CGRP在人骨髓间充质干细胞增殖中的作用研究
摘要
降钙素基因相关肽(CGRP)是一种在骨组织中广泛分布的神经肽,已发现,CGRP与骨组织的增殖、塑形密切相关。通过组织学研究,在骨折愈合试验中观察到,骨组织中,含CGRP的神经纤维主要分布在骨生成和塑形活跃的区域,例如在长骨的干骺端分布就比骨干部位高约10倍,且神经分布及CGRP含量的变化与骨折痊愈的过程紧密相关。研究证实,CGRP能显著促进成骨细胞增殖,增强成骨活性。
骨髓间充质干细胞(MSCs)是成骨细胞重要的来源干细胞,也是骨组织工程的主要种子细胞来源。MSCs的增值能力,直接影响骨的生长、塑形和修复,也对组织工程骨的构建效能和成骨能力起着决定性的作用。因此,对影响MSCs增殖的因素的研究,一直是骨科界和组织工程学界研究的重点。
目前的研究已经发现,MSCs在骨组织的分布,主要集中在干骺端的红骨髓中,这与CGRP能的神经分布相符合,相关研究也发现,CGRP能促进骨髓有核细胞集落的形成。但是关于CGRP对MSCs的增殖的影响及其机制,仍有大量研究需要深入。
第一部分人骨髓间充质干细胞表面CGRP受体存在证据
目的:寻找人骨髓MSCs表面存在CGRP受体的确切证据(从基因及蛋白质表达水平)
方法:原材料采自健康志愿者骨髓,应用梯度离心法及贴壁培养筛选,获得MSCs细胞。体外培养扩增后,取对数生长期细胞,采用RT-PCR技术进行细胞CGRP受体mRNA表达检测;采用杂交瘤技术,获得兔抗人CGRP受体蛋白抗体,利用该抗体,使用Westernblot技术进行MSCs表达CGRP受体蛋白检测。
结果:采用梯度离心及贴壁培养筛选,细胞纯度较高,表面标记稳定,且与文献报道相符;经RT-PCR检测结果,证实人骨髓MSCs表达CGRP受体mRNA;采用杂交瘤技术获得的兔抗人CGRP受体抗体,结构稳定,滴度较高,能较好的满足Westernblot试验需要;采用Westernblot的DAB显色法,检测出人MSCs表达CGRP受体蛋白,且表达量较大。
结论:采用梯度离心及贴壁培养法获得MSCs纯度较高,增殖效果稳定;经过RT-PCR检测,证实MSCs表达CGRP受体mRNA;Western-blot检测证实,MSCs表达CGRP受体蛋白。
第二部分CGRP对人骨髓间充质干细胞增殖的作用
目的:观察在添加外源性CGRP的情况下,MSCs增殖的改变,并证实这种改变与添加CGRP间的关联性
方法:MSCs获取仍采自健康志愿者骨髓,应用梯度离心法及贴壁培养筛选,获得MSCs细胞。分组采用对照组、CGRP组(根据CGRP添加的终浓度分为10-7mol/ L、10-8mol/ L和10-9mol/ L三组),以MTT法检测细胞增殖曲线变化;体外培养扩增后,取相同时相点,光镜下进行细胞形态观察;将各组细胞在第三代传代后72小时的对数生长期,进行细胞周期检测,观察各组细胞处在DNA合成期及细胞分裂前期的比例。
结果:经过对照培养,细胞增殖同时相点,对照组细胞细胞密度低于各实验组,细胞形态各组均较为典型、规则;细胞周期检测,各实验组处于细胞分裂前期及DNA合成期细胞比例明显高于对照组,各实验组间该比例为,10-8mol/ L>10-9mol/ L组>10-7mol/ L,但各实验组间差异无统计学意义;MTT组检测,在对数生长期,各实验组增殖速率高于对照组,实验组间细胞增殖速率为10-8mol/ L>10-9mol/ L组>10-7mol/ L。10-8mol/ L速率与另外两实验组间差异有统计学意义,其余两组间无显著性差异
结论:采用MTT法,证实添加外源性CGRP能促进对数增殖期MSCs细胞增殖速度;采用流失细胞法检测,证实,外源性CGRP能提高MSCs细胞处于DNA合成和有丝分裂前期的比例。
第三部分: CGRP对人骨髓间充质干细胞细胞信号传导的变化的影响的研究
目的:观察在添加外源性CGRP的情况下,MSCs胞间通讯连接的改变,并验证改变与CGRP的关联性
方法:MSCs获取仍采自健康志愿者骨髓,应用梯度离心法及贴壁培养筛选,获得MSCs细胞。分组采用对照组、CGRP组和拮抗剂组,放射免疫法检测各组胞间信号分子cAMP含量改变;使用CFDA染料,应用激光共聚焦技术观察细胞间缝隙连接和胞间信号传导能力变化;采用荧光定量PCR技术检测缝隙连接分子Cx43mRNA表达变化。
结果:放免法检测结果显示,各组间,以CGRP组胞间cAMP含量最高,与其余两组间存在统计学差异;CGRP结合拮抗剂组的含量高于对照组,后两者间无统计学差异。采用激光共聚焦技术,结合CFDA生物活性染料,显示CGRP组荧光信号恢复幅度较对照组及拮抗剂组大,差异有显著性(P<0.05);拮抗剂组恢复幅度较对照组大,但两组间差异不具有显著性(P>0.05)。三组细胞Cx43mRNA表达,CGRP组表达量高于抑制剂组及对照组,差异有显著性(P<0.05);抑制剂组表达量高于对照组,两者间差异无显著性。
结论:CGRP能促进MSCs胞间缝隙连接,促进缝隙连接蛋白的基因表达。
第四部分:CGRP对人骨髓间充质干细胞细胞增殖相关因子的作用
目的:研究在添加外源性CGRP的情况下,与MSCs增殖及分化相关的细胞因子IGF-1、BMP-2的受体mRNA表达变化;研究外源性CGRP是否造成MSCs对成骨诱导因子BMP-2的mRNA表达。
方法:MSCs获取、分离及培养方法同前。实验分组为对照组、CGRP组和拮抗剂组,采用荧光定量PCR技术分别检测增殖相关因子IGF-1及其受体mRNA表达;以及成骨分化因子BMP-2及其受体mRNA表达。
结果:采用相对定量技术,CGRP组MSCs表达IGF-1、IGF-1受体以及BMP-2受体mRNA量高于拮抗剂组及对照组,且差异有统计学意义。而三组在表达BMP-2 mRNA无显著差异,且Ct值≥35。
结论:外源性CGRP能够提高MSCs表达IGF-1、IGF-1受体以及BMP-2受体mRNA,而不能诱导MSCs表达BMP-2mRNA。三组BMP-2的mRNA平均Ct值均在35左右,可以认为阴性表达。
Calcitonin gene-related peptide(CGRP) is a kind of peptide that existes widely in the bone tissue.It has been proved that CGRP has affect on the proliferation and formation .In the period of bone fracture healing ,nervouses fiber containing CGRP were observed in the histological way.Nervous fiber containing CGRP distribute mainly in the area that bone grows and remodifies actively.For example ,the content of nervous containing CGRP in the metaphysis of long bone is 10 times higher than the content in the backbone.And the nervous distribut and the content of CGRP have high correlation with the period of fracture healing.There has evidence that CGRP can significantly stimulate the proliferation of osteoblast,and promote the avtivety of osteoblast.
Mesenchymal stem cells(MSCs)is one of the sources stem cell of osteoblast.And it is also be the main seed cell in the tissue engineering bone.The ability of proliferation of MSCs will affect the growth , modify and repairation of bone directely,and it also has decisive affect on construction and osteogenic ability of tissue engineering bone.So ,study in this field ,is always be the emphersize on the orthopedic field and tissue engineering field.
As all know,MSCs distribute mostly in the red bone marrow.which coincide the nervous distribution which contains CGRP.People has found that CGRP can promote colony of the bone marrow karyocyte.But there still has much work should do in this field. Part I The evidence of CGRP receptor on MSCs
Objective:To get specifical evidence that CGRP receptor exist on human MSCs
Methods:Bone marrow is come from healthy young volunteers.MSCs were colleced by gradientcentrifugation and adherent culture.After prolifed in vivo,RT-PCR was used to detect CGRP receptor in MSCs in logarithmic growth phase. Hybridoma technique was used to produce rabbit-anti-human CGRP receptor,which was used in the Westernblot test to detect CGRP receptor protein produced in human MSCs.
Result:MSCs collected by gradientcentrifugation and cultured by adherent culture has high purity and proliferation effect.It was proved through RT-PCR that MSCs express CGRP-receptor mRNA ,and it was also be proved through Western-blot that MSCs express CGRP-receptor protein.
Conclusion:Combined gradientcentrifugation with adherent culture,high purity MSCs could be collected.With RT-PCR test ,it was proved that MSCs express CGRP receptor mRNA. With Westernblot test ,it was proved that MSCs express CGRP receptor protein.
Part II The effect of CGRP on proliferation of MSCs
Objective:To observe the change of proliferation of MSCs with exogenous CGRP,and to explor the correlation. Methods:Human MSCs were aparted into 3 group decided by the concentration of
CGRP.Cell proliferatinon was detected through MTT test.Cell form in each group was detected through optical microscope,in the same time point.Cell cycle was detected with Flowcytometry to analyze the ratio of cell in the mitotic time.
Result:According to the contrast culture test ,cell in all the groups has regular and typical cellular morphology.In the same detect time point ,the cell density of control group was lower than density of each experimental group.In the cell cycle test ,in the logarithmic growth phase,the rates order of cells in the prophase and DNA synthesis phase in each group was 10-8mol/ LCGRP group,10-9mol/ LCGRP group,10-7mol/ LCGRP group and the control group.But between each group,there was no stastical difference.The MTT test showed similar result ,the 10-8mol/ LCGRP group had the highest proliferation speed ,and the control group had the lowest.There had stastical difference between experimental group and control group.There also had stastic differenc between the 10-8mol/ LCGRP group and the other two experimental group.
Conclusion:With MTT test ,it had been proved that exogenous CGRP can accelerate the proliferation speed in the logarithmic growth phase.With flowcytometry,it had been proved that exogenous CGRP can raise the ratio of the cell in the DNA synthesis period and mitosis prophase.
Part III Effect of CGRP on intracellular communication of MSCs
Objective:To observe the intracellular communication of MSCs with exogenous CGRP and to expleor the relationship between the change of intracellular communication of MSCs and exogenous CGRP。
Methods:MSCs collected from healthy volunteer was aparted into 3 group,the control group ,the antagon group and the experimental group. Intracellular communication medium molecule was detected through radioimmunoassay;Intracellular communication and signal conduction were dected through CFDA fluorescent dye The espression of Cx43mRNA was dected through Realtime-PCR.
Result:The result of radioimmunoassay showed that cAMP content in the CGRP group was higher than the other two groups,with ststical difference. Confocal laser-CFDA test had shown that recovery of florescent light of the experimental group is much higher than the other two groups with stastic difference. The recovery range of antagon group was larger than that of control group ,but,without stastic difference.Expression of Cx43mRNA in experimental group was higher than the other two groups , but,without stastic difference.
Conclusion:CGRP can promote not only intracellular communication of MSCs but also the expression of Cx43mRNA.
Part IV effect of CGRP on proliferation related biological factor of MSCs
Objective:To observe the change of IGF-1、BMP-2 and their receptor in the MSCs cell with exogenous CGRP.To explor whether exogenous CGRP will induce MSCs to express the osteo-induced factor and it’s receptor.
Methods:MSCs collected from healthy volunteer was aparted into 3 group,the control group ,the antagon group and the experimental group. The mRNA expressions of proliferation related biological factor of MSCs were detected through Real-time PCR.
Result:It was proved that the mRNA expressions of IGF-1,IGF-1receptor and BMP-2 receptor in experimental group were higher than that in control group with stastic difference.The the mRNA expressions of BMP-2 in all the groups had no stastic difference.And Ct index in all the groups were higner than 35.
Result:MSCs collected by gradientcentrifugation and cultured by adherent culture has high purity and proliferation effect.It was proved through RT-PCR that MSCs express CGRP-receptor mRNA ,and it was also be proved through Western-blot that MSCs express CGRP-receptor protein.
Conclusion:The exogenous CGRP can increase the mRNA expression of IGF-1,IGF-1receptor and BMP-2 receptor of MSCs.In all the grops ,the Ct indexes of BMP-2mRNA were higher than 35,which could be considered as negative expression.
骨髓间充质干细胞(MSCs)是成骨细胞重要的来源干细胞,也是骨组织工程的主要种子细胞来源。MSCs的增值能力,直接影响骨的生长、塑形和修复,也对组织工程骨的构建效能和成骨能力起着决定性的作用。因此,对影响MSCs增殖的因素的研究,一直是骨科界和组织工程学界研究的重点。
目前的研究已经发现,MSCs在骨组织的分布,主要集中在干骺端的红骨髓中,这与CGRP能的神经分布相符合,相关研究也发现,CGRP能促进骨髓有核细胞集落的形成。但是关于CGRP对MSCs的增殖的影响及其机制,仍有大量研究需要深入。
第一部分人骨髓间充质干细胞表面CGRP受体存在证据
目的:寻找人骨髓MSCs表面存在CGRP受体的确切证据(从基因及蛋白质表达水平)
方法:原材料采自健康志愿者骨髓,应用梯度离心法及贴壁培养筛选,获得MSCs细胞。体外培养扩增后,取对数生长期细胞,采用RT-PCR技术进行细胞CGRP受体mRNA表达检测;采用杂交瘤技术,获得兔抗人CGRP受体蛋白抗体,利用该抗体,使用Westernblot技术进行MSCs表达CGRP受体蛋白检测。
结果:采用梯度离心及贴壁培养筛选,细胞纯度较高,表面标记稳定,且与文献报道相符;经RT-PCR检测结果,证实人骨髓MSCs表达CGRP受体mRNA;采用杂交瘤技术获得的兔抗人CGRP受体抗体,结构稳定,滴度较高,能较好的满足Westernblot试验需要;采用Westernblot的DAB显色法,检测出人MSCs表达CGRP受体蛋白,且表达量较大。
结论:采用梯度离心及贴壁培养法获得MSCs纯度较高,增殖效果稳定;经过RT-PCR检测,证实MSCs表达CGRP受体mRNA;Western-blot检测证实,MSCs表达CGRP受体蛋白。
第二部分CGRP对人骨髓间充质干细胞增殖的作用
目的:观察在添加外源性CGRP的情况下,MSCs增殖的改变,并证实这种改变与添加CGRP间的关联性
方法:MSCs获取仍采自健康志愿者骨髓,应用梯度离心法及贴壁培养筛选,获得MSCs细胞。分组采用对照组、CGRP组(根据CGRP添加的终浓度分为10-7mol/ L、10-8mol/ L和10-9mol/ L三组),以MTT法检测细胞增殖曲线变化;体外培养扩增后,取相同时相点,光镜下进行细胞形态观察;将各组细胞在第三代传代后72小时的对数生长期,进行细胞周期检测,观察各组细胞处在DNA合成期及细胞分裂前期的比例。
结果:经过对照培养,细胞增殖同时相点,对照组细胞细胞密度低于各实验组,细胞形态各组均较为典型、规则;细胞周期检测,各实验组处于细胞分裂前期及DNA合成期细胞比例明显高于对照组,各实验组间该比例为,10-8mol/ L>10-9mol/ L组>10-7mol/ L,但各实验组间差异无统计学意义;MTT组检测,在对数生长期,各实验组增殖速率高于对照组,实验组间细胞增殖速率为10-8mol/ L>10-9mol/ L组>10-7mol/ L。10-8mol/ L速率与另外两实验组间差异有统计学意义,其余两组间无显著性差异
结论:采用MTT法,证实添加外源性CGRP能促进对数增殖期MSCs细胞增殖速度;采用流失细胞法检测,证实,外源性CGRP能提高MSCs细胞处于DNA合成和有丝分裂前期的比例。
第三部分: CGRP对人骨髓间充质干细胞细胞信号传导的变化的影响的研究
目的:观察在添加外源性CGRP的情况下,MSCs胞间通讯连接的改变,并验证改变与CGRP的关联性
方法:MSCs获取仍采自健康志愿者骨髓,应用梯度离心法及贴壁培养筛选,获得MSCs细胞。分组采用对照组、CGRP组和拮抗剂组,放射免疫法检测各组胞间信号分子cAMP含量改变;使用CFDA染料,应用激光共聚焦技术观察细胞间缝隙连接和胞间信号传导能力变化;采用荧光定量PCR技术检测缝隙连接分子Cx43mRNA表达变化。
结果:放免法检测结果显示,各组间,以CGRP组胞间cAMP含量最高,与其余两组间存在统计学差异;CGRP结合拮抗剂组的含量高于对照组,后两者间无统计学差异。采用激光共聚焦技术,结合CFDA生物活性染料,显示CGRP组荧光信号恢复幅度较对照组及拮抗剂组大,差异有显著性(P<0.05);拮抗剂组恢复幅度较对照组大,但两组间差异不具有显著性(P>0.05)。三组细胞Cx43mRNA表达,CGRP组表达量高于抑制剂组及对照组,差异有显著性(P<0.05);抑制剂组表达量高于对照组,两者间差异无显著性。
结论:CGRP能促进MSCs胞间缝隙连接,促进缝隙连接蛋白的基因表达。
第四部分:CGRP对人骨髓间充质干细胞细胞增殖相关因子的作用
目的:研究在添加外源性CGRP的情况下,与MSCs增殖及分化相关的细胞因子IGF-1、BMP-2的受体mRNA表达变化;研究外源性CGRP是否造成MSCs对成骨诱导因子BMP-2的mRNA表达。
方法:MSCs获取、分离及培养方法同前。实验分组为对照组、CGRP组和拮抗剂组,采用荧光定量PCR技术分别检测增殖相关因子IGF-1及其受体mRNA表达;以及成骨分化因子BMP-2及其受体mRNA表达。
结果:采用相对定量技术,CGRP组MSCs表达IGF-1、IGF-1受体以及BMP-2受体mRNA量高于拮抗剂组及对照组,且差异有统计学意义。而三组在表达BMP-2 mRNA无显著差异,且Ct值≥35。
结论:外源性CGRP能够提高MSCs表达IGF-1、IGF-1受体以及BMP-2受体mRNA,而不能诱导MSCs表达BMP-2mRNA。三组BMP-2的mRNA平均Ct值均在35左右,可以认为阴性表达。
Calcitonin gene-related peptide(CGRP) is a kind of peptide that existes widely in the bone tissue.It has been proved that CGRP has affect on the proliferation and formation .In the period of bone fracture healing ,nervouses fiber containing CGRP were observed in the histological way.Nervous fiber containing CGRP distribute mainly in the area that bone grows and remodifies actively.For example ,the content of nervous containing CGRP in the metaphysis of long bone is 10 times higher than the content in the backbone.And the nervous distribut and the content of CGRP have high correlation with the period of fracture healing.There has evidence that CGRP can significantly stimulate the proliferation of osteoblast,and promote the avtivety of osteoblast.
Mesenchymal stem cells(MSCs)is one of the sources stem cell of osteoblast.And it is also be the main seed cell in the tissue engineering bone.The ability of proliferation of MSCs will affect the growth , modify and repairation of bone directely,and it also has decisive affect on construction and osteogenic ability of tissue engineering bone.So ,study in this field ,is always be the emphersize on the orthopedic field and tissue engineering field.
As all know,MSCs distribute mostly in the red bone marrow.which coincide the nervous distribution which contains CGRP.People has found that CGRP can promote colony of the bone marrow karyocyte.But there still has much work should do in this field. Part I The evidence of CGRP receptor on MSCs
Objective:To get specifical evidence that CGRP receptor exist on human MSCs
Methods:Bone marrow is come from healthy young volunteers.MSCs were colleced by gradientcentrifugation and adherent culture.After prolifed in vivo,RT-PCR was used to detect CGRP receptor in MSCs in logarithmic growth phase. Hybridoma technique was used to produce rabbit-anti-human CGRP receptor,which was used in the Westernblot test to detect CGRP receptor protein produced in human MSCs.
Result:MSCs collected by gradientcentrifugation and cultured by adherent culture has high purity and proliferation effect.It was proved through RT-PCR that MSCs express CGRP-receptor mRNA ,and it was also be proved through Western-blot that MSCs express CGRP-receptor protein.
Conclusion:Combined gradientcentrifugation with adherent culture,high purity MSCs could be collected.With RT-PCR test ,it was proved that MSCs express CGRP receptor mRNA. With Westernblot test ,it was proved that MSCs express CGRP receptor protein.
Part II The effect of CGRP on proliferation of MSCs
Objective:To observe the change of proliferation of MSCs with exogenous CGRP,and to explor the correlation. Methods:Human MSCs were aparted into 3 group decided by the concentration of
CGRP.Cell proliferatinon was detected through MTT test.Cell form in each group was detected through optical microscope,in the same time point.Cell cycle was detected with Flowcytometry to analyze the ratio of cell in the mitotic time.
Result:According to the contrast culture test ,cell in all the groups has regular and typical cellular morphology.In the same detect time point ,the cell density of control group was lower than density of each experimental group.In the cell cycle test ,in the logarithmic growth phase,the rates order of cells in the prophase and DNA synthesis phase in each group was 10-8mol/ LCGRP group,10-9mol/ LCGRP group,10-7mol/ LCGRP group and the control group.But between each group,there was no stastical difference.The MTT test showed similar result ,the 10-8mol/ LCGRP group had the highest proliferation speed ,and the control group had the lowest.There had stastical difference between experimental group and control group.There also had stastic differenc between the 10-8mol/ LCGRP group and the other two experimental group.
Conclusion:With MTT test ,it had been proved that exogenous CGRP can accelerate the proliferation speed in the logarithmic growth phase.With flowcytometry,it had been proved that exogenous CGRP can raise the ratio of the cell in the DNA synthesis period and mitosis prophase.
Part III Effect of CGRP on intracellular communication of MSCs
Objective:To observe the intracellular communication of MSCs with exogenous CGRP and to expleor the relationship between the change of intracellular communication of MSCs and exogenous CGRP。
Methods:MSCs collected from healthy volunteer was aparted into 3 group,the control group ,the antagon group and the experimental group. Intracellular communication medium molecule was detected through radioimmunoassay;Intracellular communication and signal conduction were dected through CFDA fluorescent dye The espression of Cx43mRNA was dected through Realtime-PCR.
Result:The result of radioimmunoassay showed that cAMP content in the CGRP group was higher than the other two groups,with ststical difference. Confocal laser-CFDA test had shown that recovery of florescent light of the experimental group is much higher than the other two groups with stastic difference. The recovery range of antagon group was larger than that of control group ,but,without stastic difference.Expression of Cx43mRNA in experimental group was higher than the other two groups , but,without stastic difference.
Conclusion:CGRP can promote not only intracellular communication of MSCs but also the expression of Cx43mRNA.
Part IV effect of CGRP on proliferation related biological factor of MSCs
Objective:To observe the change of IGF-1、BMP-2 and their receptor in the MSCs cell with exogenous CGRP.To explor whether exogenous CGRP will induce MSCs to express the osteo-induced factor and it’s receptor.
Methods:MSCs collected from healthy volunteer was aparted into 3 group,the control group ,the antagon group and the experimental group. The mRNA expressions of proliferation related biological factor of MSCs were detected through Real-time PCR.
Result:It was proved that the mRNA expressions of IGF-1,IGF-1receptor and BMP-2 receptor in experimental group were higher than that in control group with stastic difference.The the mRNA expressions of BMP-2 in all the groups had no stastic difference.And Ct index in all the groups were higner than 35.
Result:MSCs collected by gradientcentrifugation and cultured by adherent culture has high purity and proliferation effect.It was proved through RT-PCR that MSCs express CGRP-receptor mRNA ,and it was also be proved through Western-blot that MSCs express CGRP-receptor protein.
Conclusion:The exogenous CGRP can increase the mRNA expression of IGF-1,IGF-1receptor and BMP-2 receptor of MSCs.In all the grops ,the Ct indexes of BMP-2mRNA were higher than 35,which could be considered as negative expression.
引文
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[1] Liese S, Schinke T, Catala-Lehnen P, Priemel M, Rueger JM, Emeson RB, Amling M. Decreased bone formation and osteopenia in mice lacking aCGRP [J]. Journal of Bone and Mineral Research. 2003, 18: S14-S14.
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[1] Ma HZ, Zeng BF, Li XL, Chai YM. Temporal and spatial expression of BMP-2 in sub-chondral bone of necrotic femoral heads in rabbits by use of extracorporeal shock waves. Acta Orthopaedica, 2008; 79 (1):98-105.
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