三七總皂苷溫敏凝膠通過上調VEGF表達促進大鼠拔牙創(chuàng)愈合的研究
發(fā)布時間：2018-08-27 11:13
【摘要】：拔牙創(chuàng)愈合的本質是骨修復改建。骨組織高度血管化,血管生成表現在骨生長代謝的整個過程。而血管內皮細胞的分化增殖和促血管生成生長因子的局部調節(jié)是血管生成的兩個重要因素,VEGF是促進血管化的重要因素。VEGF在骨形成的早期階段由血管內皮細胞及成骨細胞生成,與血管內皮細胞表面特異性受體結合,向血管內皮細胞分化,聚集進而形成毛細血管網。VEGF同時也影響成骨細胞的成骨活動。成骨細胞對VEGF的趨化性,表現在對細胞表面的特異性受體結合,從而促進細胞募集,提高新生骨的質量。VEGF促進血管內皮細胞和成纖維細胞分泌IGF-I,進而促進成骨細胞的生長與聚集。此外,VEGF與BMP-2存在協(xié)同作用,調節(jié)局部骨形成。動物實驗表明,將VEGF與BMP-2同時作用于骨缺損局部,治療效果明顯優(yōu)于單純使用BMP-2。殼聚糖廣泛存在于自然界中,由于易獲得、生物相容性好、造價低廉等特點被廣泛應用于載藥緩釋系統(tǒng)。在適宜的條件下,殼聚糖溶液與β-甘油磷酸鈉二者表面之間具有的性質相反的離子相互作用形成復合物,這種復合物具有溫敏特性,即在室溫呈流體狀態(tài),待溫度接近37℃時呈不流動的固體狀態(tài)。這種特性的優(yōu)點在于可以在注射后均勻分布在局部,尤其適用于范圍小且形狀不規(guī)則的骨缺損。而拔牙的過程由于病因、手術難易程度、患者依從性等各種因素,往往使拔牙創(chuàng)呈現不規(guī)則的形態(tài),因此將具有促進局部骨愈合作用的藥物載入殼聚糖溫敏水凝膠體系中,可以使藥物均勻充分的作用于各個部分,從而提高骨愈合質量。三七總皂苷是傳統(tǒng)中藥三七的主要活性成分,具有上調某些成骨相關細胞生長因子的表達,如BMP-2、IGF及VEGF等,從而提高成骨活性。同時,三七總皂苷還能誘導大鼠骨髓間充質干細胞和人牙周膜干細胞向成骨細胞分化。然而,三七總皂苷是否能促進拔牙創(chuàng)的愈合,是否以促進VEGF的表達來實現,目前尚未有報道。目的:為了進一步研究三七總皂苷對于拔牙創(chuàng)愈合的作用及合理載藥方式,通過制備3種不同藥物濃度三七總皂苷殼聚糖溫控凝膠,作用于大鼠右側中切牙拔牙創(chuàng),HE染色及VEGF免疫組織化學染色觀察骨愈合情況,測定VEGF陽性細胞計數及平均光度值,以探究三七總皂苷對拔牙創(chuàng)愈合作用。材料和方法:分別制備不同濃度(0.5mg/ml,1.0mg/ml,1.5mg/ml)的殼聚糖溫敏凝膠;選取SD雄性大鼠45只,拔除右側中切牙后注入殼聚糖溫敏凝膠,隨機分成5組(0.5mg/ml組、1.0mg/ml組、1.5mg/ml組,0.0mg/ml組和對照組);分別于術后1周、2周、4周處死大鼠,獲取術區(qū)標本;HE染色和VEGF免疫組化染色觀察拔牙創(chuàng)愈合情況,測定VEGF陽性細胞計數和平均光密度值。結果:HE染色組織學觀察1.給藥1周時,載藥組局部可見血管內皮細胞大量遷移聚集,新生血管數目多,毛細血管擴張,血管化程度高。2.給藥2周時,載藥組局部可見明顯的成骨細胞遷移聚集于骨小梁周圍,形態(tài)成熟,成骨活躍。新生毛細血管數量增多,形態(tài)更為成熟,血管化程度達到頂峰。其中1.0mg/ml組血管化及成骨最為活躍。3.給藥4周時,載藥組新生血管數目有所減少,但形態(tài)更為成熟,骨小梁形態(tài)成熟,排列整齊。載藥組血管化及成骨均強于對照組。VEGF陽性細胞計數檢測及平均光度值統(tǒng)計分析1.給藥1周時,VEGF陽性表達主要集中于血管周圍聚集的血管內皮細胞之上,以1.0mg/ml組最明顯,呈陽性,0.5mg/ml組和1.5mg/ml組呈弱陽性。載藥組相對于0.0mg/ml組及對照組明顯。VEGF陽性細胞計數和平均光度值結果顯示,0.0mg/ml組與對照組差異不顯著(P0.05);載藥組與對照組差異顯著(p0.05),其中以1.Omg/ml組最高。2.給藥2周時,各組血管內皮細胞和成骨細胞VEGF陽性表達明顯提高,VEGF陽性表達達到頂峰。其中1.0mg/ml組呈強陽性,較0.5mg/ml組及1.5mg/ml組顯著,載藥組VEGF陽性表達均較空白殼聚糖凝膠組和對照組顯著。VEGF陽性細胞計數和平均光度值結果顯示,0.0mg/ml組與對照組差異不顯著(P0.05);載藥各組與對照組差異顯著(p0.05),1.0mg/ml組最高。3.給藥4周時,血管化的程度降低,VEGF陽性表達較第2周有所降低,主要表現在血管內皮細胞的表達,載藥組較O.Omg/ml組和對照組明顯。VEGF陽性細胞計數和平均光度值結果顯示,O.Omg/ml組與對照組差異不顯著(P0.05);載藥各組與對照組差異顯著(p0.05),其中以1.Omg/ml組最高。結論1.三七總皂苷在通過上調大鼠拔牙創(chuàng)VEGF表達,誘導血管內皮細胞分化、增殖,發(fā)揮血管生成作用;2.三七總皂苷可能通過促進大鼠拔牙創(chuàng)VEGF表達促進成骨細胞聚集,提高成骨活性;3.在本實驗環(huán)境下,載藥濃度為1.Omg/ml時血管化程度最高,成骨最活躍。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R782.1
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