PD-L1单抗加强紫杉醇联合香菇多糖体外抗人乳腺癌MDA-MB-231作用

目的 探讨程序性细胞死亡-配体1(PD-L1)单抗、紫杉醇(PTX)联合香菇多糖(LNT)体外对人乳腺癌细胞(MDA-MB-231)的作用。方法 将MDA-MB-231、人外周血单个核细胞(PBMC)和MDA-MB-231+PBMC共培养,随机分为对照组、PTX组、LNT组、MPDL3280A(PD-L1单抗)组、PTX+LNT组和PTX+LNT+MPDL3280A组。采用CCK8检测细胞的活性;流式细胞术检测MHC-I和PD-L1的表达;ELISA试剂盒检测IFN-γ和TNF-α的含量。结果 与对照组相比,PTX组、MPDL3280A组、PTX+LNT组及PTX+LNT+MPDL3280A组显著抑制MDA-MB-231的活性(P<0.01);LNT组和PTX+LNT+MPDL3280A组显著促进PBMC的免疫作用(P<0.05,P<0.01);PTX+LNT+MPDL3280A组显著抑制MDA-MB-231+PBMC共培养MDA-MB-231的活性(0.56±0.16 vs. 0.39±0.13,P<0.05);LNT显著促进MDA-MB-231上PD-L1的表达和PBMC分泌IFN-γ(P<0.05)。结论 PD-L1单抗通过阻断PD-L1与PD-1之间的作用,提高免疫,促进PTX联合LNT的体外抗三阴性乳腺癌作用。

Methanolic extract of Abrus precatorius promotes breast cancer MDA-MB-231 cell death by inducing cell cycle arrest at G0/G1 and upregulating Bax

Objective:To determine the anti-proliferative activity of Abrus precatorius(A.precatorius)leaf extracts and their effect on cell death.Methods:A.precatorius leaves were extracted successively with hexane,ethyl acetate and methanol by Soxhlet extraction.Aqueous extract was prepared by decoction at 50 ℃.Extracts of A.precatorius leaves were used to treat selected cancer and normal cell lines for72 h.Furthermore,3-(4,5-dimethyl thiazol-2-yl)2,5-diphenyl tetrazolium bromide assay was performed to determine cell viability.Analysis of cell cycle arrest,apoptosis assay and apoptosis protein expressions were determined by flow cytometry.Results:Methanolic extract of A.precatorius leaves showed the lowest IC50 on MDA-MB-231 cells at(26.40±5.40)μg/mL.Flow cytometry analysis revealed that cell arrest occurred at G0/G1 phase and the apoptosis assay showed the occurrence of early apoptosis at 48 h in MDAMB-231 cells treated with methanolic extract of A.precatorius leaves.Methanolic extract of A.precatorius leaves induced apoptosis by upregulation of Bax,p53 and caspase-3 and downregulation of Bcl-2.Conclusions:Methanolic extract of A precatorius leaves promotes MDA-MB-231 cell death by inducing cell cycle arrest and apoptosis possibly via the mitochondrial-related pathway.

Anticancer property of sediment actinomycetes against MCF-7 and MDA-MB-231 cell lines

ObjectiveTo investigate the anticancer property of marine sediment actinomycetes against two different breast cancer cell lines.MethodsIn vitro anticancer activity was carried out against breast (MCF-7 and MDA-MB-231) cancer cell lines. Partial sequences of the 16s rRNA gene, phylogenetic tree construction, multiple sequence analysis and secondary structure analysis were also carried out with the actinomycetes isolates.ResultsOf the selected five actinomycete isolates, ACT01 and ACT02 showed the IC50 value with (10.13±0.92) and (22.34±5.82) μg/mL concentrations, respectively for MCF-7 cell line at 48 h, but ACT01 showed the minimum (18.54±2.49 μg/mL) level of IC50 value with MDA-MB-231 cell line. Further, the 16s rRNA partial sequences of ACT01, ACT02, ACT03, ACT04 and ACT05 isolates were also deposited in NCBI data bank with the accession numbers of GQ478246, GQ478247, GQ478248, GQ478249 and GQ478250, respectively. The phylogenetic tree analysis showed that, the isolates of ACT02 and ACT03 were represented in group I and III, respectively, but ACT01 and ACT02 were represented in group II. The multiple sequence alignment of the actinomycete isolates showed that, the maximum identical conserved regions were identified with the nucleotide regions of 125 to 221st base pairs, 65 to 119th base pairs and 55, 48 and 31st base pairs. Secondary structure prediction of the 16s rRNA showed that, the maximum free energy was consumed with ACT03 isolate (-45.4 kkal/mol) and the minimum free energy was consumed with ACT04 isolate (?7.6 kkal/mol).ConclusionsThe actinomycete isolates of ACT01 and ACT02 (GQ478246 and GQ478247) which are isolated from sediment sample can be further used as anticancer agents against breast cancer cell lines.

Effect of Guizhi Fuling capsule and its extracts on human breast cancer cells proliferation

OBJECTIVE To evaluate the effect of Guizhi Fuling Capsule active pharmaceutical ingredient(API)and its fractions on human breast cancer cells proliferation by high-throughput screening assay.METHODS The crude fractions were obtained from the extraction and elution of the API of Guizhi Fuling Capsule,and 929 standard fractions were obtained by the optimal separation conditions.Sulforhodamine B(SRB)method was used to evaluate the effects of the Guizhi Fuling capsule API and929 kinds of fractions on the proliferation of human breast cancer cells MCF-7 and MDA-MB-231.RESULTS The Guizhi Fuling capsule API had a strong ability to inhibit the proliferation of MCF-7 cells at high concentration and the ability to inhibit MDA-MB-231 cells' proliferate at low concentration following 72 h treatment;some samples of 929 fractions(5μg·mL^(-1))was found to have a breast cancer cell growth inhibition rate above 50%,without toxicity on HUVECs proliferation.CONCLUSION The API of Guizhi Fuling capsule had significant cytotoxicity effects on these two human breast cancer cells,with significant concentration-and time-dependent manner.

不同浓度吗啡复合罗哌卡因对乳腺癌MDA-MB-231细胞增殖、迁移、侵袭和细胞周期的作用影响

目的 观察不同浓度的吗啡复合罗哌卡因对乳腺癌MDA-MB-231细胞增殖、转移侵袭和细胞周期的影响。方法 人乳腺癌细胞MDA-MB-231细胞接种于培养板24h,随机分为8组:对照组(C组)、罗哌卡因400μg/ml组(R组)、吗啡3μg/ml组(LM组)、吗啡30μg/ml组(MM组)、吗啡300μg/ml组(HM组)、罗哌卡因400μg/ml组+吗啡3μg/ml组(R+LM组)、罗哌卡因400μg/ml+吗啡30μg/ml组(R+MM组)和罗哌卡因400μg/ml+吗啡300μg/ml组(R+HM组)。处理乳腺癌细胞MDA-MB-231细胞24h后,检测其增殖能力、迁移能力、侵袭能力和细胞周期。结果 人乳腺癌细胞MDA-MB-231细胞的增殖抑制情况:单独应用吗啡时,LM组、MM组、HM组均对人乳腺癌细胞MDA-MB-231细胞的增殖具有抑制作用(P<0.05),并且抑制率随着吗啡浓度的升高而依次增加。单独应用罗哌卡因时对人乳腺癌细胞MDA-MB-231细胞的增殖具有抑制作用(P<0.05)。吗啡与罗哌卡因联合应用时,高浓度吗啡组与罗哌卡因具有协同作用。人乳腺癌细胞MDA-MB-231细胞的迁移情况:单独应用吗啡时,LM组、MM组、HM组均能够抑制细胞迁移率(P<0.05),迁移率随着吗啡浓度的升高而依次降低。单独应用罗哌卡因能够抑制细胞迁移率(P<0.05)。吗啡与罗哌卡因联合应用时,低浓度和中浓度吗啡组与罗哌卡因具有协同作用(P<0.05)。人乳腺癌细胞MDA-MB-231细胞的侵袭情况:单独应用吗啡时,MM组、HM组均能够抑制细胞侵袭能力(P<0.05),并且侵袭能力随着吗啡浓度的升高而依次降低,单独应用罗哌卡因时能够抑制细胞的侵袭能力(P<0.05)。吗啡与罗哌卡因联合应用时,中、高浓度组吗啡和罗哌卡因具有协同作用。人乳腺癌细胞MDA-MB-231细胞的细胞周期情况:单独应用高浓度吗啡,能够抑制细胞进入G2/M期(P<0.05)。单独应用罗哌卡因,能够抑制细胞进入G2/M期(P<0.05),低浓度吗啡与罗哌卡因联合应用对于将细胞抑制在G0/G1期和S期具有协同作用(P<0.05)。结论 吗啡复合罗哌卡因能够抑制乳腺癌MDA-MB-231细胞的增殖、迁移和侵袭,具有联合作用并呈剂量依赖性。

基于ERK/NF-κB通路探讨苦杏仁苷对乳腺癌MDA-MB-231细胞增殖、侵袭、迁移的影响

目的探讨苦杏仁苷对乳腺癌MDA-MB-231细胞增殖、侵袭、迁移及细胞外信号调节激酶(ERK)/核因子(NF)-κB通路的影响。方法将乳腺癌MDA-MB-231细胞随机分成对照组、紫杉醇组(10μg/ml)、苦杏仁苷低剂量组(20μg/ml)、苦杏仁苷高剂量组(40μg/ml);以上各组每孔设6个平行样,培养72 h。实验结束后,噻唑蓝(MTT)法测定乳腺癌MDA-MB-231细胞增殖水平,Transwell小室测定乳腺癌MDA-MB-231细胞侵袭迁移水平,实时荧光定量聚合酶链反应(PCR)及Western印迹法测定乳腺癌MDA-MB-231细胞ERK、NF-κB mRNA和蛋白表达水平。结果与对照组比较,紫杉醇组、苦杏仁苷低、高剂量组光密度(OD)值、细胞存活率、穿膜数、ERK、NF-κB mRNA和蛋白表达水平显著降低(P<0.05);与紫杉醇组比较,苦杏仁苷低剂量组上述指标显著升高(P<0.05),苦杏仁苷高剂量组上述指标无显著差异(P>0.05);与苦杏仁苷低剂量组比较,苦杏仁苷高剂量组上述指标显著降低(P<0.05)。结论苦杏仁苷能明显抑制乳腺癌MDA-MB-231细胞增殖、侵袭、迁移,其机制可能与苦杏仁苷抑制ERK、NF-κB mRNA和蛋白表达进而抑制ERK/NF-κB通路的激活有关。

Loss of the vitamin D receptor in human breast and prostate cancers strongly induces cell apoptosis through downregulation of Wnt/β-catenin signaling

Vitamin D co-regulates cell proliferation, differentiation and apoptosis in numerous tissues, including cancers. The known anti-proliferative and pro-apoptotic actions of the active metabolite of vitamin D, 1,25-dihydroxy-vitamin D [1,25（OH）2D] are mediated through binding to the vitamin D receptor （VDR）. Here, we report on the unexpected finding that stable knockdown of VDR expression in the human breast and prostate cancer cell lines, MDA-MB-231 and PC3, strongly induces cell apoptosis and inhibits cell proliferation in vitro. Implantation of these VDR knockdown cells into the mammary fat pad （MDA-MB-231）, subcutaneously （PC3） or intra-tibially （both cell lines） in immune-incompetent nude mice resulted in reduced tumor growth associated with increased apoptosis and reduced cell proliferation compared with controls. These growth-retarding effects of VDR knockdown occur in the presence and absence of vitamin D and are independent of whether cells were grown in bone or soft tissues. Transcriptome analysis of VDR knockdown and non-target control cell lines demonstrated that loss of the VDR was associated with significant attenuation in the Wnt/0-catenin signaling pathway. In particular, cytoplasmic and nuclear β-catenin protein levels were reduced with a corresponding downregulation of downstream genes such as Axin2, Cyclin D1, interleukin-6 （IL-6）, and IL-8. Stabilization of 0-catenin using the GSK-3β inhibitor BIO partly reversed the growth-retarding effects of VDR knockdown. Our results indicate that the unliganded VDR possesses hitherto unknown functions to promote breast and prostate cancer growth, which appear to be operational not only within but also outside the bone environment. These novel functions contrast with the known anti-proliferative nuclear actions of the liganded VDR and may represent targets for new diagnostic and therapeutic approaches in breast and prostate cancer.

Meclizine Chloridrate and Methyl-β-Cyclodextrin Associated with Monophosphoester Synthetic Phosphoethanolamine Modulating Proliferative Potential in Triple-Negative Breast Cancer Cells

Synthetic phosphoethanolamine(Pho-s)is a monophosphoester ester with anti-inflammatory and pro-apoptotic properties.Meclizine chloridrate(MC)is a histamine H1 receptor blocker that is also able to inhibit cellular respiration.However,MC does not inhibit cellular respiration in isolated mitochondria such as antimycin and rotenone.Methyl-β-cyclodextrin(MβCD)belongs to theβ-cyclodextrin family,which is capable of removing cholesterol from the plasma membrane.The aim of this study was to evaluate the proliferative effects of meclizine chloridrate and methyl-β-cyclodextrin compounds associated with synthetic phosphoethanolamine in a triple-negative human breast tumor line,MDA-MB-231 Cell viability of the tumor line and normal cells FN1 was evaluated by MTT colorimetric test;the production of free radicals was determined by lipoperoxidation(LPO)test;and the percentage of cell cycle phases and proliferative index was evaluated by flow cytometry.Cell viability demonstrated a significant decrease with the treatments of MβCD,MC and Pho-s associated with MC.The production of free radicals decreases significantly in all treatments.In addition,a significant increase of DNA fragment and decrease in G0/G1 cell cycle phase were observed in cellular percentage with concentrations of 20 and 30 mM of Pho-s in association with MC and MβCD,respectively.

Sensitivity Evaluation of Two Human Breast Cancer Cell Lines to Tamoxifen through Apoptosis Induction

Tamoxifen citrate (TAM) has been used to treat breast cancer in women for many years. The com-parative effects of TAM in inducing apoptosis were evaluated in estrogen receptor-positive (ER- positive MCF-7) and estrogen receptor-negative (ER-negative MDA-MB-231) human breast cancer cell lines in vitro in order to determine if these two cell lines differ in their sensitivity to TAM. Mi-tochondrial membrane permeability potential disruption was assessed in both cell lines by a lip-ophilic cationic dye (DePsipher assay, Trevigen, Inc.) utilizing fluorescence microscopy. Using this specific fluorochrome, we were able to associate mitochondrial membrane disruption to early, mid-, and late apoptotic cells. TAM induced cell death via apoptosis in both ER-positive and ER- negative cells, however, apoptosis induction was more pronounced in ER-positive MCF-7 compared to ER-negative MDA-MB-231 breast cancer cells. These findings may have some therapeutic use in the treatment of estrogen dependent and estrogen independent breast cancer.

羊肚菌多糖抑制人乳腺癌细胞MDA-MB-231增殖和诱导细胞凋亡研究

以黑脉羊肚菌多糖为原料,研究羊肚菌多糖对人乳腺癌细胞MDA-MB-231(以下简称MDA)增殖和凋亡的影响。结果表明:在无细胞毒性范围内,羊肚菌多糖能显著抑制人乳腺癌细胞MDA的增殖,半数有效浓度(median effective concentration,EC_(50))值为0.096 mg/m L,同时人乳腺癌细胞MDA表现出多种细胞凋亡的形态学变化。免疫印迹检测实验结果显示,羊肚菌多糖能抑制B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)蛋白表达,促进Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)表达,同时增加Bax/Bcl-2蛋白表达比值,并呈现出剂量依赖效应。说明羊肚菌多糖能抑制人乳腺癌细胞MDA增殖,促进细胞凋亡。

Celecoxib下调NF-кB信号通路促进人乳腺癌MDA-MB-231细胞凋亡的体外研究

背景与目的:Celecoxib可以抑制多种肿瘤细胞增殖、诱导其凋亡,但具体的作用机制尚不明确。本研究探讨Celecoxib是否通过阻断NF-кB信号途径诱导人乳腺癌MDA-MB-231细胞的凋亡。方法:RT-PCR法检测MDA-MB-231细胞中COX-2mRNA的表达。MTT法检测Celecoxib、PGE2与Celecoxib联合应用对MDA-MB-231细胞增殖的影响。流式细胞术检测细胞周期、凋亡的变化。Westernblot法检测0、40、80、120μmol/LCelecoxib作用MDA-MB-231细胞24h后细胞中Caspase-3、p65蛋白的表达变化。结果:RT-PCR检测显示MDA-MB-231细胞中COX-2mRNA的表达随着Celecoxib浓度的增加而逐渐降低。Celecoxib能够显著地抑制MDA-MB-231细胞增殖(P<0.05),但Celecoxib联合PGE2与单独应用Celecoxib对细胞增殖的影响差异无统计学意义(P>0.05)。流式细胞术结果显示Celecoxib可使MDA-MB-231细胞阻滞在G0/G1期,并可诱发细胞凋亡。Western blot检测发现Celecoxib作用MDA-MB-231细胞24h,随着Celecoxib浓度的增加,Caspase-3蛋白表达增加,P65蛋白表达下调。结论:Celecoxib可以通过下调P65蛋白的表达从而阻断NF-кB信号途径,抑制人乳腺癌MDA-MB-231细胞增殖,促进其凋亡。

桔梗皂苷D诱导乳腺癌MDA-MB-231细胞凋亡

目的:研究来自桔梗的天然单体化合物桔梗皂苷D(platycodin D,PD)对高转移性乳腺癌细胞MDA-MB-231凋亡的影响,初步探索其可能的作用机制。方法:以不同浓度(0、2.5、5、10μmol/L)PD处理乳腺癌MDA-MB-231细胞后,采用MTT法检测细胞增殖率并计算IC50值,流式细胞术检测细胞凋亡情况,Western blotting检测凋亡相关蛋白的表达水平。结果:PD呈剂量依赖性显著抑制MDA-MB-231细胞的增殖(P<0.01),作用72 h的IC50值为(7.30±2.67)μmol/L。与对照组相比,10μmol/L的PD可显著促进MDA-MB-231细胞的凋亡(P<0.05)。PD激活了caspase家族蛋白,上调有活性的cleaved caspase-3、cleaved caspase-8和cleaved caspase-9的表达,下调无活性的caspase-8和caspase-9的表达;PD同时减少Bcl-2的表达,增加Bax的表达,使Bcl-2/Bax的比值降低。研究还发现PD使突变型P53蛋白的表达减少、E2F1的表达增加。结论:PD抑制乳腺癌细胞增殖具有明显的抗肿瘤效应,而诱导凋亡的发生可能是其发挥抗肿瘤效应的机制之一。

塞来昔布通过阻断NF-кB信号通路诱导人乳腺癌细胞MDA-MB-231细胞周期阻滞的相关研究

背景与目的:研究表明塞来昔布能抑制人乳腺癌细胞增殖、诱导其凋亡,但其对肿瘤细胞周期的作用机制尚不明确。本实验研究塞来昔布是否通过阻断NF-кB信号通路对人乳腺癌细胞MDA-MB-231增殖及周期产生影响。方法:MTT法和流式细胞术观察塞来昔布对MDA-MB-231细胞增殖、周期分布的影响;应用RT-PCR和Western印迹法检测经塞来昔布干预该细胞24h后,细胞周期相关因子及NF-кB信号途径中p-IκBα的变化。结果:塞来昔布可显著抑制MDA-MB-231细胞生长,呈时间剂量依赖性。高浓度塞来昔布可改变细胞进程,将其阻滞于G0/G1期。塞来昔布作用24h后,细胞周期相关蛋白CyclinD1、CDK4呈剂量依赖性表达下降(P<0.05)。同时,细胞中p-IκBα表达随药物浓度增加而下降(P<0.05)。结论:塞来昔布能显著抑制MDA-MB-231细胞增殖,诱导细胞G0/G1期阻滞,其作用机制可能是通过抑制IκBα的磷酸化阻断NF-кB信号通路,进而下调其下游基因CyclinD1对细胞周期进行调控。

Livin靶向RNA干扰对MDA-MB-231细胞增殖和凋亡的影响

目的研究凋亡抑制蛋白Livin靶向RNA干扰MDA-MB-231对细胞增殖和凋亡的影响,探讨其可能的机制。方法构建3个靶向Livin的siRNA真核表达载体,分别转染乳腺癌细胞MDA-MB-231后,通过RT-PCR、Western blot技术检测干扰前后MDA-MB-231细胞Livin、Caspase-3和Smac/DIABLO在mRNA、蛋白水平的表达;采用MTT法检测对细胞增殖的抑制作用,流式细胞法测细胞周期,TUNEL法检测细胞凋亡以及观察细胞形态变化。结果成功构建了针对Livin基因的3个特异性siRNA表达载体。它们不同程度地抑制了MDA-MB-231细胞Livin基因的表达,以si-Livin2抑制效率最高,它对Livin的mRNA和蛋白的抑制率分别达76.4%,70.2%。si-Livin2转染48 h后,与未转染组相比,细胞增殖活力受到抑制,转染48 h抑制率达36.1%。细胞周期重新分布,G0/G1期上升为(71.75±1.31)%,S期下降为(18.06±1.46)%;细胞凋亡率上升为(13.28±1.65)%;电镜下可见典型的凋亡细胞特征。细胞内Caspase-3, Smac/DIABLO的mRNA、蛋白表达有所上升。结论 Livin靶向RNA干扰通过上调Caspase-3, Smac/DIABLO表达有效地抑制MDA-MB-231细胞增殖,促进凋亡。

黄芩素对人乳腺癌MDA-MB-231细胞株SATB1表达的影响

目的:观察黄芩素对人乳腺癌MDA-MB-231细胞SATB1蛋白表达的影响。方法:用MTT法、划痕愈合实验观察不同浓度黄芩素干预后MDA-MB-231细胞增殖和运动迁移能力的变化;用Western Blot法检测黄芩素干预后MDA-MB-231细胞SATB1蛋白表达的变化。结果:随作用时间的延长和药物浓度的增加,黄芩素对MDA-MB-231细胞增殖和运动迁移的抑制作用逐渐增强,呈明显的时间-剂量依赖性(P<0.05);黄芩素可显著降低MDA-MB-231细胞中SATB1蛋白的表达,而且随着药物浓度增加,SATB1蛋白表达量逐渐减少(P<0.05)。结论:黄芩素可通过抑制SATB1的表达抑制肿瘤细胞的恶性增殖、侵袭和迁移能力。

莲心碱对乳腺癌细胞MDA-MB-231自噬功能的影响

目的观察莲心碱作用于乳腺癌细胞MDA-MB-231后,自噬表型的变化,初步论证莲心碱对乳腺癌细胞MDA-MB-231自噬功能的调控作用。方法透射电子显微镜观察莲心碱处理后MDAMB-231的亚细胞结构变化,激光共聚焦显微镜观察p EGFP-LC3或ptf LC3(mRFP-EGFP-LC3)转染后自噬小体的形成和自噬流的变化,Western blot检测自噬相关蛋白LC3B,SQSTM1/p62,Beclin1的表达,以及论证自噬流的变化。结果透射电镜显示,与对照组比较,莲心碱处理后MDA-MB-231细胞内有较多自噬空泡出现,部分分裂的线粒体,但未见到高密度的嗜锇性的自噬溶酶体结构;EGFP-LC3转染后,莲心碱组自噬体数量显著多于对照组(P<0.01);Western blot检测到自噬相关蛋白LC3B-Ⅱ,SQSTM1/p62表达呈现量效性和时效性的增多[20μmol/L组或24 h组与对照组比较(P<0.01)],Beclin1未见显著变化[20μmol/L组与对照组比较(P>0.05)];mRFP-EGFP-LC3转染后,对照组可见少量自噬体和自噬溶酶体,莲心碱和Bafilomycin A1处理组均可见大量自噬体结构,未见显著自噬溶酶体结构;Western blot证实莲心碱处理后增多的LC3B-Ⅱ不受Bafilomycin A1预处理的影响[莲心碱组LC3B-Ⅱ,SQSTM1/p62蛋白水平与Bafilomycin A1预处理加莲心碱组比较(P>0.05)]。结论莲心碱抑制自噬小体成熟,阻断乳腺癌细胞MDA-MB-231自噬降解作用。

甘精胰岛素和人胰岛素对人乳腺癌MDA-MB-231细胞的增殖作用

目的探讨甘精胰岛素(insulin glargine)和人胰岛素(human insulin)对人乳腺癌细胞株MDA-MB-231增殖的影响及细胞外信号调节激酶(ERK)的调节作用。方法使用不同浓度的甘精胰岛素和人胰岛素刺激人乳腺癌MDA-MB-231细胞株,检测不同时间段的细胞增殖作用,探讨抑制ERK1/2激活对细胞增殖的影响。使用cell counting kit-8(CCK-8)试剂检测细胞增殖,流式细胞术检测细胞周期分布。结果甘精胰岛素和人胰岛素在1、10、100IU.L-1刺激MDA-MB-231细胞96h后均可轻度促进MDA-MB-231细胞增殖,相同条件下甘精胰岛素和人胰岛素的促增殖效应无差异。甘精胰岛素与人胰岛素以10IU.L-1分别刺激48、72、96h后两药均诱导细胞增殖,比较两种药物间增殖效应的差异无统计学意义。甘精胰岛素和人胰岛素均使S+G2/M期细胞比例增加,但两处理组间差异无显著性。ERK1/2抑制剂PD98059可抑制MDA-MB-231细胞增殖;但PD98059不影响甘精胰岛素和人胰岛素对MDA-MB-231细胞增殖的促进作用。结论甘精胰岛素和人胰岛素对人乳腺癌细胞株MDA-MB-231增殖有类似的轻度促进作用,该作用不依赖于ERK的激活。

阿霉素对二氢卟吩e6声动力抑制乳腺癌细胞MDA-MB-231生长体外增敏作用

目的声动力治疗(SDT)是通过超声波激活聚集在肿瘤细胞内声敏剂治疗肿瘤的一种新方法。本文研究观测阿霉素对二氢卟吩e6为声敏剂声动力抑制乳腺癌细胞MDA-MB-231生长的作用,旨在探讨阿霉素对二氢卟吩e6声动力是否具增敏作用。方法二氢卟吩e6声动力单独及联合阿霉素处理MDA-MB-231细胞,45min后四甲基偶氮唑盐(MTT)显色分光光度法检测细胞增殖。结果 1.1.0MHz频率超声强度0.5～2.0W/cm2范围作用60s呈强度依赖性抑制MDA-MB-231细胞生长;二氢卟吩e6和阿霉素分别在0.05及0.1μg/ml～0.4μg/ml范围内浓度依赖性抑制MDA-MB-231细胞生长。2.与超声(0.5W/cm2×1.0 MHz×60s)和二氢卟吩e6(0.05～0.2mg/ml)单独作用相比,超声和二氢卟吩e6两者联合作用抑制MDA-MB-231细胞生长作用显著增强(P<0.05)。3.与二氢卟吩e6声动力(超声:0.5W/cm2×1.0MHz×60s;二氢卟吩e6:0.1mg/ml)和阿霉素(0.1～0.4g/ml)单独作用相比,声动力联合阿霉素抑制MDA-MB-231细胞生长作用显著增强(P<0.05),其抑制作用与联合时序相关,声动力作用后用阿霉素比先用阿霉素后进行声动力更显著抑制乳腺癌细胞的生长(P<0.05)。结论阿霉素对二氢卟吩e6声动力抑制乳腺癌细胞MDA-MB-231生长具增敏作用。

虫草素对MDA-MB-231细胞增殖的影响与TMSG-1表达的研究

目的研究虫草素对乳腺癌细胞株MDA-MB-231的作用与人肿瘤转移抑制基因TMSG-1mRNA表达的影响,探讨其抗癌作用机制。方法体外培养乳腺癌细胞株MDA-MB-231,加入虫草素干扰,免疫组化技术检测TMSG-1蛋白表达;半定量RT-PCR检测MDA-MB-231细胞中TMSG-1mRNA表达状况;使用MTT法检测对肿瘤细胞增殖的抑制作用。结果虫草素可使乳腺癌MDA-MB-231细胞中TMSG-1mRNA与蛋白表达上调,同时对乳腺癌细胞生长有抑制作用。结论虫草素能抑制乳腺癌细胞的增殖,可以上调TMSG-1mRNA与蛋白的表达发挥潜在的抗肿瘤细胞转移的作用,为国产新型抗肿瘤药物的临床研发及运用提供实验室依据。

用于活体成像的人三阴性乳腺癌MDA-MB-231细胞系的构建

目的构建能稳定表达荧光素酶和绿色荧光蛋白的人三阴性乳腺癌MDA-MB-231细胞系以建立可用于活体成像的三阴性乳腺癌裸鼠移植瘤模型。方法采用磷酸钙共沉淀的方法构建表达荧光素酶和绿色荧光蛋白的慢病毒载体,体外感染MDA-MB-231细胞,经G418筛选得到稳定细胞系后,通过活体成像评价感染后细胞表达荧光素酶的情况,并通过MTT法、transwell肿瘤侵袭模型、细胞划痕实验等评价细胞的增殖、侵袭及转移能力是否改变;此外将细胞接种至雌性BALB/c-nu//nu裸小鼠的右侧第2对乳房垫内,构建乳腺癌原位肿瘤模型。利用活体成像系统监测体内肿瘤的生长及转移情况,并通过冰冻切片、HE染色评价细胞系在活体内的稳定性及成瘤能力。结果成功构建能稳定表达荧光素酶和绿色荧光蛋白的MDA-MB-231细胞系,荧光素酶的活性达到每细胞9689 photons/s,慢病毒的整合没有改变细胞的增殖、迁移及侵袭能力;成功建立了乳腺癌裸鼠原位移植瘤模型。结论(1)慢病毒以其高效稳定的感染率,应作为标记荧光素酶基因的首选载体;(2)带有荧光素酶的MDA-MB-231细胞在动物体内可被快速而灵敏的检测到,这将为人三阴性乳腺癌转移机制的研究及抗肿瘤药物的研发提供一个直观、方便及灵敏的平台;(3)荧光素酶和荧光蛋白基因双标记优于单标记。