Hsp70在小鼠体外早期胚胎发育中的表达
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摘要
为探究热休克蛋白Hsp70在小鼠体外早期胚胎发育中的表达情况以及Hsp70在胚胎发育中的作用机理,本试验采集8～12周龄SPF小白鼠的卵巢,在体式显微镜下采集卵母细胞并培养成熟,用0. 1%透明质酸酶消化卵丘卵母细胞复合体后,放入SrCl_2+CB液中进行5 h(37℃)的孤雌激活,然后移入G1培养液进行培养。取培养至2细胞期、4～8细胞期、9～16细胞期、桑椹期和囊胚期的小鼠胚胎提取总RNA,通过实时荧光定量PCR检测Hsp70在各个时期的表达情况,免疫荧光染色检测其在各个时期小鼠胚胎的表达定位。实时荧光定量PCR结果表明,Hsp70在小鼠体外早期胚胎的2细胞期、4～8细胞期、9～16细胞期、桑椹期、囊胚期都有表达,但在2细胞期胚胎和4～8细胞期胚胎的相对表达水平较高,在9～16细胞期胚胎、桑椹期胚胎和囊胚期胚胎的相对表达水平较低。免疫荧光染色结果显示,Hsp70定位于各个时期胚胎的细胞核和细胞质中,但9～16细胞期以后Hsp70主要定位于细胞核中。综上所述,Hsp70对小鼠体外早期胚胎的发育具有潜在的调控作用,这为进一步明确热休克蛋白Hsp70在胚胎抗热应激中的作用及机理研究提供了理论依据。
To research the expression of heat shock protein Hsp70 in early embryonic development of mice in vitro,and to explore the effect and mechanism of Hsp70 in embryonic development. The ovaries of SPF mice aged 8-12 weeks were collected in this study,then oocytes were collected under the microscope and cultivated to maturity,we placed COCs into the SrCl_2+CB solution for parthenogenetic activation for 5 hours( 37℃) after digesting it with 0. 1% hyaluronidase,then we transferred it into G1 culture fluid for cultivating.We extracted the total RNA from the mouse embryos that had been cultivated to 2 cell embryo,4-8 cell embryo,9-16 cell embryo,morula and blastocyst,then we detected the expression of Hsp70 in each period by real-time Q-PCR,and the expression and location of Hsp70 at various stages of mouse embryos by immunofluorescence staining. The result of the real-time Q-PCR shows that Hsp70 expressed in the 2 cell embryo,4-8 cell embryo,9-16 cell embryo,morula and blastocyst in early extracorporeal embryo of mice,but the expression of Hsp70 is higher in the 2 cell embryo and 4-8 cell embryo,and lower in the 9-16 cell embryo,morula and blastocyst.The result of immunofluorescence staining shows that Hsp70 locates in the nucleus and cytoplasm of mouse embryos at various stages,but Hsp70 mainly locates in the nucleus after the 9-16 cell embryo. In summary,Hsp70 has the potential regulatory effects in early embryonic development of mice in vitro,and this study provides a theoretical basis for further clarifying the role and mechanism of heat shock protein Hsp70 in embryo heat stress resistance.
To research the expression of heat shock protein Hsp70 in early embryonic development of mice in vitro,and to explore the effect and mechanism of Hsp70 in embryonic development. The ovaries of SPF mice aged 8-12 weeks were collected in this study,then oocytes were collected under the microscope and cultivated to maturity,we placed COCs into the SrCl_2+CB solution for parthenogenetic activation for 5 hours( 37℃) after digesting it with 0. 1% hyaluronidase,then we transferred it into G1 culture fluid for cultivating.We extracted the total RNA from the mouse embryos that had been cultivated to 2 cell embryo,4-8 cell embryo,9-16 cell embryo,morula and blastocyst,then we detected the expression of Hsp70 in each period by real-time Q-PCR,and the expression and location of Hsp70 at various stages of mouse embryos by immunofluorescence staining. The result of the real-time Q-PCR shows that Hsp70 expressed in the 2 cell embryo,4-8 cell embryo,9-16 cell embryo,morula and blastocyst in early extracorporeal embryo of mice,but the expression of Hsp70 is higher in the 2 cell embryo and 4-8 cell embryo,and lower in the 9-16 cell embryo,morula and blastocyst.The result of immunofluorescence staining shows that Hsp70 locates in the nucleus and cytoplasm of mouse embryos at various stages,but Hsp70 mainly locates in the nucleus after the 9-16 cell embryo. In summary,Hsp70 has the potential regulatory effects in early embryonic development of mice in vitro,and this study provides a theoretical basis for further clarifying the role and mechanism of heat shock protein Hsp70 in embryo heat stress resistance.
引文
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[6]王荣青,万红建,李志邈,叶青静,阮美颖,周国治,姚祝平,杨悦俭.番茄Hsp70基因鉴定及系统发育关系分析[J].核农学报,2014,28(3):378-385
[7]Manucha W.HSP70 Family in the renal inflammatory response[J].Inflamm Allergy Drug Targets,2014,13(4):235-240
[8]Drini S,Criscuolo A,Lechat P,Imamura H,Skalicky T,Rachidi N,Lukes J,Dujardin J C,Spath G F.Species and strain-specific adaptation of the HSP70 super family in pathogenic trypanosomatids[J].Genome Biology and Evolution,2016,8(6):1980-1995
[9]Caty C.GRP78 at the centre of the stage in cancer and neuroprotection[J].Frontiers in Neuroscience,2017,11:177
[10]郑妩媚,王福文.热休克蛋白70及其在运动中作用研究进展[J].食品与药品,2015,17(3):223-227
[11]王新兴,孔瑞瑞,钱令嘉.葡萄糖调节蛋白75在线粒体中的功能[J].生命的化学,2009,29(1):33-36
[12]杨茹洁.热休克蛋白-70调控公畜繁殖机能的研究进展[J].畜禽业,2016(3):35-36
[13]Qin H,Cheng C,Tang X,Bian Z.Impact of psychological stress on irritable bowel syndrome[J].World Journal of Gastroenterology,2014,20(39):14126-14131
[14]Lan C,Sun X N,Zhou X C,Yang B,Huang B L,Deng T Z,He Z T,Han X Y.Preinduced intestinal HSP70 improves visceral hypersensitivity and abnormal intestinal motility in PI-IBS mouse model[J].Asian Pacific Journal of Tropical Medicine,2016,9(3):297-300
[15]Qi X N,Li H T,Cong X,Wang X,Jiang Z L,Cao R F,Tian W R.Baicalin increases developmental competence of mouse embryos in vitro by inhibiting cellular apoptosis and modulating HSP70 and DNMT expression[J].The Journal of reproduction and development,2016,62(6):561-569
[16]Santos T M M,Sinzato Y K,Gallego F Q,IessiⅠL,Volpato G T,Dallaqua B,Damasceno D C.Extracellular HSP70 levels in diabetic environment in rats[J].Cell Stress and Chaperones,2015,20(4):595-603
[17]屈平平,李涛,胡士林.哺乳动物早期胚胎热休克蛋白70的研究进展[J].现代畜牧兽医,2018(3):49-53
[18]Li X H,Pang H Q,Qin L,Jin S,Zeng X,Bai Y,Li S W.HSP70overexpression may play a protective role in the mouse embryos stimulated by CUMS[J].Reproductive Biology and Endocrinology,2015,13(1):125
[19]王伟.FAK在小鼠早期胚胎发育中的定位表达及功能研究[D].济南:山东师范大学,2014:2
[20]Zhu Y F,Ren C L,Wan X Y,Zhu Y P,Zhu J B,Zhou H Y,Zhang TB.Gene expression of Hsp70,Hsp90 and Hsp110 families in normal palate and cleft palate during mouse embryogenesis[J].Toxicology&Industrial Health,2013,29(10):915-930
[21]钟小毛,韩宝生,汪莹.孤雌激活与人类辅助生殖技术[J].牡丹江医学院学报,2017,38(4):119-122
[22]钟小毛.小鼠孤雌激活胚胎干细胞体外发育潜能的探讨[D].唐山:华北理工大学,2017:19
[23]王栋娜.小鼠孤雌激活胚胎葡萄糖及主要氨基酸代谢水平的分析[D].唐山:华北理工大学,2015:12
[24]姜同泉.小鼠Hsp70基因序列分析及其在囊胚内表达的研究[D].青岛:青岛农业大学,2007:1
[25]Cheng S L,Lai Y L,Lee M C,Shen P C,Liu S S,Liu B T.Effects of deer velvet extract from Formosan sika deer on the embryonic development and anti-oxidative enzymes mRNA expression in mouse embryos[J].Journal of Ethnopharmacology,2014,154(3):600-605
[26]Fleming J V,Fontanier N,Harries D N,Rees W D.The growth arrest genes gas5,gas6,and CHOP-10(gadd153)are expressed in the mouse preimplantation embryo[J].Molecular Reproduction&Development,2015,48(3):310-316
[27]Lin P F,Jin Y P,Lan X L,Yang Y Z,Chen F L,Wang N,Li X,Sun Y J,Wang A H.GRP78 expression and regulation in the mouse uterus during embryo implantation[J].Journal of Molecular Histology,2014,45(3):259-268
[28]Suo J J,Cao R F,Cui X N,Jiang Z L,Cong X,Cui K,Tian W R.Expression of HSP72 in mouse preimplantation embryos with heat shock[J].Journal of Northeast Agricultural University,2014,21(2):38-45
[29]朱勇飞,张天宝.热休克蛋白与胚胎发育的关系[J].环境卫生学杂志,2004,31(5):287-292
[30]张宝.黄牛HSP家族基因差异表达及经济性状相关基因遗传变异研究[D].杨凌:西北农林科技大学,2011:14
[31]Edwards J L,Ealy A D,Monterroso V H,Hansen P J.Ontogeny of temperature-regulated heat shock protein 70 synthesis in preimplantation bovine embryos[J].Molecular Reproduction&Development,1997,48(1):25-33
[32]姜同泉,任登良,姜忠玲,田文儒,朱绍辉.热应激处理鼠囊胚Hsp70基因表达的RT-PCR检测[J].中国畜牧杂志,2007(11):8-10
[33]Matwee C,Kamaruddin M,Betts D H,Basrur P K,Allan King W.The effects of antibodies to heat shock protein 70 in fertilization and embryo development[J].Molecular Human Reproduction,2001,7(9):829-837
[34]魏世宝,金一.热休克蛋白对胚胎早期发育的影响[J].黑龙江畜牧兽医,2006(9):23-25
[35]Hunter E S,Dix D J.Heat shock proteins Hsp70-1 and Hsp70-3are necessary and sufficient to prevent arsenite-induced dysmorphology in mouse embryos[J].Molecular Reproduction&Development,2010,59(3):285
[2]何翃闳,崔燕,潘阳阳,樊江峰,胡威,张译夫,刘鹏刚,李秦,余四九.牦牛HSP27基因的克隆及其在雌性生殖器官中的表达[J].中国农业科学,2015,48(20):4178-4187
[3]Yan Z L,Wei H M,Ren C L,Yuan S S,Fu H,Lv Y,Zhu Y F,Zhang T B.Gene expression of Hsps in normal and abnormal embryonic development of mouse hindlimbs[J].Human and Experimental Toxicology,2015,34(6):563-574
[4]Park C J,Seo Y S.Heat shock proteins:A review of the molecular chaperones for plant immunity[J].Plant Pathology Journal,2015,31(4):323-333
[5]Alderson T,Kim J,Markley J.Dynamical structures of Hsp70 and Hsp70-Hsp40 complexes[J].Structure,2016,2(7):1014-1030
[6]王荣青,万红建,李志邈,叶青静,阮美颖,周国治,姚祝平,杨悦俭.番茄Hsp70基因鉴定及系统发育关系分析[J].核农学报,2014,28(3):378-385
[7]Manucha W.HSP70 Family in the renal inflammatory response[J].Inflamm Allergy Drug Targets,2014,13(4):235-240
[8]Drini S,Criscuolo A,Lechat P,Imamura H,Skalicky T,Rachidi N,Lukes J,Dujardin J C,Spath G F.Species and strain-specific adaptation of the HSP70 super family in pathogenic trypanosomatids[J].Genome Biology and Evolution,2016,8(6):1980-1995
[9]Caty C.GRP78 at the centre of the stage in cancer and neuroprotection[J].Frontiers in Neuroscience,2017,11:177
[10]郑妩媚,王福文.热休克蛋白70及其在运动中作用研究进展[J].食品与药品,2015,17(3):223-227
[11]王新兴,孔瑞瑞,钱令嘉.葡萄糖调节蛋白75在线粒体中的功能[J].生命的化学,2009,29(1):33-36
[12]杨茹洁.热休克蛋白-70调控公畜繁殖机能的研究进展[J].畜禽业,2016(3):35-36
[13]Qin H,Cheng C,Tang X,Bian Z.Impact of psychological stress on irritable bowel syndrome[J].World Journal of Gastroenterology,2014,20(39):14126-14131
[14]Lan C,Sun X N,Zhou X C,Yang B,Huang B L,Deng T Z,He Z T,Han X Y.Preinduced intestinal HSP70 improves visceral hypersensitivity and abnormal intestinal motility in PI-IBS mouse model[J].Asian Pacific Journal of Tropical Medicine,2016,9(3):297-300
[15]Qi X N,Li H T,Cong X,Wang X,Jiang Z L,Cao R F,Tian W R.Baicalin increases developmental competence of mouse embryos in vitro by inhibiting cellular apoptosis and modulating HSP70 and DNMT expression[J].The Journal of reproduction and development,2016,62(6):561-569
[16]Santos T M M,Sinzato Y K,Gallego F Q,IessiⅠL,Volpato G T,Dallaqua B,Damasceno D C.Extracellular HSP70 levels in diabetic environment in rats[J].Cell Stress and Chaperones,2015,20(4):595-603
[17]屈平平,李涛,胡士林.哺乳动物早期胚胎热休克蛋白70的研究进展[J].现代畜牧兽医,2018(3):49-53
[18]Li X H,Pang H Q,Qin L,Jin S,Zeng X,Bai Y,Li S W.HSP70overexpression may play a protective role in the mouse embryos stimulated by CUMS[J].Reproductive Biology and Endocrinology,2015,13(1):125
[19]王伟.FAK在小鼠早期胚胎发育中的定位表达及功能研究[D].济南:山东师范大学,2014:2
[20]Zhu Y F,Ren C L,Wan X Y,Zhu Y P,Zhu J B,Zhou H Y,Zhang TB.Gene expression of Hsp70,Hsp90 and Hsp110 families in normal palate and cleft palate during mouse embryogenesis[J].Toxicology&Industrial Health,2013,29(10):915-930
[21]钟小毛,韩宝生,汪莹.孤雌激活与人类辅助生殖技术[J].牡丹江医学院学报,2017,38(4):119-122
[22]钟小毛.小鼠孤雌激活胚胎干细胞体外发育潜能的探讨[D].唐山:华北理工大学,2017:19
[23]王栋娜.小鼠孤雌激活胚胎葡萄糖及主要氨基酸代谢水平的分析[D].唐山:华北理工大学,2015:12
[24]姜同泉.小鼠Hsp70基因序列分析及其在囊胚内表达的研究[D].青岛:青岛农业大学,2007:1
[25]Cheng S L,Lai Y L,Lee M C,Shen P C,Liu S S,Liu B T.Effects of deer velvet extract from Formosan sika deer on the embryonic development and anti-oxidative enzymes mRNA expression in mouse embryos[J].Journal of Ethnopharmacology,2014,154(3):600-605
[26]Fleming J V,Fontanier N,Harries D N,Rees W D.The growth arrest genes gas5,gas6,and CHOP-10(gadd153)are expressed in the mouse preimplantation embryo[J].Molecular Reproduction&Development,2015,48(3):310-316
[27]Lin P F,Jin Y P,Lan X L,Yang Y Z,Chen F L,Wang N,Li X,Sun Y J,Wang A H.GRP78 expression and regulation in the mouse uterus during embryo implantation[J].Journal of Molecular Histology,2014,45(3):259-268
[28]Suo J J,Cao R F,Cui X N,Jiang Z L,Cong X,Cui K,Tian W R.Expression of HSP72 in mouse preimplantation embryos with heat shock[J].Journal of Northeast Agricultural University,2014,21(2):38-45
[29]朱勇飞,张天宝.热休克蛋白与胚胎发育的关系[J].环境卫生学杂志,2004,31(5):287-292
[30]张宝.黄牛HSP家族基因差异表达及经济性状相关基因遗传变异研究[D].杨凌:西北农林科技大学,2011:14
[31]Edwards J L,Ealy A D,Monterroso V H,Hansen P J.Ontogeny of temperature-regulated heat shock protein 70 synthesis in preimplantation bovine embryos[J].Molecular Reproduction&Development,1997,48(1):25-33
[32]姜同泉,任登良,姜忠玲,田文儒,朱绍辉.热应激处理鼠囊胚Hsp70基因表达的RT-PCR检测[J].中国畜牧杂志,2007(11):8-10
[33]Matwee C,Kamaruddin M,Betts D H,Basrur P K,Allan King W.The effects of antibodies to heat shock protein 70 in fertilization and embryo development[J].Molecular Human Reproduction,2001,7(9):829-837
[34]魏世宝,金一.热休克蛋白对胚胎早期发育的影响[J].黑龙江畜牧兽医,2006(9):23-25
[35]Hunter E S,Dix D J.Heat shock proteins Hsp70-1 and Hsp70-3are necessary and sufficient to prevent arsenite-induced dysmorphology in mouse embryos[J].Molecular Reproduction&Development,2010,59(3):285