胶原/壳聚糖—明胶微球支架缓释三七皂甙Rg1促血管化研究
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摘要
目的：
探索中药单体三七皂甙Rgl对血管内皮细胞生物活性影响及机制,为其促血管化、协同治疗提供依据。构建有效的复合三七皂甙Rgl的胶原／壳聚糖-明胶微球支架缓释体系,评价与探索三七皂甙Rgl在促进组织工程皮肤血管化中的作用及相关机制,为组织工程促血管化策略提供新的思路。
方法：
各种急、慢性损伤造成的皮肤缺损是亟待有效解决的重大临床问题之一。组织工程化皮肤为皮肤创面的修复和愈合提供了一崭新的治疗途径,但是目前各种组织工程化皮肤移植存活率较低,其中一个主要原因是组织工程化皮肤移植后血管化速度较慢。因此寻求有效促进组织工程移植物血管化的方法尤为重要。三七作为一种常见的活血化瘀中药具有良好的促血管生成作用,这一作用主要是由其有效成分三七皂甙Rgl主导的。前期研究证实胶原／壳聚糖支架具有良好的机械支持作用和促进组织、血管长入的作用,而明胶微球作为一种缓释载体技术已经非常成熟。因此我们首先通过对内皮细胞增殖和凋亡,迁移,小管形成的作用,流式细胞术检测细胞周期和凋亡率,HUVEC细胞的VEGF表达探索三七皂甙Rg1对HUVEC细胞血管生成的影响及机制。为了使Rg1能最好的应用于组织工程,我们构建了缓释三七皂甙Rgl的胶原／壳聚糖-明胶微球(CC-GMS)支架,并通
过体外实验对这种支架的微观形貌、缓释性能以及缓释出的Rg1对HUVEC细胞增殖能力影响进行评价。为进一步表征缓释Rg1的CC-GMS支架对血管化及诱导再生的影响,我们采用SD大鼠埋植实验模型,分别采用组织病理技术、免疫组化和免疫荧光技术及分子生物学技术对组织反应性和促血管化因子的表达进行了评价。结论：
三七皂甙Rgl有良好的促进HUVEC细胞增殖,迁移,管形成作用。Rg1增加HUVEC细胞周期中G2/M期和S期的比例,并未引起内皮细胞的凋亡。同时三七皂甙Rgl可上调HUVEC细胞VEGF表达。构建的CC-GMS支架具有很好的组织相容性并能持续释放活性的Rg1。构建的缓释Rg1的CC-GMS支架在体内具有良好的促血管化作用,作为组织工程皮肤替代物有一定应用前景。
Objective:
The purpose of this manuscript is to evaluate the effects of Panax notoginseng saponin Rgl on HUVEC proliferation, migration and VEGF expression in vitro and to develop a collagen/chitosan-gelatin microspheres scaffold controlled release Rgl and to evaluate its effect on tissue angiogenesis and wound regeneration in vivo.
Methods:
The consistent and steady release of angiogenic factors is an important factor in the promotion of angiogenesis in dermal substitutes, which usually lack, yet need, a vascular network. Rgl, isolated from Panax notoginseng (PNS) saponins, has been proven to be a candidate drug for angiogenesis, whereas its applications in tissue engineering have been rarely reported. In this study, the effect of Rgl on the biological behavior of human umbilical vein endothelial cells (HUVECs) was investigated in vitro. Furthermore, to develop a local delivery and subsequent controlled release system, PNS Rgl was first introduced into gelatin microspheres (GMS), which serve as drug carriers, and then incorporated into collagen/chitosan scaffolds to fabricate a collagen/chitosan-GMS (CC-GMS) scaffold. The morphology of the CC-GMS scaffold was investigated in vitro. To characterize the effect of the CC-GMS scaffold on angiogenesis and tissue regeneration, the CC-GMS scaffold was subcutaneously embedded in Sprague-Dawley (SD) rats. At days7,10,14, and21after implantation, the implants were imaged to analyze the newly formed blood vessels, and tissue specimens were harvested for histology, immunofluorescence, real-time quantitative PCR (RT-qPCR), and western blotting analyses.
Conclusions:
The results demonstrated that Rgl promoted the proliferation of HUVECs in a time-and dose-dependent manner. Moreover, the CC-GMS scaffold showed more rapid angiogenesis than did the control groups in vivo. Our data suggested that Rgl could be a candidate for accelerating angiogenesis in skin tissue engineering, and the ability of the CC-GMS scaffold to promote angiogenesis demonstrates its potential in skin tissue engineering.
探索中药单体三七皂甙Rgl对血管内皮细胞生物活性影响及机制,为其促血管化、协同治疗提供依据。构建有效的复合三七皂甙Rgl的胶原／壳聚糖-明胶微球支架缓释体系,评价与探索三七皂甙Rgl在促进组织工程皮肤血管化中的作用及相关机制,为组织工程促血管化策略提供新的思路。
方法：
各种急、慢性损伤造成的皮肤缺损是亟待有效解决的重大临床问题之一。组织工程化皮肤为皮肤创面的修复和愈合提供了一崭新的治疗途径,但是目前各种组织工程化皮肤移植存活率较低,其中一个主要原因是组织工程化皮肤移植后血管化速度较慢。因此寻求有效促进组织工程移植物血管化的方法尤为重要。三七作为一种常见的活血化瘀中药具有良好的促血管生成作用,这一作用主要是由其有效成分三七皂甙Rgl主导的。前期研究证实胶原／壳聚糖支架具有良好的机械支持作用和促进组织、血管长入的作用,而明胶微球作为一种缓释载体技术已经非常成熟。因此我们首先通过对内皮细胞增殖和凋亡,迁移,小管形成的作用,流式细胞术检测细胞周期和凋亡率,HUVEC细胞的VEGF表达探索三七皂甙Rg1对HUVEC细胞血管生成的影响及机制。为了使Rg1能最好的应用于组织工程,我们构建了缓释三七皂甙Rgl的胶原／壳聚糖-明胶微球(CC-GMS)支架,并通
过体外实验对这种支架的微观形貌、缓释性能以及缓释出的Rg1对HUVEC细胞增殖能力影响进行评价。为进一步表征缓释Rg1的CC-GMS支架对血管化及诱导再生的影响,我们采用SD大鼠埋植实验模型,分别采用组织病理技术、免疫组化和免疫荧光技术及分子生物学技术对组织反应性和促血管化因子的表达进行了评价。结论：
三七皂甙Rgl有良好的促进HUVEC细胞增殖,迁移,管形成作用。Rg1增加HUVEC细胞周期中G2/M期和S期的比例,并未引起内皮细胞的凋亡。同时三七皂甙Rgl可上调HUVEC细胞VEGF表达。构建的CC-GMS支架具有很好的组织相容性并能持续释放活性的Rg1。构建的缓释Rg1的CC-GMS支架在体内具有良好的促血管化作用,作为组织工程皮肤替代物有一定应用前景。
Objective:
The purpose of this manuscript is to evaluate the effects of Panax notoginseng saponin Rgl on HUVEC proliferation, migration and VEGF expression in vitro and to develop a collagen/chitosan-gelatin microspheres scaffold controlled release Rgl and to evaluate its effect on tissue angiogenesis and wound regeneration in vivo.
Methods:
The consistent and steady release of angiogenic factors is an important factor in the promotion of angiogenesis in dermal substitutes, which usually lack, yet need, a vascular network. Rgl, isolated from Panax notoginseng (PNS) saponins, has been proven to be a candidate drug for angiogenesis, whereas its applications in tissue engineering have been rarely reported. In this study, the effect of Rgl on the biological behavior of human umbilical vein endothelial cells (HUVECs) was investigated in vitro. Furthermore, to develop a local delivery and subsequent controlled release system, PNS Rgl was first introduced into gelatin microspheres (GMS), which serve as drug carriers, and then incorporated into collagen/chitosan scaffolds to fabricate a collagen/chitosan-GMS (CC-GMS) scaffold. The morphology of the CC-GMS scaffold was investigated in vitro. To characterize the effect of the CC-GMS scaffold on angiogenesis and tissue regeneration, the CC-GMS scaffold was subcutaneously embedded in Sprague-Dawley (SD) rats. At days7,10,14, and21after implantation, the implants were imaged to analyze the newly formed blood vessels, and tissue specimens were harvested for histology, immunofluorescence, real-time quantitative PCR (RT-qPCR), and western blotting analyses.
Conclusions:
The results demonstrated that Rgl promoted the proliferation of HUVECs in a time-and dose-dependent manner. Moreover, the CC-GMS scaffold showed more rapid angiogenesis than did the control groups in vivo. Our data suggested that Rgl could be a candidate for accelerating angiogenesis in skin tissue engineering, and the ability of the CC-GMS scaffold to promote angiogenesis demonstrates its potential in skin tissue engineering.
引文
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