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糖鞘脂GM1调控生长密度相关的乳腺细胞体外增殖抑制

  • 卓定浩1,2

    机 构：

    1. 江南大学糖化学与生物技术教育部重点实验室，无锡 214122

    2. 西北大学生命科学学院，西安 710069

    ×
  • 关锋1,2

    机 构：

    1. 江南大学糖化学与生物技术教育部重点实验室，无锡 214122

    2. 西北大学生命科学学院，西安 710069

    ×

1. 江南大学糖化学与生物技术教育部重点实验室，无锡 214122； 2. 西北大学生命科学学院，西安 710069

中图分类号: Q81

DOI:10.16476/j.pibb.2018.0238

Regulation of Glycosphingolipid GM1 on The Density Dependent Inhibition of Human Breast Cell Growth In Vitro

  • ZHUO Ding-Hao1,2

    Affiliation：

    1. Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122， China

    2. The College of Life Sciences, Northwestern University，Xi’an 710069, China

    ×
  • ） GUAN Feng1,2

    Affiliation：

    1. Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122， China

    2. The College of Life Sciences, Northwestern University，Xi’an 710069, China

    ×

1. Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122， China； 2. The College of Life Sciences, Northwestern University，Xi’an 710069, China

CLC: Q81

DOI:10.16476/j.pibb.2018.0238

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目录 contents

    摘要

    细胞在体内增殖受到制约，以确保器官的正常大小和组织稳态的维持，体外培养的细胞也存在接触抑制生长现象. 分布于细胞膜上的糖鞘脂具有调控细胞增殖的作用. 本研究探讨了糖鞘脂GM1对人乳腺细胞MCF-10A、人乳腺癌细胞BT-549和SK-BR3增殖的影响. 通过对细胞不同接种量研究细胞增殖的变化；利用流式细胞术检测细胞在不同密度生长时GM1的表达差异；探索细胞在不同密度生长时外源添加GM1对细胞增殖的影响；构建GM1干扰和过表达细胞并检测转染细胞株的增殖差异. 结果显示，相较于常规密度，在低密度和高密度生长时，细胞增殖受到抑制，GM1表达量提高；GM1处理抑制低密度和高密度生长时细胞增殖，对常规密度生长细胞没有显著影响. 在低密度和高密度生长时，GM1干扰细胞增殖能力提高，而GM1过表达细胞增殖能力下降. 综上，本实验研究证实GM1抑制乳腺细胞MCF-10A、乳腺癌细胞BT-549和SK-BR-3在体外低密度和高密度生长时的细胞增殖，为研究GM1抑制细胞增殖分子机理提供了工作基础.

    Abstract

    Regulation of cell proliferation is essential for controlling organ size and maintenance of tissue homeostasis in adult organisms. Growth inhibition, termed as “contact inhibition”, in a cell-density dependent manner was common in vitro culture. In this study, the effect of glycosphingolipid GM1 on the contact inhibition of human mammary epithelial cell lines MCF-10A, human breast cancer cell lines BT-549 and SK-BR-3 were investigated. Changes of cell proliferation of MCF-10A, BT-549 and SK-BR-3 cells at low and high cell density was explored. Expression of GM1 in different cell density was detected by flow cytometry. Exogenous GM1 at different cell density was added to explore the effect on cell proliferation. B3GALT4, the GM1 synthase, was knocked down or overexpressed in BT-549 and SK-BR-3 cells by using lentiviral vectors. Then, proliferating ability were tested by cell counting and related pathways was assayed by Western blot, in stable-transfected cell lines. Results showed cell proliferation was inhibited and GM1 expression increased at low and high cell density compared with normal density. Exogenous addition of GM1 at low and high density cells inhibited cell growth, but it had no influence on cell growth at normal density. Down regulation of GM1 promoted cell proliferation at low and high cell density, and overexpression of GM1 had the opposite effect. Together, these results indicate that GM1 inhibit MCF-10A, BT-549 and SK-BR-3 cells proliferation at low and high cell density in vitro, which may potentially provide the experimental basis for further research on its molecular mechanisms.

    在多细胞生物中，各种器官中细胞的生长在生物发育过程中受到严格调控，以确保器官的大小处在合适的范围内. 器官大小的调节对组织稳态的维持起到至关重要的作用. 正常细胞在体内的增殖受到制约以维持细胞数量的相对稳定，而癌细胞能够抵抗生长调控从而获得无限增殖的能力[1,2]. 体外培养的正常贴壁细胞也存在接触抑制(contact inhibition)现象，即相互汇合接触时，细胞移动和生长受到抑制. 细胞发生癌变后对接触抑制的敏感性显著下降，这种能力的改变也是体外细胞转化的一个广泛应用标准[3]. 细胞增殖涉及很多信号通路，其中比较重要的是表皮生长因子受体（EGFR）和细胞外调节蛋白激酶（ERK）1/2(p44/42)参与的EGFR/MAPK信号通路[4]. 虽然近年来的研究提出了多种关于接触抑制机理的假说，但是接触抑制所涉及的调控因子纷繁复杂，至今仍未形成一套完整的理论体系[5]. 体外培养细胞在低密度时的生长也受到一定的抑制，可能原因是生长物质弥散细胞外所达到的浓度低于细胞生长最低限度，然而确切的分子调控机制仍不清楚.

    糖鞘脂(glycosphingolipids)是一类分布于所有动物细胞膜表面的具有重要生物学功能的糖脂(glycolipids)，是由糖链头部和神经酰胺(ceramide)尾部组成的两亲性物质[6]. 糖鞘脂参与和调控了细胞增殖、运动、黏附及信号转导等细胞活动[7]. 此外，在许多癌症中都检测到了糖鞘脂的异常表达，并已证实某些糖鞘脂在癌症的发生发展过程中发挥了重要的功能[8]. 神经节苷脂(ganglioside)是一类广泛分布于脑组织神经细胞中含有唾液酸的糖鞘脂，介导了神经的生长、发育、分化和成熟过程[9]. 研究发现，当细胞密度增大时，糖鞘脂特别是神经节苷脂的含量大幅提高[10]，例如：神经节苷脂GM3与人肺成纤维细胞WI38在高密度时的生长抑制有关[11]；神经节苷脂GD1b能促使乳腺癌细胞MCF-7凋亡[12]. GM1是最重要的神经节苷脂之一，其结构为Galβ1-3GalNAcβ1-4(NeuAcα2-3)Galβ1-4GlcβCer，主要分布于细胞膜上的脂筏区(lipid raft)，具有调节脂筏物理化学性质，调控细胞运动、黏附和信号传导等重要作用[13]. 然而GM1对细胞增殖作用的影响鲜有报道，尤其是GM1对乳腺癌细胞增殖的作用未见研究.

    因此，本文选取了人乳腺细胞MCF-10A(通过在低钙离子浓度的无血清培养液中长时间培养而得到的非致瘤的上皮细胞株)、人乳腺管癌细胞BT-549(来源于乳头状侵入性导管癌)和人乳腺腺癌细胞SK-BR-3(分离于一位43岁的白人女性乳腺癌患者的胸腔积液中)，通过研究不同细胞生长密度时GM1对细胞增殖的影响，探索GM1调控乳腺癌细胞增殖的具体方式. 本研究不仅为GM1在乳腺癌细胞增殖中的作用提供实验基础，也为研究神经节苷脂在癌症中的作用提供更多的思路.

  • 1　材料与方法

  • 1.1　细胞培养

    人乳腺细胞MCF-10A、人乳腺管癌细胞BT-549和人乳腺腺癌细胞SK-BR-3购于上海中国科学院细胞库. MCF-10A细胞生长使用含5%马血清(Gibco)、20 μg/L EGF(Peprotech)、0.5 mg/L 氢化可的松、100 μg/L霍乱毒素(Sigma-Aldrich)、 10 mg/L人胰岛素、100 mg/L青霉素和100 mg/L链霉素的DMEM/F12(Hyclone)培养基；BT-549和SK-BR-3细胞生长使用含10%胎牛血清(Biological Industries)、100 mg/L青霉素和 100 mg/L链霉素的DMEM高糖培养基；BT-549细胞生长使用含10%胎牛血清、2.5 g/L葡萄糖、0.023 U/ml牛胰岛素的RPMI 1640培养基. 三株细胞均在37℃，5% CO2条件下培养. 不同细胞密度的获得方式为，以5×103/cm2接种培养2 d获得低密度生长细胞，以2×104/cm2接种培养2 d获得常规密度生长细胞，以1.5×105/cm2(MCF-10A细胞为 1×105/cm2)接种培养2 d获得高密度生长细胞.

  • 1.2　细胞计数

    将细胞按特定接种量接种至48孔板，培养过夜后添加GM1(本实验室从猪脑中提取)孵育 36 h或者直接培养48 h，用胰蛋白酶消化成细胞悬液，充分混匀后取20 μl加至细胞计数板，利用细胞计数器BioLab(Counterstar)进行细胞计数并统计分析.

  • 1.3　细胞增殖检测

    将细胞按特定接种量接种至12孔板，培养2 d后添加5 μmol/L 5-乙炔基-2‘脱氧尿嘧啶核苷(EdU)继续培养12 h. 胰蛋白酶消化细胞并转移至流式管，PBS清洗2次，4%多聚甲醛室温固定 15 min，PBS清洗2次，0.2%Triton X-100通透 20 min，PBS清洗2次，荧光染料室温避光孵育 30 min，PBS清洗2次后上流式细胞仪(BD Biosciences)检测.

  • 1.4　细胞转染

    以下提到质粒除pLVX-AcGFP1-N1购于Takara公司（https://www.takarabio.com/），其余均购于Addgene质粒库（http://www.addgene.org/）. 通过慢病毒转染法构建B3GALT4干扰(正义链：TGACGGACGATGATGTGTATTTCAAGAGAATACACATCATCGTCCGTCTTTTTG；反义链：TCGACAAAAAGACGGACGATGATGTGTATTCTCTTGAAATACACATCATCGTCCGTCA)和过表达细胞株(上游引物：GGAATTCGCCACCATG-CAGCTCAGGCTCTTCC；下游引物：GCTCTA-GAGTCCTGTTCCTGCCTTTCCT). 干扰慢病毒载体为pSicoR-pGK-Puro，过表达慢病毒载体为pLVX-AcGFP1-N1，病毒包装所需工具载体为psPAX2和pMD2.G, 病毒包装细胞为293T细胞. 利用Lipofectamine2000（Invitrogen）将3个质粒共转染293T细胞，48 h后收获病毒液. 将病毒液加至BT-549或SK-BR-3细胞，并通过嘌呤霉素(puro)抗生素筛选最终获得稳定转染细胞株.

  • 1.5　实时荧光定量PCR

    用动物组织/细胞RNA提取试剂盒（Cwbiotech）从细胞中提取RNA，反转录试剂盒（TOYOBO）合成cDNA. 根据GeneBank中B3GALT4基因序列，设计并合成实时荧光定量PCR（QPCR）所需引物序列. 结合UltraSYBR Mixture试剂盒（Cwbiotech），使用荧光定量PCR仪CFX96 Touch（Bio-Rad）进行PCR反应，并通过2-∆∆Ct法计算转染细胞株目的基因的相对表达差异.

  • 1.6　蛋白质免疫印迹

    将细胞按特定接种量接种，培养过夜后添加GM1孵育36 h或者直接培养2 d，用含蛋白酶抑制剂(Selleck)和磷酸酶抑制剂(Sigma-Aldrich)的RIPA裂解液提取细胞蛋白质. BCA蛋白质浓度测定试剂盒(Beyotime Biotechnology)测定蛋白质浓度后，取30 μg上样SDS-PAGE蛋白质凝胶电泳. 通过湿转法将蛋白质转移到PVDF膜(Millipore)上，经5% BSA或牛奶37℃封闭1 h后，4℃一抗孵育过夜(一抗anti-EGFR、anti-p-EGFR、anti-ERK1/2、anti-p-ERK1/2购于Cell Signaling Technology，anti-B3GALT4购于Abcam，anti-Tubulin购于Sigma-Aldrich)，TBST清洗3次. 添加辣根过氧化物酶(HRP)标记的二抗(Beyotime Biotechnology)室温孵育1 h，TBST清洗5次. 利用Pro-Light HRP化学发光检测试剂(Tiangen)显色并通过ChemiDocTMXRS+成像系统(Bio-Rad)采集图像，进行3次独立重复实验，结果用Image J软件进行相对定量分析，并用GraphPad Prism进行显著性分析.

  • 1.7　流式细胞术检测GM1含量

    将细胞按特定接种量接种至6孔板，培养2 d后胰蛋白酶消化细胞并转移至流式管，经过PBS清洗、多聚甲醛固定后，添加FITC标记的霍乱毒素B亚基(Sigma-Aldrich)室温避光孵育30 min，PBS清洗2次后上流式细胞仪检测.

  • 2　结果与分析

  • 2.1　人乳腺细胞生长受到细胞密度调控

    细胞生长受到诸多因素的调控，细胞密度是其中重要的影响条件之一. 将人乳腺细胞MCF-10A、人乳腺癌细胞BT-549和SK-BR-3分别以低接种量(5×103/cm2)和高接种量(1×105/cm2)接种，连续培养4 d. 以高接种量接种培养到第4天时，3株细胞均表现出明显的生长抑制现象(图1b). 此外，以低接种量接种培养前2天，两株细胞的生长都受到一定的抑制，之后才进入快速生长期(图1a).

    Fig. 1 Density dependent growth inhibition of human mammary cell

    NOTE: Cells were seeded at low density (a)or high density (b)and cultured for 4 days. Cells were digested by trypsin and counted using an Automate Cell Counter.

  • 2.2　高密度生长时细胞增殖受到抑制

    细胞在高密度时存在接触抑制现象，即相互接触的细胞能抑制细胞生长和运动. 根据图1实验结果，MCF-10A细胞分别以常规密度(2×104/cm2)和高密度(1×105/cm2)接种，BT-549和SK-BR-3分别以常规密度(2×104/cm2)和高密度(1.5× 105/cm2)接种，然后连续培养2 d，获得常规密度生长细胞和高密度生长细胞，并基于EdU染色法对这两种细胞进行细胞增殖检测. 流式细胞术实验结果表明，与常规密度生长的细胞相比，MCF-10A(图2a)、BT-549(图2b)和SK-BR-3(图2c)细胞的增殖能力在高密度培养下均减弱. 此外，蛋白质印迹(Western blot)检测发现，MCF-10A(图2d)、BT-549(图2e)和SK-BR-3(图2f)细胞生长相关蛋白EGFR与ERK1/2的磷酸化程度都下调，该结果与细胞计数及流式细胞实验一致，表明3株细胞在高密度时生长受到抑制.

    Fig. 2 Growth inhibition of human mammary cells in high density

    NOTE: Cells proliferation were assessed on the basis of EdU incorporation followed by flow cytometry analysis (a–c). Phosphorylation level of EGFR and ERK1/2 in cells were analyzed by Western blotting (d–f). ***P< 0.001.

  • 2.3　外源添加GM1抑制低密度和高密度生长时细胞增殖

    糖鞘脂GM1能够与霍乱毒素B亚基(cholera toxin B subunit，CTB)结合，利用FITC标记的CTB，通过流式细胞检测了低密度(5×103/cm2)、常规密度和高密度接种培养2 d后细胞内GM1含量的变化. 发现相较于常规密度，低密度和高密度接种培养细胞中GM1的表达水平都有所升高 (图3a). 添加不同浓度的GM1处理3种密度接种过夜培养的细胞，36 h后消化细胞计数. MCF-10A(图3b)、BT-549(图3c)和SK-BR-3(图3d)细胞在高密度接种并添加100 μmol/L GM1处理的培养条件下细胞增殖被抑制. 在低密度接种条件下，GM1对细胞增殖的抑制效果更加显著. 而在常规密度接种条件下，GM1对细胞增殖没有明显的抑制作用.

    Fig. 3 Exogenous addition of GM1 to low and high density cells inhibits cell growth

    NOTE: GM1 on the surface of low,normal and high density cells were analyzed by flow cytometry(a). MCF-10A (b),BT-549(c)and SK-BR-3 (d)cells were incubated with different concentration of GM1 for 36 h and followed by cell counting. *P< 0.05,**P< 0.01,***P< 0.001.

  • 2.4　GM1干扰和过表达细胞构建

    糖基转移酶B3GALT4是糖鞘脂GM1的合成酶，为了进一步研究GM1对BT-549和SK-BR-3细胞增殖的调控作用，利用慢病毒转染技术在两株细胞中构建B3GALT4干扰和过表达细胞，以达到改变细胞中GM1含量的目的. 实时荧光定量PCR实验结果证实，在转染细胞中B3GALT4的mRNA水平相比于野生型细胞有显著的下调或提高(图4a). 同时，B3GALT4蛋白水平在转染细胞株中也显著下调或提高(图4b). 最后，通过流式细胞仪检测了转染细胞株中GM1的表达，从图中可以看到(图4c)，B3GALT4干扰或过表达细胞株中，GM1的含量也发生相应变化.

    Fig. 4 Construction of GM1 knockdown and overexpression cells in BT-549 and SK-BR-3

    NOTE: (a)Quantitative real-time RT-PCR determination of B3GALT4 mRNA levels in transfected cells. Values presented as mean ± SD (n= 3). (b) Western blotting analysis of B3GALT4. (c)GM1 expression assayed by flow cytometry.

  • 2.5　转染细胞低密度和高密度生长时细胞增殖变化

    为了近一步研究GM1干扰或过表达后细胞的增殖差异，将野生型细胞和转染细胞按低密度，常规密度和高密度接种后培养2 d并计数. 计数结果表明，无论是BT-549细胞(图5a)，还是SK-BR-3细胞(图5b)，GM1干扰细胞在低密度和高密度生长时增殖能力显著增强，GM1过表达细胞在低密度和高密度生长时增殖能力显著下降，而在常规密度培养时，GM1干扰和过表达细胞增殖能力无显著变化(图5).

    Fig. 5 Proliferation changes of transfected cells in low and high density

    NOTE: BT-549 (a), SK-BR-3 (b) and transfected cell were seeded at different density and cultured for 2 d followed by cell counting. *P< 0.05; **P< 0.01.

  • 2.6　转染细胞低密度和高密度生长时EGFR和ERK1/2磷酸化水平变化

    转染细胞在高密度生长时细胞增殖能力变化表明GM1干扰细胞在高密度生长时接触抑制现象减弱，而过表达细胞在高密度生长时接触抑制现象增强. 为了进一步证明在高密度培养时GM1对细胞增殖的调控作用，将BT-549细胞(图6a)和SK-BR-3细胞(图6b)及其转染株细胞以常规密度和高密度接种，培养2 d后检测生长相关蛋白EGFR和ERK1/2磷酸化水平变化. Western blotting结果表明，GM1干扰细胞在高密度生长时EGFR和ERK1/2磷酸化增强，GM1过表达细胞在高密度生长时EGFR和ERK1/2磷酸化减弱.

    Fig. 6 Phosphorylation level of EGFR and ERK1/2 in transfected cells

    NOTE: Phosphorylation level of EGFR and ERK1/2 in transfected BT-549 (a) and SK-BR-3 (b) cells were analyzed by Western blotting. *P< 0.05; **P< 0.01;***P< 0.001.

  • 2.7　转染细胞高密度生长时EGFR配体激活能力检测

    在高密度培养时，转染细胞EGFR与ERK1/2磷酸化水平有显著差异，为了更进一步研究转染细胞EGFR被生长因子激活能力的变化，将BT-549(图7a)和SK-BR-3(图7b)及其转染株细胞以高密度接种并培养2 d，换无血清培养基饥饿处理 12 h，用100 μg/L EGF刺激10 min后提取蛋白质检测. 结果表明，在高密度培养及EGF刺激下，GM1干扰细胞的EGFR和ERK1/2的磷酸化水平增强，GM1过表达细胞的EGFR和ERK1/2的磷酸化水平减弱，说明在高密度培养时，干扰GM1能增强EGFR被配体激活的能力，过表达GM1能抑制EGFR被配体激活的能力.

    Fig. 7 Phosphorylation level of EGFR and ERK1/2 in EGF-treated transfected cells

    NOTE: Phosphorylation level of EGFR and ERK1/2 in transfected BT-549 (a) and SK-BR-3 (b) cells after EGF stimulation were analyzed by Western blotting. **P< 0.01; ***P< 0.001.

  • 3　讨论

    早在20世纪50年代，研究人员就发现细胞生长受到密度的调控，当细胞生长到相互汇合接触时，其增殖能力受到显著抑制[14]，随后在60年代正式提出了接触抑制的概念[15]. 进一步研究表明，细胞接触抑制受到很多信号通路的调控，其中报道较多的有Hippo信号通路[16]和钙黏蛋白（cadherin）介导的细胞黏附信号通路[17]. 细胞发生癌变后能够抵抗接触抑制，这种能力的转变为研究细胞接触抑制机理提供很好的契机，同时也为癌症的治疗提供不同的研究思路. 某些糖鞘脂被证实能够调控细胞增殖，如GM3能够通过与EGFR的相互作用来抑制许多癌细胞的增殖[18]，GD3和Gb3能够增强人乳腺细胞MCF-10A的接触抑制生长能力[19]. 然而，作为在细胞表面普遍表达的神经节苷脂GM1，对其在细胞增殖中的作用研究较少.

    本文选取人乳腺细胞MCF-10A、人乳腺癌细胞BT-549和SK-BR-3，通过研究细胞不同密度生长时增殖能力的变化、外源添加GM1对细胞增殖的影响、以及构建GM1干扰和过表达细胞株并检测增殖差异等实验，初步探讨了GM1对细胞增殖的调控作用. 实验结果表明，细胞在低密度和高密度生长时细胞增殖均受到抑制且GM1的表达升高. 外源添加GM1能够显著抑制低密度和高密度生长时细胞的增殖能力，而对常规密度生长的细胞没有显著作用. GM1干扰细胞株在低密度和高密度生长时增殖能力有所提高，而GM1过表达细胞株则表现出相反的特性. 此外，无论是干扰还是过表达GM1，在常规密度生长时，其增殖水平均没有表现出明显的差异. 对细胞增殖信号通路相关蛋白EGFR和ERK1/2的检测结果表明，GM1能够抑制EGFR配体激活的磷酸化从而抑制细胞的增殖. 然而不同于GM3通过与EGFR上的N-糖链互作直接抑制EGFR信号通路的激活[20]，GM1对EGFR的调控作用可能更加复杂. 一般来讲，细胞膜上的脂筏区可以分为富含胆固醇和糖鞘脂(包括GM1)的糖脂富集区(glycolipid-enriched microdomains，GEM)和富含小窝蛋白（caveolin）的小窝区(caveolae)[21]. EGFR主要分布在GEM区，少部分在caveolae区，配体介导的EGFR磷酸化主要发生在GEM区，而在caveolae区，EGFR的磷酸化会被抑制[22,23]. 文献表明，caveolae标志蛋白caveolin-1在乳腺癌中表达下调，在乳腺癌细胞中过表达caveolin-1能够抑制细胞增殖[24,25]，caveolin-1能够与EGFR激酶区结合抑制EGFR激活[26]，而过表达GM3能够促进caveolin-1与EGFR共定位，抑制EGFR激活[27]. 而GM1不能与EGFR直接结合抑制其激活[28]，但过表达GM1能将血小板衍生生长因子受体PDGFR赶出GEM区使其不能激活，表明GM1更可能与EGFR存在一定的排斥作用[29]. 另外，研究表明GM1能够抑制人恶性胶质瘤细胞增殖[30].

    综合文献报道及以上实验数据，本文推测：当细胞在常规密度生长时，EGFR在GEM的分布和激活不受GM1的影响，无论是外源添加GM1还是在细胞中过表达GM1，细胞膜上的GEM区仍有足够的空间容纳EGFR. 而当细胞处在高密度生长时，单个细胞的细胞膜面积急剧缩小，此时外源添加GM1或者过表达GM1都会让GEM区填满GM1，迫使EGFR被赶出GEM区至caveolae区或非脂筏区，导致EGFR不能正常激活，细胞增殖被抑制.当然，针对这个假设，目前的研究还有诸多不足，有待于在后续的工作中加以验证.

    总之，本研究首次发现，GM1能够抑制人乳腺细胞MCF-10A、人乳腺癌细胞BT-549和 SK-BR-3在体外低密度和高密度生长时的细胞增殖，推测细胞高密度生长时GM1抑制细胞增殖的可能机理是GM1表达丰富时将EGFR赶出GEM区从而抑制EGFR激活，而对这个机理的阐明将是本课题组研究的重点.

  • 参 考 文 献

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      Hakomori S I. Structure, organization, and function of glycosphingolipids in membrane. Curr Opin Hematol, 2003, 10(1): 16-24

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  • 基本信息

    DOI:10.16476/j.pibb.2018.0238

    中图分类号: Q81

    引用信息

    卓定浩,关锋.糖鞘脂GM1调控生长密度相关的乳腺细胞体外增殖抑制[J].生物化学与生物物理进展,2019,46(05):485-495.

    ZHUO Ding-Hao,） GUAN Feng.Regulation of Glycosphingolipid GM1 on The Density Dependent Inhibition of Human Breast Cell Growth In Vitro[J].Progress in Biochemistry and Biophysics,2019,46(05):485-495.

    基金信息

    国家自然科学基金面上项目(81672537)资助.

    This work was supported by a grant from The National Natural Science Foundation of China (81672537).

    稿件历史

    纸刊出版 : 2019-05-20
    收稿日期 : 2018-11-23
    录用日期 : 2019-03-28

    • 卓定浩

    机 构：

    1. 江南大学糖化学与生物技术教育部重点实验室，无锡 214122

    2. 西北大学生命科学学院，西安 710069

    Affiliation：

    1. Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122， China

    2. The College of Life Sciences, Northwestern University，Xi’an 710069, China

    关锋

    机 构：

    1. 江南大学糖化学与生物技术教育部重点实验室，无锡 214122

    2. 西北大学生命科学学院，西安 710069

    Affiliation：

    1. Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122， China

    2. The College of Life Sciences, Northwestern University，Xi’an 710069, China

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    Fig. 1 Density dependent growth inhibition of human mammary cell

    Fig. 2 Growth inhibition of human mammary cells in high density

    Fig. 3 Exogenous addition of GM1 to low and high density cells inhibits cell growth

    Fig. 4 Construction of GM1 knockdown and overexpression cells in BT-549 and SK-BR-3

    Fig. 5 Proliferation changes of transfected cells in low and high density

    Fig. 6 Phosphorylation level of EGFR and ERK1/2 in transfected cells

    Fig. 7 Phosphorylation level of EGFR and ERK1/2 in EGF-treated transfected cells

    image /

    Cells were seeded at low density (a)or high density (b)and cultured for 4 days. Cells were digested by trypsin and counted using an Automate Cell Counter.

    Cells proliferation were assessed on the basis of EdU incorporation followed by flow cytometry analysis (a–c). Phosphorylation level of EGFR and ERK1/2 in cells were analyzed by Western blotting (d–f). ***P< 0.001.

    GM1 on the surface of low,normal and high density cells were analyzed by flow cytometry(a). MCF-10A (b),BT-549(c)and SK-BR-3 (d)cells were incubated with different concentration of GM1 for 36 h and followed by cell counting. *P< 0.05,**P< 0.01,***P< 0.001.

    (a)Quantitative real-time RT-PCR determination of B3GALT4 mRNA levels in transfected cells. Values presented as mean ± SD (n= 3). (b) Western blotting analysis of B3GALT4. (c)GM1 expression assayed by flow cytometry.

    BT-549 (a), SK-BR-3 (b) and transfected cell were seeded at different density and cultured for 2 d followed by cell counting. *P< 0.05; **P< 0.01.

    Phosphorylation level of EGFR and ERK1/2 in transfected BT-549 (a) and SK-BR-3 (b) cells were analyzed by Western blotting. *P< 0.05; **P< 0.01;***P< 0.001.

    Phosphorylation level of EGFR and ERK1/2 in transfected BT-549 (a) and SK-BR-3 (b) cells after EGF stimulation were analyzed by Western blotting. **P< 0.01; ***P< 0.001.

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      Yu F X, Zhao B, Guan K L. Hippo pathway in organ size control, tissue homeostasis, and cancer. Cell, 2015, 163(4): 811-828

    • 3

      Abercrombie M. Contact inhibition and malignancy. Nature, 1979, 281(5729): 259-262

    • 4

      Zhang W, Liu H T. MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res, 2002, 12(1): 9-18

    • 5

      Ribatti D. A revisited concept: contact inhibition of growth. From cell biology to malignancy. Experimental Cell Research, 2017, 359(1): 17-19

    • 6

      Hakomori S I. Structure, organization, and function of glycosphingolipids in membrane. Curr Opin Hematol, 2003, 10(1): 16-24

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      Hakomori S I. Structure and function of glycosphingolipids and sphingolipids: recollections and future trends. Biochimica et Biophysica Acta, 2008, 1780(3): 325-346

    • 8

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