Glycodelin blocks progression to S phase and inhibits cell growth: a possible progesterone-induced regulator for endometrial epithelial cell growth

Prolonged exposure to unopposed estrogen in the absence of progesterone gives rise to endometrial hyperplasia and carcinoma. Post-ovulatory progesterone is necessary for the proper growth and differentiation of endometrial epithelial cells (EECs). Progesterone exposure induces the endometrial production of numerous bioactive substances, one of which is the glycoprotein, glycodelin (Gd). We investigated the role of Gd in cell cycle progression and cell growth to better understand how Gd affects EEC behavior and endometrial cancer pathogenesis. Ishikawa cells, a well-differentiated human endometrial epithelial cancer cell line, were transfected with expression plasmids encoding enhanced green fluorescent protein (EGFP) or EGFP-fused Gd (EGFP-Gd). They were then subjected to a cell proliferation assay, flow cytometry cell cycle analysis and RT-PCR analysis of cyclin-dependent kinase inhibitors (CDKIs) including p21, p27 and p16. Overexpression of EGFP-Gd resulted in a reduction of cell proliferation activity, an accumulation of G1-phase cells and up-regulation of p21, p27 and p16 mRNAs. Furthermore, progesterone-induced inhibition of Ishikawa cell growth was partially attenuated by Gd knockdown using siRNA. These results indicate that Gd causes inhibition of G1/S progression together with up-regulation of CDKIs thereby reducing cell growth. Thus, progesterone-induced expression of Gd may, at least in part, contribute to the suppression of endometrial epithelial growth observed during the secretory phase.     

小檗碱对人卵巢癌细胞SKOV3 增殖及凋亡机制的研究

目的:探讨小檗碱对人卵巢癌细胞(SKOV3)增殖及凋亡的影响。方法:MTT 法检测细胞增殖;流式细胞仪Annexin V/ PI 双染色法和透射电子显微镜检测细胞凋亡情况;甲基化特异性PCR 分析hMLH1 基因启动子区CpG 岛的甲基化状态;实时荧光定量RT-PCR 检测Bcl-2、Bax、Survivin 和hMLH1 mRNA 基因的表达。结果:小檗碱对卵巢癌SKOV3 细胞增殖有明显的抑制作用(P<0.05),呈剂量和时间依赖性。当与顺铂联用时,小檗碱对卵巢癌细胞有协同抗癌作用。小檗碱可明显诱导SKOV3 细胞凋亡,并下调Bcl-2、Survivin 基因及上调Bax 基因的表达。此外,小檗碱能恢复hMLH1 启动子的甲基化状态及增强hMLH1 mRNA 的表达。结论:小檗碱可抑制卵巢癌细胞增殖及诱导细胞凋亡,小檗碱可协同增强抗癌药物顺铂的抗肿瘤作用。     

EGCG通过调控SIRT1-P53通路诱导人卵巢癌SKOV-3细胞凋亡

 目的: 研究表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)调控人卵巢癌SKOV-3细胞活力和凋亡的分子机制。方法: SKOV-3细胞给予EGCG(0~50 μmol/L)、SIRT1激动剂SRT1720(1 μmol/L)和SIRT1抑制剂EX527(1 μmol/L)处理后,用CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,real-time PCR检测Bax和Bcl-2 mRNA的表达水平;采用SIRT1去乙酰化酶活性检测试剂盒检测SIRT1酶活性;使用Western blot法检测SIRT1、乙酰化P53和P53的蛋白表达变化。结果: 与正常对照组相比,单独给予EGCG或EX527处理之后SKOV-3细胞活力下降,凋亡率增加;SIRT1的酶活性和蛋白表达水平均明显下降;P53的乙酰化水平显著增加。与EGCG组相比,SRT1720预处理组的细胞活力上升,凋亡率下降,Bax/Bcl-2的相对比值及激活型caspase-3的蛋白水平明显下降,并且SIRT1的酶活性和蛋白表达水平显著增加,P53的乙酰化水平下降。结论: EGCG可通过调控SIRT1-P53通路抑制卵巢癌SKOV-3细胞活力并诱导其凋亡。     

亚硒酸钠对子宫内膜癌细胞增殖能力的影响

目的: 研究亚硒酸钠(Na₂SeO₃)对子宫内膜癌细胞增殖能力的影响,并探讨其作用机制。方法: 用Na₂SeO₃作用于子宫内膜癌Ishikawa细胞和HEC-1A细胞,MTT法检测细胞增殖能力,流式细胞法测定Na₂SeO₃作用后细胞周期的变化及凋亡情况,Western blotting检测周期蛋白cyclin A的表达。结果: Na₂SeO₃对子宫内膜癌Ishikawa细胞和HEC-1A细胞的增殖均有抑制作用,抑制率在一定浓度范围内与Na₂SeO₃浓度呈正相关,对Ishikawa细胞作用48 h的IC₅₀为3.26 μmol/L,对HEC-1A细胞作用48 h的IC₅₀为4.77 μmol/L。Na₂SeO₃作用后两种细胞G₀/G₁期减少,S期细胞增多,G₂/M期细胞有所增加。作用48 h后两种细胞凋亡率增加。Na₂SeO₃使子宫内膜癌Ishikawa细胞和HEC-1A细胞的cyclin A表达增加。结论: 亚硒酸钠对子宫内膜癌Ishikawa细胞和HEC-1A细胞增殖有抑制作用,其作用机制与上调cyclin A表达,引起细胞周期阻滞和诱导凋亡有关。     

Tanshinone IIA inhibits human breast cancer cells through increased Bax to Bcl-xL ratios

Tanshinone IIA (C19H18O3) was extracted from danshen (Salviae miltiorrhizae radix). It has cytotoxic properties and induces apoptosis in many human cancer cells. The molecular mechanisms are poorly understood, therefore, in the present study, we aimed to elucidate its anticancer activity on human breast cancer MDA-MB-231 cells. The cytotoxic effects of tanshinone IIA on MDA-MB-231 cells were measured by MTT assay. The percentages of cells in different cell cycle phases were determined by flow cytometry. The protein expression of Bax and Bcl-2 was examined using Western blotting. The results showed that tanshinone IIA inhibits the proliferation of MDA-MB-231 cells in a dose- and time-dependent manner. Tanshinone IIA induces apoptosis in a dose-dependent manner and the percentage of cells in sub-G1 phase. It increases the protein expression of Bax but decreases the Bcl-2 expression in MDA-MB-231 cells. Our findings suggest that tanshinone IIA can inhibit the proliferation of MDA-MB-231 cells by active apoptosis. One of the mechanisms may be through up-regulating the expression of Bax but down-regulating Bcl-2 expression and then inducing apoptosis. In conclusion, tanshinone IIA has therapeutic potential in breast cancer patients.     

A novel histone deacetylase inhibitor, Scriptaid, induces growth inhibition, cell cycle arrest and apoptosis in human endometrial cancer and ovarian cancer cells

Histone deacetylase inhibitors (HDACIs) can inhibit cell proliferation, induce cell cycle arrest, and stimulate the apoptosis of cancer cells. We investigated the effects of a novel HDACI, Scriptaid, on the Ishikawa endometrial cancer cell line, SK-OV-3 ovarian cancer cell line, and normal human endometrial epithelial cells. Endometrial and ovarian cancer cells were treated with various concentrations of Scriptaid, and its effect on cell growth, cell cycle, apoptosis, and related measurements was investigated. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed that all endometrial and ovarian cancer cell lines were sensitive to the growth inhibitory effect of Scriptaid, although normal endometrial epithelial cells were viable after treatment with the same doses of Scriptaid that induced the growth inhibition of endometrial and ovarian cancer cells. Cell cycle analysis indicated that their exposure to Scriptaid decreased the proportion of cells in the S phase and increased the proportion in the G0/G1 and/or G2/M phases of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with the altered expression of genes related to cell growth, malignant phenotype, and apoptosis. Furthermore, Scriptaid treatment of these cell lines increased acetylation of H3 and H4 histone tails. These results raise the possibility that Scriptaid may prove particularly effective in the treatment of endometrial and ovarian cancers.     

Apicidin, a novel histone deacetylase inhibitor, has profound anti-growth activity in human endometrial and ovarian cancer cells

Histone deacetylase inhibitors (HDACIs) can inhibit proliferation, induce cell cycle arrest and stimulate apoptosis of cancer cells. Our purpose was to investigate the antiproliferative effects of a novel HDACI, apicidin, on the Ishikawa endometrial cancer cell line, the SK-OV-3 ovarian cancer cell line and normal human endometrial epithelial cells. Endometrial and ovarian cancer cells were treated with various concentrations of apicidin, and the effects on cell growth, cell cycle, apoptosis and related measurements were investigated. MTT assays showed that all endometrial and ovarian cancer cell lines were sensitive to the growth inhibitory effect of apicidin, although normal endometrial epithelial cells were viable after the treatment with the same doses of apicidin that induced the growth inhibition of endometrial and ovarian cancer cells. Cell cycle analysis indicated that their exposure to apicidin decreased the proportion of cells in S-phase and increased the proportion in G0/G1 and/or G2/M phases of the cell cycle. Induction of apoptosis was confirmed by Annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with the altered expression of p21WAF1, p27KIP1, p16, cyclin A, and E-cadherin. Furthermore, apicidin treatment of these cell lines increased acetylation of H3 and H4 histone tails. These results suggest that apicidin exhibits the antiproliferative effects through selective induction of genes related to cell growth, malignant phenotype, and apoptosis. The findings raise the possibility that apicidin may prove particularly effective in the treatment of endometrial and ovarian cancers.     

Genistein effects on stromal cells determines epithelial proliferation in endometrial co-cultures

Background

Estrogen is the leading etiologic factor for endometrial cancer. Estrogen-induced proliferation of endometrial epithelial cells normally requires paracrine growth factors produced by stromal cells. Epidemiologic evidence indicates that dietary soy prevents endometrial cancer, and implicates the phytoestrogen genistein in this effect. However, results from previous studies are conflicting regarding the effects of genistein on hormone responsive cancers.

Methods

The effects of estrogen and genistein on proliferation of Ishikawa (IK) endometrial adenocarcinoma cells were examined in co-cultures of IK cells with endometrial stromal cells, recapitulating the heterotypic cell-to-cell interactions observed in vivo. The roles of estrogen receptor (ER)α and ERβ were evaluated using ERα and ERβ specific agonists. ER activation and cell proliferation in the IK epithelial cells were determined by alkaline phosphatase assay and Coulter counter enumeration, respectively.

Results

Both estrogen and genistein increased estrogen receptor-induced gene activity in IK cells over a range of concentrations. Estrogen alone but not genistein increased IK proliferation in co-cultures. When primed by estrogen treatment, increasing concentrations of genistein produced a biphasic effect on IK proliferation: nM concentrations inhibited estrogen-induced proliferation while μM concentrations increased proliferation. Studies with an ERβ-specific agonist produced similar results. Genistein did not influence the effects of estrogen on IK proliferation in monoculture.

Conclusions

Our study indicates that nutritionally relevant concentrations (nM) of genistein inhibit the proliferative effects of estrogen on endometrial adenocarcinoma cells presumably through activation of stromal cell ERβ. We believe that sub-micromolar concentrations of genistein may represent a novel adjuvant for endometrial cancer treatment and prevention.     

Hormonal regulation of apoptosis and the Fas and Fas ligand system in human endometrial cells

The process of apoptosis is responsible for normal cellular turnover in numerous tissues throughout the body. The endometrial layer of the uterus shows steroid-dependent cyclic changes in structure and function. After a proliferative and secretory phase, steroid support is withdrawn and the uterine epithelium is shed. We hypothesize that the apoptosis observed in endometrial cells following hormonal withdrawal is mediated by the Fas/Fas ligand (FasL) system. Normal endometrial cells and endometrial cancer cells were cultured in the presence of estrogen and progesterone. In order to mimic physiological hormonal changes, estrogen and progesterone were removed from the media. Apoptosis was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) assay and propidium iodide staining, while Fas and FasL expression were evaluated by Western blot analysis. The endometrial cells expressed Fas and low levels of FasL. Withdrawal of estrogen and/or progesterone from the culture induced apoptosis causing an approximately 50% decrease in cell viability. This coincided with increased Fas and FasL expression. Treatment of the cells with anti-FasL antibody prevented cell death following hormonal withdrawal. Estrogen and progesterone therefore represent survival factors which hamper cell death by impeding the expression of apoptotic factors. Our results indicate that Fas-mediated apoptosis is important for endometrial cycling and suggest that dysregulation of the Fas/FasL interactions may have an important role in the development of endometrial cancer.     

SOX4在子宫内膜癌组织中的表达及其生物学作用

目的 探讨性别决定区Y框蛋白4(SOX4)在子宫内膜癌组织中的表达水平及其生物学作用.方法收集156例子宫内膜癌组织和子宫内膜癌癌旁组织,另取156例子宫内膜不典型增生组织,采用免疫组织化学法检测子宫内膜癌、子宫内膜不典型增生和癌旁组织中SOX4的表达,并分析SOX4表达与子宫内膜癌患者临床特征的关系.采用慢病毒转染的...     

沉默miR-21对子宫内膜癌顺铂耐药细胞株Ishikawa/DDP的影响

目的:观察沉默miR-21对子宫内膜癌顺铂耐药细胞株Ishikawa/DDP的影响.方法:以Lipofectamine 2000介导miR-21抑制剂转染Ishikawa/DDP细胞株,同时设置阴性组和耐药组.采用反转录PCR检测miR-21、多药耐药基因MDR1、促凋亡基因Bax和抗凋亡基因Bcl-2的表达.采用蛋白印迹法检测多药耐药蛋白P-gp、促凋亡蛋白Bax和抗凋亡蛋白Bcl-2的表达.采用噻唑蓝比色法检测细胞对顺铂的敏感性.采用流式细胞术检测细胞凋亡情况.结果:与耐药组和阴性组比较,抑制剂组miR-21,MDR1和Bcl-2 mRNA表达显著下调(P＜0.0l),而Bax mRAN表达显著上调(P＜0.00l);抑制剂组P-gp和Bcl-2蛋白表达显著低下调(p＜0.05),而Bax蛋白表达显著上调(P＜0.001).与耐药组和阴性组比较,顺铂对抑制剂组的IC50值显著(P＜0.001);顺铂对抑制剂组细胞的诱导凋亡率显著增加(P＜0.00l).结论:沉默miR-21可显著提高Ishikawa/DDP细胞株对顺铂的敏感性,并促进细胞凋亡,其具体机制可能与下调MDR1,P-gp和Bcl-2表达,以及上调Bax表达有关.     

Raloxifene,a selective estrogen receptor modulator,induces mitochondria-mediated apoptosis in human endometrial carcinoma cells

Raloxifene is a nonsteroidal benzothiophene that has also been classified as a selective estrogen receptor modulator (SERM) on the basis of studies in which it produced both estrogen-agonistic effects on bone and lipid metabolism and estrogen-antagonistic effects on uterine endometrium and breast tissue. We investigated apoptotic cell death and the apoptotic pathway in human endometrial carcinoma cells (Ishikawa cells) expressing estrogen receptor treated with raloxifene. Cell viability was significantly decreased in Ishikawa cells treated with raloxifene at 20 microM and higher levels. Raloxifene at 20 microM induced 54% inhibition of cell viability after 48 h treatment. Apoptotic parameters were analyzed for determination of apoptotic pathway in Ishikawa cells treated with 20 microM or 40 microM raloxifene for 48 h. The numbers of apoptotic cells were significantly increased in cells treated with raloxifene as compared with control cells. Activities of caspase-3,-8, and-9 were significantly elevated in Ishikawa cells treated with raloxifene. A significant decrease in mitochondrial membrane potential was observed in this treatment. In addition, the levels of cytosolic cytochrome c were significantly elevated in raloxifene-treated cells. Expression of Bid was detected in both control and raloxifene-treated cells, but Bid cleavage was not observed. In caspase inhibitor experiments, cell viability was significantly increased by the caspase-9 inhibitor z-LEHD-fmk and by the caspase-3 inhibitor z-DEVD-fmk. However, cell viability was unaffected by addition of the caspase-8 inhibitor z-IETD-fmk. Thus, raloxifene induced mitochondria-mediated apoptosis in endometrial cancer cells but not via the Bid-mitochondria pathway. It is possibly that raloxifene may be useful as an adjuvant to current chemotherapies for endometrial cancer and possibly is useful as a chemopreventive agent.     

表皮生长因子促进子宫内膜腺癌细胞系Ishikawa增殖

目的研究表皮生长因子(EGF)对子宫内膜腺癌细胞系Ishikawa增殖的影响,并探讨雌激素受体α(ERα)和Ack1在其调控机制中的作用。方法无雌激素环境下,EGF作用于Ishikawa细胞,CCK-8法检测子宫内膜腺癌细胞增殖;Western blot检测细胞ERα及Ack1磷酸化;用酪氨酸激酶抑制剂达沙替尼处理细胞后,检测Ishikawa细胞增殖和ERα及Ack1磷酸化状态。结果 EGF可增强Ishikawa细胞增殖(P0.05),并促进ERαTyr-537特异位点磷酸化和Ack1磷酸化;用达沙替尼后,细胞增殖能力下降(P0.05),ERαTyr-537特异位点磷酸化和Ack1磷酸化水平下调。结论 EGF促进Ishikawa细胞增殖,其机制可能与诱导ERαTyr-537特异位点磷酸化和激活Ack1激酶通路有关。     

NS398体外对子宫内膜癌细胞的影响

目的探讨环氧合酶抑制剂NS-398对子宫内膜癌的作用机制。方法采用形态学,MTT,DNA梯度降解试验,原位末端标记及流式细胞仪等方法,对NS-398在子宫内膜癌细胞系Ishikawa中的作用进行体外研究。结果NS-398对Ishikawa细胞产生明显的生长抑制作用,且表现为浓度和剂量依赖性;原位末端标记、流式细胞仪显示NS-398诱导了Ishikawa细胞凋亡。结论NS-398对Ishikawa细胞具有显著的体外生长抑制及凋亡诱导作用。     

下调晚期糖基化终末产物受体对乳腺癌细胞 增殖、凋亡、侵袭、迁移能力的影响*

目的：研究下调晚期糖基化终末产物受体（RAGE）对乳腺癌细胞增殖、凋亡、侵袭、迁移能力的影响。 方法：培养乳腺癌BT474 细胞,分为对照组（ 不做任何处理的乳腺癌BT474 细胞）、上调组（ 乳腺癌BT474 细 胞+RAGE阴性对照）、下调组（ 乳腺癌BT474 细胞+RAGE siRNA）。用四甲基偶氮唑盐法检测乳腺癌细胞增 殖、凋亡水平;用Transwell 法检测乳腺癌细胞迁移、侵袭水平;用免疫印迹法检测细胞PI3K/Akt 信号通路蛋白 PI3K、p-PI3K、Akt、p-Akt、caspase 3、Bcl-2、Bax 表达量。结果：下调组细胞不同时间点增殖率均显著低于 上调组、对照组。下调组细胞不同时间点凋亡率均显著高于上调组、对照组。下调组细胞迁移、侵袭数均显著 低于上调组、对照组。下调组p-PI3K、PI3K、Akt、p-Akt、Bcl-2 蛋白表达量低于上调组、对照组,caspase 3、 Bax 蛋白表达量高于上调组、对照组。结论：经下调RAGE作用于PI3K/Akt 信号通路,其可抑制p-PI3K、PI3K、 Akt、p-Akt、Bcl-2 表达,促进caspase 3、Bax 表达,致乳腺癌细胞凋亡,抑制乳腺癌细胞增殖、侵袭、迁移。     

靶向ANG-2的shRNA重组载体抑制人子宫内膜癌Ishikawa生长和侵袭的研究

目的 探讨采用RNA干扰(RNA interference,RNAi)抑制血管生成素-2(angiopoietin-2,ANC-2)基因的表达对人子宫内膜癌细胞Ishikawa细胞的影响.方法 采用脂质体LipofectamineTM 2000介导的方法将针对ANG-2基因的短发卡状RNA(short hairpin RNA,ahRNA)表达载体转染到人子宫内膜癌Ishikawa细胞中,逆转录聚合酶链反应及Western印迹法检测ANG-2 mRNA及蛋白的表达;噻唑蓝比色法检测Ishikawa细胞的增殖;荧光显微镜观察细胞形态;流式细胞仪检测细胞周期及凋亡;侵袭实验检测其对侵袭能力的影响.结果 人子宫内膜癌Ishikawa细胞ANG-2 mRNA及其蛋白质表达水平均显著降低;增殖被抑制,抑制率63.11%;细胞凋亡增加,细胞凋亡率与空白对照组和阴性对照组相比明显增高;细胞生长减慢,阻滞于G1期的增多,S期细胞减少;侵袭能力明显下降.结论 靶向ANG-2的shRNA能成功下调ANG-2基因的表达,抑制人子宫内膜癌Ishikawa细胞生长和侵袭.     

靶向ANG-2的shRNA重组载体抑制人子宫内膜癌Ishikawa生长和侵袭的研究

目的 探讨采用RNA干扰(RNA interference,RNAi)抑制血管生成素-2(angiopoietin-2,ANC-2)基因的表达对人子宫内膜癌细胞Ishikawa细胞的影响.方法 采用脂质体LipofectamineTM 2000介导的方法将针对ANG-2基因的短发卡状RNA(short hairpin RNA,ahRNA)表达载体转染到人子宫内膜癌Ishikawa细胞中,逆转录聚合酶链反应及Western印迹法检测ANG-2 mRNA及蛋白的表达;噻唑蓝比色法检测Ishikawa细胞的增殖;荧光显微镜观察细胞形态;流式细胞仪检测细胞周期及凋亡;侵袭实验检测其对侵袭能力的影响.结果 人子宫内膜癌Ishikawa细胞ANG-2 mRNA及其蛋白质表达水平均显著降低;增殖被抑制,抑制率63.11%;细胞凋亡增加,细胞凋亡率与空白对照组和阴性对照组相比明显增高;细胞生长减慢,阻滞于G1期的增多,S期细胞减少;侵袭能力明显下降.结论 靶向ANG-2的shRNA能成功下调ANG-2基因的表达,抑制人子宫内膜癌Ishikawa细胞生长和侵袭.     

靶向ANG-2的shRNA重组载体抑制人子宫内膜癌Ishikawa生长和侵袭的研究

目的 探讨采用RNA干扰(RNA interference,RNAi)抑制血管生成素-2(angiopoietin-2,ANC-2)基因的表达对人子宫内膜癌细胞Ishikawa细胞的影响.方法 采用脂质体LipofectamineTM 2000介导的方法将针对ANG-2基因的短发卡状RNA(short hairpin RNA,ahRNA)表达载体转染到人子宫内膜癌Ishikawa细胞中,逆转录聚合酶链反应及Western印迹法检测ANG-2 mRNA及蛋白的表达;噻唑蓝比色法检测Ishikawa细胞的增殖;荧光显微镜观察细胞形态;流式细胞仪检测细胞周期及凋亡;侵袭实验检测其对侵袭能力的影响.结果 人子宫内膜癌Ishikawa细胞ANG-2 mRNA及其蛋白质表达水平均显著降低;增殖被抑制,抑制率63.11%;细胞凋亡增加,细胞凋亡率与空白对照组和阴性对照组相比明显增高;细胞生长减慢,阻滞于G1期的增多,S期细胞减少;侵袭能力明显下降.结论 靶向ANG-2的shRNA能成功下调ANG-2基因的表达,抑制人子宫内膜癌Ishikawa细胞生长和侵袭.     

靶向ANG-2的shRNA重组载体抑制人子宫内膜癌Ishikawa生长和侵袭的研究

目的 探讨采用RNA干扰(RNA interference,RNAi)抑制血管生成素-2(angiopoietin-2,ANC-2)基因的表达对人子宫内膜癌细胞Ishikawa细胞的影响.方法 采用脂质体LipofectamineTM 2000介导的方法将针对ANG-2基因的短发卡状RNA(short hairpin RNA,ahRNA)表达载体转染到人子宫内膜癌Ishikawa细胞中,逆转录聚合酶链反应及Western印迹法检测ANG-2 mRNA及蛋白的表达;噻唑蓝比色法检测Ishikawa细胞的增殖;荧光显微镜观察细胞形态;流式细胞仪检测细胞周期及凋亡;侵袭实验检测其对侵袭能力的影响.结果 人子宫内膜癌Ishikawa细胞ANG-2 mRNA及其蛋白质表达水平均显著降低;增殖被抑制,抑制率63.11%;细胞凋亡增加,细胞凋亡率与空白对照组和阴性对照组相比明显增高;细胞生长减慢,阻滞于G1期的增多,S期细胞减少;侵袭能力明显下降.结论 靶向ANG-2的shRNA能成功下调ANG-2基因的表达,抑制人子宫内膜癌Ishikawa细胞生长和侵袭.     

靶向ANG-2的shRNA重组载体抑制人子宫内膜癌Ishikawa生长和侵袭的研究

目的 探讨采用RNA干扰(RNA interference,RNAi)抑制血管生成素-2(angiopoietin-2,ANC-2)基因的表达对人子宫内膜癌细胞Ishikawa细胞的影响.方法 采用脂质体LipofectamineTM 2000介导的方法将针对ANG-2基因的短发卡状RNA(short hairpin RNA,ahRNA)表达载体转染到人子宫内膜癌Ishikawa细胞中,逆转录聚合酶链反应及Western印迹法检测ANG-2 mRNA及蛋白的表达;噻唑蓝比色法检测Ishikawa细胞的增殖;荧光显微镜观察细胞形态;流式细胞仪检测细胞周期及凋亡;侵袭实验检测其对侵袭能力的影响.结果 人子宫内膜癌Ishikawa细胞ANG-2 mRNA及其蛋白质表达水平均显著降低;增殖被抑制,抑制率63.11%;细胞凋亡增加,细胞凋亡率与空白对照组和阴性对照组相比明显增高;细胞生长减慢,阻滞于G1期的增多,S期细胞减少;侵袭能力明显下降.结论 靶向ANG-2的shRNA能成功下调ANG-2基因的表达,抑制人子宫内膜癌Ishikawa细胞生长和侵袭.