon transduction of ad-UG, the rate of cell growth and the ability to produce colonies in soft agar were evaluated. Cell cycle analysis, Western blot for cell cycle-related proteins and annexin V staining for apoptosis were carried out to see if they were associated with the changes in cell growth. All the tested lung cancer cell lines did not express the uteroglobin gene. The growth rates, and colony-forming ability of transformed cells, were significantly inhibited by the induction of uteroglobin gene expression. The DNA histogram showed that the cell fraction of the G2/M phase was increased, and this G2/M phase arrest was related to a decrease of cdk1 and cyclin A. However, a fraction of apoptotic cells were same as the control. From these results, uteroglobin is thought to have an inhibitory effect on the growth of lung cancer cells. This suggests a potential role for uteroglobin in gene therapy for lung cancer.