运动对骨质疏松小鼠骨长链非编码RNA表达的影响

发布时间：2018-01-03 15:44

本文关键词：运动对骨质疏松小鼠骨长链非编码RNA表达的影响　出处：《上海体育学院》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的:骨质疏松是一种以全身性骨量减少以及骨组织微观结构退化为特征的疾病,会导致骨强度降低,脆性增加,骨折风险增加。采用运动方法用于骨质疏松的预防和治疗可以有效的减少因药物治疗带来的家庭经济负担和社会医疗保障系统的负担及其毒副作用。运动可以通过机械应力刺激、细胞因子、骨代谢信号通路等对骨质疏松的防治发挥关键作用,其作用机制是当下一大研究热点。最新研究表明非编码RNA(long noncoding RNA,lncRNA)在骨代谢中发挥重要的调控作用。本研究拟通过建立小鼠骨质疏松模型,并进行中等强度的运动干预,探究运动对骨质疏松小鼠骨lncRNA表达的影响,为进一步探究运动是否会通过影响骨lncRNA起到防治骨质疏松的作用提供理论依据。研究方法:8周龄雌性C57BL/6小鼠44只,随机分为假手术组(SHAM),假手术+运动组(SHAM+EX),去卵巢组(OVX),去卵巢+运动组(OVX+EX)。去卵巢小鼠麻醉后于背部脊柱旁开1cm,身长下1/3处切口去除卵巢,假手术小鼠于相同部位去除同等大小的脂肪。手术后4周,SHAM+EX组和OVX+EX组进行为期9周的跑台训练,每周5天,周六周日休息,坡度25°。第一周为运动预适应,速度6M/min,时间30min;后八周起始速度为8M/min,每周增加1M/min;时间为5min热身运动(速度:6M/min)和55min的正式训练。SHAM组和OVX组不进行任何干预,常规饲养。干预结束后取血尿以及左右股骨胫骨,左侧股骨用于检测骨密度、mcro CT以及骨生物力学指标,右侧股骨检测骨形态计量学指标,每组随机选取3只小鼠的全部股骨胫骨,提取总RNA用于lncRNA芯片检测,同时对各组差异性表达在4倍以上的lncRNA进行筛选用于GO Pathway分析。所得数据结果用SPSS 20.0软件进行统计分析。研究结果:(1)去卵巢可以使小鼠体重增加明显,运动可以减少去卵巢小鼠的体重。(2)运动对骨密度的影响:运动可以显著提高假手术与骨质疏松小鼠的BMD,与SHAM组相比,SHAM+EX组BMD显著提高(P0.05),OVX组显著降低(P0.01);与OVX组相比,OVX+EX组BMD显著提高(P0.01)。(3)运动对骨质疏松小鼠骨形态计量学的影响:SHAM组的MAR低于SHAM+EX组但没有显著差异,显著高于OVX组(P0.05),OVX组MAR显著低于OVX+EX组(P0.05),运动可以提高假手术和骨质疏松的小鼠的BFR/BV,但没有显著性差异。与SHAM组相比,SHAM+EX组小鼠的OB.S/BS显著提高(P0.05),OVX组显著降低(P0.01);与OVX组相比,OVX+EX组OB.S/BS显著提高(P0.05)。与SHAM组相比,OVX组OC.S/BS显著增加(P0.01);与OVX组相比,OVX+EX组OC.S/BS显著降低(P0.01)。(4)microCT检测:运动可以明显提高假手术与骨质疏松小鼠的BV/TV、Tb.Th、Tb.N降低Tb.Sp,但结果没有显著性差异。(5)运动对骨质疏松小鼠骨生物力学的影响:与SHAM组相比,SHAM+EX组最大载荷显著增加(P0.05),OVX组显著降低(P0.05);与OVX组相比,OVX+EX组最大载荷显著增加(P0.05)。与SHAM组相比,SHAM+EX组弯曲强度提高但无显著差异,OVX组显著降低(P0.05),与OVX相比OVX+EX组显著降低(P0.05)。与SHAM相比,SHAM+EX组弹性模量提高但无显著差异,OVX+EX组弹性模量较OVX组提高,但无显著差异。(6)运动对骨质疏松小鼠骨lncRNAs的影响:筛选出差异性表达在2倍和4倍以上,且P0.05的lncRNAs。与SHAM组相比,SHAM+EX组差异表达在2倍以上的有64条表达上调,161条表达下调,差异在4倍以上表达上调的有3条,表达下调的有3条。与SHAM组相比,OVX组差异性表达2倍以上的有437条表达上调,其中Gm30783、LOC102639568等25条差异性在4倍以上;有231条表达下调,其中Gm33272、Gm12678、Gm10248、LOC105243625等17条差异性在4倍以上。与OVX组相比,OVX+EX组差异性表达在2倍以上的有181条表达下调,其中lnc 105247265等3条lncRNA的差异性在4倍以上;56条表达上调,其中Gm31126、LOC105243692、Gm11651等8条的差异性在4倍以上。(7)通过KEGG pathway分析发现,Melanoma信号通路、mTOR信号通路、Toxoplasmosis信号通路、NF-κB信号通路、PI3K-Akt signaling pathway等信号通路可能被lncRNA调控。Regulation of cell cycle phase transition、vocalization behavior、detection of biotic stimulus等生物过程,TOR complex、synapse part等细胞组分以VEGF-A-activated receptor activity、VEGF-B-activated receptor activity等分子可能被lncRNA所调控。研究结论:中等强度的跑台运动可以显著提高骨质松小鼠的骨密度,提高其骨生成速率和骨强度;同时可以对小鼠骨lncRNA产生一定的影响,造成其表达的升高或降低,但其机制有待进一步研究。
[Abstract]:Objective: osteoporosis is a decrease and degradation of the microstructure of bone tissue disease characterized by systemic bone, will lead to reduced bone strength, brittleness increases, increase the risk of fracture. The exercise method for the prevention and treatment of osteoporosis due to drug treatment can effectively reduce the financial burden of the family and social medical security system the burden and side effects. They can move through mechanical stress, cell factor, prevention and treatment of bone metabolic pathways on osteoporosis play a key role in the mechanism is a hot research topic. The latest research shows that non encoding RNA (long noncoding RNA, lncRNA) play an important role in the regulation of bone metabolism in the present study was to establish a mouse model for osteoporosis, exercise intervention and moderate intensity, explore the impact of exercise on bone osteoporosis lncRNA expression in mice, as in Further study whether exercise would provide a theoretical basis for the effect on the bone lncRNA contributes to the prevention and treatment of osteoporosis. Methods: 8 week old female C57BL/6 44 mice were randomly divided into sham operation group (SHAM), sham operation + exercise group (SHAM+EX), ovariectomized group (OVX), OVX + exercise group (OVX+EX) in ovariectomized mice. After anesthesia 1cm on the back of paravertebral, length 1/3 incision at the removal of the ovaries, sham operated mice in the same part of the same size to remove fat. 4 weeks after surgery, SHAM+EX group and OVX+EX group for 9 weeks of treadmill training, 5 days a week, Saturday Sunday rest, slope 25 degrees. The first week of exercise preconditioning, speed 6M/min, time 30min; eight weeks after the initial velocity is 8M/min, the weekly increase 1M/min; time is 5min warm-up (speed: 6M/min) and 55min.SHAM formal training group and OVX group without any intervention, regular feeding. After intervention for hematuria and The left and right femur tibia, left femur for the detection of bone mineral density and bone biomechanics, mcro CT index, index of right femur bone histomorphometry detection, each group of 3 mice were randomly selected and all femoral and tibial total RNA extraction, lncRNA chip for detection, at the same time to each expression of more than 4 times in lncRNA were screened for GO Pathway analysis the data obtained by SPSS 20. The results were analyzed. Results: (1) ovariectomy can make the body weight of mice increased significantly, exercise can reduce the weight of ovariectomized mice. (2) the effects of exercise on bone mineral density: exercise can significantly improve the sham operation and osteoporosis in mice BMD, compared with the SHAM group SHAM+EX group BMD significantly increased (P0.05), OVX group significantly decreased (P0.01); compared with OVX group, OVX+EX group BMD significantly increased (P0.01). (3) effects of exercise on bone histomorphometry in osteoporosis mice: group SHAM MAR Lower than the SHAM+EX group but no significant differences, significantly higher than that of group OVX (P0.05), OVX MAR group was significantly lower than that of OVX+EX group (P0.05), sham operation and exercise can improve the osteoporosis in mice BFR/BV, but no significant difference. Compared with the SHAM group, SHAM+EX group of mice OB.S/BS significantly increased (P0.05), OVX group significantly decreased (P0.01); compared with OVX group, OVX+EX group OB.S/BS significantly increased (P0.05). Compared with SHAM group, OVX group, OC.S/BS increased significantly (P0.01); compared with OVX group, OVX+EX group, OC.S/BS decreased significantly (P0.01). (4) microCT detection: exercise can significantly improve the sham operation and osteoporosis in mice BV/TV, Tb.Th, Tb.N decreased Tb.Sp, but the results had no significant difference. (5) the effects of exercise on bone biomechanics in osteoporosis mice: compared with SHAM group, SHAM+EX group significantly increased the maximum load (P0.05), OVX group significantly decreased (P0.05); compared with OVX group, OVX+EX group, the maximum load significantly Increased (P0.05). Compared with the SHAM group, but no significant difference between the SHAM+EX group to improve the bending strength of OVX group decreased significantly (P0.05), compared with OVX OVX+EX group decreased significantly (P0.05). Compared with SHAM, but there was no significant difference between the SHAM+EX group to improve the elastic modulus, elastic modulus of OVX+EX group than in OVX group increased, but no significant difference. (6) the effects of exercise on bone lncRNAs osteoporosis mice: screening travelheterosexual expression in 2 times and 4 times, P0.05 and lncRNAs. compared with group SHAM, group SHAM+EX differentially expressed in more than 2 times the 64 up-regulated genes and 161 downregulated and up-regulated the expression of differences in more than 4 times of the 3, down regulated 3. Compared with SHAM group, OVX group, differential expression of 2 times more than the 437 up-regulated genes including Gm30783, LOC102639568, etc. 25 differences in more than 4 times; 231 expression, including Gm33272, Gm12678, Gm10248, LOC105243625 and other 17 difference specific In more than 4 times. Compared with the OVX group, OVX+EX group differentially expressed in more than 2 times of the 181 expression, and the difference of LNC 105247265 and 3 lncRNA in more than 4 times; 56 up-regulated genes including Gm31126, LOC105243692, Gm11651, etc. 8 of the differences in the (more than 4 times. 7) by KEGG pathway analysis showed that the Melanoma signaling pathway, mTOR signaling pathway, Toxoplasmosis signaling pathway, B signaling pathway NF-, PI3K-Akt signaling pathway signaling pathway may be regulated by lncRNA.Regulation of cell cycle phase transition, vocalization behavior, detection of biotic stimulus TOR complex synapse, biological process, and cellular component in part VEGF-A-activated receptor activity, VEGF-B-activated receptor activity and other molecules may be regulated lncRNA. Conclusion: moderate intensity treadmill exercise can significantly improve the osteoporosis mice bone Density can increase the rate of bone formation and bone strength, meanwhile, it can produce certain effects on the bone lncRNA in mice, resulting in the increase or decrease of its expression, but its mechanism needs further study.

【学位授予单位】：上海体育学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R580

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