本文主要研究内容
作者张辉（2019）在《福美双致肉鸡胫骨软骨发育不良的分子机制及淫羊藿苷的治疗作用》一文中研究指出：肉鸡胫骨软骨发育不良(Tibial Dyschondroplasia,TD)是指快速生长、体型较大的肉鸡胫骨软骨细胞发育异常,其临床表现为行走能力部分甚至完全丧失、运动障碍、站立困难、骨骼发育畸形、生长性能下降等。肉鸡TD主要引起胫骨近干骺端干骺面生长板内出现无血管、无钙化的半透明状“病理软骨栓”。大量研究显示,肉鸡TD的发生主要由于肉鸡胫骨软骨区血管生成减少,TD软骨生长板肥大区血管数量分布密度降低,导致骨生成的相关细胞所需养分供应不足,尤其是成骨细胞、破骨细胞和间充质干细胞;同时血管的减少直接导致钙化区骨沉积受到破坏、钙化终止。肉鸡TD的临床发病率较高,达10%-30%,其中多表现为亚临床症状。TD的发生会降低肉鸡抗病能力,导致胸部肌肉受损、囊肿,严重危及肉鸡健康,影响肉鸡产品品质和性能,给肉鸡养殖业带来较为严重的打击。我国家禽养殖业中,表现为临床症状或亚临床症状TD的发病率约为10%,给我国肉鸡业造成较大的经济损失。为了全面系统的了解近几年我国肉鸡TD的发病形势、发病原因、发病过程、病理变化、相关分子机制以及防治措施,本研究采用福美双诱导肉鸡TD的方法构建动物模型,通过肉鸡生长性能、TD发病情况、胫骨指数、胫骨生长板病理切片观察、Wnt/β-catenin通路中关键因子的表达情况、TD发生后差异miRNA的筛选及靶基因表达情况等来评估肉鸡TD的发病机制以及淫羊藿苷对肉鸡TD的治疗作用机制。主要研究内容及结果如下:1我国部分地区肉鸡TD发病普调查及其诊断近年来,肉鸡TD已成为影响我国家禽养殖主要骨骼疾病之一,对家禽养殖业的发展造成了重大的经济损失。为了查明我国家禽养殖的主产省份肉鸡TD发病情况,本研究采用网络问卷调查的方法对我国部分省市规模化肉鸡养殖场进行肉鸡养殖过程中TD发病情况调查。同时,本研究采用病理剖检和组织病理学方法相结合,对肉鸡TD发生过程中的病理变化进行比较研究。结果显示,河北省肉鸡TD发病率最高,其次是山西、河南、江苏、山东、辽宁等省份。此外,我国近几年肉鸡表现TD临床症状的发病率均小于5%。本研究通过对TD病变“白色软骨栓”进行测量,按照其与干骺端面积比值,对TD病程进行量化。本次研究将为我国肉鸡TD的发病率、诊断方法、TD病程的量化提供参考。2福美双在诱导肉鸡胫骨软骨发育不良中的机制研究福美双是一种重要的农药,在农业生产中广泛使用;如果福美双与细胞膜结合,则会引起细胞膜损伤,从而阻止血管生成。福美双被认为是导致TD发生的常见原因,福美双在农业上的过度使用及其在环境中的残留,成为其污染饲料的重要原因。但是,福美双引起肉鸡TD的相关机理较为复杂,尚不明确。因此,本研究利用福美双诱导肉鸡TD模型,评估福美双对肉鸡内脏器官指数、生长性能、组织病理变化、成骨细胞分化、血管分化、胫骨发育相关基因表达等影响。结果显示:福美双组肉鸡多表现为精神沉郁、运动障碍、站立困难、跛行。与对照组相比,福美双组肉鸡生长性能、肝脏指数和胫骨长度显著降低,脏器指数(脾、心)、胫骨指数和TD发病的严重程度显著提高;福美双组肉鸡血清中钙、磷浓度升高,胫骨密度降低,但差异不显著。胫骨和肝脏的组织病理学显示,福美双可导致严重的肝脏毒性作用、软骨细胞凋亡、血管分布减少。此外,免疫组织化学分析结果显示,福美双组肉鸡HIF-1α和VEGF的表达量显著增加,WNT4的表达量减少。研究结果表明福美双可以促进软骨细胞凋亡,诱导血管内皮细胞损伤,使得胫骨生长板血管分布减少,伴随大量死亡的软骨细胞聚集,最终形成“软骨栓”,伴随成骨失败。3肉鸡胫骨软骨发育不良差异miRNA的筛选及miRNA与Wnt/β-catenin通路关联性分析肉鸡TD是许多禽类常见的骨骼疾病,但其涉及miRNA的分子机制尚未完全清楚。为此,我们通过高通量RNA测序(RNA-seq)对TD肉鸡差异表达的miRNA(DEGs)进行筛选,并对差异表达miRNAs进行功能注释,通过差异表达miRNA与mRNA的关联分析,进而阐明影响肉鸡TD的发病机制。结果表明:与对照组相比,TD组肉鸡软骨栓明显增加,软骨细胞萎缩、排列不规则。本研究共鉴定了375(P<0.1)、340(P<0.05)和266(P<0.01)个显著差异表达miRNAs,其中上调的分别有168、166和141个,下调的分别有207、174和125个。差异表达基因GO分析和KEGG富集分析显示,碳代谢、缬氨酸-亮氨酸和异亮氨酸降解、细胞周期等在肉鸡TD发生过程中扮演重要角色。其中15种差异表达的miRNA涉及调控5种Wnt蛋白,分别为Wnt4、Wnt6、Wnt8c、Wnt9A、Wnt11,暗示肉鸡TD发生后差异表达miRNAs可能通过调控Wnt/β-catenin通路相关蛋白的表达,进而影响TD的发生。本研究为今后深入了解TD发病机制提供了理论依据,为今后筛选特异性miRNA作为生物标志物以及对肉鸡TD进行有效诊断和治疗提供了新的思路。4 Wnt/β-catenin信号通路中关键因子在肉鸡TD发生过程中的作用Wnt/β-catenin通路在哺乳动物骨骼发育中起到了重要的调控作用。而在家禽TD中是否也是如此,目前未见研究报到。本研究采用RT-qPCR、免疫组织化学、Western blot等方法检测胫骨生长板Wnt/β-catenin信号通路相关关键基因及其靶基因的表达情况。结果表明:肉鸡在发生TD后,胫骨干骺端生长板中Wnt5a和PKC基因表达量显著下降,GSK-3β蛋白和p-β-catenin的表达上调,同时伴随Axin和APC的下调。另外,Wnt/β-catenin通路中CK1、Dvl、TCF和Cdk8等关键基因的表达量在肉鸡TD发生过程中发生不同程度的下降,而sFRP的表达量升高;TD肉鸡中,Wnt/β-catenin通路的靶基因Sox9、MMP9、Runx2、BMP2均出现不同程度的下降。研究结果表明Wnt/β-catenin信号通路及其调控的下游相关因子在肉鸡TD发生过程中发挥着重要作用。5淫羊藿苷对肉鸡胫骨软骨发育不良的作用研究淫羊藿苷是一种传统的中药,广泛应用于各种骨骼疾病的治疗,但淫羊藿苷对TD是否有治疗作用尚无报道。为了探讨淫羊藿苷对肉鸡TD的治疗效果,将180只AA肉鸡随机分为对照组、TD组、淫羊藿苷组(10 mg/kg/day);其中,TD组由日粮中添加福美双诱导。通过RT-qPCR和Western blot分析WNT4、VEGF、P2RX7基因的表达;通过胫骨各参数、软骨细胞发育、血管生成等评估淫羊藿苷的治疗作用。结果表明:与TD组相比,淫羊藿苷组WNT4和P2RX7的表达显著上调(P<0.05),VEGF的表达显著下调(P<0.05);同时生长板的宽度恢复,软骨栓变小、软骨细胞损伤降低,促进生长板血管的发育,肉鸡的生长性能显著提高,且肉鸡的跛行程度减轻。本研究系统地从临床症状、生理生化指标、胫骨参数及基因表达的差异等角度评估了淫羊藿苷对肉鸡TD的治疗效果。该研究将为今后TD的传统中药预防和治疗提供参考。
Abstract
rou ji jing gu ruan gu fa yo bu liang (Tibial Dyschondroplasia,TD)shi zhi kuai su sheng chang 、ti xing jiao da de rou ji jing gu ruan gu xi bao fa yo yi chang ,ji lin chuang biao xian wei hang zou neng li bu fen shen zhi wan quan sang shi 、yun dong zhang ai 、zhan li kun nan 、gu ge fa yo ji xing 、sheng chang xing neng xia jiang deng 。rou ji TDzhu yao yin qi jing gu jin gan hou duan gan hou mian sheng chang ban nei chu xian mo xie guan 、mo gai hua de ban tou ming zhuang “bing li ruan gu shuan ”。da liang yan jiu xian shi ,rou ji TDde fa sheng zhu yao you yu rou ji jing gu ruan gu ou xie guan sheng cheng jian shao ,TDruan gu sheng chang ban fei da ou xie guan shu liang fen bu mi du jiang di ,dao zhi gu sheng cheng de xiang guan xi bao suo xu yang fen gong ying bu zu ,you ji shi cheng gu xi bao 、po gu xi bao he jian chong zhi gan xi bao ;tong shi xie guan de jian shao zhi jie dao zhi gai hua ou gu chen ji shou dao po huai 、gai hua zhong zhi 。rou ji TDde lin chuang fa bing lv jiao gao ,da 10%-30%,ji zhong duo biao xian wei ya lin chuang zheng zhuang 。TDde fa sheng hui jiang di rou ji kang bing neng li ,dao zhi xiong bu ji rou shou sun 、nang zhong ,yan chong wei ji rou ji jian kang ,ying xiang rou ji chan pin pin zhi he xing neng ,gei rou ji yang shi ye dai lai jiao wei yan chong de da ji 。wo guo jia qin yang shi ye zhong ,biao xian wei lin chuang zheng zhuang huo ya lin chuang zheng zhuang TDde fa bing lv yao wei 10%,gei wo guo rou ji ye zao cheng jiao da de jing ji sun shi 。wei le quan mian ji tong de le jie jin ji nian wo guo rou ji TDde fa bing xing shi 、fa bing yuan yin 、fa bing guo cheng 、bing li bian hua 、xiang guan fen zi ji zhi yi ji fang zhi cuo shi ,ben yan jiu cai yong fu mei shuang you dao rou ji TDde fang fa gou jian dong wu mo xing ,tong guo rou ji sheng chang xing neng 、TDfa bing qing kuang 、jing gu zhi shu 、jing gu sheng chang ban bing li qie pian guan cha 、Wnt/β-catenintong lu zhong guan jian yin zi de biao da qing kuang 、TDfa sheng hou cha yi miRNAde shai shua ji ba ji yin biao da qing kuang deng lai ping gu rou ji TDde fa bing ji zhi yi ji yin yang huo gan dui rou ji TDde zhi liao zuo yong ji zhi 。zhu yao yan jiu nei rong ji jie guo ru xia :1wo guo bu fen de ou rou ji TDfa bing pu diao cha ji ji zhen duan jin nian lai ,rou ji TDyi cheng wei ying xiang wo guo jia qin yang shi zhu yao gu ge ji bing zhi yi ,dui jia qin yang shi ye de fa zhan zao cheng le chong da de jing ji sun shi 。wei le cha ming wo guo jia qin yang shi de zhu chan sheng fen rou ji TDfa bing qing kuang ,ben yan jiu cai yong wang lao wen juan diao cha de fang fa dui wo guo bu fen sheng shi gui mo hua rou ji yang shi chang jin hang rou ji yang shi guo cheng zhong TDfa bing qing kuang diao cha 。tong shi ,ben yan jiu cai yong bing li pou jian he zu zhi bing li xue fang fa xiang jie ge ,dui rou ji TDfa sheng guo cheng zhong de bing li bian hua jin hang bi jiao yan jiu 。jie guo xian shi ,he bei sheng rou ji TDfa bing lv zui gao ,ji ci shi shan xi 、he na 、jiang su 、shan dong 、liao ning deng sheng fen 。ci wai ,wo guo jin ji nian rou ji biao xian TDlin chuang zheng zhuang de fa bing lv jun xiao yu 5%。ben yan jiu tong guo dui TDbing bian “bai se ruan gu shuan ”jin hang ce liang ,an zhao ji yu gan hou duan mian ji bi zhi ,dui TDbing cheng jin hang liang hua 。ben ci yan jiu jiang wei wo guo rou ji TDde fa bing lv 、zhen duan fang fa 、TDbing cheng de liang hua di gong can kao 。2fu mei shuang zai you dao rou ji jing gu ruan gu fa yo bu liang zhong de ji zhi yan jiu fu mei shuang shi yi chong chong yao de nong yao ,zai nong ye sheng chan zhong an fan shi yong ;ru guo fu mei shuang yu xi bao mo jie ge ,ze hui yin qi xi bao mo sun shang ,cong er zu zhi xie guan sheng cheng 。fu mei shuang bei ren wei shi dao zhi TDfa sheng de chang jian yuan yin ,fu mei shuang zai nong ye shang de guo du shi yong ji ji zai huan jing zhong de can liu ,cheng wei ji wu ran si liao de chong yao yuan yin 。dan shi ,fu mei shuang yin qi rou ji TDde xiang guan ji li jiao wei fu za ,shang bu ming que 。yin ci ,ben yan jiu li yong fu mei shuang you dao rou ji TDmo xing ,ping gu fu mei shuang dui rou ji nei zang qi guan zhi shu 、sheng chang xing neng 、zu zhi bing li bian hua 、cheng gu xi bao fen hua 、xie guan fen hua 、jing gu fa yo xiang guan ji yin biao da deng ying xiang 。jie guo xian shi :fu mei shuang zu rou ji duo biao xian wei jing shen chen yu 、yun dong zhang ai 、zhan li kun nan 、bo hang 。yu dui zhao zu xiang bi ,fu mei shuang zu rou ji sheng chang xing neng 、gan zang zhi shu he jing gu chang du xian zhe jiang di ,zang qi zhi shu (pi 、xin )、jing gu zhi shu he TDfa bing de yan chong cheng du xian zhe di gao ;fu mei shuang zu rou ji xie qing zhong gai 、lin nong du sheng gao ,jing gu mi du jiang di ,dan cha yi bu xian zhe 。jing gu he gan zang de zu zhi bing li xue xian shi ,fu mei shuang ke dao zhi yan chong de gan zang du xing zuo yong 、ruan gu xi bao diao wang 、xie guan fen bu jian shao 。ci wai ,mian yi zu zhi hua xue fen xi jie guo xian shi ,fu mei shuang zu rou ji HIF-1αhe VEGFde biao da liang xian zhe zeng jia ,WNT4de biao da liang jian shao 。yan jiu jie guo biao ming fu mei shuang ke yi cu jin ruan gu xi bao diao wang ,you dao xie guan nei pi xi bao sun shang ,shi de jing gu sheng chang ban xie guan fen bu jian shao ,ban sui da liang si wang de ruan gu xi bao ju ji ,zui zhong xing cheng “ruan gu shuan ”,ban sui cheng gu shi bai 。3rou ji jing gu ruan gu fa yo bu liang cha yi miRNAde shai shua ji miRNAyu Wnt/β-catenintong lu guan lian xing fen xi rou ji TDshi hu duo qin lei chang jian de gu ge ji bing ,dan ji she ji miRNAde fen zi ji zhi shang wei wan quan qing chu 。wei ci ,wo men tong guo gao tong liang RNAce xu (RNA-seq)dui TDrou ji cha yi biao da de miRNA(DEGs)jin hang shai shua ,bing dui cha yi biao da miRNAsjin hang gong neng zhu shi ,tong guo cha yi biao da miRNAyu mRNAde guan lian fen xi ,jin er chan ming ying xiang rou ji TDde fa bing ji zhi 。jie guo biao ming :yu dui zhao zu xiang bi ,TDzu rou ji ruan gu shuan ming xian zeng jia ,ruan gu xi bao wei su 、pai lie bu gui ze 。ben yan jiu gong jian ding le 375(P<0.1)、340(P<0.05)he 266(P<0.01)ge xian zhe cha yi biao da miRNAs,ji zhong shang diao de fen bie you 168、166he 141ge ,xia diao de fen bie you 207、174he 125ge 。cha yi biao da ji yin GOfen xi he KEGGfu ji fen xi xian shi ,tan dai xie 、xie an suan -liang an suan he yi liang an suan jiang jie 、xi bao zhou ji deng zai rou ji TDfa sheng guo cheng zhong ban yan chong yao jiao se 。ji zhong 15chong cha yi biao da de miRNAshe ji diao kong 5chong Wntdan bai ,fen bie wei Wnt4、Wnt6、Wnt8c、Wnt9A、Wnt11,an shi rou ji TDfa sheng hou cha yi biao da miRNAske neng tong guo diao kong Wnt/β-catenintong lu xiang guan dan bai de biao da ,jin er ying xiang TDde fa sheng 。ben yan jiu wei jin hou shen ru le jie TDfa bing ji zhi di gong le li lun yi ju ,wei jin hou shai shua te yi xing miRNAzuo wei sheng wu biao zhi wu yi ji dui rou ji TDjin hang you xiao zhen duan he zhi liao di gong le xin de sai lu 。4 Wnt/β-cateninxin hao tong lu zhong guan jian yin zi zai rou ji TDfa sheng guo cheng zhong de zuo yong Wnt/β-catenintong lu zai bu ru dong wu gu ge fa yo zhong qi dao le chong yao de diao kong zuo yong 。er zai jia qin TDzhong shi fou ye shi ru ci ,mu qian wei jian yan jiu bao dao 。ben yan jiu cai yong RT-qPCR、mian yi zu zhi hua xue 、Western blotdeng fang fa jian ce jing gu sheng chang ban Wnt/β-cateninxin hao tong lu xiang guan guan jian ji yin ji ji ba ji yin de biao da qing kuang 。jie guo biao ming :rou ji zai fa sheng TDhou ,jing gu gan hou duan sheng chang ban zhong Wnt5ahe PKCji yin biao da liang xian zhe xia jiang ,GSK-3βdan bai he p-β-cateninde biao da shang diao ,tong shi ban sui Axinhe APCde xia diao 。ling wai ,Wnt/β-catenintong lu zhong CK1、Dvl、TCFhe Cdk8deng guan jian ji yin de biao da liang zai rou ji TDfa sheng guo cheng zhong fa sheng bu tong cheng du de xia jiang ,er sFRPde biao da liang sheng gao ;TDrou ji zhong ,Wnt/β-catenintong lu de ba ji yin Sox9、MMP9、Runx2、BMP2jun chu xian bu tong cheng du de xia jiang 。yan jiu jie guo biao ming Wnt/β-cateninxin hao tong lu ji ji diao kong de xia you xiang guan yin zi zai rou ji TDfa sheng guo cheng zhong fa hui zhao chong yao zuo yong 。5yin yang huo gan dui rou ji jing gu ruan gu fa yo bu liang de zuo yong yan jiu yin yang huo gan shi yi chong chuan tong de zhong yao ,an fan ying yong yu ge chong gu ge ji bing de zhi liao ,dan yin yang huo gan dui TDshi fou you zhi liao zuo yong shang mo bao dao 。wei le tan tao yin yang huo gan dui rou ji TDde zhi liao xiao guo ,jiang 180zhi AArou ji sui ji fen wei dui zhao zu 、TDzu 、yin yang huo gan zu (10 mg/kg/day);ji zhong ,TDzu you ri liang zhong tian jia fu mei shuang you dao 。tong guo RT-qPCRhe Western blotfen xi WNT4、VEGF、P2RX7ji yin de biao da ;tong guo jing gu ge can shu 、ruan gu xi bao fa yo 、xie guan sheng cheng deng ping gu yin yang huo gan de zhi liao zuo yong 。jie guo biao ming :yu TDzu xiang bi ,yin yang huo gan zu WNT4he P2RX7de biao da xian zhe shang diao (P<0.05),VEGFde biao da xian zhe xia diao (P<0.05);tong shi sheng chang ban de kuan du hui fu ,ruan gu shuan bian xiao 、ruan gu xi bao sun shang jiang di ,cu jin sheng chang ban xie guan de fa yo ,rou ji de sheng chang xing neng xian zhe di gao ,ju rou ji de bo hang cheng du jian qing 。ben yan jiu ji tong de cong lin chuang zheng zhuang 、sheng li sheng hua zhi biao 、jing gu can shu ji ji yin biao da de cha yi deng jiao du ping gu le yin yang huo gan dui rou ji TDde zhi liao xiao guo 。gai yan jiu jiang wei jin hou TDde chuan tong zhong yao yu fang he zhi liao di gong can kao 。
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论文作者分别是来自华中农业大学的张辉，发表于刊物华中农业大学2019-07-15论文，是一篇关于胫骨软骨发育不良论文,福美双论文,淫羊藿苷论文,通路论文,华中农业大学2019-07-15论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自华中农业大学2019-07-15论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：胫骨软骨发育不良论文; 福美双论文; 淫羊藿苷论文; 通路论文; 华中农业大学2019-07-15论文;