厄貝沙坦對AGEs誘導成骨細胞損傷的影響
發(fā)布時間：2018-08-06 19:43
【摘要】：研究背景近年來,我國的糖尿病患病率在迅速增長,其造成的糖尿病相關性骨質疏松,可使骨折等并發(fā)癥顯著升高,嚴重影響著糖尿病患者的健康和生活質量。既往研究發(fā)現(xiàn),在糖尿病中,晚期糖基化終產物(advanced glycation end products,AGEs)的形成和積累加速,是導致多種糖尿病慢性并發(fā)癥發(fā)生的原因,AGEs在糖尿病性骨質疏松中亦起者重要作用。使用有效的干預途徑抑制AGEs的對成骨的損傷,改善骨形成,為臨床防治糖尿病性骨質疏松提供理論根據和有效的方法。目的本課題擬以成骨細胞為研究對象,觀察厄貝沙坦對AGEs誘導下的成骨細胞增殖、凋亡、成骨分化、氧化應激、RAGE表達等方面的影響。進而明確厄貝沙坦能否通過阻斷AGEs-RAGE的作用預防或治療糖尿病骨質疏松癥。內容本研究分為以下兩部分:第一部分AGEs誘導成骨細胞損傷目的本部分實驗以成骨細胞為研究對象,采用不同濃度及作用時間的AGEs對成骨細胞進行處理,篩選出AGEs誘導成骨細胞損傷的合適的作用濃度及時間。方法1、成骨細胞的分離與培養(yǎng)采用顱骨溶解法,分離新生SD大鼠的成骨細胞,并通過傳代進行純化,置于5%C02、飽和濕度、37℃孵箱中培養(yǎng)。2、實驗分組探討不同濃度AGEs對成骨細胞增殖的影響時,分為正常對照組、BSA組、AGEs組(50、100、200、400μg/ml),分別作用于細胞3d。探討AGEs作用不同時間對成骨細胞增殖的影響時,分為空白對照組、1d、3d、5d、7d、9d組,以100μg/mlAGEs作用于細胞。3、采用CCK-8法檢測不同濃度、不同作用時間AGEs對成骨細胞增殖的影響。統(tǒng)計學處理計量資料以均數(shù)±標準差(x±S)表示,統(tǒng)計學分析采用SPSS20.0統(tǒng)計軟件進行,方差齊時,多樣本比較采用單向方差分析(One-WayANOVA)檢驗,組間兩兩比較采用LSD檢驗;方差不齊時,用校正welch檢驗,組間兩兩比較采用Dunnett T3法。P0.05具有統(tǒng)計學意義。結果1、不同濃度的AGEs對成骨細胞增殖的影響50、100、200、400μg/ml AGEs作用成骨細胞3d后,AGEs組的OD值與空白對照組及BSA組比較,差異有統(tǒng)計學意義(P0.01),其中10μg/mlAGEs組細胞的OD值與50μg/ml AGEs組比較,差異有統(tǒng)計學意義(P0.01)。2、AGEs作用不同時間對成骨細胞增殖的影響100μg/mlAGEs分別作用成骨細胞1d、3d、5d、7d、9d后,結果發(fā)現(xiàn)3d組細胞的OD值與空白對照組、BSA組及其他作用時間組相比明顯降低,差異統(tǒng)計學意義(P0.01)。結論篩選出AGEs抑制成骨細胞增殖合適的濃度為100μg/ml,作用時間為3d。第二部分厄貝沙坦對AGEs誘導成骨細胞損傷的影響目的用厄貝沙坦與AGEs聯(lián)合處理成骨細胞,觀察厄貝沙坦對AGEs誘導成骨細胞損傷的影響。方法1、成骨細胞的分離培養(yǎng)同上。2、成骨細胞的增殖、凋亡:MTT法檢測成骨細胞的增殖,流式細胞儀測定細細胞凋亡率、胞生長周期、JC-1熒光探針檢測線粒體膜電位。3、成骨細胞分化檢測:鈣化結節(jié)染色,RT-PCR法檢測成骨分化標志物mRNA表達情況。4、氧化應激的檢測:熒光探針檢測細胞內氧活性。5、RAGE表達情況:Quantitation RT-PCR法檢測RAGE的mRNA表達情況。統(tǒng)計學處理同第一部分。結果1、AGEs組成骨細胞活力減低,細胞增殖能力下降,聯(lián)合厄貝沙坦處理組成骨細胞增殖較AGEs組改善,差異有統(tǒng)計學意義(P0.01)。2、AGEs作用于成骨細胞后導致細胞周期停滯,聯(lián)合厄貝沙坦處理組G1期及S期的細胞比例較AGEs組減少。3、與AGES組相比,聯(lián)合厄貝沙坦處理組能顯著減少成骨細胞的凋亡,差異有統(tǒng)計學意義(P0.01)。4、AGEs造成成骨細胞線粒體膜電位下降。熒光顯微鏡下觀察到AGEs組紅色熒光減少,綠色熒光增加,聯(lián)合厄貝沙坦處理組,能夠改變上述趨勢。5、采用茜素紅染色,與正常對照組及BSA組相比,AGEs處理組幾乎看不到被染成深紅色的鈣結節(jié),聯(lián)合厄貝沙坦處理組,細胞外可以見到被茜素紅染成深紅色的鈣結節(jié)。6、AGEs組能顯著抑制成骨細胞中ALP、Collagen Ⅰ、RUNX2的表達,差異有顯著統(tǒng)計學意義(P0.01),而聯(lián)合厄貝沙坦處理組,ALP、RUNX2的表達與AGEs組相比具有統(tǒng)計學差異(P0.05),Collagen Ⅰ的表達與AGEs組相比具有顯著差異(P0.01)。7、熒光顯微鏡下觀察細胞內活性氧水平,AGEs組細胞的熒光強度與空白對照組相比明顯增強,而聯(lián)合厄貝沙坦處理組細胞的熒光強度比AGEs組顯著降低。8、AGEs組能顯著增加成骨細胞中RAGEmRNA的表達,與空白對照組相比,差異有顯著統(tǒng)計學意義(P0.01)。聯(lián)合厄貝沙坦處理后,RAGEmRNA的表達較AGEs組顯著下調,差異有顯著統(tǒng)計學意義(P0.01)。結論本實驗證實厄貝沙坦對AGEs誘導的成骨細胞損傷起保護作用。厄貝沙坦可下調RAGE的表達,抑制AGEs誘導的氧化應激,在一定程度上能夠改善AGEs誘導的成骨細胞的損傷,促進成骨細胞增殖,減少其凋亡,并促進成骨細胞的分化,增加骨形成相關標志物mRNA的表達。
[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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.1;R580
本文編號：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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.1;R580
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