三七总皂苷温敏凝胶通过上调VEGF表达促进大鼠拔牙创愈合的研究
发布时间：2018-08-27 11:13
【摘要】：拔牙创愈合的本质是骨修复改建。骨组织高度血管化,血管生成表现在骨生长代谢的整个过程。而血管内皮细胞的分化增殖和促血管生成生长因子的局部调节是血管生成的两个重要因素,VEGF是促进血管化的重要因素。VEGF在骨形成的早期阶段由血管内皮细胞及成骨细胞生成,与血管内皮细胞表面特异性受体结合,向血管内皮细胞分化,聚集进而形成毛细血管网。VEGF同时也影响成骨细胞的成骨活动。成骨细胞对VEGF的趋化性,表现在对细胞表面的特异性受体结合,从而促进细胞募集,提高新生骨的质量。VEGF促进血管内皮细胞和成纤维细胞分泌IGF-I,进而促进成骨细胞的生长与聚集。此外,VEGF与BMP-2存在协同作用,调节局部骨形成。动物实验表明,将VEGF与BMP-2同时作用于骨缺损局部,治疗效果明显优于单纯使用BMP-2。壳聚糖广泛存在于自然界中,由于易获得、生物相容性好、造价低廉等特点被广泛应用于载药缓释系统。在适宜的条件下,壳聚糖溶液与β-甘油磷酸钠二者表面之间具有的性质相反的离子相互作用形成复合物,这种复合物具有温敏特性,即在室温呈流体状态,待温度接近37℃时呈不流动的固体状态。这种特性的优点在于可以在注射后均匀分布在局部,尤其适用于范围小且形状不规则的骨缺损。而拔牙的过程由于病因、手术难易程度、患者依从性等各种因素,往往使拔牙创呈现不规则的形态,因此将具有促进局部骨愈合作用的药物载入壳聚糖温敏水凝胶体系中,可以使药物均匀充分的作用于各个部分,从而提高骨愈合质量。三七总皂苷是传统中药三七的主要活性成分,具有上调某些成骨相关细胞生长因子的表达,如BMP-2、IGF及VEGF等,从而提高成骨活性。同时,三七总皂苷还能诱导大鼠骨髓间充质干细胞和人牙周膜干细胞向成骨细胞分化。然而,三七总皂苷是否能促进拔牙创的愈合,是否以促进VEGF的表达来实现,目前尚未有报道。目的:为了进一步研究三七总皂苷对于拔牙创愈合的作用及合理载药方式,通过制备3种不同药物浓度三七总皂苷壳聚糖温控凝胶,作用于大鼠右侧中切牙拔牙创,HE染色及VEGF免疫组织化学染色观察骨愈合情况,测定VEGF阳性细胞计数及平均光度值,以探究三七总皂苷对拔牙创愈合作用。材料和方法:分别制备不同浓度(0.5mg/ml,1.0mg/ml,1.5mg/ml)的壳聚糖温敏凝胶;选取SD雄性大鼠45只,拔除右侧中切牙后注入壳聚糖温敏凝胶,随机分成5组(0.5mg/ml组、1.0mg/ml组、1.5mg/ml组,0.0mg/ml组和对照组);分别于术后1周、2周、4周处死大鼠,获取术区标本;HE染色和VEGF免疫组化染色观察拔牙创愈合情况,测定VEGF阳性细胞计数和平均光密度值。结果:HE染色组织学观察1.给药1周时,载药组局部可见血管内皮细胞大量迁移聚集,新生血管数目多,毛细血管扩张,血管化程度高。2.给药2周时,载药组局部可见明显的成骨细胞迁移聚集于骨小梁周围,形态成熟,成骨活跃。新生毛细血管数量增多,形态更为成熟,血管化程度达到顶峰。其中1.0mg/ml组血管化及成骨最为活跃。3.给药4周时,载药组新生血管数目有所减少,但形态更为成熟,骨小梁形态成熟,排列整齐。载药组血管化及成骨均强于对照组。VEGF阳性细胞计数检测及平均光度值统计分析1.给药1周时,VEGF阳性表达主要集中于血管周围聚集的血管内皮细胞之上,以1.0mg/ml组最明显,呈阳性,0.5mg/ml组和1.5mg/ml组呈弱阳性。载药组相对于0.0mg/ml组及对照组明显。VEGF阳性细胞计数和平均光度值结果显示,0.0mg/ml组与对照组差异不显著(P0.05);载药组与对照组差异显著(p0.05),其中以1.Omg/ml组最高。2.给药2周时,各组血管内皮细胞和成骨细胞VEGF阳性表达明显提高,VEGF阳性表达达到顶峰。其中1.0mg/ml组呈强阳性,较0.5mg/ml组及1.5mg/ml组显著,载药组VEGF阳性表达均较空白壳聚糖凝胶组和对照组显著。VEGF阳性细胞计数和平均光度值结果显示,0.0mg/ml组与对照组差异不显著(P0.05);载药各组与对照组差异显著(p0.05),1.0mg/ml组最高。3.给药4周时,血管化的程度降低,VEGF阳性表达较第2周有所降低,主要表现在血管内皮细胞的表达,载药组较O.Omg/ml组和对照组明显。VEGF阳性细胞计数和平均光度值结果显示,O.Omg/ml组与对照组差异不显著(P0.05);载药各组与对照组差异显著(p0.05),其中以1.Omg/ml组最高。结论1.三七总皂苷在通过上调大鼠拔牙创VEGF表达,诱导血管内皮细胞分化、增殖,发挥血管生成作用;2.三七总皂苷可能通过促进大鼠拔牙创VEGF表达促进成骨细胞聚集,提高成骨活性;3.在本实验环境下,载药浓度为1.Omg/ml时血管化程度最高,成骨最活跃。
[Abstract]:The essence of tooth extraction wound healing is bone repair and reconstruction.Bone tissue is highly vascularized and angiogenesis is manifested in the whole process of bone growth and metabolism.The differentiation and proliferation of vascular endothelial cells and the local regulation of angiogenic growth factors are two important factors of angiogenesis.VEGF is an important factor of promoting vascularization. Vascular endothelial cells and osteoblasts are formed at the stage of osteogenesis. They bind to specific receptors on the surface of vascular endothelial cells, differentiate into vascular endothelial cells, aggregate and form capillary networks. VEGF also affects the osteogenic activity of osteoblasts. VEGF promotes the secretion of IGF-I by vascular endothelial cells and fibroblasts, and then promotes the growth and aggregation of osteoblasts. In addition, there is a synergistic effect between VEGF and BMP-2 to regulate local bone formation. Pure use of BMP-2. Chitosan is widely found in nature. It is widely used in drug delivery systems because of its easy availability, good biocompatibility and low cost. Temperature-sensitive properties, i.e. fluid at room temperature and non-fluid solid at temperatures approaching 37 C. The advantage of this property is that it can be uniformly distributed locally after injection, especially for small and irregular bone defects. Because of the irregular shape of the extraction wound, the drug loaded into the chitosan thermosensitive hydrogel system can make the drug act on each part evenly and sufficiently to improve the quality of bone healing. Panax notoginseng saponins are the main active components of traditional Chinese medicine Panax notoginseng, which can up-regulate some osteogenetic phases. At the same time, Panax notoginseng saponins can induce rat bone marrow mesenchymal stem cells and human periodontal ligament stem cells to differentiate into osteoblasts. However, whether Panax notoginseng saponins can promote the healing of tooth extraction wounds and whether they can promote the expression of VEGF has not yet been found. Objective: To study the effect of Panax notoginseng saponins (PNS) on the healing of tooth extraction wounds and the rational drug-loading method, three different concentrations of PNS-chitosan temperature-controlled gels were prepared, which acted on rat right central incisor extraction wounds, HE staining and VEGF immunohistochemical staining were used to observe the bone healing, and the positive cytometer of VEGF was determined. Materials and Methods: Chitosan thermosensitive gels with different concentrations (0.5mg/ml, 1.0mg/ml, 1.5mg/ml) were prepared, and 45 SD male rats were selected and injected with chitosan thermosensitive gel after extraction of the right central incisor. G / ml group and control group; rats were sacrificed one week, two weeks and four weeks after operation to obtain the operation area specimens; HE staining and VEGF immunohistochemical staining were used to observe the healing of tooth extraction wounds, and the number of VEGF positive cells and the average optical density were measured. At 2 weeks of administration, osteoblasts migrated and aggregated around the trabeculae. The morphology of osteoblasts was mature and active. The number of new capillaries increased, the morphology became more mature and the degree of vascularization reached the peak. The vascularization and osteogenesis of 1.0 mg/ml group were the most active. Vascularization and osteogenesis were stronger in the drug-loaded group than in the control group. The positive expression of VEGF was mainly concentrated in the aggregated vessels around the vessels at 1 week of administration. The number of VEGF positive cells and the average luminosity value showed no significant difference between the 0.0mg/ml group and the control group (P 0.05); the difference between the drug-loaded group and the control group was significant (P 0.05); the drug-loaded group and the control group was the most significant (P 0.05). The positive expression of VEGF in vascular endothelial cells and osteoblasts was significantly increased and reached the peak at 2 weeks after administration. The positive expression of VEGF in 1.0mg/ml group was significantly higher than that in 0.5mg/ml group and 1.5mg/ml group. The positive expression of VEGF in Drug-loaded group was significantly higher than that in blank chitosan gel group and control group. The results showed that there was no significant difference between 0.0mg/ml group and control group (P 0.05), and there was significant difference between drug-loaded groups and control group (P 0.05). The highest level was found in 1.0mg/ml group. At 4 weeks of administration, the degree of vascularization decreased, and the expression of VEGF decreased, mainly in vascular endothelial cells. The expression of VEGF positive cells in Drug-loaded group was more obvious than that in O.Omg/ml group and control group. The results of counting and average luminosity showed that there was no significant difference between O.Omg/ml group and control group (P 0.05); there was significant difference between drug-loaded groups and control group (P 0.05). Among them, 1.Omg/ml group was the highest. Conclusion 1.Panax notoginseng saponins could induce vascular endothelial cell differentiation, proliferation and angiogenesis by up-regulating the expression of VEGF in rat tooth extraction wound. It may promote osteoblast aggregation and enhance osteogenic activity by promoting the expression of VEGF in rat tooth extraction wound. 3. In this experimental environment, when the drug concentration was 1.Omg/ml, the degree of vascularization was the highest and osteogenesis was the most active.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R782.1
[Abstract]:The essence of tooth extraction wound healing is bone repair and reconstruction.Bone tissue is highly vascularized and angiogenesis is manifested in the whole process of bone growth and metabolism.The differentiation and proliferation of vascular endothelial cells and the local regulation of angiogenic growth factors are two important factors of angiogenesis.VEGF is an important factor of promoting vascularization. Vascular endothelial cells and osteoblasts are formed at the stage of osteogenesis. They bind to specific receptors on the surface of vascular endothelial cells, differentiate into vascular endothelial cells, aggregate and form capillary networks. VEGF also affects the osteogenic activity of osteoblasts. VEGF promotes the secretion of IGF-I by vascular endothelial cells and fibroblasts, and then promotes the growth and aggregation of osteoblasts. In addition, there is a synergistic effect between VEGF and BMP-2 to regulate local bone formation. Pure use of BMP-2. Chitosan is widely found in nature. It is widely used in drug delivery systems because of its easy availability, good biocompatibility and low cost. Temperature-sensitive properties, i.e. fluid at room temperature and non-fluid solid at temperatures approaching 37 C. The advantage of this property is that it can be uniformly distributed locally after injection, especially for small and irregular bone defects. Because of the irregular shape of the extraction wound, the drug loaded into the chitosan thermosensitive hydrogel system can make the drug act on each part evenly and sufficiently to improve the quality of bone healing. Panax notoginseng saponins are the main active components of traditional Chinese medicine Panax notoginseng, which can up-regulate some osteogenetic phases. At the same time, Panax notoginseng saponins can induce rat bone marrow mesenchymal stem cells and human periodontal ligament stem cells to differentiate into osteoblasts. However, whether Panax notoginseng saponins can promote the healing of tooth extraction wounds and whether they can promote the expression of VEGF has not yet been found. Objective: To study the effect of Panax notoginseng saponins (PNS) on the healing of tooth extraction wounds and the rational drug-loading method, three different concentrations of PNS-chitosan temperature-controlled gels were prepared, which acted on rat right central incisor extraction wounds, HE staining and VEGF immunohistochemical staining were used to observe the bone healing, and the positive cytometer of VEGF was determined. Materials and Methods: Chitosan thermosensitive gels with different concentrations (0.5mg/ml, 1.0mg/ml, 1.5mg/ml) were prepared, and 45 SD male rats were selected and injected with chitosan thermosensitive gel after extraction of the right central incisor. G / ml group and control group; rats were sacrificed one week, two weeks and four weeks after operation to obtain the operation area specimens; HE staining and VEGF immunohistochemical staining were used to observe the healing of tooth extraction wounds, and the number of VEGF positive cells and the average optical density were measured. At 2 weeks of administration, osteoblasts migrated and aggregated around the trabeculae. The morphology of osteoblasts was mature and active. The number of new capillaries increased, the morphology became more mature and the degree of vascularization reached the peak. The vascularization and osteogenesis of 1.0 mg/ml group were the most active. Vascularization and osteogenesis were stronger in the drug-loaded group than in the control group. The positive expression of VEGF was mainly concentrated in the aggregated vessels around the vessels at 1 week of administration. The number of VEGF positive cells and the average luminosity value showed no significant difference between the 0.0mg/ml group and the control group (P 0.05); the difference between the drug-loaded group and the control group was significant (P 0.05); the drug-loaded group and the control group was the most significant (P 0.05). The positive expression of VEGF in vascular endothelial cells and osteoblasts was significantly increased and reached the peak at 2 weeks after administration. The positive expression of VEGF in 1.0mg/ml group was significantly higher than that in 0.5mg/ml group and 1.5mg/ml group. The positive expression of VEGF in Drug-loaded group was significantly higher than that in blank chitosan gel group and control group. The results showed that there was no significant difference between 0.0mg/ml group and control group (P 0.05), and there was significant difference between drug-loaded groups and control group (P 0.05). The highest level was found in 1.0mg/ml group. At 4 weeks of administration, the degree of vascularization decreased, and the expression of VEGF decreased, mainly in vascular endothelial cells. The expression of VEGF positive cells in Drug-loaded group was more obvious than that in O.Omg/ml group and control group. The results of counting and average luminosity showed that there was no significant difference between O.Omg/ml group and control group (P 0.05); there was significant difference between drug-loaded groups and control group (P 0.05). Among them, 1.Omg/ml group was the highest. Conclusion 1.Panax notoginseng saponins could induce vascular endothelial cell differentiation, proliferation and angiogenesis by up-regulating the expression of VEGF in rat tooth extraction wound. It may promote osteoblast aggregation and enhance osteogenic activity by promoting the expression of VEGF in rat tooth extraction wound. 3. In this experimental environment, when the drug concentration was 1.Omg/ml, the degree of vascularization was the highest and osteogenesis was the most active.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R782.1
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