MMP2在人脑胶质瘤中的表达及姜黄素对胶质瘤细胞和HUVEC生物学特性的影响
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摘要
背景:胶质瘤是最常见的原发性颅内肿瘤,传统治疗手段不能将其治愈,总的来说患者疗效不理想。因而深入研究胶质瘤发生发展的细胞及分子机制具有重大现实意义。血管新生是生理性及病理性血管生成的主要形式,研究发现实体性肿瘤的生长高度依赖于血管的新生。绝大部分脑胶质瘤属于实体性肿瘤,研究表明它亦具有高度血管化的的特征,它通过获得新生血管供应的O_2及营养物质,不断生长增殖并向周围组织侵袭。因此,抗血管生成是治疗胶质瘤极具希望的策略之一,但其血管新生的调节机制目前尚不清楚,寻找和识别在血管生成过程中的调控因子将是抗胶质瘤血管生成的有效途径,具有重要意义。
基质金属蛋白酶-2(MMP2)是蛋白水解酶家族的成员之一,MMP2以无活性酶原的形式由多种细胞(成纤维细胞、巨噬细胞、内皮细胞和恶性肿瘤细胞等)分泌,在激活剂的作用下转变为成熟的酶。MMP2不仅可以降解细胞外基质,使细胞间质的空隙增大,为肿瘤血管的形成提供有利的空间,还可以增强内皮细胞移动的能力,通过提高促血管生成因子间接参与血管的形成,以及作为促进血管形成的因子直接参与新生血管的形成。MMP2将有望成为抗血管生成治疗的靶点。目前已经有一些关于各种肿瘤MMP2表达的报道,人脑胶质瘤中也有报道表达,但其与胶质瘤病理性血管的关系,目前国内外无相关研究报道。
姜黄素是姜黄中提取的一种植物多酚,也是姜黄发挥药理作用最重要的活性成分。近年的研究不仅证明了姜黄的传统作用,如抗炎、抗氧化、清除氧自由基、抗人类免疫缺陷病毒、保护肝脏和肾脏、抗纤维化等;而且还揭示出一些新的药理作用,如防癌抗癌、抗血管生成等作用。
目的:探讨MMP2在人脑胶质瘤组织和细胞中的表达情况;探讨姜黄素对胶质瘤细胞MMP2表达和对胶质瘤细胞、HUVEC生物学特性的影响。
方法:收集38例新鲜胶质瘤标本,其中包括18例低度恶性(Ⅰ-Ⅱ级)及20例高度恶性者(Ⅲ-Ⅳ级);另取术前无瞳孔散大的脑外伤病人行内减压手术的新鲜脑组织7例作为对照。用RT-PCR方法检测其MMP2 mRNA的表达。另收集这些胶质瘤组织和脑组织的石蜡标本,用免疫组织化学检测MMP2蛋白、Ⅷ因子、Ki-67的表达情况。并分析MMP2蛋白的表达与胶质瘤的病理分级、胶质瘤Ki-67标记指数(LI)、微血管密度(MVD)的关系。
用姜黄素干预胶质瘤细胞,检测姜黄素对胶质瘤细胞MMP2表达的影响;再用姜黄素干预胶质瘤细胞和HUVEC,用MTT法检测胶质瘤细胞细胞及HUVEC的增殖活性;结晶紫染色法检测HUVEC的粘附及迁移能力。
结果:①检测到MMP2 mRNA及其蛋白在人脑胶质瘤中存在表达,MMP2蛋白表达主要定位于胶质瘤细胞的胞浆,且其表达与肿瘤的恶性程度(χ~2=3.993,P＜0.01),Ki-67(r=0.661,P＜0.01)及MVD(r=0.557,P＜0.01)有正相关性;MMP2在正常脑组织中没有表达。②胶质瘤细胞中存在MMP2mRNA的表达。姜黄素对胶质瘤细胞中MMP2 mRNA表达有显著的下调作用(P＜0.01)。③加入姜黄素干预后,胶质瘤细胞的增殖活性亦明显低于对照组(P＜0.01);姜黄素对单纯和混合培养的HUVEC增殖活性、迁移能力有明显影响(P＜0.01)。
结论:
1.人脑胶质瘤组织和细胞中可检测出MMP2表达。MMP2的表达与胶质瘤恶性程度、Ki-67及MVD具有正相关性。提示MMP2在胶质瘤的生长、侵袭中发挥重要作用。
2.姜黄素可以下调胶质瘤细胞MMP2 mRNA的表达。
3.姜黄素可以明显抑制胶质瘤细胞的增殖活性。
4.姜黄素可以抑制HUVEC的增殖、粘附、迁徙能力。提示姜黄素还可以抑制血管生成,起到抗肿瘤和抗血管生成的双重作用。
BACKGROUND:Gliomas are the most common brain malignancy tumor and are marked by an extremely poor prognosis,despite advances in the traditional treatment.Thus,gliomas require much further research to understand the molecular and cellular clinical basis.Angiogenesis is needed in several physiological and pathological processes.The growth of solid tumors depends on new blood vessels.The most of gliomas is a kind of solid tumors which is among the best vascularized human tumors.The proliferation and invasion of them are dependent on the angiogenesis in order to acquire O2 and nutritive material.Furthermore,angiogenesis is crucial to the growth of malignant gliomas too.Therefore,antiangiogenic treatment may be a promising therapeutic modality for gliomas.But little is known about its regulatory mechanism of angiogenesis.To look for and identify a key regulator in this process may be very important.
Matrix metalloproteinase -2(MMP-2) is a member of the protease family. MMP-2 is secreted by a variety of cells such as fibroblasts,Macrophages, endothelial cells and malignant cells and so on,as an inactive zymogen.Actived by activators,MMP-2 may turn into a mature enzyme.MMP-2 can not only degrade extracellular matrix,increase the gap of stromal cells in order to provide enough space for the formation of tumor blood vessels,but also can enhance the movement ability of endothelial cells,and promote angiogenesis indirectly by increasing angiogenesis factor,as well as a factor directly involved in the formation of neovascularization.So,MMP2 is expected to become a key target for the antiangiogenic therapy.At present,there are already some reports on the expression of MMP2 in kinds of tumors.But the relationship between MMP2 and angiogenesis is currently not reported in gliomas.We can only surmise that MMP2 play an important role in gliomas angiogenesis.
Curcumin extracted from Curcuma longa is a kind of plant polyphenols, which is the most important ingredient to exert pharmacological effects.It is reported in recent years that the curcumin not only has traditional roles,such as antiinflammatory,antioxidant,anti human immunodeficiency virus,the protection of liver and kidney,antifibrosis,etc;but also has some new pharmacological effects,such as anticancer,antiangiogenesis.
OBJECTIVE:The present study was carried out to investigate the expression of MMP2 in human glioma tissues and cells.Explore the effect of curcumin on the expression of MMP2 in glioma cells.Discuss the effect of curcumin on glioma cells and HUVEC biological characteristics.
METHODS:Fresh tissues of 38 cases of glioma,including low malignance 18 (gradeⅠ～Ⅱ) and high malignance 20(gradeⅢ～Ⅳ),and fresh tissues of 7 cases of cerebral trauma were used in the present study.The expression of MMP2 mRNA by these glioma tissues was examined by RT-PCR.Another collection of these paraffin-embedded glioma specimens,were used to assess the expression status of MMP2 protein,FactorⅧand Ki-67 by immunohistochemical staining.The Ki-67 labeling index(LI) and microvessel density(MVD) were calculated.The correlation between MMP2 expression,the Ki-67 LI or the MVD and the pathological grade of glioma was investigated.
Changes of the expression of MMP2 mRNA on glioma cells infected with curcumin was examined by RT-PCR.Transwell method in vitro to mimic the effect of tumor cells on endothelial cells in microecological environment of glioma in vivo.Infected with curcumin cell proliferative activity of glioma cells and HUVECs was measured by MTT assay.Adhesion and migration capability of HUVECs were evaluated by Methyl Violet staining.
RESULTS:①The expression of MMP2 mRNA and protein in human gliomas was detected.MMP2 protein located in plasm of tumor cells and its expression correlated directly with the degree of malignancy(χ~2 = 3.993, P＜0.01),Ki-67 LI(r=0.661,P＜0.01) and MVD(r=0.557,P＜0.01).However,it was not detected in normal brain.②MMP2 mRNA exist in glioma cells.Effect of curcumin on the expression of glioma cells MMP2 mRNA has significant down regulation(P＜0.01).③Cell proliferation activity of glioma cells was inhibited significantly by curcumin group compared with that in negative control group(P＜0.01);Proliferative activity,migration capability were different between the two groups(P＜0.01) in culture system.
CONCLUSION:
1.Human glioma cells and glioma tissues can express MMP2.The level of MMP2 expression directly correlates with the degree of malignancy,Ki-67 LI and MVD.We prompt MMP2 play an important role in glioma growth, invasion.
2.Curcumin can lower the expression of MMP2 mRNA.
3.Curcumin can inhibit the proliferation of glioma cells.
4.Curcumin can inhibit the proliferation,adhesion and migration of HUVECs.Curcumin has the role of antiangiogenesis.
基质金属蛋白酶-2(MMP2)是蛋白水解酶家族的成员之一,MMP2以无活性酶原的形式由多种细胞(成纤维细胞、巨噬细胞、内皮细胞和恶性肿瘤细胞等)分泌,在激活剂的作用下转变为成熟的酶。MMP2不仅可以降解细胞外基质,使细胞间质的空隙增大,为肿瘤血管的形成提供有利的空间,还可以增强内皮细胞移动的能力,通过提高促血管生成因子间接参与血管的形成,以及作为促进血管形成的因子直接参与新生血管的形成。MMP2将有望成为抗血管生成治疗的靶点。目前已经有一些关于各种肿瘤MMP2表达的报道,人脑胶质瘤中也有报道表达,但其与胶质瘤病理性血管的关系,目前国内外无相关研究报道。
姜黄素是姜黄中提取的一种植物多酚,也是姜黄发挥药理作用最重要的活性成分。近年的研究不仅证明了姜黄的传统作用,如抗炎、抗氧化、清除氧自由基、抗人类免疫缺陷病毒、保护肝脏和肾脏、抗纤维化等;而且还揭示出一些新的药理作用,如防癌抗癌、抗血管生成等作用。
目的:探讨MMP2在人脑胶质瘤组织和细胞中的表达情况;探讨姜黄素对胶质瘤细胞MMP2表达和对胶质瘤细胞、HUVEC生物学特性的影响。
方法:收集38例新鲜胶质瘤标本,其中包括18例低度恶性(Ⅰ-Ⅱ级)及20例高度恶性者(Ⅲ-Ⅳ级);另取术前无瞳孔散大的脑外伤病人行内减压手术的新鲜脑组织7例作为对照。用RT-PCR方法检测其MMP2 mRNA的表达。另收集这些胶质瘤组织和脑组织的石蜡标本,用免疫组织化学检测MMP2蛋白、Ⅷ因子、Ki-67的表达情况。并分析MMP2蛋白的表达与胶质瘤的病理分级、胶质瘤Ki-67标记指数(LI)、微血管密度(MVD)的关系。
用姜黄素干预胶质瘤细胞,检测姜黄素对胶质瘤细胞MMP2表达的影响;再用姜黄素干预胶质瘤细胞和HUVEC,用MTT法检测胶质瘤细胞细胞及HUVEC的增殖活性;结晶紫染色法检测HUVEC的粘附及迁移能力。
结果:①检测到MMP2 mRNA及其蛋白在人脑胶质瘤中存在表达,MMP2蛋白表达主要定位于胶质瘤细胞的胞浆,且其表达与肿瘤的恶性程度(χ~2=3.993,P＜0.01),Ki-67(r=0.661,P＜0.01)及MVD(r=0.557,P＜0.01)有正相关性;MMP2在正常脑组织中没有表达。②胶质瘤细胞中存在MMP2mRNA的表达。姜黄素对胶质瘤细胞中MMP2 mRNA表达有显著的下调作用(P＜0.01)。③加入姜黄素干预后,胶质瘤细胞的增殖活性亦明显低于对照组(P＜0.01);姜黄素对单纯和混合培养的HUVEC增殖活性、迁移能力有明显影响(P＜0.01)。
结论:
1.人脑胶质瘤组织和细胞中可检测出MMP2表达。MMP2的表达与胶质瘤恶性程度、Ki-67及MVD具有正相关性。提示MMP2在胶质瘤的生长、侵袭中发挥重要作用。
2.姜黄素可以下调胶质瘤细胞MMP2 mRNA的表达。
3.姜黄素可以明显抑制胶质瘤细胞的增殖活性。
4.姜黄素可以抑制HUVEC的增殖、粘附、迁徙能力。提示姜黄素还可以抑制血管生成,起到抗肿瘤和抗血管生成的双重作用。
BACKGROUND:Gliomas are the most common brain malignancy tumor and are marked by an extremely poor prognosis,despite advances in the traditional treatment.Thus,gliomas require much further research to understand the molecular and cellular clinical basis.Angiogenesis is needed in several physiological and pathological processes.The growth of solid tumors depends on new blood vessels.The most of gliomas is a kind of solid tumors which is among the best vascularized human tumors.The proliferation and invasion of them are dependent on the angiogenesis in order to acquire O2 and nutritive material.Furthermore,angiogenesis is crucial to the growth of malignant gliomas too.Therefore,antiangiogenic treatment may be a promising therapeutic modality for gliomas.But little is known about its regulatory mechanism of angiogenesis.To look for and identify a key regulator in this process may be very important.
Matrix metalloproteinase -2(MMP-2) is a member of the protease family. MMP-2 is secreted by a variety of cells such as fibroblasts,Macrophages, endothelial cells and malignant cells and so on,as an inactive zymogen.Actived by activators,MMP-2 may turn into a mature enzyme.MMP-2 can not only degrade extracellular matrix,increase the gap of stromal cells in order to provide enough space for the formation of tumor blood vessels,but also can enhance the movement ability of endothelial cells,and promote angiogenesis indirectly by increasing angiogenesis factor,as well as a factor directly involved in the formation of neovascularization.So,MMP2 is expected to become a key target for the antiangiogenic therapy.At present,there are already some reports on the expression of MMP2 in kinds of tumors.But the relationship between MMP2 and angiogenesis is currently not reported in gliomas.We can only surmise that MMP2 play an important role in gliomas angiogenesis.
Curcumin extracted from Curcuma longa is a kind of plant polyphenols, which is the most important ingredient to exert pharmacological effects.It is reported in recent years that the curcumin not only has traditional roles,such as antiinflammatory,antioxidant,anti human immunodeficiency virus,the protection of liver and kidney,antifibrosis,etc;but also has some new pharmacological effects,such as anticancer,antiangiogenesis.
OBJECTIVE:The present study was carried out to investigate the expression of MMP2 in human glioma tissues and cells.Explore the effect of curcumin on the expression of MMP2 in glioma cells.Discuss the effect of curcumin on glioma cells and HUVEC biological characteristics.
METHODS:Fresh tissues of 38 cases of glioma,including low malignance 18 (gradeⅠ～Ⅱ) and high malignance 20(gradeⅢ～Ⅳ),and fresh tissues of 7 cases of cerebral trauma were used in the present study.The expression of MMP2 mRNA by these glioma tissues was examined by RT-PCR.Another collection of these paraffin-embedded glioma specimens,were used to assess the expression status of MMP2 protein,FactorⅧand Ki-67 by immunohistochemical staining.The Ki-67 labeling index(LI) and microvessel density(MVD) were calculated.The correlation between MMP2 expression,the Ki-67 LI or the MVD and the pathological grade of glioma was investigated.
Changes of the expression of MMP2 mRNA on glioma cells infected with curcumin was examined by RT-PCR.Transwell method in vitro to mimic the effect of tumor cells on endothelial cells in microecological environment of glioma in vivo.Infected with curcumin cell proliferative activity of glioma cells and HUVECs was measured by MTT assay.Adhesion and migration capability of HUVECs were evaluated by Methyl Violet staining.
RESULTS:①The expression of MMP2 mRNA and protein in human gliomas was detected.MMP2 protein located in plasm of tumor cells and its expression correlated directly with the degree of malignancy(χ~2 = 3.993, P＜0.01),Ki-67 LI(r=0.661,P＜0.01) and MVD(r=0.557,P＜0.01).However,it was not detected in normal brain.②MMP2 mRNA exist in glioma cells.Effect of curcumin on the expression of glioma cells MMP2 mRNA has significant down regulation(P＜0.01).③Cell proliferation activity of glioma cells was inhibited significantly by curcumin group compared with that in negative control group(P＜0.01);Proliferative activity,migration capability were different between the two groups(P＜0.01) in culture system.
CONCLUSION:
1.Human glioma cells and glioma tissues can express MMP2.The level of MMP2 expression directly correlates with the degree of malignancy,Ki-67 LI and MVD.We prompt MMP2 play an important role in glioma growth, invasion.
2.Curcumin can lower the expression of MMP2 mRNA.
3.Curcumin can inhibit the proliferation of glioma cells.
4.Curcumin can inhibit the proliferation,adhesion and migration of HUVECs.Curcumin has the role of antiangiogenesis.
引文
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