青蒿琥酯通过调控PI3K/Akt信号通路抑制肝癌细胞增殖促进凋亡

目的 探讨青蒿琥酯对人肝癌细胞系HepG2的增殖和凋亡的影响及可能机制.方法 体外培养人肝癌细胞系HepG2,分别用浓度为12.5、25、50、100mg/L青蒿琥酯作用不同时间后,采用CCK-8实验检测细胞增殖情况;随后选择最佳浓度的青蒿琥酯作用HepG2细胞24h后,流式细胞术检测细胞凋亡,Western blot检测Bax、Bcl-2、PI3K、p-PI3K、Akt和p-Akt的水平.结果 不同浓度的青蒿琥酯处理细胞24h后,青蒿琥酯均能显著抑制肝癌细胞HepG2的增殖,且该作用呈现剂量依赖性.流式细胞仪检测发现青蒿琥酯能显著促进肝癌细胞HepG2的凋亡,同时Western blot也进一步证实,青蒿琥酯处理后HepG2细胞中的抑凋亡蛋白Bcl-2显著下调,促凋亡蛋白Bax表达显著上调;同时发现青蒿琥酯能够降低p-PI3K和p-Akt的水平,但未磷酸化的PI3K和未磷酸化的Akt含量变化无显著性差异.结论 青蒿琥酯可以抑制肝癌细胞HepG2的增殖,促进细胞凋亡,该作用与调控PI3K和Akt的磷酸化水平密切相关.     

Fas及FasL和Caspase-3在青蒿琥酯诱导人胚肺成纤维细胞凋亡中的表达

背景:青蒿琥酯具有减轻肺纤维化的作用,但相关机制的研究罕见报道。 目的：探讨青蒿琥酯对人胚肺成纤维细胞凋亡的作用及其与Fas,FasL,Caspase-3表达的关系。 方法：用1,10,100 mg/L青蒿琥酯分别干预体外培养的人胚肺成纤维细胞。采用CCK-8法检测青蒿琥酯对人胚肺成纤维细胞增殖的影响,流式细胞术测定细胞凋亡率,RT-PCR法测定Fas,FasL,Caspase-3 的mRNA的表达。 结果与结论：青蒿琥酯呈浓度依赖性抑制人胚肺成纤维细胞增殖,细胞经青蒿琥酯作用后凋亡率明显增加(P  < 0.05或P  < 0.01),Fas,FasL,Caspase-3 mRNA的表达显著高于对照组(P < 0.05)。结果证实,青蒿琥酯可通过上调Fas,FasL,Caspase-3 mRNA的表达抑制人胚肺成纤维细胞增殖、并促进细胞凋亡,发挥抗肺纤维化作用。     

青蒿琥酯对人肝癌HepG2细胞增殖和凋亡的影响及联合化疗药物的抗肝癌效应

目的: 研究青蒿琥酯对人肝癌HepG2细胞增殖和凋亡的作用,并检测其是否影响HepG2细胞对化疗药物的敏感性。方法: 采用CCK-8法观察不同浓度青蒿琥酯对HepG2细胞生长增殖的影响;克隆形成实验检测青蒿琥酯对HepG2细胞克隆形成的影响;Hoechst 33258染色观察青蒿琥酯作用HepG2细胞后细胞形态的变化;PI单染流式细胞术检测青蒿琥酯对HepG2细胞周期以及亚二倍体率的影响;Annexin V-PI双染测定青蒿琥酯对HepG2细胞凋亡的影响;CCK-8法检测青蒿琥酯对HepG2细胞化疗药物敏感性的影响。结果: (1)青蒿琥酯作用于HepG2细胞48 h后,能有效抑制其增殖,随着浓度的升高,增殖抑制率越高,IC₅₀值为19.2 μmol/L。(2)青蒿琥酯作用HepG2细胞7 d后,能有效抑制HepG2细胞形成的克隆数。(3)青蒿琥酯作用于HepG2细胞48 h后,Hoechst 33258染色可见实验组细胞胞核固缩、边聚和裂解、凋亡小体形成等凋亡形态学变化。(4)青蒿琥酯作用于HepG2细胞48 h后,PI单染流式细胞术检测细胞周期发现实验组细胞明显阻滞于G₂期。(5)青蒿琥酯作用于HepG2细胞48 h后,PI单染实验检测实验组出现明显的亚二倍体凋亡峰;Annexin V-PI 双染实验检测实验组细胞出现明显的早期凋亡细胞群。(6)青蒿琥酯分别联合化疗药物5-氟尿嘧啶、卡铂和表柔比星作用于HepG2细胞48 h后,能明显增强化疗药物对肿瘤细胞的抑制作用,增敏倍数分别为3.33、2.02和1.71。结论: (1)青蒿琥酯对人肝癌HepG2细胞有增殖抑制作用,并诱导其凋亡。(2)青蒿琥酯能提高人肝癌HepG2细胞对5-氟尿嘧啶、卡铂和表柔比星的敏感性。     

诱导细胞凋亡青蒿琥酯抗食管癌作用机制研究

 [摘要] 目的 研究青蒿琥酯诱导食管癌细胞凋亡作用及探讨青蒿琥酯抗食管癌作用机制。方法 不同浓度的青蒿琥酯(Artesunate, Art)(0、10、20、40μg/ml) 作用Eca109细胞24h,流式细胞术（Flow cytometry, FCM）方法检测细胞凋亡、周期及细胞中bcl-2和bax蛋白的表达量。结果 青蒿琥酯作用Eca109细胞24h后,与对照组相比,细胞凋亡率显著增高P<0.05,且具有剂量依赖性。青蒿琥酯组与对照组相比,Eca109细胞中bcl-2蛋白表达水平及细胞增殖指数显著降低P<0.05,而bax蛋白表达量显著增高P<0.05,且具有剂量依赖性。结论 青蒿琥酯可以通过调节Eca109细胞中bcl-2、bax蛋白表达水平和细胞增殖,从而诱导Eca109细胞产生凋亡,起到抗食管癌作用。     

龙血素A对体外培养人乳腺癌MCF-7细胞增殖、凋亡的影响

观察龙血素A对体外培养人乳腺癌MCF-7细胞增殖、凋亡的影响。采用龙血素A与MCF-7细胞体外共培养(药物组)干预手段,运用四甲基偶氮唑蓝(MTT)法检测细胞的增殖情况;免疫荧光检测细胞增殖标志性蛋白Ki67的表达;免疫印迹检测凋亡调节蛋白Bax、Bcl-XL/S的表达;annexin V-FITC/PI双染流式细胞仪检测细胞的凋亡率。与对照组相比,药物组细胞增殖受到明显抑制,且存在浓度及时间依赖性;药物组Ki67表达显著减少,Bax表达明显增加,Bcl-XL/S表达明显减少;细胞凋亡率增高,上述指标间比较差异均有统计学意义。龙血素A对体外培养的人乳腺癌MCF-7细胞具有抑制增殖和促进凋亡的作用。     

青蒿琥酯对吉西他滨体外抗胰腺癌活性的作用

目的:探讨青蒿琥酯辅助治疗对吉西他滨抗胰腺癌活性的影响和机制。方法:分别采用分子克隆及RNA干扰方法于人胰腺癌细胞Capan-2中过表达和干扰鼠双微体蛋白2(MDM2),MTT实验检测p53野生型胰腺癌细胞系Capan-2的细胞活力。Western blot实验检测Capan-2细胞中MDM2、p53、Noxa和Puma的表达水平,细胞色素C和凋亡诱导因子的释放,以及caspase-9和caspase-3的活化。流式细胞术检测Capan-2细胞的线粒体膜电位和凋亡水平。结果:吉西他滨联合青蒿琥酯组Capan-2的相对细胞活力显著低于吉西他滨单处理组(P 0. 05)。青蒿琥酯处理显著抑制Capan-2细胞中MDM2的表达水平(P 0. 05)。吉西他滨联合青蒿琥酯组的p53、Noxa及Puma表达水平均明显高于吉西他滨单处理组(P 0. 05)。青蒿琥酯明显促进吉西他滨依赖的Capan-2细胞线粒体膜电位的下降,细胞色素C和凋亡诱导因子的释放,caspase-9和caspase-3的活化,以及凋亡的发生。转染MDM2表达质粒后,青蒿琥酯联合吉西他滨对Capan-2细胞的凋亡诱导途径受到显著抑制。结论:青蒿琥酯通过MDM2/p53途径提高吉西他滨的抗胰腺癌活性。     

雌激素通过ERK-FAK信号通路调节MCF-7乳腺癌细胞失巢凋亡

目的探讨雌激素(E2)对MCF-7乳腺癌细胞抗失巢凋亡能力的影响及细胞外信号调节激酶(ERK)-局部黏着斑激酶(FAK)通路在其中的作用,以加深对E2促癌作用信号机制的认识。方法用MCF-7乳腺癌细胞,聚甲基丙烯酸羟乙基酯(poly-Hema)涂层培养细胞诱导失巢凋亡,E2刺激及MEK和FAK抑制剂预处理细胞。用蛋白印迹法检测ERK和FAK磷酸化,台盼蓝染色细胞计数法检测细胞存活力,Hoechst荧光染色法验证细胞凋亡。结果用Poly-Hema悬浮培养可显著降低细胞存活力(P0.01),E2处理则可明显增强悬浮培养细胞的存活力(P0.05),同时减少细胞凋亡;E2可引起ERK和FAK磷酸化,MEK抑制剂则可显著抑制E2诱导的ERK和FAK磷酸化,并使E2处理的悬浮培养细胞存活率下降57.48%(P0.01);FAK抑制剂能明显抑制FAK和ERK的磷酸化,同时使E2处理的悬浮培养细胞存活率下降53.59%(P0.01)。结论 E2可明显提高MCF-7乳腺癌细胞对抗失巢凋亡的能力,该细胞保护效应可能与E2激活胞内ERK-FAK信号活动有关。     

13-MTD通过激活MAPK途径和抑制Akt存活途径诱导乳腺癌MCF-7细胞凋亡

目的： 探讨侧链饱和脂肪酸13-甲基十四烷酸(13-MTD)诱导人乳腺癌MCF-7细胞凋亡的作用机制。方法：140 mg/L 13-MTD处理体外培养的人乳腺癌MCF-7细胞和人乳腺正常细胞,采用流式细胞仪检测技术观察13-MTD对人乳腺癌MCF-7细胞凋亡的影响,免疫印迹法检测13-MTD处理后细胞内c-Jun氨基末端激酶(JNK）,p38, Fas 相关死亡结构域蛋白(FADD)和丝氨酸/苏氨酸蛋白激酶(Akt)等蛋白磷酸化变化。结果：流式细胞仪实验结果显示13-MTD能有效地诱导人乳腺癌MCF-7细胞凋亡,但不引起正常人乳腺上皮细胞凋亡。免疫印迹检测显示经13-MTD处理后的人乳腺癌MCF-7细胞JNK和p38磷酸化蛋白明显增加,Akt磷酸化蛋白明显减少。结论：13-MTD是一个新的安全高效抗肿瘤药物,其作用机制可能是通过激活MAPK途径和抑制Akt存活途径来诱导肿瘤细胞凋亡。     

黄芩素和U0126体外抗人乳腺癌的作用及机制研究

目的:探讨黄芩素和U0126体外对乳腺癌的作用及其机制。方法:用黄芩素、U0126单独处理以及二者联合处理人乳腺癌细胞株MCF-7细胞,采用CCK8检测细胞增殖变化;流式细胞术检测MCF-7细胞周期以及凋亡变化;显微镜观察细胞数量变化;TUNEL法检测细胞凋亡改变;划痕实验分析细胞迁移情况;Western blot实验检测细胞凋亡相关因子的蛋白水平变化,分析黄芩素与U0126对乳腺癌细胞的增殖、凋亡和迁移的影响。结果:黄芩素或U0126可抑制细胞的增殖且具有浓度依赖性(P0.05);黄芩素阻滞MCF-7细胞周期进入S期,增加G0~G1期细胞比例(P0.05),降低S期细胞比例(P0.05),诱导细胞凋亡(P0.05);降低ERK1/2和JNK蛋白的磷酸化水平和CyclinD 1的蛋白水平表达;黄芩素与U0126联合处理MCF-7细胞,与黄芩素单独处理组相比较,S期细胞比例明显降低(P0.05),细胞凋亡更加明显(P0.05),ERK1/2和JNK的磷酸化水平(P0.05)、CyclinD 1的蛋白表达量降低更加明显(P0.05);同时黄芩素可有效抑制人乳腺癌细胞株MCF-7细胞的迁移(P0.05)。结论:黄芩素和U0126通过降低ERK1/2、JNK的磷酸化水平表达,降低CyclinD 1的水平表达,有效抑制MCF-7细胞增殖和迁移,诱导MCF-7细胞凋亡并且二者具有协同作用。因此,黄芩素和U0126联合有望用于临床治疗乳腺癌。     

毛蕊异黄酮对人乳腺癌细胞增殖及凋亡的作用及机制研究

目的:探讨毛蕊异黄酮对人乳腺癌细胞MCF-7、T47D增殖及凋亡的作用及其相关机制。方法:体外培养MCF-7、T47D细胞,将其均分为对照组和药物组,利用CCK8生长曲线检测不同浓度毛蕊异黄酮(0、25、50、100μM)对MCF-7、T47D细胞增殖的影响,从而确定毛蕊异黄酮最佳给药浓度;采用Hoechst 33258荧光染色法、流式细胞术评价不同浓度毛蕊异黄酮(0、50、100μM)对MCF-7、T47D细胞凋亡的调节作用;通过Western blotting法检测MCF-7细胞中ERK1/2与p-ERK1/2蛋白表达水平。结果:毛蕊异黄酮能够有效抑制MCF-7、T47D细胞的增殖,并具有剂量和时间依赖性(P0.05)。CCK8实验表明,与对照组(0μM)相比,不同浓度的毛蕊异黄酮药物组(25、50、100μM)分别作用72 h后,100μM毛蕊异黄酮处理后的MCF-7、T47D细胞的增殖率明显被抑制(P0.05);Hoechst 33258荧光染色法和流式细胞术显示,与对照组(0μM)相比,50μM和100μM毛蕊异黄酮药物组皆能促进MCF-7、T47D细胞凋亡,其中100μM药物组凋亡效果最为明显(P0.05);同时Western blotting法检测到50μM和100μM毛蕊异黄酮能够显著抑制MCF-7细胞中p-ERK1/2蛋白表达水平(P0.05)。结论:毛蕊异黄酮能够剂量依赖性抑制MCF-7、T47D细胞的增殖并诱导其凋亡。     

8-硝基白杨素对人宫颈癌Hela细胞增殖和凋亡的影响

目的：观察8-硝基白杨素(8-NOChR)抑制体外培养人宫颈癌Hela细胞增殖和诱导凋亡作用。方法：体外培养人宫颈癌Hela细胞系细胞。MTT比色法测定Hela细胞增殖活性。软琼脂培养克隆形成法检测Hela细胞集落形成能力。AO/EB染色荧光显微镜观察Hela细胞凋亡形态学改变。DNA凝胶电泳观察梯形DNA条带。结果：MTT比色测定显示,8-NOChR抑制Hela细胞增殖,呈剂量依赖性。软琼脂培养克隆形成法检测表明,8-NOChR显著抑制Hela细胞集落形成,呈剂量依赖性。AO/EB染色荧光显微镜观察发现,8-NOChR诱导Hela细胞呈现典型凋亡细胞形态特征。8-NOChR（30μmol/L）处理Hela细胞72h,琼脂糖凝胶电泳出现“梯形”DNA条带。结论：8-NOChR具有抑制人宫颈癌Hela细胞增殖和诱导细胞凋亡作用     

USP37 downregulation elevates the Chemical Sensitivity of Human Breast Cancer Cells to Adriamycin

Background: The evolution of adriamycin (ADR) resistance in the treatment of breast cancer often leads to a poor prognosis in patients. Ubiquitin-specific peptidase 37 (USP37) has been recently identified as a modulator in regulating the stemness of breast cancer cells, but its underlying mechanism remains unclear. In this study, we investigated whether USP37 knockdown could hamper the chemical resistance of MCF-7 and MCF-7/ADR cells to adriamycin and elucidated the potential mechanism.Methods: Immunohistochemistry, western blotting, and RT-qPCR assays were performed to detect the USP37 expression in MCF-7 and MCF-7/ADR cells. The efficiency of USP37 knockdown in breast cancer cells was confirmed by western blotting and RT-qPCR assays. We also performed CCK-8 assay, flow cytometry, western blotting, and TUNEL assays to evaluate cell viability and apoptosis in breast cancer cells. In vivo study was performed to detect the tumorigenicity of MCF-7/ADR cells transfected with shScramble or shUSP37#1 under adriamycin treatment.Results: Bioinformatic analysis indicated that USP37 overexpression was positively correlated with adriamycin resistance. The expression levels of USP37 in both MCF-7 and MCF-7/ADR cells increased significantly with the exposure to adriamycin in a dose-dependent manner. It was verified by the observation that USP37 downregulation elevated the inhibitory effects of adriamycin on breast cancer cells, suppressed cell proliferation caused by cell cycle arrest in G1/S transition, as well as induced apoptosis. Furthermore, in vivo study showed that knockdown of USP37 expression also decreased tumorigenicity of MCF-7/ADR cells in mice. TUNEL assay and observation of cell morphology magnified USP37 knockdown synergized with Adriamycin could elevate the apoptosis of MCF-7 and MCF-7/ADR cells. Western blotting assay illustrated that the combination of USP37 knockdown with adriamycin treatment significantly upregulated the expression levels of cleaved caspase 3 and Bax, whereas the expression level of Bcl-2 was inhibited.Conclusion: Knockdown of USP37 gene expression can reverse the resistance of breast cancer cells to adriamycin, and down-regulating USP37 might be a valuable strategy against ADR resistance in breast cancer therapy.     

PKCα inhibited apoptosis by decreasing the activity of JNK in MCF-7/ADR cells

The development of multidrug resistance (MDR) in breast cancer patients is a serious therapeutic problem. The role of signal transduction in the development of MDR has drawn intensive attention recently. In this study, the role of c-Jun N-terminal kinase (JNK) pathway in MDR, specifically regulated by PKCα, was investigated in MCF-7/ADR cells. MTT, DNA ladder and flow cytometry were used to detect cell growth inhibition or apoptosis while Western blot was used to detect the activation of proteins. Compared with MCF-7 cells, the cell growth inhibition and apoptosis induced by tamoxifen (TAM) could not be detected in MCF-7/ADR cells, but the expression of PKCα in MCF-7/ADR cells was higher. And, Western blot results showed that JNK was activated by TAM in MCF-7 cells while not in MCF-7/ADR cells, even at very high doses. In addition, sp600125, the inhibitor of JNK, decreased the percentage of apoptosis induced by TAM in MCF-7 cells. These data showed that PKCα and JNK were key regulators in the apoptosis of MCF-7/ADR cells. Furthermore, PKCα being the upstream of JNK in inhibiting apoptosis was suggested by using Go6976, the specific PKCα inhibitor, in the presence or absence of sp600125. This study highlighted an important signaling pathway involved in MDR regulated by PKCα in MCF-7/ADR breast cancer cells and implied that JNK might be an important downstream target of PKCα in this cellular context.     

DMSO诱导MCF-7细胞凋亡的研究

目的研究二甲基亚砜(DMSO)对MCF-7细胞凋亡的诱导作用。方法用不同浓度DMSO处理体外培养的MCF-7细胞,应用倒置光显微镜观察细胞形态学变化,用MTT比色法检测细胞存活率;Hoechst33258/PI荧光染色,用荧光显微镜分析凋亡细胞比率;琼脂糖凝胶电泳检测DNA梯状条带。结果在倒置光学显微镜下观察1%DMSO处理细胞12h后细胞形态发生变化。约有50%以上的细胞变圆,细胞内有多泡小体形成。随DMSO浓度增加和作用时间的延长,细胞存活率明显下降,经MTT检测其IC_(50)值为1%;荧光显微镜下可见60%以上细胞核染色质凝集,核碎裂等凋亡细胞的形态学变化;琼脂糖凝胶电泳呈现梯状条带(DNA ladder)。结论适当浓度的DMSO能够抑制乳腺癌细胞增殖并诱导其凋亡。     

Phenotypical Reversion and Differentiation of Breast Cancer in vitro by Sodium Phenylacetate

Objective To evaluate the effect of a nontoxic differentiation inducer, sodium phenylacetate (NaPA) on breast cancer MCF-7 and MDA-453 cells in vitro. Methods The anchorage growth assay, ELISA, immunofluorescence analysis and electron microscopy were used to study the effect of NaPA on MCF-7 and MDA-453 cells. Results NaPA treatment resulted in a dose-dependent inhibition of MCF-7 or MDA-453 cell growth, including anchorage-dependent and anchorage-independent growth, but il did not significantly inhibit the growth of normal amnion tissue Wish cells and adult hepatic cells L-02. In addition, NaPA could enhance the expressions of intercellular adhesion molecule-1(ICAM-1), human leukocyte antigen ( HLA ) class Ⅰ and Ⅱ molecules on the surface of MCF-7 and MDA-453 cells. Electron micrographs demonstrated richer rough endoplasmic reticulum,mitochondria in NaPA-treated MCF-7 and MDA-453 cells, in contrast to the scant before-mentwned structures but scattered cytoplasmic polyribosomes in the untreated ones. Conclusion NaPA is effective in inducing phenotype reversion and differentiation of breast cancer, and potential in cancer intervention.     

Molecular Mechanism of SAHA on Regulation of Autophagic Cell Death in Tamoxifen-Resistant MCF-7 Breast Cancer Cells

Objective: Tamoxifen is currently used for the treatment of estrogen receptor-positive breast cancer patients, but acquired resistance to tamoxifen is a critical problem in breast cancer therapy. Suberoylanilide hydroxamic acid (SAHA) is a prototype of the newly developed HDAC inhibitor. The aim of this study is to investigate the anticancer effects of SAHA in tamoxifen-resistant MCF-7 (TAMR/MCF-7) cells.Methods: Cytotoxicity, apoptosis and autophagic cell death induced by SAHA were studied. A TAMR/MCF-7 cells xenograft model was established to investigate the inhibitory effect of SAHA on tumor growth in vivo.Results: SAHA inhibited the proliferation of TAMR/MCF-7 cells in a dose-dependent manner. SAHA significantly reduced the expression of HDAC1, 2, 3, 4 and 7 and increased acetylated histone H3 and H4. Although SAHA induced G2/M phase arrest of cell cycle, apoptotic cell death was very low, which is correlated with the slight change in the activation of caspases and PARP cleavage. Interestingly, expression of the autophagic cell death markers, LC3-II and beclin-1, was significantly increased in TAMR/MCF-7 cells treated with SAHA. Autophagic cell death induced by SAHA was confirmed by acridine orange staining and transmission electron microscopy (TEM) in TAMR/MCF-7 cells. In mice bearing the TAMR/MCF-7 cell xenografts, SAHA significantly reduced the tumor growth and weight, without apparent side effects.Conclusion: These results suggest that SAHA can induce caspase-independent autophagic cell death rather than apoptotic cell death in TAMR/MCF-7 cells. SAHA-mediated autophagic cell death is a promising new strategy to treatment of tamoxifen-resistant human breast cancer.     

全反式维甲酸通过DOK1/PPARγ信号通路抑制人乳腺癌细胞MCF-7的增殖

目的:探讨全反式维甲酸(ATRA)对人乳腺癌细胞MCF-7增殖的影响及可能机制。方法:MCF-7细胞培养于含10%胎牛血清、1%青霉素/链霉素的DMEM培养液,蛋白免疫印迹检测蛋白质的表达,MTT法和溴脱氧尿嘧啶核苷(Brd U)掺入法测定细胞增殖,TUNEL法测定细胞凋亡,携带目标基因shRNA的慢病毒用于沉默目标基因,过氧化物酶体增殖物激活受体γ(PPARγ)的活性采用商品化的试剂盒测定。结果:ATRA处理可抑制MCF-7细胞的增殖,促进其凋亡;同时,ATRA以时间依赖方式刺激停靠蛋白1(DOK1)的表达。沉默DOK1可降低ATRA对MCF-7细胞增殖和凋亡的影响。此外,DOK1基因沉默可抑制PPARγ的表达和活性。而PPARγ选择性抑制剂GW9662可减轻ATRA对MCF-7细胞增殖的抑制和对细胞凋亡的促进作用。结论:ATRA通过抑制细胞增殖、促进细胞凋亡而抑制MCF-7细胞的生存,这一作用经DOK1激活的PPARγ介导。     

百里醌抑制乳腺癌血管生长的实验研究

 目的: 探讨百里醌对乳腺癌血管生长的作用于及其机制。方法: 不同浓度百里醌作用于人脐静脉内皮细胞(HUVECs)及人乳腺癌细胞株MCF-7后,应用CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,小管形成实验检测内皮细胞体外小管形成能力,Western blot法检测内皮细胞中cleaved caspase-3、p-ERK及p-AKT的蛋白水平;建立裸鼠乳腺癌移植瘤模型,完全随机法分成对照组和百里醌组,免疫组织化学法检测乳腺肿瘤组织中CD31的表达,TUNEL法检测移植瘤细胞凋亡。结果: 百里醌(20~80 nmol/L)对MCF-7细胞生长无明显抑制作用,20 nmol/L、40 nmol/L和80 nmol/L百里醌作用于内皮细胞24 h后,细胞存活率分别为(74.3±5.9)％、(68.7±5.6)％和(47.9±4.3)％;20 nmol/L、40 nmol/L和80 nmol/L百里醌作用于MCF-7细胞24 h后,细胞凋亡率分别为(2.6±0.3)％、(2.4±0.3)％和(4.6±0.4)％,而相应的内皮细胞凋亡率分别为(21.5±3.7)％、(23.8±2.9)％和(27.8±3.1)％,内皮细胞较乳腺癌细胞对百里醌反应更敏感(P<0.05);20 nmol/L、40 nmol/L和80 nmol/L百里醌作用于内皮细胞1 h后,内皮细胞小管形成长度分别为(0.88±0.12)mm、(0.76±0.10)mm和(0.54±0.08)mm,与对照组相比,差异有统计学意义(P<0.05);80 nmol/L百里醌作用于内皮细胞24 h后,内皮细胞中cleaved caspase-3的水平上调, AKT和ERK磷酸化被抑制,与对照组比较差异有统计学意义(P<0.05);免疫组化检测移植瘤组织中CD31结果显示,百里醌组IA值明显少于对照组(P<0.05);TUNEL检测结果显示,百里醌组IA值明显增大(P<0.05)。结论: 百里醌可抑制体内外乳腺癌血管生长,降低p-ERK及p-AKT的水平可能是其作用机制之一。     

蛴螬提取物对MCF-7人乳腺癌细胞株凋亡的影响

目的：研究蛴螬提取物对MCF-7人乳腺癌细胞株凋亡通路的影响，为天然药物蛴螬的开发提供实验和理论依据。方法:应用MTT法检测蛴螬提取物对MCF-7人乳腺癌细胞株的抗增殖作用及细胞毒活性；通过倒置显微镜、HE染色、吖碇橙（AO）/溴化乙啶（EB）荧光染色方法观察肿瘤细胞的形态学改变；应用流式细胞仪检测细胞凋亡率的变化；应用免疫细胞化学技术（S-P法）检测用药前后凋亡通路中Bcl-2、Fas、caspase-9、caspase-3的表达改变，探讨蛴螬提取物对凋亡通路的影响。结果:(1)用MTT法检测结果表明，蛴螬提取物在体外对MCF-7人乳腺癌细胞株有明显的抑制增殖作用，实验组与对照组相比其生长抑制率有显著差异（P<0.01），而且这种抑制作用呈现浓度和时间的依赖性；(2)倒置显微镜观察表明，实验组可见胞核固缩、碎裂、凋亡小体形成等凋亡形态学变化；(3)HE染色表明实验组胞核浓缩呈蓝黑色、胞浆呈淡红色、核染色质浓缩、呈碎块状，有凋亡小体形成；(4)经AO／EB荧光染色观察结果表明，实验组出现凋亡细胞；(5)流式细胞仪结果表明实验组凋亡率明显增加，呈时间依赖性；(6)免疫细胞化学染色结果表明，实验组 Bcl-2表达下调，Fas、caspase-3、caspase-9表达均上调，与对照组比差异显著（P<0.01）。结论:①蛴螬提取物在体外显著抑制MCF-7人乳腺癌细胞株增殖；②蛴螬提取物对MCF-7人乳腺癌细胞株凋亡通路的影响机制可能是通过下调Bcl-2，上调Fas、caspase-3 、caspase-9的蛋白表达而起作用，是经由细胞凋亡的线粒体途径和死亡受体途径来完成凋亡的启动和执行。     

双硫仑螯合铜对MCF-7和MCF-10A细胞超微结构和力学特性的影响

目的:基于原子力显微镜(atomic force microscope,AFM)在纳米级分辨率和皮牛级力学测量探讨双硫仑(disulfiram,DSF)螯合氯化铜复合物(DSF/Cu)对人乳腺癌MCF-7和正常乳腺上皮MCF-10A细胞膜超微结构及力学特性的影响。方法:采用流式细胞术比较DSF/Cu作用于2种细胞后凋亡率和细胞周期的改变。AFM探测细胞表面形貌、超微结构、细胞高度、宽度及粗糙度的变化。利用AFM纳米压痕技术检测DSF/Cu对2种细胞硬度(杨氏模量)的影响。结果:流式细胞术结果显示:DSF/Cu可显著诱导MCF-7细胞凋亡,且呈浓度依赖性;而对MCF-10A细胞影响不大。细胞周期检测显示DSF/Cu对2种细胞的细胞周期影响不同。AFM成像观察发现,400和800 nmol/L的DSF/Cu作用于MCF-7细胞6 h后,细胞形态明显皱缩,体积变小,表面变得平整,丝状伪足明显回缩、变短,有些变成了板状伪足,甚至完全消失。进一步定量分析发现细胞宽度变小,高度增高,均方根粗糙度和平均粗糙度均减小,且呈浓度依赖性。而对MCF-10A细胞则影响甚微。最后,单细胞水平生物力学检测显示:DSF/Cu作用于MCF-7和MCF-10A细胞6 h后,2种细胞的杨氏模量随药物浓度的增加均有所增加,且呈浓度依赖性,但MCF-7细胞增加的比例要远远高于MCF-10A细胞。结论:DSF/Cu可能通过特异性影响MCF-7细胞的生物力学特性发挥高效低毒的抗乳腺肿瘤作用。