本文選題：缺氧再?gòu)?fù)氧　切入點(diǎn)：缺氧　出處：《皖南醫(yī)學(xué)院》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:探討缺氧及缺氧再?gòu)?fù)氧環(huán)境下新生大鼠成骨細(xì)胞細(xì)胞活性、凋亡率及I型膠原(Collagen type I)、骨形態(tài)發(fā)生蛋白2(BMP-2)、核心結(jié)合因子2(RUNX2)、轉(zhuǎn)化生長(zhǎng)因子β1(TGF-β1)基因及蛋白表達(dá)的變化。方法:采用骨組織塊培養(yǎng)法、酶消化法及差速貼壁法獲得純凈的新生SD大鼠成骨細(xì)胞,采用茜素紅染色(Alizarin Red staining)和堿性磷酸酶染色(Alkaline phosphatase staining)對(duì)成骨細(xì)胞進(jìn)行鑒定。取第3-4代成骨細(xì)胞,分別常氧培養(yǎng)36h,缺氧培養(yǎng)24h,缺氧培養(yǎng)36h,缺氧培養(yǎng)24h再?gòu)?fù)氧培養(yǎng)12h后,利用茜素紅S染色法檢測(cè)四組成骨細(xì)胞鈣鹽沉積;采用MTT法檢測(cè)四組成骨細(xì)胞增值率;采用流式細(xì)胞技術(shù)檢測(cè)四組成骨細(xì)胞的凋亡率;采用實(shí)時(shí)熒光定量PCR檢測(cè)成骨細(xì)胞成骨特異性基因I型膠原(Collagen type I)、骨形態(tài)發(fā)生蛋白2(BMP-2)、核心結(jié)合因子α1(RUNX-2)、轉(zhuǎn)化生長(zhǎng)因子β1(TGF-β1)mRNA表達(dá)情況,利用蛋白免疫技術(shù)(Western-blot)法檢測(cè)四組成骨細(xì)胞I型膠原、骨形態(tài)發(fā)生蛋白2、核心結(jié)合因子α1、轉(zhuǎn)化生長(zhǎng)因子β1蛋白表達(dá)情況。結(jié)果:原代提取并純化的新生SD大鼠成骨細(xì)胞24h后完全貼壁,7d可以長(zhǎng)滿培養(yǎng)瓶。酶消化法傳代培養(yǎng)后細(xì)胞貼壁較原代細(xì)胞快,約6h可完全貼壁,2-3天可長(zhǎng)滿培養(yǎng)瓶。在10×40倍顯微鏡下,單個(gè)成骨細(xì)胞形態(tài)呈梭形、多邊形,待成骨細(xì)胞長(zhǎng)滿培養(yǎng)瓶時(shí),呈卵石路樣,符合成骨細(xì)胞形態(tài)特征;用茜素紅S染色法鑒定14d培養(yǎng)細(xì)胞,可見(jiàn)培養(yǎng)瓶底遍布染成紅色的鈣結(jié)節(jié);用偶氮偶聯(lián)法堿性磷酸酶染色鑒定2d培養(yǎng)細(xì)胞,顯示細(xì)胞內(nèi)紫褐色顆粒,鑒定細(xì)胞內(nèi)可合成堿性磷酸酶。由此鑒定所培養(yǎng)的細(xì)胞為成骨細(xì)胞。MTT法檢測(cè)成骨細(xì)胞活性:缺氧及缺氧復(fù)氧環(huán)境下成骨細(xì)胞活性降低,常氧培養(yǎng)組活性為(99.12±8.33)%,缺氧24h組為(90.87±9.41)%,缺氧36h組為(79.86±8.72)%,缺氧24h復(fù)氧12h組為(72.95±8.16)%,缺氧24h復(fù)氧12h組組缺氧36h組組缺氧24h組常氧培養(yǎng)組(F=37.886,p=0.000);成骨細(xì)胞凋亡率增加,常氧培養(yǎng)組為(1.86±1.33)%,缺氧24h組為(16.33±2.48)%,缺氧36h組(28.17±4.16)%,缺氧24h復(fù)氧12h組為(33.52±3.62)%,缺氧24h復(fù)氧12h組缺氧36h組缺氧24h組常氧培養(yǎng)組(F=26.198,p=0.000);BMP-2、RUNX-2、Collagen type I、TGF-β1 mRNA表達(dá)量降低,且缺氧24h復(fù)氧12h組缺氧36h組缺氧24h組常氧培養(yǎng)組(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蛋白表達(dá)量降低,且缺氧24h復(fù)氧12h組缺氧36h組缺氧24h組常氧培養(yǎng)組(F=8.114,p=0.013;F=28.935,p=0.000;F=9.857,p=0.007;F=46.541,p=0.000)。結(jié)論:缺氧及缺氧再?gòu)?fù)氧環(huán)境會(huì)降低成骨細(xì)胞活性,增加成骨細(xì)胞凋亡率,下調(diào)成骨特異性基因的表達(dá)。同時(shí)缺氧再?gòu)?fù)氧環(huán)境會(huì)加重缺氧狀態(tài)下成骨細(xì)胞的損傷。
[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 偽 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 偽 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 鹵8.33, that of hypoxia 24h group was 90.87 鹵9.41, that of hypoxia group was 79.86 鹵8.72, that of 12h group was 72.95 鹵8.16, that of hypoxia 24h reoxygenated 12h group was 72.95 鹵8.16, that of hypoxia 24h reoxygenation group was 36h hypoxia, that of normoxic culture group was 90.87 鹵9.41, and that of osteoblast apoptosis was increased. The normal oxygen culture group (1.86 鹵1.33), hypoxia 24h group (16.33 鹵2.48), hypoxia 36h group (28.17 鹵4.16), hypoxia 24h reoxygenation 12h group (33.52 鹵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.
【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R580;R683

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