缺氧及缺氧复氧环境对成骨细胞影响的研究

发布时间：2018-03-22 13:00

  本文选题：缺氧再复氧　切入点：缺氧　出处：《皖南医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:探讨缺氧及缺氧再复氧环境下新生大鼠成骨细胞细胞活性、凋亡率及I型胶原(Collagen type I)、骨形态发生蛋白2(BMP-2)、核心结合因子2(RUNX2)、转化生长因子β1(TGF-β1)基因及蛋白表达的变化。方法:采用骨组织块培养法、酶消化法及差速贴壁法获得纯净的新生SD大鼠成骨细胞,采用茜素红染色(Alizarin Red staining)和碱性磷酸酶染色(Alkaline phosphatase staining)对成骨细胞进行鉴定。取第3-4代成骨细胞,分别常氧培养36h,缺氧培养24h,缺氧培养36h,缺氧培养24h再复氧培养12h后,利用茜素红S染色法检测四组成骨细胞钙盐沉积;采用MTT法检测四组成骨细胞增值率;采用流式细胞技术检测四组成骨细胞的凋亡率;采用实时荧光定量PCR检测成骨细胞成骨特异性基因I型胶原(Collagen type I)、骨形态发生蛋白2(BMP-2)、核心结合因子α1(RUNX-2)、转化生长因子β1(TGF-β1)mRNA表达情况,利用蛋白免疫技术(Western-blot)法检测四组成骨细胞I型胶原、骨形态发生蛋白2、核心结合因子α1、转化生长因子β1蛋白表达情况。结果:原代提取并纯化的新生SD大鼠成骨细胞24h后完全贴壁,7d可以长满培养瓶。酶消化法传代培养后细胞贴壁较原代细胞快,约6h可完全贴壁,2-3天可长满培养瓶。在10×40倍显微镜下,单个成骨细胞形态呈梭形、多边形,待成骨细胞长满培养瓶时,呈卵石路样,符合成骨细胞形态特征;用茜素红S染色法鉴定14d培养细胞,可见培养瓶底遍布染成红色的钙结节;用偶氮偶联法碱性磷酸酶染色鉴定2d培养细胞,显示细胞内紫褐色颗粒,鉴定细胞内可合成碱性磷酸酶。由此鉴定所培养的细胞为成骨细胞。MTT法检测成骨细胞活性:缺氧及缺氧复氧环境下成骨细胞活性降低,常氧培养组活性为(99.12±8.33)%,缺氧24h组为(90.87±9.41)%,缺氧36h组为(79.86±8.72)%,缺氧24h复氧12h组为(72.95±8.16)%,缺氧24h复氧12h组组缺氧36h组组缺氧24h组常氧培养组(F=37.886,p=0.000);成骨细胞凋亡率增加,常氧培养组为(1.86±1.33)%,缺氧24h组为(16.33±2.48)%,缺氧36h组(28.17±4.16)%,缺氧24h复氧12h组为(33.52±3.62)%,缺氧24h复氧12h组缺氧36h组缺氧24h组常氧培养组(F=26.198,p=0.000);BMP-2、RUNX-2、Collagen type I、TGF-β1 mRNA表达量降低,且缺氧24h复氧12h组缺氧36h组缺氧24h组常氧培养组(F=13.082,p=0.006;F=7.088,p=0.017;F=6.857,p=0.038;F=51.368,p=0.000);BMP-2、RUNX-2、Collagen type I、TGF-β1蛋白表达量降低,且缺氧24h复氧12h组缺氧36h组缺氧24h组常氧培养组(F=8.114,p=0.013;F=28.935,p=0.000;F=9.857,p=0.007;F=46.541,p=0.000)。结论:缺氧及缺氧再复氧环境会降低成骨细胞活性,增加成骨细胞凋亡率,下调成骨特异性基因的表达。同时缺氧再复氧环境会加重缺氧状态下成骨细胞的损伤。
[Abstract]:Objective: to investigate the osteoblast activity of newborn rats under hypoxia and hypoxia reoxygenation. Apoptosis rate and changes of gene and protein expression of collagen I type, bone morphogenetic protein 2BMP 2, core binding factor 2rUNX2, transforming growth factor 尾 1 (TGF- 尾 1). Methods: bone tissue culture method was used to investigate the expression of TGF- 尾 1, TGF- 尾 1, TGF- 尾 1, TGF- 尾 1, TGF- 尾 1, TGF- 尾 1 and TGF- 尾 1. Pure neonatal SD rat osteoblasts were obtained by enzyme digestion and differential adherence method. The osteoblasts were identified by alizarin red staining (Alizarin Red staining) and alkaline phosphatase staining (Alkaline phosphatase staininging). The calcium deposition of osteoblasts was detected by alizarin red S staining after hypoxia culture for 36 h, hypoxia culture for 24 h, hypoxia culture for 36 h and reoxygenation for 24 h, and osteoblast proliferation rate for 4 groups was detected by MTT method. The apoptosis rate of osteoblasts in four groups was detected by flow cytometry, and the expression of osteoblast specific gene type I collagen type, bone morphogenetic protein 2 (BMP-2), core binding factor 伪 1RUNX-2and transforming growth factor 尾 1 (TGF- 尾) were detected by real-time fluorescence quantitative PCR. Type I collagen of osteoblasts in four groups was detected by Western blot method. Expression of bone morphogenetic protein 2, core binding factor 伪 1 and transforming growth factor 尾 1. Results: osteoblasts of newborn SD rats extracted and purified in primary culture could be cultured in culture flask for 7 days after 24 hours. After culture, the cells adhered to the wall faster than the primary cells. Under 10 脳 40 times microscope, the single osteoblast was fusiform and polygonal. When the osteoblast was filled with the flask, the osteoblast appeared as pebbles, which was in accordance with the morphological characteristics of osteoblasts. The cultured cells were identified by alizarin red S staining for 14 days, and red calcium nodules were found on the bottom of the culture bottle, and alkaline phosphatase staining with azo coupling method was used to identify the cultured cells for 2 days. The osteoblasts were identified as osteoblasts. MTT assay was used to detect the activity of osteoblasts: the activity of osteoblasts was decreased under hypoxia and hypoxia reoxygenation. The activity of normal oxygen culture group was 99.12 卤8.33, that of hypoxia 24h group was 90.87 卤9.41, that of hypoxia group was 79.86 卤8.72, that of 12h group was 72.95 卤8.16, that of hypoxia 24h reoxygenated 12h group was 72.95 卤8.16, that of hypoxia 24h reoxygenation group was 36h hypoxia, that of normoxic culture group was 90.87 卤9.41, and that of osteoblast apoptosis was increased. The normal oxygen culture group (1.86 卤1.33), hypoxia 24h group (16.33 卤2.48), hypoxia 36h group (28.17 卤4.16), hypoxia 24h reoxygenation 12h group (33.52 卤3.62), hypoxia 24h reoxygenation group (36h) hypoxia group (36h) hypoxia group (36h) hypoxia group (F26.198p0.000) decreased the expression of BMP-2RUNX-2Collagen type IGF- 尾 1 mRNA. And the protein expression of BMP-2Collagen type ITGF- 尾 1 protein decreased in hypoxia 24h reoxygenation 12h group hypoxia 36h group hypoxia 36h hypoxia group hypoxia 36h group hypoxia culture group normal oxygen culture group F13.082p0.006F7.088p 0.017F6.857p0.038FU 51.368p0.000m BMP-2Collagen type ITGF- 尾 1 protein expression, and hypoxia 24h reoxygenation 12h hypoxia group hypoxia 36h group hypoxia 36h group hypoxia culture group normal oxygen culture group F8.114p0.013F28.935p0.000F9.857F0. 007FN 46.541p0.000. Conclusion: hypoxia and reoxygenation can reduce osteoblast activity. The apoptosis rate of osteoblasts was increased and the expression of osteoblast-specific genes was down-regulated. At the same time, hypoxia and reoxygenation environment would aggravate the damage of osteoblasts under anoxic condition.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R580;R683

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