5'-Aza-CdR对HT-29和LoVo结直肠癌细胞株中CDX2基因甲基化状态、mRNA表达及蛋白表达的影响

目的:探讨甲基化酶抑制剂5'-氮杂-2'-脱氧胞苷(5'-Aza-2'-deoxycytidine,5'-Aza-CdR)对结直肠癌细胞株HT-29和LoVo中CDX2基因甲基化水平、mRNA及蛋白表达的影响.方法:用不同浓度5'-Aza-CdR处理结直肠癌细胞株HT-29和LoVo.应用TaqMan探针为基础的实时定量PCR(Methylight)方法、SYBR Green PCR方法及蛋白印迹实验(Western blot)检测药物处理前后HT-29和LoVo细胞中CDX2基因的甲基化状态、mRNA和蛋白表达.结果:Methylight检测HT-29和LoVo细胞中C D X2蛋白在药物作用异常甲基化未得到逆转;实时荧光定量PCR和Western blot检测到0.5、1.0、1.5μmol/L 5'-Aza-CdR处理后C D X2基因m R N A和蛋白重新表达,实时荧光定量PCR检测对照组和不同浓度实验组在HT-29细胞株mRNA相对表达量分别为1.000±0.000、0.973±0.024、1.014±0.019和1.094±0.020;在LoVo细胞株相对表达量分别为1.000±0.000、0.966±0.038、1.050±0.029和1.007±0.019.Western blot检测CDX2蛋白在对照组和不同浓度实验组中HT-29细胞株相对表达量分别为0.454±0.049、0.501±0.041、0.340±0.050和0.531±0.046;LoVo细胞株相对表达量分别为0.527±0.037、0.415±0.037、0.432±0.040和0.626±0.046.以上作用无时间、剂量依赖性,但CDX2基因mRNA在HT-29细胞株表达(F=25.146,P=0.000)和LoVo细胞株表达(F=5.470,P=0.024)具有统计学差异,C D X2蛋白表达在HT-29细胞株表达(F=9.700,P=0.005)和LoVo细胞株表达(F=17.701,P=0.001)亦具有统计学差异.结论:结直肠癌细胞株HT-29和LoVo中CDX2基因mRNA和蛋白表达不受中DNA甲基化的表观遗传修饰的调控.

5-aza-2’deoxycytidine联合trichostatin A抑制乳腺癌细胞增殖能力的研究

背景与目的:晚近报道应用DNA甲基转移酶抑制剂5-aza-2’deoxycytid ine(AZA)联合组蛋白去乙酰化酶抑制剂trichostatin A(TSA)作用于多种肿瘤,可以达到治疗肿瘤的目的。本文在此探讨通过AZA联合TSA作用,抑制乳腺癌细胞株增殖能力。方法:联合应用两种药物作用于肿瘤细胞,应用RT-PCR的方法检测MDA-MB-435细胞株p21和p27的mRNA转录水平的改变。通过W ST方法来检测乳腺癌细胞的增殖能力变化,将MDA-MB-435细胞根据药物处理共分四组:①对照组;②AZA+TSA组;③AZA+TSA+4-OH TAM组;④4-OH TAM组。并且采用流式细胞仪来分析肿瘤细胞周期分布的改变,将MDA-MB-435分成另外四组:①对照组;②AZA+TSA组;③AZA+TSA+E2组;④AZA+TSA+E2+4-OH TAM组。结果:TSA以及AZA联合应用能使肿瘤细胞MDA-MB-435的p27的mRNA水平增加,而p21mRNA水平略减弱。增殖分析的四个组中:AZA+TSA组细胞增殖能力减弱(P<0.01),同时出现细胞阻滞于S期,加入雌激素后细胞阻滞稍减弱。AZA+TSA+4-OH TAM组增殖能力进一步减弱(P<0.01)。4-OH TAM组增殖能力没有改变。细胞周期分析的四个组中:AZA+TSA组出现细胞阻滞于S期,AZA+TSA+E2组细胞阻滞稍减弱。AZA+TSA+E2+4-OH TAM组细胞阻滞再次提高。结论:两种药物联合应用能使得乳腺癌肿瘤细胞增殖能力下降,对于肿瘤细胞有一定抑制作用。

5-Aza-2′-dC通过上调DR4与DR5表达增加TRAIL诱导乳腺癌细胞凋亡的敏感性

目的探讨5-Aza-2′-dC增加肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导乳腺癌细胞凋亡敏感性的作用及其可能机制。方法 5μmol/L 5-Aza-2′-dC预处理细胞96 h,TRAIL(0,50,100,200 ng/mL)处理细胞12 h,MTT检测细胞增殖抑制率,Annexin V-FITC/PI法检测细胞凋亡,流式细胞技术检测TRAIL受体DR4及DR5的表达,Western blot检测caspase-8蛋白表达水平,Real-time PCR检测细胞DR4、DR5和caspase-8 mRNA表达。结果 5-Aza-2′-dC预处理组与对照组相比,TRAIL对细胞增殖抑制率和诱导凋亡率明显高于对照组(P<0.05)。5-Aza-2′-dC能增加细胞表面DR4和DR5表达,促进凋亡通路关键蛋白caspase-8活化。5-Aza-2′-dC在mRNA水平增加了DR4、DR5和caspase-8的表达。结论 5-Aza-2′-dC增加了TRAIL诱导乳腺癌细胞凋亡的敏感性,其可能机制是通过上调DR4、DR5和caspase-8表达实现。

5-aza-2’-deoxycitydine诱导胃癌细胞系TIMP3基因去甲基化和转录上调的实验性探讨

目的:研究人类胃癌细胞系中5-aza-2’-deoxycitydine诱导肿瘤抑制基因TIMP3mRNA和蛋白表达。方法:DNA甲基化抑制剂5-aza-2’-deoxycitydine处理人类胃癌细胞系SGC-7901。应用RT-PCR和Western-Blot方法检测TIMP3基因mRNA和蛋白表达。应用MSP分析TIMP3基因启动子甲基化状态。结果:5-aza-2’-deoxycitydine诱导TIMP3基因启动子去甲基化,上调TIMP3mRNA和蛋白表达。结论:甲基化异常是胃癌细胞TIMP3基因失活的原因之一,并可受去甲基化制剂调控表达。

5-Aza-2’-CdR对K562细胞甲基化酶及miR-146a、miR-29b表达的影响

目的观察甲基化酶抑制剂5-Aza-2’-CdR作用后K562细胞中miR-146a及miR-29b的表达水平变化及DNMT1、DNMT3a、DNMT3b三种甲基化酶水平的改变。方法 MTT法检测5-Aza-2’-CdR作用于K562细胞时的IC50浓度。通过茎环引物法及荧光定量PCR的方法检测miRNAs以及甲基化酶的水平。结果 5-Aza-2’-CdR作用于K562细胞时的IC50浓度随药物作用的时间不同而异。药物作用后72h进行检测,凋亡细胞比例上升,miR-146a、miR-29b的水平较对照组升高,而DNMT1、DNMT3a、DNMT3b的水平与对照组相比呈降低的趋势,DNMT3a在11μM 5-Aza-2’-CdR处理72h后虽表现出了升高的趋势,但差异无统计学意义。结论 5-Aza-2’-CdR能促进K562细胞的凋亡,5-Aza-2’-CdR作用后miR-146a、miR-29b表现出了上升的趋势而三种甲基化酶表达水平有所降低。

5-aza-2’-deoxycytidine重新激活MDA-MB-231乳腺癌细胞雌激素受体基因的表达

目的 探讨 5 AZA CdR重新激活MDA MB 2 3 1乳腺癌细胞雌激素受体基因的表达。方法 采用免疫组织化学的方法 ,对用浓度为 2mg/L的 5 AZA CdR处理MDA MB 2 3 1乳腺癌细胞前后和阳性对照MCF 7乳腺癌细胞雌激素受体基因的表达情况进行检测分析。结果 5 AZA CdR处理过的MDA MB 2 3 1乳腺癌细胞ER的阳性表达率明显增高 (P <0 .0 5 )。结论 5 AZA CdR可以重新激活MDA MB 2 3

Anti-tumor effect of 5-aza-2'-deoxycytidine by inhibiting telomerase activity in hepatocellular carcinoma cells

AIM:To investigate the effect of the demethylating reagent 5-aza-2'-deoxycitidine(DAC) on telomerase activity in hepatocellular carcinoma(HCC) cell lines,SMMC-7721 and HepG2.METHODS:The related gene expression in cell lines was examined by real-time reverse transcription-polymerase chain reaction and Western blotting analysis.The telomerase activity was examined by telomeric repeat amplification protocol-enzyme-linked immunosorbent assay and DNA methylation was determined by methylation-specific polymerase chain reaction.RESULTS:The telomerase activity was significantly reduced in both cell lines treated with DAC,accompanied by downregulation of telomerase reverse transcriptase(hTERT).We also observed the effect of DAC on the methylation status of hTERT promoter and the expression of regulatory genes,such as c-myc,p15,p16,p21,E2F1,and WT1.The methylation status of hTERT promoter could be reversed in SMMC-7721 by DAC,but not in HepG2 cells.However,p16 expression could be reactivated by demethylation of its promoter,and c-Myc expression was repressed in both cell lines.Moreover,DAC could enhance the sensitivity to the chemotherapeutic agents,such as cisplatin,by induction of apoptosis of HCC cells.CONCLUSION:The DAC exerts its anti-tumor effects in HCC cells by inhibiting the telomerase activity.

Effect of 5-Aza-2’-deoxycytidine on immune-associated proteins in exosomes from hepatoma

AIM: To study the effect of 5-Aza-2’-deoxycytidine (5-Aza-CdR) on heat shock protein 70 (HSP70), human leucocyte antigen-Ⅰ (HLA-Ⅰ) and NY-ESO-1 proteins in exosomes produced by hepatoma cells, HepG2 and Hep3B. METHODS: Exosomes derived from HepG2 and Hep3B cells treated with or without 5-aza-CdR were isolated and purified by ultrafiltration centrifugation and sucrose gradient ultracentrifugation. The number of exosomes was counted under electron microscope. Concentration of proteins in exosomes was measured by bicinchoninic acid protein assay. Expression of HSP70, HLA-Ⅰ and NY-ESO-1 proteins in exosomes was detected by Western blotting and immunoelectron microscopy. mRNA expression of p53 gene was detected by reverse transcription polymerase chain reaction.RESULTS: The mRNA expression of p53 gene was increased in both hepatoma cell lines after treatment with 5-Aza-CdR. The number of exosomes and the concentration of total proteins in exosomes were increased signifi cantly after treatment with 5-aza-CdR (P < 0.05). After treatment with 5-Aza-CdR, immunoelectron microscopy and Western blotting showed that the HSP70, HLA-Ⅰ and NY-ESO-1 proteins were increased in exosomes produced by both hepatoma cell lines. CONCLUSION: 5-aza-CdR, an inhibitor of DNA methyltransferase, can increase exosomes produced by hepatoma cells and immune-associated protein component of exosomes, which may be mediated by p53 gene upregulation and 5-Aza-CdR demethylation.

5-Aza-2'-deoxycytidine inhibits retinoblastoma cell by reactivating epigenetically silenced RASSF1A gene

AIM: To investigate the effect of 5-Aza-2’-deoxycytidine(5-Aza-CdR),a DNA methyltransferase(DNMT) inhibitor,on the growth and survival of the Chinese retinoblastoma(RB) cell line HXO-RB44. ·METHODS: The DNA methylation status of the Ras association domain family(RASSF1A) promoter in the presence of 5-Aza-CdR at different concentrations was analyzed by methylation-specific polymerase chain reaction(MSP). RASSF1A mRNA and protein levels were measured by semiquantitative RT-PCR and immunohistochemistry staining,respectively,when cells were treated with 5.0μmol/L of 5-Aza-CdR. The effect of 5.0μmol/L 5-Aza-CdR on the proliferation and viability of HXO-RB44 cells was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and flow cytometry. ·RESULTS: 5-Aza-CdR efficiently induced cell cycle arrest at G0 /G1 and apoptotic death in HXO-RB44 cells. MSP analysis showed that unmethylated RASSF1A DNA increased and methylated RASSF1A decreased in a dose-dependent manner in a range of 0.5-5.0μmol/L 5-Aza-CdR. Accordingly,RASSF1A expression was reactivated at both mRNA and protein levels. Incubation time of 5-Aza-CdR treatment also functioned as a factor for the demethylation status of RASSF1A promoter DNA,with a plateau on day four. 5-Aza-CdR at 5.0μmol/L completely demethylated the RASSF1A promoter in HXORB44 cells on day four,and as a result,RASSF1A expression increased significantly from day 4 to day 7.·CONCLUSION: 5-Aza-CdR inhibits the growth of the HXO-RB44 RB cell line and induces apoptosis by demethylating the RASSF1A gene.

Induction of HLA-G expression in a melanoma cell line OCM-1A following the treatment with 5-aza-2'-deoxycytidine

The non-classical HLA class Ⅰ antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells （DC）. As a result, HLA-G expression in malignant cells may provide them with a mechanism to escape the immune surveillance. In melanoma, HLA-G antigen expression has been found in 30% of surgically removed lesions but in less than 1% of established cell lines. One possible mechanism underlying the differential HLA-G expression in vivo and in vitro is that the HLA-G gene is epigenetically repressed in melanoma cells in vitro. To test this hypothesis, we treated the HLA-G negative melanoma cell line OCM-1A with the DNA methyltransferase inhibitor 5-aza-2＇-deoxycytidine （5-AC） and analyzed whether HLA-G expression can be restored. Our data strongly suggest that HLA-G is silenced as a result of CpG hypermethylation within a 5＇ regulatory region encompassing 220 bp upstream of the start codon. After treatment, HLA-G mRNA expression was dramatically increased. Western blot and flow cytometry showed that HLA-G protein was induced. Interestingly, HLA-G cell surface expression on the 5-AC treated OCM-1A cells is much less than that on the HLA-G positive JEG-3 cells while a similar amount of total HLA-G was observed. Possible mechanisms for the difference were analyzed in the study such as cell cold-treatment, peptide loading and antigen processing machinery components （APM） as well as β2 microglobulin （β2-m） expression. Data revealed that the APM component calreticulin might be involved in the lower HLA-G surface expression on OCM-1A cells. Taken together, our results indicated that DNA methylation is an important epigenetic mechanism by which HLA-G antigen expression is modulated in melanoma cells in vitro. Furthermore, to the first time, we hypothesized that the deficiency of calreticulin might be involved in the low HLA-G surface expression on the 5-AC treated OCM-1A cells.

Inhibitory Effects of 5-Aza-2'-Deoxycytidine and Trichostatin A in Combination with p53-Expressing Adenovirus on Human Laryngocarcinoma Cells

Objective： To investigate the effects of 5-Aza-2＇-deoxycytidine （5-Aza-Cdr） and trichostatin A （TSA） combined with p53-expressing adenovirus （Ad-p53） on Hep-2 cell line in vivo and in vitro, in order to explore its possibility in biological treatment of laryngocarcinoma. Methods： Effects of 5-Aza-Cdr and TSA in combination with Ad-p53 on Hep-2 cell line in vivo were determined by Cell Counting Kit-8 （CCK-8） assay. The effect of drug combination was calculated by Jin＇s formula. Effects on the cell line in vitro were investigated by establishing the nude mice model. Results： 5-Aza-Cdr and TSA showed inhibitory effects on the proliferation of Hep-2 cells in dose- and time-dependent manner. Ad-p53 can inhibit the growth of Hep-2 cells in vivo and in vitro. However, the combination of epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 was less effective than individual use of Ad-p53. 5-Aza-Cdr and Ad-p53 inhibited the growth of transplanted tumors and reduced the volume of tumors, and the tumor volume of Ad-p53 group was significantly smaller than that of the control group （P0.05）. Conclusion： Both epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 can suppress cell proliferation on Hep-2 in vivo and in vitro and there may be some antagonistic mechanism between Ad-p53 and epigenetic reagents （5-Aza-Cdr/ TSA）.

Effects of 5-aza-2’-deoxyctidine on the development of porcine parthenogenetic and nuclear transfer embryos

The current study was to investigate whether embryo or fetal fibroblast cells treated with 5-aza-2’-deoxyctidine (5-aza-dC) have a positive effect on the in vitro development of porcine parthenogenetic and cloned embryos. To this end, porcine fetal fibroblast cells were treated with different concentrations (5 nM, 50 nM and 500 nM) of 5-aza-dC for different exposure times (1, 6 and 20 hours), the results showed that DNA methylation in PRE-1 SINE region was gradually reduced over time in cells treated with 5-aza-dC. To determine the effect of 5-aza-dC on in vitro development of porcine activated oocytes, the parthenogenetic embryo was treated with 5-aza- dC. Notably, treatment with 5 nM 5-aza-dC for 1 hour led to a significant improvement in blastocyst development, compared with the control group. The effects of donor cell treatment with 5-aza-dC on porcine cloned embryos development were further examined by treating fetal fibroblast cells with various concentrations (5 nM, 50 nM and 500 nM) of 5-aza-dC for different exposure times (1, 6 and 20 hours). Exposure of cells in 5 nM 5-aza-dC for 1 - 20 hours led to a significant improvement in the percentage of developed blastocysts, while treatment with 500 nM 5-aza-dC did not affect blastocyst development, compared to untreated controls. These findings indicate that treatment of fetal fibro-blast cells with relatively low concentrations of 5-aza-dC for short exposure times improves subsequent blastocyst development of porcine cloned embryos.

5-Aza-2dc通过表观修饰对Naive T细胞诱导的研究

30多年前，研究者发现了大量具有解除特异抗原的免疫应答的淋巴细胞。在1975年，这些细胞被命名为抑制细胞，即目前的Treg细胞，它可抑制器官移植的排斥反应。Treg细胞表面高表达CD25，而胞内Foxp3基因是其一个相对特异标志和功能分子。高表达Foxp3基因的Treg细胞对于维持免疫系统的内环境的稳定和预防自身免疫疾病有极其重要的作用。而Foxp3基因的稳定表达可通过表观修饰方式调节，

Comparative Evaluation of the Effects of 5-Aza-2'-deoxycytidine and Trichostatin A on Reactivation of hMLH1 in COC1/DDP Ovarian Cancer Cell Line

Objective： hMLH1 protein serves to detect the DNA damage caused by cisplatin （DDP） and destroys the cell. The absence of hMLH1 expression has been correlated with acquired resistance of ovarian cancer cells to platinum. The aim of this study was to determine the possible role of DNA methylation and histone H3 lysine 9 （H3-K9） acetylation on the loss of hMLH1 expression, and to evaluate the reversal effects of 5-Aza-2＇-deoxycytidine （5-Aza-dC） and Trichostatin A （TSA） on DDP-resistance in ovarian cancer cell lines. Methods： Two human ovarian cancer cell lines, COC1 and its DDP-resistant subline, COCI/DDP were cultured. The two cancer cells were treated with 5-Aza-dC or TSA. Using COC1 cells as a control, we used methylation-specific PCR （MSP） to analyze DNA methylation at hMLHI gene promoter, hMLH1 mRNA and protein expressions were analyzed by reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blot. Chromatin immunoprecipitation assay （CHIP） was used to test the levels of histone H3-K9 acetylation at hMLH1 gene promoter. Results： In COC1 cells, there was no DNA methylation at hMLH1 gene promoter, while there were hMLH1 mRNA and protein expression. In COC1/DDP cells, there was DNA hypermethylation at hMLH1 gene promoter, while there was no hMLH1 mRNA or protein expression. The treatment with 5-Aza-dC resulted in DNA demethylation at the promoter region, as well as restoration of hMLH1 expression in COC1/DDP cells. The treatment with TSA had no effects on DNA demethylation or restoration of hMLH1 expression in COC1/DDP cells. Conclusion： Hypermethylation of DNA at the promoter is related to the silencing of hMLH1 in COC1/DDP ovarian cancer cells. DNA methylation at hMLH1 promoter could play a significant role in determining the sensitivity of ovarian cancer to DDP. The drug resistance mediated by methylation of hMLH1 could be overcome by 5-Aza-dC.

Effect of 5-Aza-2'-deoxycytidine on the expression of p16 in hepatocellular carcinoma cells in vitro

Objective: To study the effect of 5-Aza-2’ -deoxycytidine (5-Aza-cdR) on tumour suppressor gene p16 expres- sion in hepatocellular carcinoma cells. Method: Expression of pl6 mRNA and protein in hepatocellular carcinoma cell lines SMMC-7721 and HePG2 before and after treatment with 5-Aza-cdR were analyzed via reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistrty Results: The expression levels of p16 mRNA and protein were increased dramatically after treatment with 5-Aza-cdR. Conclusion: Our data show that, 5-Aza-2’ -deoxycytidine can increase the expression of pl6 gene both at transcription and translation. The findings suggested that 5-Aza-cdR may reactivate the pl6 gene by demethylation.

Gene Expression Profiling of Human Myeloid Leukemic MV4-11 Cells Treated with 5-Aza-2’-deoxycytidine

The pyrimidine analog, 5-aza-2’-deoxycytidine (5-aza-dC) is a DNA methyltransferase inhibitor that triggers DNA demethylation leading to the reactivation of epigenetically silenced tumor suppressor genes. To understand the shift in gene expression which mediates the beneficial 5-aza-dC effects in leukemia, we have treated human myeloid derived leukemic cells with 5-aza-dC. Target genes were identified first in MV4-11 cells using a genome-wide gene expression profiling assay to detect differences in treated and untreated cells. From this analysis six genes were identified (HOXA4, HOXD4, HOXA8, HOXD12, CD9 and RGS2) as being significantly different expressed after treatment. To validate microarray data, we performed quantitative PCR on these genes from multiple leukemic cells. The results suggest that these genes are epigenetically regulated indicating that dysregulation of HOXA4, HOXD4, HOXA8, HOXD12, CD9 and RGS2 expression may play an important role in establishing the malignant phenotype in AML.

Synthesis and Crystal Structure of (2S,5S)-1-Aza-2- (2-pyridyl)-3-oxa-4,4-diphenylbicyclo [3.3.0] Octane

The title compound (C23H22N2O) 3 has been synthesized by the reaction of (S)- (-)-1,1-diphenyl-2-pyrrolidinemethanol 1 with 2-pyridinecarboxaldehyde 2 in refluxing benzene catalyzed by TsOH, and its structure was determined by single-crystal X-ray diffraction. The crystal belongs to orthorhombic, space group P212121 with a = 8.4747(8), b = 10.8829(10), c = 19.777(2) , Mr = 342.43, V =1824.0(3) 3, Dc = 1.247 g/cm3, Z = 4, m = 0.077 mm-1 and F(000) = 728. The structure was solved by direct methods and refined by full-matrix least-squares techniques to the final R = 0.0497 and wR = 0.0927.

5-氮杂-2'-脱氧胞苷预处理致敏百草枯对V79细胞的毒性观察

目的观察DNA甲基化调控对预处理致敏百草枯V79细胞的影响.方法采用DNA甲基化特异性抑制剂5-氮杂-2'-脱氧胞苷(5-Aza-2'-dc)对V79细胞进行预处理12 h,然后加入百草枯作用12 h,采用显微镜观察V79细胞形态结构,应用MTT法、台盼蓝染色法计数检测各实验组中细胞存活状况.结果显微镜检查观察到5-Aza-2'-dc与百草枯联合作用组比5-Aza-2'-dc组、百草枯组V79细胞贴壁能力减弱、细胞体积变小,噻唑蓝(MTT)比色法检测到联合作用组细胞存活率(54.47±3.04)%显著低于5-Aza-2'-dc组(95.52±0.90)%、百草枯组(89.68±4.26)%(P<0.05),台盼蓝染色计数算出联合作用组死细胞率(53.58±1.57)%显著高于5-Aza-2'-dc组(7.44±2.31)%、百草枯组(12.90±1.21)%(P<0.05).结论 5-Aza-2'-dc能够促进百草枯对V79细胞的损伤.

DNA甲基化酶抑制剂5-Aza-CdR对人牙髓细胞增殖活性及矿化能力的影响

目的：研究DNA甲基化酶抑制剂5-氮杂脱氧胞苷（5-Aza-2＇-deoxycytidine,5-Aza-CdR）对人牙髓细胞（human dental pulp cells,hDPCs）增殖活性和矿化能力的影响.方法：以酶消化联合组织块法体外培养人牙髓细胞,取第3代细胞并随机分为常规培养组（对照组）、矿化诱导组、5-Aza-CdR组及5-Aza-CdR联合矿化诱导液组（联合组）；分别于培养不同时间后,采用CCK8法检测各组hDPCs细胞的增殖活性；碱性磷酸酶活性检测法以及茜素红矿化结节染色法观察各组hDPCs的矿化能力.结果：与对照组相比,矿化诱导组、5-Aza-CdR组及联合组在培养第3～7天时,hDPCs的增殖活性明显降低（P＜0.05）；第3～14天时,碱性磷酸酶活性明显升高（P＜0.05）.第14天时,除对照组外各组均有矿化结节形成,其中5-Aza-CdR组矿化结节较少,矿化诱导组及联合组可见大片矿化结节,特别是联合组,矿化结节的密度更大、颜色更深.结论：体外培养条件下,DNA甲基化酶抑制剂5-Aza-CdR可降低hDPCs的增殖活性,增强其矿化能力.

Effects of 5-Aza-CdR on Cell Proliferation of Breast Cancer Cell Line MDA-MB-435S and Expression of maspin Gene

The effects of 5-Aza-2＇-deoxycytidine （5-Aza-CdR） on the proliferation of MDA-MB-435S cells and the expression of tumor suppressor gene maspin were investigated. Human breast cancer cell line MDA-MB-435S was treated with 5 μmol/L 5-Aza-CdR, a specific demethylating agent for 0 to 8 days. The growth of MDA-MB-435S cells was observed by MTT assay before and after 5-Aza-CdR treatment, respectively. The expression of maspin mRNA was detected by reverse transcfiption-polymerase chain reaction （RT-PCR）. The cell cycle of MDA-MB-435S cells was analyzed by flow cytometry. The results showed that the growth of MDA-MB-435S cells treated with 5-Aza-CdR for 8 days was significantly suppressed as compared with the control groups, and the inhibition rate increased sharply from 5 day to 8 day （35.42% to 71.29%）. Flow cytometry showed that 5 μmol/L 5-Aza-CdR could induce G2/M cell cycle arrest and decrease the percentage of mitosis cell number in this cell line. Maspin mRNA was expressed in MDA-MB-435S cells after 5-Aza-CdR treatment, but it was weakly detectable before the treatment. It was concluded that Maspin gene might be transcriptional silencing by hypermethylation and the re-expression of maspin gene by 5-Aza-CdR can inhibit the proliferation and induce the G2/M arrest of MDA-MB-435S breast cancer cells.