本文主要研究内容
作者郭庆（2019）在《黄芩苷对猪卵母细胞体外成熟和胚胎体外发育能力的影响》一文中研究指出：黄芩苷(baicalin)是一种从黄芩植物根部提取的传统中药单体,因其具有抗氧化、抗凋亡、抗炎症等功能,被用于治疗多种由炎症因子引起的疾病,同时在生殖上具有治疗不孕不育,保胎的作用。然而,baicalin对于卵母细胞体外成熟(IVM),胚胎体外发育的影响及其机制尚不清楚。卵母细胞IVM和胚胎体外培养会受到高浓度活性氧(ROS)的破坏作用使其效率降低,这使卵母细胞和胚胎相关机理的研究及二者在辅助生殖技术上的应用都受到很大限制。因此本实验在猪卵母细胞IVM培养液和胚胎IVC培养液中添加baicalin可以通过抑制凋亡显著提高卵母细胞IVM能力、卵母细胞质量、胚胎体外发育能力、胚胎质量。同时,利用成年雄性昆明鼠体内实验验证baicalin对酒精诱导精子损伤的保护作用。一、Baicalin对猪卵母细胞IVM及其后胚胎体外发育能力的影响1.本实验探索不同浓度的baicalin对猪卵母细胞IVM能力的影响,发现0.1μg/mL和0.5 μg/mL的baicalin均可以显著的提高猪卵母细胞IVM 能力(68.1,70.2 vs.59.7;P<0.05)。Baicalin 具有显著的抗氧化能力,因此,通过荧光染色的方法发现0.05,0.1和0.5μg/mL baicalin处理组中卵母细胞内产生活性氧(ROS)水平均显著的低于对照组(P<0.05)。在最小试验药物浓度获得最佳实验效果的原则下,后续实验选择0.1 μg/mL的baicalin作为最佳处理浓度。同时RT-PCR结果显示baicalin处理组中卵母细胞和卵丘细胞的促凋亡相关基因(Bax)mRNA表达量显著低于对照组(P<0.05)。2.荧光染色和RT-PCR方法进一步检测baicain处理后卵母细胞的质量,结果显示0.1μg/mL的baicalin处理组中线粒体潜能性(△Ψm)和ATP含量均显著的高于对照组(P<0.05)。同时,RT-PCR检测到baicalin处理组中卵母细胞成熟相关基因(BMP15,GDF9,CyclinB,CDK1)和卵丘细胞扩增相关基因(PTGS1,PTX3,TNFIP6,SHAS2)的mRNA表达量显著的高于对照组(P<0.05)。3.检测baicalin处理的卵母细胞经孤雌激活(PA)后胚胎的体外发育能力。结果显示处理组获得的胚胎体外发育能力显著的高于对照组(49.5 vs.27.2;P<0.05)。同时,baicalin处理后获得的胚胎发育到囊胚阶段时细胞数也显著的高于对照组(P<0.05)。RT-PCR检测到baicalin处理组中胚胎达到囊胚阶段时全能性相关基因(Nanog,Sox2)和Bcl2基因的mRNA表达量显著的高于对照组(P<0.05),而Ba基因mRNA表达量显著的低于对照组(P<0.05)。同时,Tunel染色发现baicalin处理组囊胚细胞凋亡数显著的低于对照组(P<0.05)。二、Baicalin对PA,体细胞核移植(SCNT)和体外受精(IVF)胚胎发育能力的影响1.检测baicalin对猪PA胚胎体外发育能力的影响。结果表明0.1μg/mL的baicalin处理可以显著提高猪PA胚胎的体外发育能力(21.7 vs.36.7;P<0.05),囊胚细胞数也显著高于对照组(P<0.05)。Tunel染色检测了囊胚的质量,结果显示baicalin处理组囊胚凋亡细胞数显著的低于对照组(P<0.05)。RT-PCR结果表明全能性基因(Oct4,Sox2)的mRNA表达量显著高于对照组(P<0.05)。Bax和Bcl2基因的mRNA表达量显著低于和高于对照组(P<0.05)。2.检测baicalin对猪PA胚胎ROS表达量,Bax和Bcl2基因mRNA表达量,△Ψm,ATP含量的影响。利用荧光染色检测0.1μg/mL baicalin处理对猪PA胚胎质量的影响,结果显示baicalin处理组2/4-cell时期ROS的表达量显著低于对照组和其他浓度处理组(P<0.05)。同时RT-PCR结果显示胚胎在2/4-cell时期Bax基因的mRNA表达量均显著的低于对照组(P<0.05),Bcl2基因在2-cell时期的mRNA表达没有显著的变化,而在4-cell时期mRNA的表达量显著高于对照组(P<0.05)。同时,荧光染色显示baicalin处理组中△Ψm和ATP含量显著高于对照组(P<0.05)。3.检测baicalin处理对猪PA胚胎中sonic hedgehog(SHH)信号途径的影响。RT-PCR结果显示,baicalin处理组中SHH信号途径相关基因(SHH,PTCH1,GdI1,SMO)的mRNA表达量均显著高于对照组(P<0.05)。免疫荧光染色检测发现baicalin处理可以显著的提高SHH信号途径相关蛋白(SHH,GLI1)的表达量。为了进一步检测baicalin处理对胚胎体外发育能力影响的机制,我们将胚胎分为三个组:对照组;baicalin处理组;baicalin和SHH信号抑制剂(Cyclopamine,Cy)共同处理组,结果显示Cy可以恢复baicalin对胚胎产生ROS和凋亡的抑制作用及baicalin对△Ψm和ATP含量的增加。最后,我们检测三个处理组胚胎的体外发育能力,结果显示Cy可以抑制baicalin对胚胎体外发育能力的提高。4.检测0.1 μg/mL baicalin对猪SCNT和IVF胚胎的影响。结果表明0.1 μg/mL的biacalin可以显著提高猪SCNT和IVF胚胎的发育能力(14.9 vs.25.9;14.9 vs.25.9;P<0.05)。三、Baicalin对酒精诱导的成年雄性昆明鼠精子损伤的保护作用1.本实验建立了酒精诱导成年雄性昆明鼠精子损伤模型。实验分为两组,10只同窝出生,表型一致的成年雄性昆明鼠随机分为实验组和对照组,每组各5只。实验组进行每天连续四周酒精灌胃实验,灌胃剂量按酒精质量和小鼠体重比如下:第一周每天2g/kg、第二周每天4g/kg、第三周每天6 g/kg、第四周每天8 g/kg、对照组饲喂等量的生理盐水。第四周结束时对小鼠进行剖检,检测到小鼠体重下降,死亡率增加,精子密度显著的低于对照组(P<0.05)。2.检测不同浓度baicalin处理对酒精诱导成年雄性小鼠精子损伤的保护作用。实验分为对照组(等量生理盐水)、酒精组、酒精和25 mg/kg baicalin共同处理组、酒精和50 mg/kg baicalin共同处理组(mg/kg即baicalin重量和小鼠体重比值,酒精剂量按照实验一)。结果显示,50 mg/kg的biacalin灌胃量对酒精诱导的小鼠精子密度下降和精子畸形率升高等具有部分保护作用(P<0.05)。3.检测不同浓度baicalin对酒精诱导的成年雄性昆明鼠睾丸组织凋亡和炎症因子相关基因mRNA表达量的影响。结果显示,酒精处理会显著的增加睾丸组织中凋亡基因(Caspse3,Bax)和炎症因子(IL-6,TNF-a)mRNA表达量,同时降低Bcl2基因mRNA的表达量(P<0.05)。相比于酒精组,50 mg/kg的biacalin处理可以显著降低caspase3,Bax,IL-6,TNF-a基因的mRNA表达量,同时提高Bcl2基因的mRNA表达量(P<0.05)。结论:本研究确定了 0.1 μg/mL baicalin可以显著提高猪卵母细胞IVM和PA,IVF及SCNT胚胎体外发育能力。主要表现为降低卵母细胞和胚胎中ROS水平,提高△Ψm和ATP含量等。同时证明baicalin是通过激活SHH信号途径来影响猪胚胎体外发育潜能。此外,本研究也进一步利用体内实验证明了 baicalin对酒精诱导雄性昆明鼠精子损伤具有潜在的保护作用。
Abstract
huang qin gan (baicalin)shi yi chong cong huang qin zhi wu gen bu di qu de chuan tong zhong yao chan ti ,yin ji ju you kang yang hua 、kang diao wang 、kang yan zheng deng gong neng ,bei yong yu zhi liao duo chong you yan zheng yin zi yin qi de ji bing ,tong shi zai sheng shi shang ju you zhi liao bu yun bu yo ,bao tai de zuo yong 。ran er ,baicalindui yu luan mu xi bao ti wai cheng shou (IVM),pei tai ti wai fa yo de ying xiang ji ji ji zhi shang bu qing chu 。luan mu xi bao IVMhe pei tai ti wai pei yang hui shou dao gao nong du huo xing yang (ROS)de po huai zuo yong shi ji xiao lv jiang di ,zhe shi luan mu xi bao he pei tai xiang guan ji li de yan jiu ji er zhe zai fu zhu sheng shi ji shu shang de ying yong dou shou dao hen da xian zhi 。yin ci ben shi yan zai zhu luan mu xi bao IVMpei yang ye he pei tai IVCpei yang ye zhong tian jia baicalinke yi tong guo yi zhi diao wang xian zhe di gao luan mu xi bao IVMneng li 、luan mu xi bao zhi liang 、pei tai ti wai fa yo neng li 、pei tai zhi liang 。tong shi ,li yong cheng nian xiong xing kun ming shu ti nei shi yan yan zheng baicalindui jiu jing you dao jing zi sun shang de bao hu zuo yong 。yi 、Baicalindui zhu luan mu xi bao IVMji ji hou pei tai ti wai fa yo neng li de ying xiang 1.ben shi yan tan suo bu tong nong du de baicalindui zhu luan mu xi bao IVMneng li de ying xiang ,fa xian 0.1μg/mLhe 0.5 μg/mLde baicalinjun ke yi xian zhe de di gao zhu luan mu xi bao IVM neng li (68.1,70.2 vs.59.7;P<0.05)。Baicalin ju you xian zhe de kang yang hua neng li ,yin ci ,tong guo ying guang ran se de fang fa fa xian 0.05,0.1he 0.5μg/mL baicalinchu li zu zhong luan mu xi bao nei chan sheng huo xing yang (ROS)shui ping jun xian zhe de di yu dui zhao zu (P<0.05)。zai zui xiao shi yan yao wu nong du huo de zui jia shi yan xiao guo de yuan ze xia ,hou xu shi yan shua ze 0.1 μg/mLde baicalinzuo wei zui jia chu li nong du 。tong shi RT-PCRjie guo xian shi baicalinchu li zu zhong luan mu xi bao he luan qiu xi bao de cu diao wang xiang guan ji yin (Bax)mRNAbiao da liang xian zhe di yu dui zhao zu (P<0.05)。2.ying guang ran se he RT-PCRfang fa jin yi bu jian ce baicainchu li hou luan mu xi bao de zhi liang ,jie guo xian shi 0.1μg/mLde baicalinchu li zu zhong xian li ti qian neng xing (△Ψm)he ATPhan liang jun xian zhe de gao yu dui zhao zu (P<0.05)。tong shi ,RT-PCRjian ce dao baicalinchu li zu zhong luan mu xi bao cheng shou xiang guan ji yin (BMP15,GDF9,CyclinB,CDK1)he luan qiu xi bao kuo zeng xiang guan ji yin (PTGS1,PTX3,TNFIP6,SHAS2)de mRNAbiao da liang xian zhe de gao yu dui zhao zu (P<0.05)。3.jian ce baicalinchu li de luan mu xi bao jing gu ci ji huo (PA)hou pei tai de ti wai fa yo neng li 。jie guo xian shi chu li zu huo de de pei tai ti wai fa yo neng li xian zhe de gao yu dui zhao zu (49.5 vs.27.2;P<0.05)。tong shi ,baicalinchu li hou huo de de pei tai fa yo dao nang pei jie duan shi xi bao shu ye xian zhe de gao yu dui zhao zu (P<0.05)。RT-PCRjian ce dao baicalinchu li zu zhong pei tai da dao nang pei jie duan shi quan neng xing xiang guan ji yin (Nanog,Sox2)he Bcl2ji yin de mRNAbiao da liang xian zhe de gao yu dui zhao zu (P<0.05),er Baji yin mRNAbiao da liang xian zhe de di yu dui zhao zu (P<0.05)。tong shi ,Tunelran se fa xian baicalinchu li zu nang pei xi bao diao wang shu xian zhe de di yu dui zhao zu (P<0.05)。er 、Baicalindui PA,ti xi bao he yi zhi (SCNT)he ti wai shou jing (IVF)pei tai fa yo neng li de ying xiang 1.jian ce baicalindui zhu PApei tai ti wai fa yo neng li de ying xiang 。jie guo biao ming 0.1μg/mLde baicalinchu li ke yi xian zhe di gao zhu PApei tai de ti wai fa yo neng li (21.7 vs.36.7;P<0.05),nang pei xi bao shu ye xian zhe gao yu dui zhao zu (P<0.05)。Tunelran se jian ce le nang pei de zhi liang ,jie guo xian shi baicalinchu li zu nang pei diao wang xi bao shu xian zhe de di yu dui zhao zu (P<0.05)。RT-PCRjie guo biao ming quan neng xing ji yin (Oct4,Sox2)de mRNAbiao da liang xian zhe gao yu dui zhao zu (P<0.05)。Baxhe Bcl2ji yin de mRNAbiao da liang xian zhe di yu he gao yu dui zhao zu (P<0.05)。2.jian ce baicalindui zhu PApei tai ROSbiao da liang ,Baxhe Bcl2ji yin mRNAbiao da liang ,△Ψm,ATPhan liang de ying xiang 。li yong ying guang ran se jian ce 0.1μg/mL baicalinchu li dui zhu PApei tai zhi liang de ying xiang ,jie guo xian shi baicalinchu li zu 2/4-cellshi ji ROSde biao da liang xian zhe di yu dui zhao zu he ji ta nong du chu li zu (P<0.05)。tong shi RT-PCRjie guo xian shi pei tai zai 2/4-cellshi ji Baxji yin de mRNAbiao da liang jun xian zhe de di yu dui zhao zu (P<0.05),Bcl2ji yin zai 2-cellshi ji de mRNAbiao da mei you xian zhe de bian hua ,er zai 4-cellshi ji mRNAde biao da liang xian zhe gao yu dui zhao zu (P<0.05)。tong shi ,ying guang ran se xian shi baicalinchu li zu zhong △Ψmhe ATPhan liang xian zhe gao yu dui zhao zu (P<0.05)。3.jian ce baicalinchu li dui zhu PApei tai zhong sonic hedgehog(SHH)xin hao tu jing de ying xiang 。RT-PCRjie guo xian shi ,baicalinchu li zu zhong SHHxin hao tu jing xiang guan ji yin (SHH,PTCH1,GdI1,SMO)de mRNAbiao da liang jun xian zhe gao yu dui zhao zu (P<0.05)。mian yi ying guang ran se jian ce fa xian baicalinchu li ke yi xian zhe de di gao SHHxin hao tu jing xiang guan dan bai (SHH,GLI1)de biao da liang 。wei le jin yi bu jian ce baicalinchu li dui pei tai ti wai fa yo neng li ying xiang de ji zhi ,wo men jiang pei tai fen wei san ge zu :dui zhao zu ;baicalinchu li zu ;baicalinhe SHHxin hao yi zhi ji (Cyclopamine,Cy)gong tong chu li zu ,jie guo xian shi Cyke yi hui fu baicalindui pei tai chan sheng ROShe diao wang de yi zhi zuo yong ji baicalindui △Ψmhe ATPhan liang de zeng jia 。zui hou ,wo men jian ce san ge chu li zu pei tai de ti wai fa yo neng li ,jie guo xian shi Cyke yi yi zhi baicalindui pei tai ti wai fa yo neng li de di gao 。4.jian ce 0.1 μg/mL baicalindui zhu SCNThe IVFpei tai de ying xiang 。jie guo biao ming 0.1 μg/mLde biacalinke yi xian zhe di gao zhu SCNThe IVFpei tai de fa yo neng li (14.9 vs.25.9;14.9 vs.25.9;P<0.05)。san 、Baicalindui jiu jing you dao de cheng nian xiong xing kun ming shu jing zi sun shang de bao hu zuo yong 1.ben shi yan jian li le jiu jing you dao cheng nian xiong xing kun ming shu jing zi sun shang mo xing 。shi yan fen wei liang zu ,10zhi tong wo chu sheng ,biao xing yi zhi de cheng nian xiong xing kun ming shu sui ji fen wei shi yan zu he dui zhao zu ,mei zu ge 5zhi 。shi yan zu jin hang mei tian lian xu si zhou jiu jing guan wei shi yan ,guan wei ji liang an jiu jing zhi liang he xiao shu ti chong bi ru xia :di yi zhou mei tian 2g/kg、di er zhou mei tian 4g/kg、di san zhou mei tian 6 g/kg、di si zhou mei tian 8 g/kg、dui zhao zu si wei deng liang de sheng li yan shui 。di si zhou jie shu shi dui xiao shu jin hang pou jian ,jian ce dao xiao shu ti chong xia jiang ,si wang lv zeng jia ,jing zi mi du xian zhe de di yu dui zhao zu (P<0.05)。2.jian ce bu tong nong du baicalinchu li dui jiu jing you dao cheng nian xiong xing xiao shu jing zi sun shang de bao hu zuo yong 。shi yan fen wei dui zhao zu (deng liang sheng li yan shui )、jiu jing zu 、jiu jing he 25 mg/kg baicalingong tong chu li zu 、jiu jing he 50 mg/kg baicalingong tong chu li zu (mg/kgji baicalinchong liang he xiao shu ti chong bi zhi ,jiu jing ji liang an zhao shi yan yi )。jie guo xian shi ,50 mg/kgde biacalinguan wei liang dui jiu jing you dao de xiao shu jing zi mi du xia jiang he jing zi ji xing lv sheng gao deng ju you bu fen bao hu zuo yong (P<0.05)。3.jian ce bu tong nong du baicalindui jiu jing you dao de cheng nian xiong xing kun ming shu gao wan zu zhi diao wang he yan zheng yin zi xiang guan ji yin mRNAbiao da liang de ying xiang 。jie guo xian shi ,jiu jing chu li hui xian zhe de zeng jia gao wan zu zhi zhong diao wang ji yin (Caspse3,Bax)he yan zheng yin zi (IL-6,TNF-a)mRNAbiao da liang ,tong shi jiang di Bcl2ji yin mRNAde biao da liang (P<0.05)。xiang bi yu jiu jing zu ,50 mg/kgde biacalinchu li ke yi xian zhe jiang di caspase3,Bax,IL-6,TNF-aji yin de mRNAbiao da liang ,tong shi di gao Bcl2ji yin de mRNAbiao da liang (P<0.05)。jie lun :ben yan jiu que ding le 0.1 μg/mL baicalinke yi xian zhe di gao zhu luan mu xi bao IVMhe PA,IVFji SCNTpei tai ti wai fa yo neng li 。zhu yao biao xian wei jiang di luan mu xi bao he pei tai zhong ROSshui ping ,di gao △Ψmhe ATPhan liang deng 。tong shi zheng ming baicalinshi tong guo ji huo SHHxin hao tu jing lai ying xiang zhu pei tai ti wai fa yo qian neng 。ci wai ,ben yan jiu ye jin yi bu li yong ti nei shi yan zheng ming le baicalindui jiu jing you dao xiong xing kun ming shu jing zi sun shang ju you qian zai de bao hu zuo yong 。
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