發(fā)布時(shí)間：2018-04-10 07:50

  本文選題：Collagen　切入點(diǎn)：type　出處：《北京協(xié)和醫(yī)學(xué)院》2012年碩士論文

【摘要】：大量研究表明骨組織對(duì)輻射作用非常敏感,輻射可造成骨損傷。本實(shí)驗(yàn)從基因和蛋白水平研究了輻射對(duì)成骨細(xì)胞(osteoblasts,OB)的影響及輻射后的成骨細(xì)胞對(duì)破骨細(xì)胞(osteoclasts,OC)的影響。 本實(shí)驗(yàn)首先研究了成骨細(xì)胞輻射后Ⅰ型膠原蛋白的變化。實(shí)驗(yàn)的第一步是抽取小鼠骨髓,然后將骨髓基質(zhì)細(xì)胞在體外誘導(dǎo)成成骨細(xì)胞,進(jìn)而對(duì)其特性進(jìn)行分析,例如觀察細(xì)胞形態(tài)特征、堿性磷酸酶染色等。結(jié)果表明骨髓基質(zhì)細(xì)胞在誘導(dǎo)培養(yǎng)基的作用下,分化成了成骨細(xì)胞。根據(jù)培養(yǎng)階段的不同,成骨細(xì)胞分為早期成骨細(xì)胞和晚期成骨細(xì)胞。分別提取RNA,進(jìn)行逆轉(zhuǎn)錄,用RT-PCR方法分析了1-4Gy照射的早期和晚期成骨細(xì)胞的Collagen type Ⅰ表達(dá)。揭示輻射對(duì)于成骨細(xì)胞分泌Collagen type Ⅰ能力的影響。 實(shí)驗(yàn)結(jié)果表明,與對(duì)照組相比,1-3Gy劑量照射后的早期成骨細(xì)胞Collagen typeⅠ的表達(dá)增強(qiáng)(P0.05),但培養(yǎng)10d后的晚期成骨細(xì)胞Collagen type Ⅰ的表達(dá)降低；在4Gy劑量照射下,晚期成骨細(xì)胞Collagen type Ⅰ的表達(dá)明顯高于早期成骨細(xì)胞的表達(dá)(P0.01)。總之,在1-3Gy輻射后,早期成骨細(xì)胞Collagen type Ⅰ的表達(dá)增強(qiáng),成骨能力增強(qiáng)；晚期成骨細(xì)胞Collagen type Ⅰ的表達(dá)降低,成骨能力減弱。而4Gy輻射后,晚期成骨細(xì)胞Collagen type Ⅰ的表達(dá)增強(qiáng),有可能是其代償功能表現(xiàn)。 同時(shí),探索了輻射損傷的成骨細(xì)胞對(duì)破骨細(xì)胞生長(zhǎng)和功能的影響。在正常生理情況下,骨重建的更新過(guò)程依賴(lài)于破骨細(xì)胞和成骨細(xì)胞的相互制約,使骨吸收與骨形成處于平衡狀態(tài)。破骨細(xì)胞、成骨細(xì)胞之間功能和數(shù)量的失調(diào)將導(dǎo)致骨骼形態(tài)結(jié)構(gòu)的異常。我們初步觀察了輻射損傷的成骨細(xì)胞對(duì)破骨細(xì)胞功能的調(diào)節(jié)。 本研究采用小鼠巨噬細(xì)胞系(RAW264.7)模擬破骨前體細(xì)胞,用輻射后的成骨細(xì)胞上清對(duì)RAW264.7細(xì)胞進(jìn)行培養(yǎng),分析RAW264.7細(xì)胞克隆,判定其增值能力。應(yīng)用實(shí)時(shí)定量聚合酶鏈反應(yīng)(Real-time polymerase chain reaction,Real-time PCR)方法,檢測(cè)RAW264.7細(xì)胞的抗灑石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP).核因子-κB受體活化因子(Receptor activator of nuclear Factor-κB,RANK)、及Notch-2通路中相關(guān)因子表達(dá)的變化情況。 結(jié)果顯示,經(jīng)過(guò)照射損傷的成骨細(xì)胞所提取的上清液培養(yǎng)RAW264.7細(xì)胞生長(zhǎng)克隆數(shù)隨著成骨細(xì)胞受照射劑量的增加而減少；與對(duì)照組相比,RANK、TRAP基因及Notch-2信號(hào)相關(guān)分子的表達(dá)也明顯降低(p0.01)。因此我們推測(cè)照射后的成骨細(xì)胞的上清液通過(guò)幾種信號(hào)途徑對(duì)破骨前體細(xì)胞RAW264.7的增值和分化起抑制作用。 總之,本研究有助于我們了解輻射后的成骨細(xì)胞是如何對(duì)破骨細(xì)胞進(jìn)行調(diào)節(jié)的。使我們進(jìn)一步認(rèn)識(shí),骨形成和骨吸收在骨的更新和改建中過(guò)程中發(fā)揮著重要作用。
[Abstract]:A large number of studies show that bone tissue is very sensitive to radiation, radiation can cause bone damage.In this study, the effects of radiation on osteoblastsof osteoblasts and osteoclasts osteoclasts OC were studied at the gene and protein levels.In this study, the changes of type I collagen protein were studied after radiation of osteoblasts.The first step of the experiment was to extract mouse bone marrow, then induce bone marrow stromal cells into osteoblasts in vitro, and then analyze their characteristics, such as observing the morphological characteristics of cells, alkaline phosphatase staining and so on.The results showed that bone marrow stromal cells differentiated into osteoblasts under induction medium.Osteoblasts are divided into early osteoblasts and late osteoblasts according to the stage of culture.The expression of Collagen type 鈪，

本文編號(hào)：1730326