内质网应激及PERK信号通路在氟骨症发病机制中的作用

发布时间：2018-04-24 04:19

本文选题：氟骨症 + 骨转换　；参考：《吉林大学》2014年博士论文

【摘要】：研究背景：慢性氟中毒时成骨细胞怎样被激活、骨转换怎样被加速，是地方性氟中毒发病机制中亟待解决的关键问题。以往研究中发现，染氟成骨细胞中与内质网应激相关的蛋白BiP、蛋白质二硫键异构酶、蛋白酶体26SATP酶和硫氧还蛋白表达增多。本实验拟体外培养成骨细胞染氟并结合整体氟中毒动物实验，观察内质网应激引发的关键因子PERK及其下游ATF4和Nrf2等基因和蛋白水平的表达变化，分析其与成骨细胞增生、分化和骨形成间的相互关系，探讨内质网应激及PERK信号通路在氟作用下成骨细胞激活、骨转换加速中的作用；检测与成骨细胞增殖、分化等相关的调控因子的变化，阐明内质网应激通过哪些因素介导氟中毒骨病变的发生、发展。本实验的完成有助于发现以往氟骨症机制研究从未涉及的新的因子和信号通路的重要作用，为氟骨症防治策略提供新的思路和理论依据。实验方法：动物实验：100只Wistar大鼠，按体重均匀分成对照组、低氟组和高氟组3组。大鼠通过灌胃给氟（双蒸水配置NaF,浓度为2.21g/L，相当于100mgF-/ml），低氟组大鼠按照每天10mg F-/kg体重给氟，高氟组大鼠每天20mg F-/kg体重给氟，对照组通过灌胃给以双蒸水。大鼠投氟1、2和3月后分别经乙醚麻醉，采血、分离血清以备生化检测。实验结束后取一侧胫骨采用Hologic Discovery WA骨密度仪检测大鼠整只胫骨骨密度变化。同时，取一侧大鼠股骨经10%EDTA脱钙，常规脱水、石蜡包埋、制作病理切片，HE染色和免疫组化；取另一侧股骨提取总RNA进行相关基因的表达分析。小鼠成骨细胞系MC3T3-E1体外实验：MC3T3-E1细胞植入96孔板，时间分别为1、3、7和14天，每个时间段分为对照组和0.1、1.0、2.0、4.0、8.0、16、20、32和64mg F-/L组9个不同氟浓度组，采用CCK-8检测细胞增殖活性。根据细胞活性的变化趋势，选取具有代表性低、中、高剂量氟作用成骨细胞。将细胞植入24孔培养板，分为对照组及2.0、8.0和20mg F-/L3个不同氟浓度组，进行成骨细胞早期分化标志ALP和后期分化成熟标志矿化结节特异性染色。免疫荧光实验检测不同染氟条件下成骨细胞BiP蛋白表达的变化。采用Thermo公司ON-TARGETplus siRNA进行BiP基因和PERK基因干扰实验，提取细胞RNA进行实时PCR定量，分析基因干扰前后成骨和内质网应激相关基因的表达变化。实验结果：动物实验结果： 1.灌胃方式给氟，，保证大鼠每日摄氟量的精确性，准确复制出骨吸收增强到骨形成加速的不同病变特征的氟中毒大鼠骨病变。 2.通过氟中毒大鼠骨病理形态、骨密度和骨组织成骨、破骨相关的基因考察结果，证明氟中毒大鼠氟骨症呈特征性的骨吸收活跃到成骨活跃的骨转换加强病变特征。 3.大鼠投氟早期引起内质网应激并激发未折叠蛋白反应关键因子PERK、ATF6和IRE1，PERK信号因子的变化与氟中毒骨吸收增强具有一致性，下游因子Nrf2的变化与成骨因子变化相一致。 MC3T3-E1体外实验结果： 1.氟对成骨细胞具有双向效应，即低剂量氟刺激细胞活性，而高剂量氟抑制细胞活性。 2.染氟成骨细胞内成骨相关基因的表达变化呈双向性，即低氟条件下升高，但在高氟条件下降低。 3.染氟明显刺激成骨细胞UPR信号通路的表达，BiP和PERK表达下调抑制UPR通路，且成骨相关转录因子表达明显减少。实验结论： 1、本实验采用灌胃给氟的方式，复制了骨转换加速表征的氟骨症动物模型，主要体现在投氟早期以骨吸收为主，后期成骨为主要特征的病理变化。 2、投氟激发大鼠内质网应激,诱导大鼠骨骼中未折叠蛋白反应的PERK、ATF6和IRE1表达，其中PERK增高与破骨关键转录因子相一致,Nrf2与成骨关键转录因子表达变化相一致，提示PERK及Nrf2与氟骨症的骨转换加速有一定关联。 3、染氟成骨细胞体外实验进一步明确了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，证明了内质网应激及其PERK信号通路在氟骨症发病机制中具有重要作用。
[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.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R599.1

【参考文献】