厄贝沙坦对AGEs诱导成骨细胞损伤的影响
发布时间：2018-08-06 19:43
【摘要】：研究背景近年来,我国的糖尿病患病率在迅速增长,其造成的糖尿病相关性骨质疏松,可使骨折等并发症显著升高,严重影响着糖尿病患者的健康和生活质量。既往研究发现,在糖尿病中,晚期糖基化终产物(advanced glycation end products,AGEs)的形成和积累加速,是导致多种糖尿病慢性并发症发生的原因,AGEs在糖尿病性骨质疏松中亦起者重要作用。使用有效的干预途径抑制AGEs的对成骨的损伤,改善骨形成,为临床防治糖尿病性骨质疏松提供理论根据和有效的方法。目的本课题拟以成骨细胞为研究对象,观察厄贝沙坦对AGEs诱导下的成骨细胞增殖、凋亡、成骨分化、氧化应激、RAGE表达等方面的影响。进而明确厄贝沙坦能否通过阻断AGEs-RAGE的作用预防或治疗糖尿病骨质疏松症。内容本研究分为以下两部分:第一部分AGEs诱导成骨细胞损伤目的本部分实验以成骨细胞为研究对象,采用不同浓度及作用时间的AGEs对成骨细胞进行处理,筛选出AGEs诱导成骨细胞损伤的合适的作用浓度及时间。方法1、成骨细胞的分离与培养采用颅骨溶解法,分离新生SD大鼠的成骨细胞,并通过传代进行纯化,置于5%C02、饱和湿度、37℃孵箱中培养。2、实验分组探讨不同浓度AGEs对成骨细胞增殖的影响时,分为正常对照组、BSA组、AGEs组(50、100、200、400μg/ml),分别作用于细胞3d。探讨AGEs作用不同时间对成骨细胞增殖的影响时,分为空白对照组、1d、3d、5d、7d、9d组,以100μg/mlAGEs作用于细胞。3、采用CCK-8法检测不同浓度、不同作用时间AGEs对成骨细胞增殖的影响。统计学处理计量资料以均数±标准差(x±S)表示,统计学分析采用SPSS20.0统计软件进行,方差齐时,多样本比较采用单向方差分析(One-WayANOVA)检验,组间两两比较采用LSD检验;方差不齐时,用校正welch检验,组间两两比较采用Dunnett T3法。P0.05具有统计学意义。结果1、不同浓度的AGEs对成骨细胞增殖的影响50、100、200、400μg/ml AGEs作用成骨细胞3d后,AGEs组的OD值与空白对照组及BSA组比较,差异有统计学意义(P0.01),其中10μg/mlAGEs组细胞的OD值与50μg/ml AGEs组比较,差异有统计学意义(P0.01)。2、AGEs作用不同时间对成骨细胞增殖的影响100μg/mlAGEs分别作用成骨细胞1d、3d、5d、7d、9d后,结果发现3d组细胞的OD值与空白对照组、BSA组及其他作用时间组相比明显降低,差异统计学意义(P0.01)。结论筛选出AGEs抑制成骨细胞增殖合适的浓度为100μg/ml,作用时间为3d。第二部分厄贝沙坦对AGEs诱导成骨细胞损伤的影响目的用厄贝沙坦与AGEs联合处理成骨细胞,观察厄贝沙坦对AGEs诱导成骨细胞损伤的影响。方法1、成骨细胞的分离培养同上。2、成骨细胞的增殖、凋亡:MTT法检测成骨细胞的增殖,流式细胞仪测定细细胞凋亡率、胞生长周期、JC-1荧光探针检测线粒体膜电位。3、成骨细胞分化检测:钙化结节染色,RT-PCR法检测成骨分化标志物mRNA表达情况。4、氧化应激的检测:荧光探针检测细胞内氧活性。5、RAGE表达情况:Quantitation RT-PCR法检测RAGE的mRNA表达情况。统计学处理同第一部分。结果1、AGEs组成骨细胞活力减低,细胞增殖能力下降,联合厄贝沙坦处理组成骨细胞增殖较AGEs组改善,差异有统计学意义(P0.01)。2、AGEs作用于成骨细胞后导致细胞周期停滞,联合厄贝沙坦处理组G1期及S期的细胞比例较AGEs组减少。3、与AGES组相比,联合厄贝沙坦处理组能显著减少成骨细胞的凋亡,差异有统计学意义(P0.01)。4、AGEs造成成骨细胞线粒体膜电位下降。荧光显微镜下观察到AGEs组红色荧光减少,绿色荧光增加,联合厄贝沙坦处理组,能够改变上述趋势。5、采用茜素红染色,与正常对照组及BSA组相比,AGEs处理组几乎看不到被染成深红色的钙结节,联合厄贝沙坦处理组,细胞外可以见到被茜素红染成深红色的钙结节。6、AGEs组能显著抑制成骨细胞中ALP、Collagen Ⅰ、RUNX2的表达,差异有显著统计学意义(P0.01),而联合厄贝沙坦处理组,ALP、RUNX2的表达与AGEs组相比具有统计学差异(P0.05),Collagen Ⅰ的表达与AGEs组相比具有显著差异(P0.01)。7、荧光显微镜下观察细胞内活性氧水平,AGEs组细胞的荧光强度与空白对照组相比明显增强,而联合厄贝沙坦处理组细胞的荧光强度比AGEs组显著降低。8、AGEs组能显著增加成骨细胞中RAGEmRNA的表达,与空白对照组相比,差异有显著统计学意义(P0.01)。联合厄贝沙坦处理后,RAGEmRNA的表达较AGEs组显著下调,差异有显著统计学意义(P0.01)。结论本实验证实厄贝沙坦对AGEs诱导的成骨细胞损伤起保护作用。厄贝沙坦可下调RAGE的表达,抑制AGEs诱导的氧化应激,在一定程度上能够改善AGEs诱导的成骨细胞的损伤,促进成骨细胞增殖,减少其凋亡,并促进成骨细胞的分化,增加骨形成相关标志物mRNA的表达。
[Abstract]:In recent years, the prevalence rate of diabetes in our country is increasing rapidly. The diabetes related osteoporosis can make the fracture and other complications significantly increase, seriously affecting the health and quality of life of diabetic patients. In the previous study, the late glycosylation end product (advanced glycation end products, AGEs) in diabetes was found. The acceleration of formation and accumulation is the cause of chronic complications of diabetes, and AGEs plays an important role in diabetic osteoporosis. Effective intervention is used to inhibit AGEs's osteogenesis damage, improve bone formation, and provide a theoretical basis and effective method for clinical prevention and treatment of diabetic osteoporosis. The aim of this study was to investigate the effects of irbesartan on osteoblast proliferation, apoptosis, osteogenesis, oxidative stress, and RAGE expression induced by AGEs induced by osteoblast. And whether erbesartan could prevent or treat diabetic osteoporosis by blocking the role of AGEs-RAGE. The contents of this study were divided into two parts: first Partial AGEs induced osteoblast injury in this experiment, the osteoblasts were used as the research object. The osteoblasts were treated with AGEs of different concentration and action time. The appropriate concentration and time of AGEs induced osteoblast injury were screened out. Method 1, the separation and culture of osteoblasts were separated and cultured with cranial dissolving method, and the new SD was separated. The rat osteoblasts were purified by passage and were placed in 5%C02, saturated humidity, and incubated in the incubator for.2 at 37. The effects of different concentrations of AGEs on the proliferation of osteoblasts were divided into normal control group, BSA group, and AGEs group (50100200400 mu g/ml), respectively, for the proliferation of osteoblasts at different time of the cell 3D. exploration of AGEs. The effects were divided into blank control group, 1D, 3D, 5D, 7d, 9D group, the effect of 100 mu g/mlAGEs on cell.3, the influence of AGEs on the proliferation of osteoblasts by CCK-8 method, and the difference of time AGEs on the proliferation of osteoblasts. Statistical analysis was expressed by mean standard deviation (x + S), statistical analysis was carried out with SPSS20.0 statistics, and the variance was Qi, and the variance was Qi, more The samples were compared with one-way ANOVA (One-WayANOVA) test, and 22 of the groups were compared with LSD test. When the variance was not homogeneous, the corrected Welch test was used, and the 22 of the groups was compared with the Dunnett T3 method.P0.05. Results 1, the effect of different concentrations of AGEs on the proliferation of osteoblasts was 50100200400 mu g/ml AGEs after 3D, Compared with the blank control group and the BSA group, there was a significant difference between the AGEs group and the control group and the BSA group (P0.01). The differences in the OD values of the 10 g/mlAGEs group cells were statistically significant (P0.01).2, and the effect of AGEs on the proliferation of osteoblasts at different times of the AGEs action was 100 mu g/ mlAGEs, respectively. The OD value of the group cells was significantly lower than that in the blank control group, BSA group and other action time groups, and the difference was statistically significant (P0.01). Conclusion the appropriate concentration of AGEs to inhibit the proliferation of osteoblasts was 100 u g/ml, and the effect time was 3D. second, the effect of erbesartan on the injury of osteoblast induced by AGEs was associated with the combination of irbesartan and AGEs. The effect of irbesartan on osteoblast induced damage induced by AGEs was observed. Methods 1, the isolation and culture of osteoblasts were.2, osteoblast proliferation, apoptosis: MTT assay was used to detect the proliferation of osteoblasts. Flow cytometry was used to determine the apoptosis rate, cell growth cycle, and JC-1 fluorescence probe to detect the mitochondrial membrane potential.3, osteoblasts Differentiation detection: calcified nodule staining, RT-PCR method to detect mRNA expression of osteogenic differentiation marker.4, oxidative stress detection: fluorescence probe detection of intracellular oxygen activity.5, RAGE expression: Quantitation RT-PCR method to detect the mRNA expression of RAGE. Statistical treatment is the same as the first part. Results 1, AGEs composition of bone cell vitality decreased, cell increase There was a significant difference between the combined erbesartan treatment and the AGEs group. The difference was statistically significant (P0.01).2, and the AGEs effect on the osteoblast resulted in the stagnation of the cell cycle. The proportion of cells in the G1 phase and S phase of the combined irbesartan treatment group was less.3 than that in the AGEs group. Compared with the AGES group, the combined irbesartan treatment group decreased significantly. The apoptosis of osteoblasts was statistically significant (P0.01).4, AGEs resulted in the decrease of mitochondrial membrane potential in osteoblasts. The red fluorescence decreased and the green fluorescence increased in the AGEs group under the fluorescence microscope. The aforementioned ebesartan treatment group could change the above trend.5 and use alizarin red staining, compared with the normal control group and the BSA group, the AGEs treatment group. It was almost impossible to see the calcium nodule dyed deep red, and in the ebesartan treatment group, the calcium nodules stained with alizarin red were seen outside the cells, and the AGEs group could significantly inhibit the expression of ALP, Collagen I, RUNX2 in the osteoblasts, and the difference was statistically significant (P0.01), while the expression of ALP, RUNX2 and AGE were combined with the treatment group of erbesartan. Compared with group s (P0.05), the expression of Collagen I was significantly different from that of the AGEs group (P0.01).7. The intracellular reactive oxygen level was observed under the fluorescence microscope. The fluorescence intensity of the cells in the AGEs group was significantly increased compared with the blank control group, but the fluorescence intensity of the cells in the combined irbesartan treatment group was significantly lower than that of the AGEs group, and the AGEs was significantly lower than that of the AGEs group. The group could significantly increase the expression of RAGEmRNA in osteoblasts. Compared with the blank control group, the difference was statistically significant (P0.01). After the treatment with irbesartan, the expression of RAGEmRNA was significantly lower than that of the AGEs group. The difference was statistically significant (P0.01). Conclusion the true verbesartan was proved to protect the injury of osteoblast induced by AGEs. Irbesartan can reduce the expression of RAGE and inhibit oxidative stress induced by AGEs. To a certain extent, it can improve the injury of osteoblast induced by AGEs, promote the proliferation of osteoblast, reduce its apoptosis, promote the differentiation of osteoblasts and increase the expression of bone formation related marker mRNA.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1;R580
本文编号：2168795
[Abstract]:In recent years, the prevalence rate of diabetes in our country is increasing rapidly. The diabetes related osteoporosis can make the fracture and other complications significantly increase, seriously affecting the health and quality of life of diabetic patients. In the previous study, the late glycosylation end product (advanced glycation end products, AGEs) in diabetes was found. The acceleration of formation and accumulation is the cause of chronic complications of diabetes, and AGEs plays an important role in diabetic osteoporosis. Effective intervention is used to inhibit AGEs's osteogenesis damage, improve bone formation, and provide a theoretical basis and effective method for clinical prevention and treatment of diabetic osteoporosis. The aim of this study was to investigate the effects of irbesartan on osteoblast proliferation, apoptosis, osteogenesis, oxidative stress, and RAGE expression induced by AGEs induced by osteoblast. And whether erbesartan could prevent or treat diabetic osteoporosis by blocking the role of AGEs-RAGE. The contents of this study were divided into two parts: first Partial AGEs induced osteoblast injury in this experiment, the osteoblasts were used as the research object. The osteoblasts were treated with AGEs of different concentration and action time. The appropriate concentration and time of AGEs induced osteoblast injury were screened out. Method 1, the separation and culture of osteoblasts were separated and cultured with cranial dissolving method, and the new SD was separated. The rat osteoblasts were purified by passage and were placed in 5%C02, saturated humidity, and incubated in the incubator for.2 at 37. The effects of different concentrations of AGEs on the proliferation of osteoblasts were divided into normal control group, BSA group, and AGEs group (50100200400 mu g/ml), respectively, for the proliferation of osteoblasts at different time of the cell 3D. exploration of AGEs. The effects were divided into blank control group, 1D, 3D, 5D, 7d, 9D group, the effect of 100 mu g/mlAGEs on cell.3, the influence of AGEs on the proliferation of osteoblasts by CCK-8 method, and the difference of time AGEs on the proliferation of osteoblasts. Statistical analysis was expressed by mean standard deviation (x + S), statistical analysis was carried out with SPSS20.0 statistics, and the variance was Qi, and the variance was Qi, more The samples were compared with one-way ANOVA (One-WayANOVA) test, and 22 of the groups were compared with LSD test. When the variance was not homogeneous, the corrected Welch test was used, and the 22 of the groups was compared with the Dunnett T3 method.P0.05. Results 1, the effect of different concentrations of AGEs on the proliferation of osteoblasts was 50100200400 mu g/ml AGEs after 3D, Compared with the blank control group and the BSA group, there was a significant difference between the AGEs group and the control group and the BSA group (P0.01). The differences in the OD values of the 10 g/mlAGEs group cells were statistically significant (P0.01).2, and the effect of AGEs on the proliferation of osteoblasts at different times of the AGEs action was 100 mu g/ mlAGEs, respectively. The OD value of the group cells was significantly lower than that in the blank control group, BSA group and other action time groups, and the difference was statistically significant (P0.01). Conclusion the appropriate concentration of AGEs to inhibit the proliferation of osteoblasts was 100 u g/ml, and the effect time was 3D. second, the effect of erbesartan on the injury of osteoblast induced by AGEs was associated with the combination of irbesartan and AGEs. The effect of irbesartan on osteoblast induced damage induced by AGEs was observed. Methods 1, the isolation and culture of osteoblasts were.2, osteoblast proliferation, apoptosis: MTT assay was used to detect the proliferation of osteoblasts. Flow cytometry was used to determine the apoptosis rate, cell growth cycle, and JC-1 fluorescence probe to detect the mitochondrial membrane potential.3, osteoblasts Differentiation detection: calcified nodule staining, RT-PCR method to detect mRNA expression of osteogenic differentiation marker.4, oxidative stress detection: fluorescence probe detection of intracellular oxygen activity.5, RAGE expression: Quantitation RT-PCR method to detect the mRNA expression of RAGE. Statistical treatment is the same as the first part. Results 1, AGEs composition of bone cell vitality decreased, cell increase There was a significant difference between the combined erbesartan treatment and the AGEs group. The difference was statistically significant (P0.01).2, and the AGEs effect on the osteoblast resulted in the stagnation of the cell cycle. The proportion of cells in the G1 phase and S phase of the combined irbesartan treatment group was less.3 than that in the AGEs group. Compared with the AGES group, the combined irbesartan treatment group decreased significantly. The apoptosis of osteoblasts was statistically significant (P0.01).4, AGEs resulted in the decrease of mitochondrial membrane potential in osteoblasts. The red fluorescence decreased and the green fluorescence increased in the AGEs group under the fluorescence microscope. The aforementioned ebesartan treatment group could change the above trend.5 and use alizarin red staining, compared with the normal control group and the BSA group, the AGEs treatment group. It was almost impossible to see the calcium nodule dyed deep red, and in the ebesartan treatment group, the calcium nodules stained with alizarin red were seen outside the cells, and the AGEs group could significantly inhibit the expression of ALP, Collagen I, RUNX2 in the osteoblasts, and the difference was statistically significant (P0.01), while the expression of ALP, RUNX2 and AGE were combined with the treatment group of erbesartan. Compared with group s (P0.05), the expression of Collagen I was significantly different from that of the AGEs group (P0.01).7. The intracellular reactive oxygen level was observed under the fluorescence microscope. The fluorescence intensity of the cells in the AGEs group was significantly increased compared with the blank control group, but the fluorescence intensity of the cells in the combined irbesartan treatment group was significantly lower than that of the AGEs group, and the AGEs was significantly lower than that of the AGEs group. The group could significantly increase the expression of RAGEmRNA in osteoblasts. Compared with the blank control group, the difference was statistically significant (P0.01). After the treatment with irbesartan, the expression of RAGEmRNA was significantly lower than that of the AGEs group. The difference was statistically significant (P0.01). Conclusion the true verbesartan was proved to protect the injury of osteoblast induced by AGEs. Irbesartan can reduce the expression of RAGE and inhibit oxidative stress induced by AGEs. To a certain extent, it can improve the injury of osteoblast induced by AGEs, promote the proliferation of osteoblast, reduce its apoptosis, promote the differentiation of osteoblasts and increase the expression of bone formation related marker mRNA.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1;R580
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