厄貝沙坦對(duì)AGEs誘導(dǎo)成骨細(xì)胞損傷的影響
發(fā)布時(shí)間：2018-08-06 19:43
【摘要】：研究背景近年來(lái),我國(guó)的糖尿病患病率在迅速增長(zhǎng),其造成的糖尿病相關(guān)性骨質(zhì)疏松,可使骨折等并發(fā)癥顯著升高,嚴(yán)重影響著糖尿病患者的健康和生活質(zhì)量。既往研究發(fā)現(xiàn),在糖尿病中,晚期糖基化終產(chǎn)物(advanced glycation end products,AGEs)的形成和積累加速,是導(dǎo)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(zhì)疏松中亦起者重要作用。使用有效的干預(yù)途徑抑制AGEs的對(duì)成骨的損傷,改善骨形成,為臨床防治糖尿病性骨質(zhì)疏松提供理論根據(jù)和有效的方法。目的本課題擬以成骨細(xì)胞為研究對(duì)象,觀察厄貝沙坦對(duì)AGEs誘導(dǎo)下的成骨細(xì)胞增殖、凋亡、成骨分化、氧化應(yīng)激、RAGE表達(dá)等方面的影響。進(jìn)而明確厄貝沙坦能否通過(guò)阻斷AGEs-RAGE的作用預(yù)防或治療糖尿病骨質(zhì)疏松癥。內(nèi)容本研究分為以下兩部分:第一部分AGEs誘導(dǎo)成骨細(xì)胞損傷目的本部分實(shí)驗(yàn)以成骨細(xì)胞為研究對(duì)象,采用不同濃度及作用時(shí)間的AGEs對(duì)成骨細(xì)胞進(jìn)行處理,篩選出AGEs誘導(dǎo)成骨細(xì)胞損傷的合適的作用濃度及時(shí)間。方法1、成骨細(xì)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(xì)胞,并通過(guò)傳代進(jìn)行純化,置于5%C02、飽和濕度、37℃孵箱中培養(yǎng)。2、實(shí)驗(yàn)分組探討不同濃度AGEs對(duì)成骨細(xì)胞增殖的影響時(shí),分為正常對(duì)照組、BSA組、AGEs組(50、100、200、400μg/ml),分別作用于細(xì)胞3d。探討AGEs作用不同時(shí)間對(duì)成骨細(xì)胞增殖的影響時(shí),分為空白對(duì)照組、1d、3d、5d、7d、9d組,以100μg/mlAGEs作用于細(xì)胞。3、采用CCK-8法檢測(cè)不同濃度、不同作用時(shí)間AGEs對(duì)成骨細(xì)胞增殖的影響。統(tǒng)計(jì)學(xué)處理計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差(x±S)表示,統(tǒng)計(jì)學(xué)分析采用SPSS20.0統(tǒng)計(jì)軟件進(jìn)行,方差齊時(shí),多樣本比較采用單向方差分析(One-WayANOVA)檢驗(yàn),組間兩兩比較采用LSD檢驗(yàn);方差不齊時(shí),用校正welch檢驗(yàn),組間兩兩比較采用Dunnett T3法。P0.05具有統(tǒng)計(jì)學(xué)意義。結(jié)果1、不同濃度的AGEs對(duì)成骨細(xì)胞增殖的影響50、100、200、400μg/ml AGEs作用成骨細(xì)胞3d后,AGEs組的OD值與空白對(duì)照組及BSA組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.01),其中10μg/mlAGEs組細(xì)胞的OD值與50μg/ml AGEs組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。2、AGEs作用不同時(shí)間對(duì)成骨細(xì)胞增殖的影響100μg/mlAGEs分別作用成骨細(xì)胞1d、3d、5d、7d、9d后,結(jié)果發(fā)現(xiàn)3d組細(xì)胞的OD值與空白對(duì)照組、BSA組及其他作用時(shí)間組相比明顯降低,差異統(tǒng)計(jì)學(xué)意義(P0.01)。結(jié)論篩選出AGEs抑制成骨細(xì)胞增殖合適的濃度為100μg/ml,作用時(shí)間為3d。第二部分厄貝沙坦對(duì)AGEs誘導(dǎo)成骨細(xì)胞損傷的影響目的用厄貝沙坦與AGEs聯(lián)合處理成骨細(xì)胞,觀察厄貝沙坦對(duì)AGEs誘導(dǎo)成骨細(xì)胞損傷的影響。方法1、成骨細(xì)胞的分離培養(yǎng)同上。2、成骨細(xì)胞的增殖、凋亡:MTT法檢測(cè)成骨細(xì)胞的增殖,流式細(xì)胞儀測(cè)定細(xì)細(xì)胞凋亡率、胞生長(zhǎng)周期、JC-1熒光探針檢測(cè)線粒體膜電位。3、成骨細(xì)胞分化檢測(cè):鈣化結(jié)節(jié)染色,RT-PCR法檢測(cè)成骨分化標(biāo)志物mRNA表達(dá)情況。4、氧化應(yīng)激的檢測(cè):熒光探針檢測(cè)細(xì)胞內(nèi)氧活性。5、RAGE表達(dá)情況:Quantitation RT-PCR法檢測(cè)RAGE的mRNA表達(dá)情況。統(tǒng)計(jì)學(xué)處理同第一部分。結(jié)果1、AGEs組成骨細(xì)胞活力減低,細(xì)胞增殖能力下降,聯(lián)合厄貝沙坦處理組成骨細(xì)胞增殖較AGEs組改善,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。2、AGEs作用于成骨細(xì)胞后導(dǎo)致細(xì)胞周期停滯,聯(lián)合厄貝沙坦處理組G1期及S期的細(xì)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(xì)胞的凋亡,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。4、AGEs造成成骨細(xì)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(shì)。5、采用茜素紅染色,與正常對(duì)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(jié)節(jié),聯(lián)合厄貝沙坦處理組,細(xì)胞外可以見(jiàn)到被茜素紅染成深紅色的鈣結(jié)節(jié)。6、AGEs組能顯著抑制成骨細(xì)胞中ALP、Collagen Ⅰ、RUNX2的表達(dá),差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01),而聯(lián)合厄貝沙坦處理組,ALP、RUNX2的表達(dá)與AGEs組相比具有統(tǒng)計(jì)學(xué)差異(P0.05),Collagen Ⅰ的表達(dá)與AGEs組相比具有顯著差異(P0.01)。7、熒光顯微鏡下觀察細(xì)胞內(nèi)活性氧水平,AGEs組細(xì)胞的熒光強(qiáng)度與空白對(duì)照組相比明顯增強(qiáng),而聯(lián)合厄貝沙坦處理組細(xì)胞的熒光強(qiáng)度比AGEs組顯著降低。8、AGEs組能顯著增加成骨細(xì)胞中RAGEmRNA的表達(dá),與空白對(duì)照組相比,差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01)。聯(lián)合厄貝沙坦處理后,RAGEmRNA的表達(dá)較AGEs組顯著下調(diào),差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01)。結(jié)論本實(shí)驗(yàn)證實(shí)厄貝沙坦對(duì)AGEs誘導(dǎo)的成骨細(xì)胞損傷起保護(hù)作用。厄貝沙坦可下調(diào)RAGE的表達(dá),抑制AGEs誘導(dǎo)的氧化應(yīng)激,在一定程度上能夠改善AGEs誘導(dǎo)的成骨細(xì)胞的損傷,促進(jìn)成骨細(xì)胞增殖,減少其凋亡,并促進(jìn)成骨細(xì)胞的分化,增加骨形成相關(guān)標(biāo)志物mRNA的表達(dá)。
[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(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.1;R580
本文編號(hào)：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(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.1;R580
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