本文選題：氟骨癥 + 骨轉(zhuǎn)換　； 參考：《吉林大學(xué)》2014年博士論文

【摘要】：研究背景： 慢性氟中毒時(shí)成骨細(xì)胞怎樣被激活、骨轉(zhuǎn)換怎樣被加速，是地方性氟中毒發(fā)病機(jī)制中亟待解決的關(guān)鍵問(wèn)題。以往研究中發(fā)現(xiàn)，染氟成骨細(xì)胞中與內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)的蛋白BiP、蛋白質(zhì)二硫鍵異構(gòu)酶、蛋白酶體26SATP酶和硫氧還蛋白表達(dá)增多。本實(shí)驗(yàn)擬體外培養(yǎng)成骨細(xì)胞染氟并結(jié)合整體氟中毒動(dòng)物實(shí)驗(yàn)，觀察內(nèi)質(zhì)網(wǎng)應(yīng)激引發(fā)的關(guān)鍵因子PERK及其下游ATF4和Nrf2等基因和蛋白水平的表達(dá)變化，分析其與成骨細(xì)胞增生、分化和骨形成間的相互關(guān)系，探討內(nèi)質(zhì)網(wǎng)應(yīng)激及PERK信號(hào)通路在氟作用下成骨細(xì)胞激活、骨轉(zhuǎn)換加速中的作用；檢測(cè)與成骨細(xì)胞增殖、分化等相關(guān)的調(diào)控因子的變化，闡明內(nèi)質(zhì)網(wǎng)應(yīng)激通過(guò)哪些因素介導(dǎo)氟中毒骨病變的發(fā)生、發(fā)展。本實(shí)驗(yàn)的完成有助于發(fā)現(xiàn)以往氟骨癥機(jī)制研究從未涉及的新的因子和信號(hào)通路的重要作用，為氟骨癥防治策略提供新的思路和理論依據(jù)。 實(shí)驗(yàn)方法： 動(dòng)物實(shí)驗(yàn)：100只Wistar大鼠，按體重均勻分成對(duì)照組、低氟組和高氟組3組。大鼠通過(guò)灌胃給氟（雙蒸水配置NaF,濃度為2.21g/L，相當(dāng)于100mgF-/ml），低氟組大鼠按照每天10mg F-/kg體重給氟，高氟組大鼠每天20mg F-/kg體重給氟，對(duì)照組通過(guò)灌胃給以雙蒸水。大鼠投氟1、2和3月后分別經(jīng)乙醚麻醉，采血、分離血清以備生化檢測(cè)。實(shí)驗(yàn)結(jié)束后取一側(cè)脛骨采用Hologic Discovery WA骨密度儀檢測(cè)大鼠整只脛骨骨密度變化。同時(shí)，取一側(cè)大鼠股骨經(jīng)10%EDTA脫鈣，常規(guī)脫水、石蠟包埋、制作病理切片，HE染色和免疫組化；取另一側(cè)股骨提取總RNA進(jìn)行相關(guān)基因的表達(dá)分析。 小鼠成骨細(xì)胞系MC3T3-E1體外實(shí)驗(yàn)：MC3T3-E1細(xì)胞植入96孔板，時(shí)間分別為1、3、7和14天，每個(gè)時(shí)間段分為對(duì)照組和0.1、1.0、2.0、4.0、8.0、16、20、32和64mg F-/L組9個(gè)不同氟濃度組，采用CCK-8檢測(cè)細(xì)胞增殖活性。根據(jù)細(xì)胞活性的變化趨勢(shì)，選取具有代表性低、中、高劑量氟作用成骨細(xì)胞。將細(xì)胞植入24孔培養(yǎng)板，分為對(duì)照組及2.0、8.0和20mg F-/L3個(gè)不同氟濃度組，進(jìn)行成骨細(xì)胞早期分化標(biāo)志ALP和后期分化成熟標(biāo)志礦化結(jié)節(jié)特異性染色。免疫熒光實(shí)驗(yàn)檢測(cè)不同染氟條件下成骨細(xì)胞BiP蛋白表達(dá)的變化。采用Thermo公司ON-TARGETplus siRNA進(jìn)行BiP基因和PERK基因干擾實(shí)驗(yàn)，提取細(xì)胞RNA進(jìn)行實(shí)時(shí)PCR定量，分析基因干擾前后成骨和內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)基因的表達(dá)變化。 實(shí)驗(yàn)結(jié)果： 動(dòng)物實(shí)驗(yàn)結(jié)果： 1.灌胃方式給氟，，保證大鼠每日攝氟量的精確性，準(zhǔn)確復(fù)制出骨吸收增強(qiáng)到骨形成加速的不同病變特征的氟中毒大鼠骨病變。 2.通過(guò)氟中毒大鼠骨病理形態(tài)、骨密度和骨組織成骨、破骨相關(guān)的基因考察結(jié)果，證明氟中毒大鼠氟骨癥呈特征性的骨吸收活躍到成骨活躍的骨轉(zhuǎn)換加強(qiáng)病變特征。 3.大鼠投氟早期引起內(nèi)質(zhì)網(wǎng)應(yīng)激并激發(fā)未折疊蛋白反應(yīng)關(guān)鍵因子PERK、ATF6和IRE1，PERK信號(hào)因子的變化與氟中毒骨吸收增強(qiáng)具有一致性，下游因子Nrf2的變化與成骨因子變化相一致。 MC3T3-E1體外實(shí)驗(yàn)結(jié)果： 1.氟對(duì)成骨細(xì)胞具有雙向效應(yīng)，即低劑量氟刺激細(xì)胞活性，而高劑量氟抑制細(xì)胞活性。 2.染氟成骨細(xì)胞內(nèi)成骨相關(guān)基因的表達(dá)變化呈雙向性，即低氟條件下升高，但在高氟條件下降低。 3.染氟明顯刺激成骨細(xì)胞UPR信號(hào)通路的表達(dá)，BiP和PERK表達(dá)下調(diào)抑制UPR通路，且成骨相關(guān)轉(zhuǎn)錄因子表達(dá)明顯減少。 實(shí)驗(yàn)結(jié)論： 1、本實(shí)驗(yàn)采用灌胃給氟的方式，復(fù)制了骨轉(zhuǎn)換加速表征的氟骨癥動(dòng)物模型，主要體現(xiàn)在投氟早期以骨吸收為主，后期成骨為主要特征的病理變化。 2、投氟激發(fā)大鼠內(nèi)質(zhì)網(wǎng)應(yīng)激,誘導(dǎo)大鼠骨骼中未折疊蛋白反應(yīng)的PERK、ATF6和IRE1表達(dá)，其中PERK增高與破骨關(guān)鍵轉(zhuǎn)錄因子相一致,Nrf2與成骨關(guān)鍵轉(zhuǎn)錄因子表達(dá)變化相一致，提示PERK及Nrf2與氟骨癥的骨轉(zhuǎn)換加速有一定關(guān)聯(lián)。 3、染氟成骨細(xì)胞體外實(shí)驗(yàn)進(jìn)一步明確了PERK及其下游因子Nrf2在氟刺激成骨和破骨方面扮演重要角色，證明了內(nèi)質(zhì)網(wǎng)應(yīng)激及其PERK信號(hào)通路在氟骨癥發(fā)病機(jī)制中具有重要作用。
[Abstract]:Research background:
How the osteoblasts are activated in chronic fluorosis and how the bone transformation is accelerated is the key problem to be solved in the pathogenesis of endemic fluorosis. In previous studies, it was found that protein BiP, protein two thio isomerase, proteasome 26SATP enzyme and thioredoxin were increased in fluorinated osteoblasts associated with endoplasmic reticulum stress. The experiment was designed to observe the expression changes of the key factors PERK and its downstream ATF4 and Nrf2 genes and protein levels induced by endoplasmic reticulum stress in vitro, and to analyze the interrelationship with the proliferation, differentiation and bone formation of osteoblasts, and to explore endoplasmic reticulum stress and PERK signaling pathway in fluorine. The effect of osteoblast activation, the role of bone transformation acceleration, changes in regulation factors related to osteoblast proliferation and differentiation, and the development of endoplasmic reticulum stress mediating the occurrence and development of fluorosis bone lesions. The completion of this experiment helps to find new factors and letters that have never been involved in the study of the mechanism of fluorosis in the past. The important role of signal pathway provides new ideas and theoretical basis for the prevention and treatment strategy of skeletal fluorosis.
Experimental methods:
Animal experiment: 100 Wistar rats were evenly divided into control group, low fluorine group and 3 group of high fluorine group. Rats were given fluorine by gavage (NaF, 2.21g/L, equivalent to 100mgF-/ml). The rats of low fluorine group were given fluorine per day 10mg F-/kg weight, and the rats in the high fluorine group were given fluorine 20mg F-/kg weight every day, and the control group was given by gavage. The rats fed with fluorine 1,2 and after March were anaesthetized with ether, collecting blood and separating serum for biochemical test. After the experiment, the bone mineral density of the whole tibia was detected by Hologic Discovery WA bone densitometer. The femur of one side of the rat was decalcified by 10%EDTA, routine dehydration, paraffin embedding, pathological section, HE staining Immunohistochemical staining was used to extract the total RNA from the other side of the femur to analyze the expression of the related genes.
In vitro experiment of mouse osteoblast line MC3T3-E1: MC3T3-E1 cells were implanted into 96 orifice plates for 1,3,7 and 14 days respectively. Each time period was divided into 9 different fluorine concentrations in the control group and the 0.1,1.0,2.0,4.0,8.0,16,20,32 and 64mg F-/L group. The cell proliferation activity was detected by CCK-8. According to the change trend of the cell activity, the representative low level was selected. The cells were implanted into the bone cells with high dose of fluorine. The cells were implanted into 24 hole culture plates and divided into the control group and the 2.0,8.0 and 20mg F-/L3 different fluorine concentration groups. The early differentiation marker ALP of osteoblasts and the mineralized nodule specific staining of the later differentiation maturing were carried out. The immunofluorescence test was used to detect the changes of the expression of BiP protein in the osteoblasts under different fluorine dyed conditions. The interference experiment of BiP gene and PERK gene was carried out by ON-TARGETplus siRNA of Thermo company. The real-time PCR quantification of RNA was extracted and the expression changes of the genes related to bone formation and endoplasmic reticulum stress were analyzed before and after the gene interference.
Experimental results:
Animal experiment results:
1. gavage was given to fluorine to ensure the accuracy of daily fluorine uptake in rats and to accurately replicate bone lesions in fluorosis rats with different features of bone resorption enhanced to the acceleration of bone formation.
2. through the study of bone pathological morphology, bone density, bone tissue osteogenesis and osteoclast related gene findings in fluorosis rats, it was proved that fluorosis of fluorosis rats showed characteristic bone resorption active to osteogenic active bone transformation to strengthen the lesion characteristics.
The early fluorosis of 3. rats caused endoplasmic reticulum stress and the key factors to stimulate the unfolded protein reaction PERK, ATF6 and IRE1. The change of PERK signal factor was consistent with the enhancement of bone absorption in fluorosis, and the change of downstream factor Nrf2 was in accordance with the changes of osteogenic factor.
The results of MC3T3-E1 in vitro:
1. fluoride has a bidirectional effect on osteoblasts, that is, low dose fluorine stimulates cell activity, while high dose fluoride inhibits cell viability.
2. the expression of osteogenic related genes in fluoride osteoblasts was bidirectional, that is, increased under low fluoride, but decreased under high fluoride conditions.
3. fluoride exposure significantly stimulated the expression of UPR signaling pathway in osteoblasts. Down regulated expression of BiP and PERK inhibited UPR pathway, and the expression of osteogenic related transcription factors decreased significantly.
Experimental conclusions:
1, in this experiment, the animal model of skeletal fluorosis was replicated by the method of gavage to fluorine, which was mainly manifested in the pathological changes in the early fluorosis with bone absorption and later osteogenesis as the main feature.
2, fluorine stimulated endoplasmic reticulum stress in rats and induced the expression of PERK, ATF6 and IRE1 of unfolded protein reaction in the rat skeleton, in which the increase of PERK was consistent with the key transcriptional factor of osteoclast. Nrf2 was consistent with the changes in the expression of key transcriptional factors of osteogenesis, suggesting that PERK and Nrf2 were related to the acceleration of bone transformation in skeletal fluorosis.
3, in vitro experiments of fluorinated osteoblasts further clarify that PERK and its downstream factor Nrf2 play an important role in stimulating osteogenesis and osteoclasts in fluorine. It is demonstrated that endoplasmic reticulum stress and its PERK signaling pathway play an important role in the pathogenesis of fluorosis.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R599.1

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相關(guān)期刊論文 前2條

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