舒林酸抑制结肠癌LOVO细胞增殖作用机制研究

目的 研究舒林酸对结肠癌细胞株LOVO生长的影响,并探讨其作用机制。方法 采用四甲基偶氮唑蓝（MTT）比色法检测舒林酸对结肠癌细胞增殖的抑制作用,流式细胞仪（FCM）分析其对细胞凋亡及细胞周期的影响,放免法分析其对前列腺素E2（PGE2）的影响。结果 MTT比色显示舒林酸能抑制LOVO细胞增殖,呈剂量和浓度依赖性;FCM显示舒林酸能促进细胞凋亡,使G0／G1期细胞比例升高,S期细胞比例降低;PGE2表达水平呈剂量依赖性下调。结论 舒林酸可抑制结肠癌细胞株LOVO生长,促进其凋亡;其作用机制可能与阻止细胞周期进展、抑制COX-2活性及PGE2合成有关。     

双丁酰环腺苷酸对体外HT29细胞增殖的调节

采用酸性磷酸酶细胞原位计数及流式细胞仪（ＦＣＭ）技术，观察了双丁酰环腺苷酸（ｄｂ－ｃＡＭＰ）对体外人结肠腺癌细胞系（ＨＴ２９）增殖的影响。结果：ｄｂ－ｃＡＭＰ在１０－４～１０－９Ｍ范围内可促进细胞增殖，尤其在１０－６～１０－８Ｍ时，ｄｂ－ｃＡＭＰ组ＯＤ值分别为１．５０±０．１１，１．５８±０．０７，１．５７±０．１２，分别为对照组的１０．３０％，１６．１８％和１１．７６％，差异显著（Ｐ＜０．０５或０．０１）。ｄｂ－ｃＡＭＰ浓度为１０－３Ｍ时，则抑制细胞增殖，至培养第５天，ｄｂ－ｃＡＭＰ组ＯＤ为１．０５±０．１３，对照组为１．２３±０．１７，有显著差异（Ｐ＜０．０５）。ＦＣＭ结果提示，ｄｂ－ｃＡＭＰ对细胞的抑制作用，与延长细胞Ｇ１／Ｓ期有关。结果表明，ｄｂ－ｃＡＭＰ对ＨＴ２９细胞增殖有双向调节作用。作者还对其作用机制进行了探讨。     

舒林酸抑制人胃腺癌细胞增殖及诱导凋亡

目的在体外对舒林酸抗人胃腺癌细胞增殖和凋亡诱导作用及其机制进行初步研究.方法采用人胃腺癌细胞株MKN45,MKN28作为研究对象.体外药物敏感试验(MTT)检测不同浓度和作用时间的舒林酸对细胞的增殖抑制效应;分别用Hoechst33258细胞核荧光染色、透射电镜和DNA琼脂糖凝胶电泳观察舒林酸诱导的细胞凋亡改变;应用Western斑点免疫印迹法观察经舒林酸2mmol·L-1和4mmol·L-1作用24 h后细胞内环氧化酶-2(COX-2)和凋亡抑制蛋白Bcl-2表达程度的变化.结果舒林酸对人胃腺癌细胞MKN45,MKN28的杀伤率随着剂量的增大和作用时间的延长而增加,舒林酸对不同类型的细胞杀伤率不同.舒林酸能够诱导人胃腺癌细胞MKN45,MKN28产生凋亡,凋亡诱导作用的强弱同样具有时间和剂量依赖性,而且对不同类型胃癌细胞的凋亡诱导效应有显著差异.经舒林酸2 mmol·L-1和4 mmol·L-1作用24 h后,MKN45细胞内COX-2和Bcl-2蛋白比未经舒林酸作用的细胞表达明显减少,而MKN28细胞内COX-2和Bcl-2蛋白的表达未出现明显变化.结论舒林酸在体外对人胃腺癌细胞株MKN45和MKN28有良好的增殖抑制作用,诱导凋亡是其抑制胃癌细胞增殖的重要作用机制.舒林酸对人胃腺癌细胞的抑制和凋亡诱导作用与其抑制细胞内环氧化酶-2,并进而抑制Bcl-2的表达有关,并可能与胃癌细胞的分化状态有关.     

正反义hTGF－β_1基因转染对大肠癌HT－29细胞体外生物学行为的影响

正反义ｈＴＧＦ－β＿１基因转染对大肠癌ＨＴ－２９细胞体外生物学行为的影响姜泊，周殿元，孙正基，张宏权应用基因重组技术构建了人转化生长因子（ｈＴＧＦ－β１）基因重组真核表达载体，并将其导入ＨＴ－２９细胞株，探讨对大肠癌细胞体外生物学行为的影响。材料与方...     

舒林酸对实验性高钙尿症的防治作用

目的 观察新型非类固醇类抗炎药－舒林酸对实验性高钙尿症的防治作用,探讨其可能机制。方法 建立大鼠实验性高钙尿症模型,观察舒林酸治疗后２周和４周尿Ｇａ、尿Ｃａ／Ｃｒ、尿ＮＡＧ／Ｃｒ以及肾和十二指肠Ｃａ＾２＋ＡＴＰ酶（钙泵）的变化。结果 ２周和４周时尿Ｃａ、尿Ｃａ／Ｃｒ、尿ＮＡＧ／Ｃｒ及肾和十二指肠Ｃａ＾２＋ＡＴＰ酶均显著下降（Ｐ〈０．０１）,２４ｈ尿钙定量与尿ＡＮＧ／Ｃｒ值呈正相关（Ｐ〈０．０１）。     

舒林酸诱导人肝细胞癌凋亡及对环氧合酶-2和Bcl-2蛋白表达的影响

目的 探讨舒林酸在肝细胞癌化学预防和治疗中的应用价值。方法 采用人肝细胞癌细胞株SMMC7721，HepG2作为研究对象。体外药物敏感试验检测不同浓度和作用时间的舒林酸对细胞的增殖抑制效应；分别用Hoechst33258细胞核荧光染色和透射电镜观察舒林酸诱导的细胞凋亡的形态学改变，并计算相应的细胞凋亡指数（AI）；应用Western斑点印迹法观察不同浓度舒林酸作用后细胞内环氧合酶-2（COX-2）和凋亡抑制蛋白Bcl-2表达程度的变化。结果 舒林酸对人肝细胞癌细胞SMMC7721，HepG2具有显著的增殖抑制和凋亡诱作用，并且具有时间和剂量依赖性，舒林酸对不同类型细胞的杀伤率和凋亡诱导效应有显著差异。经舒林酸2mmol/L和4mmol/L作用24h后，细胞内COX-2和Bcl-2蛋白的表达比未经舒林酸作用的细胞表达明显减少。结论 舒林酸在体外对人肝细胞癌细胞株SMMC7721和HepG2具有显著的增殖抑制和诱导凋亡作用。且与其抑制细胞内COX-2和Bcl-2蛋白的表达有关。     

表皮生长因子对HT－29细胞c－myc蛋白表达的影响

本文用细胞免疫组化法检测了表皮生长因子（ＥＧＦ）对癌基因ｃ－ｍｙｃ蛋白表达的作用，结果发现１０－６Ｍ的ＥＧＦ作用１２小时即可明显提高ＨＴ－２９细胞上ｃ－ｍｙｃ蛋白的表达，提示ＥＧＦ参与对癌基因ｃ－ｍｙｃ蛋白表达的调节。     

舒林酸对人结肠癌细胞凋亡及相关基因表达谱的作用

目的:观察舒林酸对人结肠癌LOVO细胞株凋亡的影响,以及舒林酸作用后的该细胞基因表达谱改变.方法:应用透射电镜,流式细胞仪观察舒林酸作用48 h、72 h对LOVO细胞凋亡的影响;基因芯片法检测舒林酸作用前后LOVO细胞的差异表达基因.结果:经舒林酸处理后细胞有典型的凋亡小体形成:经舒林酸0.6、0.9、1.2 mmo1/L作用后,细胞凋亡率与对照组比较明显升高(48h:4.2±1.04,4.26±0.28,7.51±2.09 vs 1.81±0.9l:72 h:6.21±0.56,7.48±1.45,10.40±1.30 vs 2.06±1.43:均P<0.05).与含有17101个cDNA的基因芯片杂交.筛选出差异表达基因1O13条,其中178条为凋亡相关基因(表达上调82条,下调96条),占所有差异表达基因的17.87%.结论:舒林酸具有诱导LOVO细胞发生凋亡的作用.上调或下调某些凋亡相关基因的表达可能是其诱导凋亡的分子机制之一.     

幽门螺杆菌相关胃溃疡的细胞增殖与凋亡研究

目的观察Ｈｐ感染对胃溃疡（ＧＵ）患者胃粘膜上皮细胞增殖与凋亡的影响,进而对溃疡发生机制进行探讨．方法标本取自内镜下胃粘膜活检,细胞凋亡检测采用Ｔｕｎｅｌ方法,细胞增殖检测采用免疫组化ＬＳＡＢ方法测定增殖细胞核抗原（ＰＣＮＡ）．结果ＧＵＨｐ＋患者细胞凋亡指数（ＡＩ）明显高于Ｈｐ－患者（Ｐ＜００５）,但比较两者的增殖细胞指数（ＰＣＮＡＬＩ）,无显著性差异（Ｐ＞００５）;慢性浅表性胃炎Ｈｐ＋患者ＡＩ,ＰＣＮＡＬＩ明显高于Ｈｐ－患者（Ｐ＜００５）,ＧＵＨｐ＋患者的ＡＩ／ＰＣＮＡＬＩ比值为１５４１,明显高于其他各组（Ｐ＜００５）．结论Ｈｐ感染能诱导ＧＵ患者胃粘膜上皮细胞凋亡增加,出现细胞凋亡与细胞增殖平衡失调,促进溃疡形成     

舒林酸经survivin/Aurora B途径对人胰腺癌细胞分裂的阻断效应

目的 观察舒林酸处理胰腺癌细胞BxPC3后对survivin、Aurora B表达及细胞周期和增殖的影响,探讨舒林酸的作用机制.方法 应用MTT法检测舒林酸对BxPC3细胞的增殖抑制作用,RT-PCR法检测survivin mRNA、Aurora B mRNA的表达,Western blot法检测survivin蛋白表达及Aurora BThr-232磷酸化水平,流式细胞仪检测细胞周期变化.结果 舒林酸呈时间和剂量依赖性抑制BxPC3细胞增殖.经500μmoL/L舒林酸作用细胞48 h后,survivin mRNA和Aurora B mRNA表达量分别从1.56和0.66下降到0.44和0.11(P<0.01);survivin蛋白表达从1.27下降到0.21(P<0.01),Aurora BThr-232磷酸化水平从0.47下降到0.25(P<0.01);G0/G1期细胞比例从(56.65±1.93)%升高到(70.58±3.21)%(P<0.01).结论 舒林酸通过下调survivin表达,降低Aurora B的活性,从而影响染色体过客复合物蛋白(CPC)对细胞染色体的排列和分离作用,使大量细胞停滞在G0/G1期,从而抑制细胞的有丝分裂.     

Inhibitory effect of sulindac on the proliferation of HT‐29 colon adenocarcinoma cells: Effects on cell cycle and apoptosis

OBJECTIVE : To investigate: (i) the effect of sulindac on the proliferation of HT‐29 cells; and (ii) the antineoplastic mechanisms of sulindac. METHODS : The MTT colorimetric assay was used to examine the effect of sulindac on the proliferation of HT‐29 cells. Flow cytometry was used for examining the cell cycle distribution. Flow cytometry, transmission electron microscopy and DNA electrophoresis were used to determine whether sulindac induces cell apoptosis. RESULTS : Sulindac inhibited cell proliferation in a time‐ and dose‐dependent manner. After treatment with 0.3, 0.6, 0.9 and 1.2 mmol/L sulindac for 72 h, the inhibition rates reached 16, 38, 62.3 and 92.2%, respectively. After treatment with 1.2 mmol/L sulindac for 24, 48 and 72 h, the inhibition rates reached 32.4, 71.3 and 92.2%, respectively (P < 0.05). Sulindac increased the proportion of cells in the G₀/G₁ phase and decreased the proportion of cells in the S phase of the cell cycle. After treatment with 1.2 mmol/L sulindac for 48 h, the proportion of cells in the G₀/G₁ phase increased from 32.5 to 70.5% and the S phase decreased from 43.1 to 11.3% compared with the control cells (P < 0.01). Flow cytometry, DNA electrophoresis and transmission electron microscopy all demonstrated that sulindac could induce apoptosis of the HT‐29 cell line. After treatment with 0.3, 0.6 and 1.2 mmol/L sulindac for 48 h, the proportion of apoptotic cells reached 5.8, 7.6 and 11.7%, compared with 2.9% in the control group; after treatment for 72 h, the proportion of apoptotic cells increased to 12.5, 15.4% and 24.2%, respectively (P < 0.05). All effects were time and dose dependent. CONCLUSIONS : The colon adenocarcinoma HT‐29 cell line can be inhibited by sulindac and the antitumor mechanism may be related to changing cell cycle distribution and inducing cell apoptosis.     

普伐他汀对人结肠癌细胞增殖及COX-2蛋白表达的影响

目的体外观察普伐他汀对人结肠癌细胞HT-29、Ls-174-T细胞增殖、细胞周期及COX-2蛋白表达的影响。方法采用噻唑蓝（MTT）法观察普伐他汀对HT-29和Ls-174-T细胞增殖的影响，流式细胞仪（FCM）研究普伐他汀对细胞周期的作用，免疫细胞化学观察COX-2蛋白的表达。结果体外普伐他汀可抑制HT-29和Ls-174-T细胞增殖，各处理组内G0／G1期细胞增多，但诱导凋亡不明显，体外普伐他汀可减少HT-29和Ls-174-T细胞株COX-2蛋白的表达。结论体外普伐他汀对HT-29和Ls-174-T细胞增殖有抑制作用，该作用可能与使细胞生长阻滞于G0／G1期及抑制COX-2蛋白表达有关。     

硒蛋氨酸诱导食管癌细胞株凋亡的实验研究

目的：探讨硒蛋氨酸对食管癌细胞系EC 9706凋亡的影响。方法：采用MTT比色法，细胞生长曲线描绘观察硒蛋氨酸对食管癌细胞系EC 9706增殖的影响。采用流式细胞仪观察硒蛋氨酸对EC 9706细胞诱导其凋亡的作用及对细胞周期的影响。琼脂糖凝胶电泳法检测DNA ladder。结果：硒蛋氨酸呈时间、剂量依赖性方式抑制EC 9706细胞增殖，改变细胞周期分布，增加G0／G1期细胞比例，诱导细胞凋亡。结论：硒蛋氨酸可能通过影响细胞周期分布和诱导细胞凋亡，从而抑制EC 9706细胞增殖。硒蛋氨酸可能是预防和治疗食管癌的一种新制剂。     

siRNA沉默p28GANK对结肠癌细胞增殖的影响

背景：研究发现p28GANK表达与结肠癌恶性程度相关,其高表达提示预后不良。然而,p28GANK在结肠癌中的作用尚未完全明确。目的：探讨沉默p28GANK对结肠癌细胞增殖能力的影响。方法：以针对p28GANK基因的慢病毒携带siRNA转染人结肠癌HT29细胞,以蛋白质印迹法检测干扰效果,以MTr法检测细胞增殖,以平板克隆实验检测细胞克隆能力,以流式细胞术检测细胞周期变化。结果：与HT29、Con—HT29细胞相比,Si—HT29细胞中p28GANK蛋白表达水平显著降低;细胞增殖速度显著减缓;细胞克隆数量显著减少;G0／G1期细胞比例显著增多,S期细胞比例显著减少,PI值显著降低（P〈0．05）。结论：沉默p28GANK在结肠癌细胞中的表达可抑制细胞增殖能力,使细胞周期阻滞。p28GANK可作为治疗结肠癌的新靶点。     

Mechanism of apoptotic effects induced selectively by ursodeoxycholic acid on human hepatoma cell lines

AIM: To investigate the effects of ursodeoxycholic acid (UDCA) on apoptosis and proliferation of hepatoma cell lines. METHODS: Human hepatoma cell lines HepG2 and BEL 7402 were cultured in medium supplemented with different concentrations of UDCA, normal human hepatic line L-02 was used as control. Cell proliferation, apoptosis and gene expression were detected using methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, Western blot, DNA ladder assay, electron microscopy, and immunocytochemistry. RESULTS: Ursodeoxycholic acid inhibited the proli- feration of HepG2 and BEL7402 cell lines in a dose- dependent manner. Ursodeoxycholic acid can change cell cycle distribution of HepG2 and BEL7402, the proportion of cells in G0-G1 phase increased whereas the proportion of S phase cells and G2-M phase cells decreased. Ursodeoxycholic acid arrested the cell cycle in G0-G1 phase by down-regulating the cell cycle related proteins cyclin D1, D3 and retinoblastoma protein (pRb). The apoptotic rates of HepG2 and BEL7402 treated with UDCA (1.0 mmol/L) were significantly higher than those of control. In the HepG2 and BEL7402 treated with UDCA, expression of bcl-2 decreased whereas expression of Bax increased, the nuclear fragmentation and chromosomal condensed, cells shrank and lost attachment, apoptotic bodies and DNA ladders appeared. UDCA had no effect in inducing apoptosis on L-02 cell lines. CONCLUSION: UDCA can selectively inhibit proliferation and induce apoptosis of HepG2 and BEL7402 cell lines by blocking cell cycle and regulating the expression of Bax/bcl-2 genes.     

波动性高糖对胰岛β细胞增殖及Skp2-p27表达的影响

目的 探讨波动性高糖对INS-1细胞增殖、凋亡及对细胞周期进程的影响,并研究其可能的分子机制.方法 采用细胞计数试剂盒(cell counting kit-8)检测细胞增殖活性,流式细胞仪测定细胞周期及细胞ROS水平,Annexin-V/PI双标流式细胞术检测细胞凋亡.应用Western印迹检测细胞周期调控蛋白p27及Skp2的表达水平.结果 (1)波动性高糖及持续性高糖均明显抑制INS-1细胞的生长,且波动性高糖对细胞增殖的抑制作用更为显著.(2)波动性高糖及持续性高糖均明显增加INS-1细胞的凋亡,且波动性高糖作用更为显著.(3)波动性高糖及持续性高糖能明显抑制细胞周期进程,使INS-1细胞周期更多滞留在G0/G1期,G2/M期与S期细胞比例下降,波动性高糖作用更显著.(4)波动性高糖及持续性高糖均能显著增强细胞周期调控蛋白p27的表达,同时减弱Skp2蛋白的表达水平.结论 波动性高糖较持续性高糖更能够抑制INS-1细胞的增殖和诱导凋亡,可能是通过减弱Skp2蛋白的表达水平,增加p27蛋白的活性,使细胞阻滞在G0/G1期,抑制细胞周期进程,从而减弱细胞的增殖活性.

Abstract：

Objective To investigate the effect of intermittent high glucose on proliferation, apoptosis, and cell cycle progression of INS-1 cells, and the possible intracellular pathways activated by intermittent high glucose. Methods Cell viability was evaluated by cell counting kit, the cell cycle was determined by flow cytometry,Annexin-V/PI double-labeled cell apoptosis detection kit was used to monitor cell apoptosis. Cell cycle related protein Skp2 and p27 expressions were detected by Western blot. Results ( 1 ) Both intermittent and constant high glucose significantly inhibited the growth of INS-1 cells, and the former effect was more significant. ( 2 ) Intermittent and constant high glucose levels significantly increased apoptosis in INS-1 cells, and the former effect was more significant. (3) Intermittent and constant high glucose levels significantly inhibited the cell process, the G0/G1 cell cycle arrest also was induced by intermittent high glucose, resulting in lowered proportion of the G2/M phase and S phase of INS-1 cells. (4) Intermittent and constant high glucose significantly decreased the level of protein Skp2 and increased the level of cell cycle related protein p27. Conclusion Intermittent high glucose levels affect INS-1 cell growth and proliferation, as well as induce cell apoptosis, probably by decreasing the level of protein Skp2 and increasing the level of p27 in the cells, resulting in arrest of progression through the G1 phase to the S phase of INS1 cells, and thus impairment of cell proliferation.     

A K-ras oncogene increases resistance to sulindac-induced apoptosis in rat enterocytes

BACKGROUND & AIMS: Mutations of c-K-ras occur commonly in colonic neoplasms. The aim of this study was to determine how c-K-ras mutations alter the responses to the chemopreventive agent sulindac. METHODS: The parental rat intestinal cell line IEC-18 and c-K-ras-transformed derivatives were treated with sulindac sulfide. Cell cycle distribution was determined by flow-cytometric analysis (fluorescence-activated cell sorter), apoptosis by DNA fragmentation (laddering), flow cytometry, and microscopy, and changes in gene expression by immunoblotting. RESULTS: Sulindac sulfide inhibited cell growth and induced apoptosis in a time- and dose-dependent manner more rapidly in and at lower concentrations in parental cells than ras-transformed cells. Expression of the sulindac sulfide arrested cells in G0/G1, but cells entered apoptosis throughout the cell cycle. Proapoptotic protein Bak was relatively high in untreated parental cells and increased markedly after sulindac sulfide but was low in untreated ras-transformed cells and did not increase after sulindac sulfide. Expression of other Bcl-2 family members was unchanged after sulindac sulfide. However, sulindac sulfide reduced levels of cyclin D1 protein and cyclin E- and cyclin D1- associated kinase activity. CONCLUSIONS: c-K-ras-transformed enterocytes are relatively resistant to sulindac sulfide-induced growth inhibition and apoptosis, which may result from specific reduction of bak expression. (Gastroenterology 1997 Dec;113(6):1892-900)     

Effect of p27mt gene on apoptosis of the colorectal cancer cell line Lovo

AIM： To construct p27mt recombinant adenovirus, transfect the colorectal cell line Lovo and observe the effects of p27mt on Lovo cell apoptosis and cell cycle inhibition. METHODS： We constructed recombinant adenovirus containing p27mt by homologous recombination in bacteria. The colorectal cancer cell line Lovo was infected with recombinant replication-defective adenovirus Ad- p27mt, and expression of p27mt was determined by Western blotting; the inhibitory effect of p27mt on Lovo cells was detected by cytometry. Cell cycle was determined by flow cytometry. DNA fragment analysis identified the occurrence of apoptosis. RESULTS： The recombinant adenovirus which already contained p27mt target gene was successfully constructed. When multiplicity of infection was ≥50, the infection efficiency was 100%. After transfection of Lovo cells with Ad-p27mt the cells had high p27 expression which was identified by immunoblotting assay. PI staining and flow cytometry showed that 77.96% of colorectal cancer cells were inhibited in phase G0/G1, while in the Ad-LacZ group and blank control group, 27.57% and 25.29% cells were inhibited in the same phase, respectively. DNA fragment analysis, flow cytometry and TUNEL assay demonstrated that p27mt is able to induce apoptosis in colorectal cancer cells. CONCLUSION： p27mt has an obvious blocking effect on colorectal cancer cell cycle, and most cells were inhibited in phase G0/G1. Therefore, p27mt can induce apoptosis in colorectal cells.     

吲哚美辛对胃癌SGC 7901细胞增殖及Cyclin D1蛋白表达的影响

目的:观察引哚美辛对胃癌SGC 7901细胞增殖及细胞周期调控蛋白Cyclin D1表达的影响,探讨吲哚美辛抑制细胞增殖的机制.方法:采用MTT法观察吲哚美辛对胃癌细胞SGC 7901增殖的影响,采用流式细胞仪观察细胞周期分布的变化,采用免疫细胞化学方法检测Cyclin D1蛋白的表达.结果:吲哚美辛呈时间、浓度依赖方式抑制胃癌SGC 7901细胞增殖,改变细胞周期的分布,增加G0/G1期细胞的比例,下调Cyclin D1蛋白的表达.结论:吲哚美辛可能通过下调Cyclin D1蛋白表达,影响细胞周期的分布来抑制胃癌SGC7901细胞增殖.