【作者】 王伟；

【导师】 何成强； 孟小倩；

【作者基本信息】 山东师范大学 ， 细胞生物学， 2014， 硕士

【摘要】 粘着斑激酶(focal adhesion kinase，FAK)是蛋白质酪氨酸激酶(protein tyrosinekinase，PTK)的一个家族，它属于非受体型酪氨酸蛋白激酶。黏着斑激酶家族的成员包括FAK和富含脯氨酸的蛋白酪氨酸激酶2／细胞粘附激酶β(proline—rich tyrosinekinase2／cellular adhesion kinaseβ，PYK2／CAKβ)。FAK是一种进化上高度保守的蛋白质,在体内分布广泛。FAK既能定位于皮质部位，又能分布在细胞核中。FAK是粘附分子超家族整合素(integrin)介导的信号转导过程中的一个关键酶，在细胞内信号转导中具有重要作用,是细胞内多条信号转导通路的交汇点，介导细胞与细胞外基质之间的作用。FAK通过其磷酸化而作用于胞质内相应的功能蛋白，从而有利于肿瘤细胞的黏附、降解和迁移。研究发现FAK对肿瘤的增殖、生存、迁移、侵袭、转移和血管形成等行为起着重要的调控作用。FAK与细胞多种信号转导有关,因此对细胞的多种生物学功能具有调节作用。研究表明, FAK对于胚胎发育有重要作用,对细胞的黏附、伸展、迁移、增殖和凋亡具有调节作用。有研究表明，在26天大的小鼠的卵巢中，FAK在卵母细胞的皮质集中表达。跨越透明带的卵丘细胞突也表达FAK，磷酸化后的FAK参与PTK的信号转导。Masahiro Sakurai等人研究了FAK在小鼠卵巢的卵泡发育过程的分布及作用，发现总FAK和pY397FAK在卵巢中高度表达，定位于卵巢卵泡的颗粒细胞，尤其是在腔前卵泡的颗粒细胞上。在闭锁卵泡的颗粒细胞中，pY397FAK减少。到目前为止，已有研究集中在对卵泡发育，卵母细胞及胚胎的后期发育中的FAK表达及功能研究，未见对FAK在小鼠受精卵及早期胚胎发育囊胚前各阶段中的表达定位及功能研究的相关报道。本文通过使用免疫荧光染色技术、激光扫描共焦显微镜技术、抑制剂干扰等方法对FAK在卵母细胞和早期胚胎发育过程中的定位及功能进行研究。并且对两种检测方法的效果进行了对比。研究发现:(1)在卵母细胞的GV期、GVBD期、MI期、MII期，FAK在卵母细胞的皮质部位定位；(2)在受精卵及2、4、8细胞各时期，FAK在皮质部位及细胞核中定位，在细胞与细胞的连接处有弱分布；在桑葚胚时期，FAK定位于细胞的顶部及基底部以及细胞与细胞的连接处和细胞核中；囊胚时期，FAK定位于细胞的顶部与基底部以及细胞与细胞的连接处和细胞核中;(3) pY397FAK在受精卵的皮质和细胞核中定位，在2细胞、4细胞和8细胞时期的皮质和细胞核中定位，在细胞与细胞的连接处也存在弱分布，在桑葚胚和囊胚时期，pY397FAK定位在细胞的顶部及基底部以及细胞与细胞的连接处；(4) pY576/577FAK在受精卵皮质及2细胞、4细胞和8细胞时期的皮质和细胞与细胞的连接处有弱分布，在囊胚时期的细胞的顶部及基底部以及细胞与细胞的连接处定位；(5) FAK与F-actin在受精卵的皮质部位共定位；(6)使用黏着斑激酶抑制剂PF573228处理受精卵，抑制了受精卵的卵裂，表明pY397FAK可能参与了受精卵的卵裂;(7)方法1（PFA+BSA）可以较好检测FAK在2、4、8细胞时期时的皮质定位与细胞连接处定位，方法2(PFA/Tritonx-100+BSA/Gly)可以清晰显示FKA在各个胚胎时期的细胞核定位以及桑葚胚与囊胚时期的皮质（顶部）及细胞连接处（基底部）的定位；方法2(PFA/Tritonx-100+BSA/Gly)对于pY397FAk与pY576/577FAK的亚细胞定位检测效果好于方法1。不论是桑葚胚还是囊胚时期，对于FAK或p-FKA的定位检测，方法2显著优于方法1。更多还原

【Abstract】 Focal adhesion kinase（FAK）,a non-receptor cytoplasmic tyrosine kinase,belongs to the focal adhesion kinase family which also includes PYK2／CAKβ.FAKis an evolutionarily conserved protein and is widespread in the body.FAK canlocalize at the cell cortex and in the nucleus.FAK is a key enzyme in the signalpathway introduced by integrin, a joint of a lot of signal pathway in the celland introduces the interaction between cell and extracellular matrix(ECM).FAK actswith corresponding functional proteins by it’s phosphorylation,facilitatingattachment,degradation and migration. FAK can regulateproliferation,survival,migration,invasion,metastasis,angiopoiesis, and soon.FAK is related with many signal pathway and can regulate many biologicalfunction.FAK is important for embryo development and regulateattachment,spreading, migration, proliferation and apotosis.One research showed that FAK expresses at the cortex of oocyte. Cumulus cellsacross ZP also express FAK and phosphorylated FAK involves signaling of PTK.Masahiro Sakurai et al revealed that total FAK and pY397FAK were highly expressedin the ovary and were localized to granulose cells of ovarian follicles,especiallypreantral follicles. In granulose cells of atretic follicles, pY397FAKdecreased.So far,researches which has carried out focus on expression and functionof FAK of follicle development,oocyte and late embryodevelopment.However,researches on localization and function of early embryodevelopment of mouse are not been seen.By using confocal microscopy,immunofluorescent staining and inhibitorsinterference, we revealed: (1) the cortex localization of FAK in the GV stage,GVBD，MI and MII stage ofoocyte;(2) the cortex localization and the nucleus localization of FAK in the fertilizedegg,2-cells,4-cells and8-cells embryo，and weak boundary distribution ofFAK in the2-cells,4-cells and8-cells embryo;the apical region distributionand the basal region distribution of superficial blastomeres and the boundarydistribution of the inside cells and the nucleus localization of FAK in themorula；the apical region distribution and the basal region distribution ofsuperficial blastomeres and the boundary distribution of the inside cells andthe nucleus distribution of FAK in the blastocyst;(3) We detected the cortex localization and the nucleus localization of pY397FAKin the fertilized egg,2-cells,4-cells and8-cells embryo，the weak boundarydistribution of pY397FAK in the2-cells,4-cells and8-cells embryo; the apicalregion distribution and the basal region distribution of superficial blastomeresand the boundary distribution of the inside cells of pY397FAK in the morula andthe blastocyst;(4) We also detected the weak cortex localization and the boundary localizationof pY576/577FAK in the fertilized egg and2-cells,4-cells and8-cells embryomorula; the apical region distribution and the basal region distribution ofsuperficial blastomeres and the boundary distribution of the inside cells ofpY576/577FAK in the morula and the blastocyst;(5) colocalization of FAK and F-actin at the cortex of the fertilized egg;(6) PF573228,the inhibitor of FAK,inhibited the cleavage of the fertilized egg;(7) Method1（PFA+BSA）could detect distinctly the cortex localization and theboundary localization of FAK in the2-cells、4-cells and8-cells embryo.Method2(PFA/Tritonx-100+BSA/Gly) could manifest distinctly the nucleus localizationof FAK at each stage and the cortex(apical) localization and theboundary(baselateral)localization of the morula and the blastocyst; In regardto the localization of pY397FAk and pY576/577FAK, the effect of method2(PFA/Tritonx-100+BSA/Gly) is better than method1（PFA+BSA）.Regardless of the morula or the blastocyst, method2(PFA/Tritonx-100+BSA/Gly) is significantlybetter than method1（PFA+BSA）for the localization detection of FAK or p-FKA。更多还原