白血病细胞株分泌的基质金属蛋白酶在中枢神经系统白血病发病机制中的作用
目的 观察白血病细胞分泌的基质金属蛋白酶（MMP）-2和MMP-9对脑微血管内皮细胞（BMVEC）紧密连接蛋白ZO-1、claudin-5、occludin表达及对血脑屏障（BBB）通透性的影响,探讨MMP-2和MMP-9在中枢神经系统白血病（CNSL）发病机制中的作用.方法 ①实时定量PCR检测SHI-1、HL-60、U937细胞MMP-2、MMP-9基因的转录水平;明胶酶谱法检测细胞培养上清中MMP-2和MMP-9蛋白表达;体外穿膜实验观察各白血病细胞株的侵袭能力.②将原代人BMVEC接种于铺有Matrigel胶和纤维黏连蛋白包被的Transwell小室系统中,建立体外BBB模型.将蛋白酶...
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| Published in | 中华血液学杂志 Vol. 37; no. 12; pp. 1070 - 1076 |
|---|---|
| Main Author | |
| Format | Journal Article |
| Language | Chinese |
| Published |
山东省千佛山医院血液病科,250014%215006,苏州大学附属第一医院江苏省血液研究所
2016
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| Subjects | |
| Online Access | Get full text |
| ISSN | 0253-2727 |
| DOI | 10.3760/cma.j.issn.0253-2727.2016.12.012 |
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| Abstract | 目的 观察白血病细胞分泌的基质金属蛋白酶（MMP）-2和MMP-9对脑微血管内皮细胞（BMVEC）紧密连接蛋白ZO-1、claudin-5、occludin表达及对血脑屏障（BBB）通透性的影响,探讨MMP-2和MMP-9在中枢神经系统白血病（CNSL）发病机制中的作用.方法 ①实时定量PCR检测SHI-1、HL-60、U937细胞MMP-2、MMP-9基因的转录水平;明胶酶谱法检测细胞培养上清中MMP-2和MMP-9蛋白表达;体外穿膜实验观察各白血病细胞株的侵袭能力.②将原代人BMVEC接种于铺有Matrigel胶和纤维黏连蛋白包被的Transwell小室系统中,建立体外BBB模型.将蛋白酶抑制剂GM6001处理或未处理的SHI-1、HL-60、U937细胞或MMP-2/MMP-9基因沉默的SHI-1细胞接种于BBB模型的Transwell小室上层与BMVEC共培养,倒置相差显微镜观察BMVEC的形态变化,激光共聚焦显微镜观察紧密连接蛋白ZO-1、claudin-5和occludin的表达,计算白血病细胞的穿膜率.结果 ①SHI-1细胞表达较高转录水平的MMP-2和MMP-9及酶活性,且侵袭能力强于HL-60、U937细胞（P＜0.01）.②与HL-60、SHI-1和U937细胞共培养后,融合致密的BMVEC之间出现间隙、细胞呈单个生长,紧密连接蛋白ZO-1、claudin-5和occludin的表达明显下调,各白血病细胞均不同程度地穿过体外BBB进入Transwell小室下层.其中SHI-1细胞对BMVEC的形态改变及3种紧密连接蛋白的下调最为明显,穿膜率最高.GM6001明显抑制白血病细胞分泌MMP-2和MMP-9,使BMVEC的形态有所恢复,同时上调ZO-1、claudin-5和occludin的表达,降低了BBB的通透性.③用siRNA分别沉默MMP-2和MMP-9基因后,SHI-1细胞分泌MMP-2和MMP-9被抑制,SHI-1细胞穿膜率较沉默前分别下降43.64％和57.30％（P＜0.01）,ZO-1、claudin-5和occludin表达上调.结论 白血病细胞株分泌的MMP-2和MMP-9能通过降解BMVEC紧密连接蛋白ZO-1、claudin-5和occludin而破坏BBB. |
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| AbstractList | 目的 观察白血病细胞分泌的基质金属蛋白酶（MMP）-2和MMP-9对脑微血管内皮细胞（BMVEC）紧密连接蛋白ZO-1、claudin-5、occludin表达及对血脑屏障（BBB）通透性的影响,探讨MMP-2和MMP-9在中枢神经系统白血病（CNSL）发病机制中的作用.方法 ①实时定量PCR检测SHI-1、HL-60、U937细胞MMP-2、MMP-9基因的转录水平;明胶酶谱法检测细胞培养上清中MMP-2和MMP-9蛋白表达;体外穿膜实验观察各白血病细胞株的侵袭能力.②将原代人BMVEC接种于铺有Matrigel胶和纤维黏连蛋白包被的Transwell小室系统中,建立体外BBB模型.将蛋白酶抑制剂GM6001处理或未处理的SHI-1、HL-60、U937细胞或MMP-2/MMP-9基因沉默的SHI-1细胞接种于BBB模型的Transwell小室上层与BMVEC共培养,倒置相差显微镜观察BMVEC的形态变化,激光共聚焦显微镜观察紧密连接蛋白ZO-1、claudin-5和occludin的表达,计算白血病细胞的穿膜率.结果 ①SHI-1细胞表达较高转录水平的MMP-2和MMP-9及酶活性,且侵袭能力强于HL-60、U937细胞（P＜0.01）.②与HL-60、SHI-1和U937细胞共培养后,融合致密的BMVEC之间出现间隙、细胞呈单个生长,紧密连接蛋白ZO-1、claudin-5和occludin的表达明显下调,各白血病细胞均不同程度地穿过体外BBB进入Transwell小室下层.其中SHI-1细胞对BMVEC的形态改变及3种紧密连接蛋白的下调最为明显,穿膜率最高.GM6001明显抑制白血病细胞分泌MMP-2和MMP-9,使BMVEC的形态有所恢复,同时上调ZO-1、claudin-5和occludin的表达,降低了BBB的通透性.③用siRNA分别沉默MMP-2和MMP-9基因后,SHI-1细胞分泌MMP-2和MMP-9被抑制,SHI-1细胞穿膜率较沉默前分别下降43.64％和57.30％（P＜0.01）,ZO-1、claudin-5和occludin表达上调.结论 白血病细胞株分泌的MMP-2和MMP-9能通过降解BMVEC紧密连接蛋白ZO-1、claudin-5和occludin而破坏BBB. 目的 观察白血病细胞分泌的基质金属蛋白酶(MMP)-2和MMP-9对脑微血管内皮细胞(BMVEC)紧密连接蛋白ZO-1、claudin-5、occludin表达及对血脑屏障(BBB)通透性的影响,探讨MMP-2和MMP-9在中枢神经系统白血病(CNSL)发病机制中的作用.方法 ①实时定量PCR检测SHI-1、HL-60、U937细胞MMP-2、MMP-9基因的转录水平;明胶酶谱法检测细胞培养上清中MMP-2和MMP-9蛋白表达;体外穿膜实验观察各白血病细胞株的侵袭能力.②将原代人BMVEC接种于铺有Matrigel胶和纤维黏连蛋白包被的Transwell小室系统中,建立体外BBB模型.将蛋白酶抑制剂GM6001处理或未处理的SHI-1、HL-60、U937细胞或MMP-2/MMP-9基因沉默的SHI-1细胞接种于BBB模型的Transwell小室上层与BMVEC共培养,倒置相差显微镜观察BMVEC的形态变化,激光共聚焦显微镜观察紧密连接蛋白ZO-1、claudin-5和occludin的表达,计算白血病细胞的穿膜率.结果 ①SHI-1细胞表达较高转录水平的MMP-2和MMP-9及酶活性,且侵袭能力强于HL-60、U937细胞(P＜0.01).②与HL-60、SHI-1和U937细胞共培养后,融合致密的BMVEC之间出现间隙、细胞呈单个生长,紧密连接蛋白ZO-1、claudin-5和occludin的表达明显下调,各白血病细胞均不同程度地穿过体外BBB进入Transwell小室下层.其中SHI-1细胞对BMVEC的形态改变及3种紧密连接蛋白的下调最为明显,穿膜率最高.GM6001明显抑制白血病细胞分泌MMP-2和MMP-9,使BMVEC的形态有所恢复,同时上调ZO-1、claudin-5和occludin的表达,降低了BBB的通透性.③用siRNA分别沉默MMP-2和MMP-9基因后,SHI-1细胞分泌MMP-2和MMP-9被抑制,SHI-1细胞穿膜率较沉默前分别下降43.64％和57.30％(P＜0.01),ZO-1、claudin-5和occludin表达上调.结论 白血病细胞株分泌的MMP-2和MMP-9能通过降解BMVEC紧密连接蛋白ZO-1、claudin-5和occludin而破坏BBB. |
| Abstract_FL | Objective To observe the effects of matrix metalloproteinases (MMP)-2 and MMP-9 secreted by leukemic cells on tight junction proteins ZO-1,claudin-5 and occluding and the permeability of the blood-brain barrier (BBB) and explore the mechanisms of MMP-2 and MMP-9 in leukemic cell infiltration of the central nervous system (CNS).Methods The rnRNA expressions of MMP-2 and MMP-9 in leukemic cell lines SHI-1,HL-60 and U937 were detected by quantitative RT-PCR.The MMP inhibitor GM6001 was used to inhibit the secretion of MMP-2 and MMP-9.RNA interference (RNAi) was used to knock down the expression of MMP-2 and MMP-9.Zymography was used to analyze the secretion of MMP-2 and MMP-9 in the supematant of different leukemia cell lines treated or untreated with drugs,as well as the RNAi-treated cells.An in vitro BBB model composed of human brain microvascular endothelial cells (BMVECs) was developed on a Matrigel-based insert.Cell invasion through a barrier of Matrigelbased human basement membrane and the BMVECs-based human BBB barrier was assayed to measure the invasive capacity and the capacity to breakdown the BBB of different leukemia cell lines treated or untreated with drugs,as well as the RNAi-treated cells.The morphologic changes of BMVECs after co-culture with different leukemia cell lines treated or untreated with drugs,as well as the RNAi-treated cells in vitro BBB models were observed by invert microscopy and tight junction proteins in these BMVECs were analyzed with a laser-scanning confocal microscope.Results (①)The mRNA expression in different leukemic cell lines shown a pronounced transcription of MMP-2 and-9,and the transcriptional level in SHI-1 cells was the highest among all leukemic cell lines tested (P＜0.01).The data of activities of MMP-2 and-9 were consistent with the results of mRNA expression and SHI-l displayed higher capacity of invasion (P＜0.01).(②)After incubation 24h with different leukemic cells,the BMVECs disrupted to loss cell-cell contacts and grew in single cell.Confocal imaging showed down-regulations of ZO-1,claudin-5 and occluding accompanied by the disruption of BBB in vitro models.SHI-1 cells had stronger alterations to BMVECs,tight junction proteins and the permeability of the BBB than HL-60 and U937 cells.However,GM6001 and the knock-down of MMP-2 and MMP-9 altered the responses of BBB.They reduced the degradation of three tight junction proteins with a decreased permeability of BBB.Conclusion MMP-2 and MMP-9 secreted by leukemic cells could disrupt the BBB by degrading the tight junction proteins ZO-1,claudin-5 and occluding,which contributed the infiltration of leukemic cell into CNS. |
| Author | 冯洒然 陈子兴 岑建农 沈宏杰 王元元 姚利 |
| AuthorAffiliation | 苏州大学附属第一医院江苏省血液研究所,215006 |
| AuthorAffiliation_xml | – name: 山东省千佛山医院血液病科,250014%215006,苏州大学附属第一医院江苏省血液研究所 |
| Author_FL | Cen Jiannong Feng Saran Wang Yuanyuan Shen Hongjie Chen Zixing Yao Li |
| Author_FL_xml | – sequence: 1 fullname: Feng Saran – sequence: 2 fullname: Chen Zixing – sequence: 3 fullname: Cen Jiannong – sequence: 4 fullname: Shen Hongjie – sequence: 5 fullname: Wang Yuanyuan – sequence: 6 fullname: Yao Li |
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| DocumentTitleAlternate | Critical roles of matrix metalloproteinases secreted by leukemic cells in the pathogenesis of central nervous system leukemia |
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| Keywords | 血脑屏障 Matrix metalloproteinases Blood-brain barrier U937 cells U937细胞 SHI-1细胞 HL-60 cells SHI-1 cells 基质金属蛋白酶类 HL-60细胞 |
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| Notes | Objective To observe the effects of matrix metalloproteinases （MMP）-2 and MMP-9 secreted by leukemic cells on tight junction proteins ZO-1,claudin-5 and occluding and the permeability of the blood-brain barrier （BBB） and explore the mechanisms of MMP-2 and MMP-9 in leukemic cell infiltration of the central nervous system （CNS）.Methods The rnRNA expressions of MMP-2 and MMP-9 in leukemic cell lines SHI-1,HL-60 and U937 were detected by quantitative RT-PCR.The MMP inhibitor GM6001 was used to inhibit the secretion of MMP-2 and MMP-9.RNA interference （RNAi） was used to knock down the expression of MMP-2 and MMP-9.Zymography was used to analyze the secretion of MMP-2 and MMP-9 in the supematant of different leukemia cell lines treated or untreated with drugs,as well as the RNAi-treated cells.An in vitro BBB model composed of human brain microvascular endothelial cells （BMVECs） was developed on a Matrigel-based insert.Cell invasion through a barrier of Matrigelbased human basement membrane and the BMVECs-based hum |
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| Publisher | 山东省千佛山医院血液病科,250014%215006,苏州大学附属第一医院江苏省血液研究所 |
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| Snippet | 目的 观察白血病细胞分泌的基质金属蛋白酶（MMP）-2和MMP-9对脑微血管内皮细胞（BMVEC）紧密连接蛋白ZO-1、claudin-5、occludin表达及对血脑屏障（BBB）通透性的影响,探... 目的 观察白血病细胞分泌的基质金属蛋白酶(MMP)-2和MMP-9对脑微血管内皮细胞(BMVEC)紧密连接蛋白ZO-1、claudin-5、occludin表达及对血脑屏障(BBB)通透性的影响,探讨MMP-2... |
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| SubjectTerms | HL-60细胞 SHI-1细胞 U937细胞 基质金属蛋白酶类 血脑屏障 |
| Title | 白血病细胞株分泌的基质金属蛋白酶在中枢神经系统白血病发病机制中的作用 |
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