CN114703121A - 一种水牛卵巢皮质体外培养用激活培养基及体外培养方法 - Google Patents
一种水牛卵巢皮质体外培养用激活培养基及体外培养方法 Download PDFInfo
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Abstract
本发明公开了一种水牛卵巢皮质体外培养用激活培养基及体外培养方法，本发明的激活培养基是在体外基础培养基中外加bpv(hopic)，其中所述bpv(hopic)的浓度为150μM。本发明的激活培养基能够显著提高原始卵泡向初级卵泡转变的卵泡数，在体外维持次级卵泡向窦性卵泡的发育趋势。
Description
技术领域
本发明涉及一种水牛卵巢皮质体外培养用激活培养基及体外培养方法。
背景技术
通过超速排卵结合活体采卵技术进行人工授精是能够快速提高水牛年存栏量的技术之一。但是相较于黄牛，水牛通过超排结合活体采卵技术可获得的成熟卵母细胞数量仅仅只有个位。研究表明哺乳动物的卵巢在出生时由一定数量的卵泡组成，大多数原始卵泡处于休眠状态，只有极少数的原始卵泡被激活并进入生长卵泡池中。动物卵巢上能利用的卵泡数量极为有限，卵巢里大量尚未得到激活的休眠卵泡只能在有限的生殖寿命里闭锁退化，造成遗传资源的极大浪费。
因此建立一个水牛原始卵泡体外激活的培养体系，对合理开发与利用水牛卵巢中的剩余休眠卵泡、增加水牛可用卵母细胞数量具有重大意义，同时也能够给水牛体外受精、克隆与转基因等基础研究提供一个获得大量优质卵母细胞的新途径。
发明内容
针对现有技术的不足，本发明提供了一种水牛卵巢皮质体外培养用激活培养基，其能够维持水牛卵巢皮质中腔前卵泡的发育，提高体外培养卵巢皮质的发育卵泡数，增加原始卵泡的激活数。
本发明所述水牛卵巢皮质体外培养用激活培养基是在体外基础培养基中外加bpv(hopic)，其中所述bpv(hopic)的浓度为150μM。bpv(hopic)为PTEN抑制剂，可以从市场上购买得到，其分子式为：K2VOO2O2C6H4NO3，分子量为：347.24g/mol。
优选地，50mL所述体外基础培养基含有以下成分：
所述液体培养基补充剂中含有浓度为1.0mg/mL的人重组胰岛素，浓度为0.55mg/mL的人转铁蛋白以及浓度为0.5μg/mL的亚硒酸钠。
本发明体外基础培养基中加入了McCoy’s 5A，可以支持多种(如骨髓、皮肤、肺和脾脏等)的原代移植物的体外生长；同时添加的20mM的HEPES能够维持所培养的水牛卵巢皮质中原始卵泡的体外发育；除此之外，激活培养基中添加的BSA(牛血清白蛋白)，能够起到保护卵母细胞、防止透明带硬化和细胞毒性的侵害，并且能够与转铁蛋白结合形成铁离子等，使卵母细胞免于伤害；添加的FSH(卵泡刺激素)则是腔前卵泡生长发育中起到关键作用的激素，可刺激体外培养的卵泡直径增加，颗粒细胞增殖、雌二醇分泌以及维持卵泡中卵丘-卵母细胞复合物的结构。ITS(液体培养基补充剂)是由1.0mg/ml人重组胰岛素，0.55mg/ml人转铁蛋白以及0.5μg/ml 100x浓度的亚硒酸钠组成。其中胰岛素与转铁蛋白协同合作，可携带和转运培养液中不利于卵泡生长发育的有毒成分，例如金属离子等；亚硒酸钠则是能够防止过氧化物对卵泡的发育造成危害。L-谷氨酰胺(L-glutamine)作为氨基酸的添加剂，能够刺激生长激素、胰岛素的分泌，参与合成谷胱甘肽，保持和增加水牛卵巢皮质中组织细胞内的GSH的储备，而提高其抗氧化能力；次黄嘌呤(hypoxanthine)：作为核苷的代谢产物，是一种重要的生物碱嘌呤；抗坏血酸(Ascorbic acid)作为高效抗氧化剂，能够清除水牛卵巢皮质培养过程中产生的自由基，减轻氧化应激反应。
本发明的另一目的是提供了一种体外培养水牛卵巢皮质的方法，具体包括以下步骤：
(1)水牛卵巢的选择及准备：从屠宰场刚屠宰的雌性水牛腹腔中采集卵巢，生理盐水清洗表面后立即放入37℃的高压灭菌处理后的生理盐水中，1小时内运回实验室；
(2)分离卵巢皮质，将水牛卵巢的周边修整干净后从韧带部分沿纵轴一分为二，去除掉髓质、白膜后，然后将卵巢皮质分离出来,将分离出的卵巢皮质切成4mm×3mm×1mm的窄条并置于分离培养基中，备用；
(3)水牛卵巢皮质的体外培养
首先将细胞培养小室放入六孔板中，加入本发明所述激活培养基，液面以正好没过培养小室底部的交换滤膜为宜；然后将细胞培养小室放入温度为39℃、5％CO2、相对湿度95％的培养箱中，备用；
取出置于分离培养基中的水牛卵巢皮质，并置于细胞培养小室的交换滤膜上，并置于上述培养箱中进行激活培养24小时；
激活培养后将细胞培养小室中的激活培养基换成体外基础培养基，然后放入上述培养箱中继续培养6天，每隔两天换一次体外培养基础液。
优选地，50mL所述分离培养基中含有以下成分：
与现有技术相比，本发明具有以下有益效果：
本发明的构建了水牛原始卵泡体外激活培养基能够维持水牛卵巢皮质中腔前卵泡体外持续发育，同时能够显著提高水牛卵巢皮质原始卵泡激活数量；
本发明的激活培养基是在基础培养基中添加了bpv(hopic)，通过调控bpv(hopic)的加入量，使得本发明的激活培养基能够显著提高原始卵泡向初级卵泡转变的卵泡数，在体外维持次级卵泡向窦性卵泡的发育趋势。
附图说明
图1-A、B分别为激活组和对照组培养的水牛卵巢皮质在倒置显微镜下的照片，比例尺为1000μm；图1-C、D为激活组和对照组的HE染色图,比例尺为400μm。
图2为对照组卵巢皮质冰冻切片HE染色图；其中，A、B、C、D中箭头指向处分别为原始卵泡、初级卵泡、次级卵泡及闭锁卵泡，比例尺为100μm。
图3为对照组和激活组水牛卵巢皮质连续切片卵泡计数结果示意图。A为不同培养天数对照组各级卵泡计数图，不同小写字母标注的相同阶段卵泡的不同组间卵泡数量百分比差异显著(p<0.05)；B为对照组和激活组各级卵泡计数图，*：P<0.05,差异显著；**：p<0.01，差异极显著。
图4为荧光定量检测对照组、激活组以及空白对照组中原始卵泡激活相关基因mRNA表达量变化情况，不同小写字母标注的不同组间相对mRNA表达量差异显著(p<0.05)。
图5为western blot检测对照组、激活组、空白对照组中原始卵泡激活相关蛋白的表达情况。
具体实施方式
本发明提供了一种水牛卵巢皮质体外培养用激活培养基是在体外基础培养基中外加bpv(hopic)，其中所述bpv(hopic)的浓度为150μM。其中50mL所述体外基础培养基含有以下成分：
其中液体培养基补充剂是由浓度为1.0mg/mL的人重组胰岛素，浓度为0.55mg/mL的人转铁蛋白以及浓度为0.5μg/mL的亚硒酸钠水溶液构成。
同时本发明还提供了一种分离培养基，50mL所述分离培养基中含有以下成分：
下面结合本发明的激活培养基以及分离培养基进行体外培养水牛卵巢皮质，具体实验方法如下：
(1)牛卵巢的选择及准备：从屠宰场刚屠宰的雌性水牛腹腔中采集卵巢，生理盐水清洗表面后立即放入37℃的高压灭菌处理后的生理盐水中，1小时内运回实验室。
(2)实验所取的新鲜水牛卵巢材料表面无突出的红体、黄体、白体及超过8mm的大卵泡，卵巢表面光滑、富有弹性且颜色呈健康的白色。卵巢表面可观察到大量直径不超过4-5mm的中小卵泡为理想的分离皮质的水牛卵巢。
从装有37℃的高压灭菌生理盐水的保温壶中取出水牛卵巢，75％酒精清洗一遍，高压灭菌生理盐水清洗两遍，用无菌剪刀剪去卵巢周围多余的结缔组织及韧带部分，之后将处理后的卵巢置于含有青链霉素(200μL/mL)的磷酸盐缓冲液PBS中备用。
选取理想的水牛卵巢，将其周边修整干净后从韧带部分沿纵轴一分为二，去除掉髓质、白膜后，用手术刀小心地将卵巢皮质分离出来，切成4×3×1mm的窄条，然后置于本发明分离培养基中，进行后续体外培养。其余的卵巢皮质窄条作为空白对照组，一部分放入液氮中速冻用于提RNA与蛋白，另一部分放入OCT包埋剂中用于制作冰冻切片。
(3)将细胞培养小室放入六孔板中，加入本发明所述激活培养基，液面以正好没过培养小室底部的交换滤膜为宜；然后将细胞培养小室放入温度为39℃、5％CO2、相对湿度95％的培养箱中，备用；
取出置于分离培养基中的水牛卵巢皮质，并置于细胞培养小室的交换滤膜上，并置于上述培养箱中进行激活培养24小时；
激活培养后将细胞培养小室中的激活培养基换成体外基础培养基，然后放入上述培养箱中继续培养6天，每隔两天换一次体外培养基础液。
体外培养7天结束后，将激活培养基处理24小时+基础培养基6天的卵巢收集起来作为激活组，基础培养基体外培养7天作为对照组。将一部分卵巢皮质组织放入液氮中速冻用于提RNA与蛋白，另一部分放入OCT包埋剂中用于制作冰冻切片。
实验一、冰冻切片的制作和HE染色
(1)组织包埋：将激活组和对照组的卵巢皮质组织用吸水纸吸干周围水分后，在卵巢皮质周围裹上适量的OCT包埋剂，在常温下浸泡2小时，之后将其转移至-20℃冰冻直至整个组织连带OCT包埋剂的颜色完全变白。
(2)连续切片：将包埋好的组织块利用OCT包埋剂黏附到冰冻切片机上的螺旋头上，以5μm进行连续切片，每五张进行贴片。
(3)HE染色：将贴片后的卵巢皮质切片用苏木精染色10分钟，伊红染色10秒，中性树胶封片。
(4)镜检及卵泡计数：显微镜观察组织切面完整，厚薄均匀，细胞形态清晰，细胞核与细胞质染色良好；按照各类水牛腔前卵泡的形态特征对卵巢皮质冰冻切片HE染色后进行计数，凡是结构破损、表面褶皱、胞质不均等判定为闭锁退化的腔前卵泡，不作计数。(原始卵泡:初级卵母细胞周围有一层扁平状的颗粒细胞；初级卵泡:由初级卵母细胞和排列在其周围的一层立方形或柱状颗粒细胞所组成；次级卵泡:有2层或2层以上的柱状颗粒细胞围绕初级卵母细胞,尚未有腔出现)。如图2显示，水牛卵巢皮质在基础培养基中进行体外培养，该培养基能够维持水牛卵巢皮质中原始卵泡、次级卵泡以及次级卵泡的形态；通过对基础培养基培养7天和激活培养基处理24小时+体外培养6天的水牛卵巢组织皮质切片进行卵泡计数，如图3-A表明基础培养基能够维持水牛卵巢皮质中腔前卵泡的发育，随着培养天数的增加，发育卵泡数也随之增加；图3-B表明150μM bpv(hopic)激活剂的添加相较于基础培养基，能够显著促进原始卵泡向初级卵泡的激活数，以及生长卵泡数。
实验二、卵巢皮质cDNA模板的获取
(1)组织样品RNA提取：将卵巢组织放在研钵中添加液氮并磨成粉末，以每50-100mg的卵巢组织加入1ml Trizol溶液剧烈摇晃进行裂解，冰上静置10分钟；静置结束后，按每1mL Trizol溶液加入0.2mL氯仿的比例添加氯仿，振荡摇匀，静置2min后转移至4℃预冷离心机中，12000rpm离心15min；取上清液于另一EP管中，按每1mL Trizol液加0.5mL异丙醇的比例添加异丙醇，轻轻颠倒EP管使其混匀，室温静置10min后转移到经4℃预冷的离心机中，12000rpm离心10min；弃去上清液，按每1mL Trizol溶液加入不少于1mL 75％乙醇的比例添加75％乙醇，充分摇匀后转移至4℃预冷的离心机中，12000rpm离心5min；然后去除上层清液，在室温或真空中干燥5-10min，然后加入适量的DEPC溶解。
(2)基因组DNA的去除：在离心管中配置如下混合液，用移液器轻轻吹打混匀。转至PCR仪中，程序为42℃，2分钟。
表1基因组DNA去除体系配制
(3)逆转录体系配置：在上一步的反应管中加入如下试剂，置于PCR仪中进行逆转录反应，程序为37℃15min，85℃5s，产物可存放在-20℃冰箱中保存备用。
表2逆转录反应体系配制
实验三、实时荧光定量检测
(1)qPCR反应体系：按下表依次加各类试剂；
表3实时荧光定量PCR反应体系配制
(2)qPCR引物序列及反应条件：按下表(注：F-Forward Primer上游，R-ReversePrimer下游)；
表4 PCR定量引物序列及其反应条件
qPCR反应程序为95℃预变性5min，95℃变性15s、60℃退火50s，变性、退火40个循环。每个样本重复3次，采用2-ΔΔCT方法计算基因的表达情况。对所得数据用GraphPad Prism8作图，用SPSS Statistics软件进行统计学分析。
如图4显示，结果表明150μM bpv(hopic)激活剂处理体外培养的水牛卵巢皮质后，RT-qPCR检测与原始卵泡激活相关基因FOXO3a、KITLG等相关基因mRNA表达量显著升高，与增殖相关基因PCNA的mRNA表达量也显著增加，表明该体外激活培养体系能够维持水牛卵巢皮质中腔前卵泡的发育并且促进原始卵泡激活相关基因的表达。
实验四、蛋白提取及Western Blot检测
(1)蛋白提取：将卵巢组织放在研钵中添加液氮并磨成粉末，以组织的10倍体积添加高效蛋白裂解液并转移至EP管中，使用超声波细胞破碎机粉碎组织使其充分裂解，之后冰上静置30分钟。4℃离心机离心，12000rpm离心10分钟。取部分上清液采用BCA法测蛋白浓度，剩余上清液加入4×上样缓冲液，使用PCR仪在99℃下变性10min。分装成小管存于-20℃冰箱用于后续实验。
(2)SDS-PAGE凝胶制备：按照下列配方分别制备6％SDS-PAGE分离胶(5ml体系)：水2.6ml，30％Acrylamide 1ml，1.5M Tris-HCL(pH8.8)1.3ml，10％SDS 0.05ml，10％过硫酸铵0.05ml，TEMED 0.004ml。10％SDS-PAGE分离胶(5ml体系)：水1.9ml，30％Acrylamide1.7ml，1.5M Tris-HCL(pH8.8)1.3ml，10％SDS 0.05ml，10％过硫酸铵0.05ml，TEMED0.002ml。SDS-PAGE浓缩胶(2ml体系)：水1.4ml，30％Acrylamide 0.33ml，1.0M Tris-HCL(pH6.8)0.25ml，10％SDS 0.02ml，10％过硫酸铵0.02ml，TEMED 0.002ml。
(3)电泳分离：将制好的凝胶放入电泳槽中，依次加上蛋白Marker和样品。mTOR蛋白分子量为用6％的SDS-PAGE凝胶；其余蛋白用10％SDS-PAGE凝胶进行电泳分离。浓缩胶电泳为恒压80V，分离胶电泳为恒压110V。
(4)转膜：根据蛋白Marker切胶，将凝胶、大小匹配的NC膜、滤纸置于转膜液中浸泡。将浸泡好转膜液的凝胶、NC膜、滤纸按照从上到下的次序纸-胶-膜-纸放好、压平，排尽气泡，放入转膜仪中以凝胶面积的1.5倍电流进行转膜。>70kda的蛋白转60-70分钟，40-70kda蛋白转40-50分钟。
(5)封闭：转膜结束后，TBST洗3次，每次5分钟。之后用5％脱脂牛奶封闭过夜
(6)免疫杂交与显色：取封闭结束的膜加入0.5％脱脂牛奶稀释后的一抗mTOR(1：1000)，AKT(1：1000)，GADPH(1：1000)，室温摇床孵育2小时；结束后，用TBST清洗3次，每次10分钟；之后加入0.5％脱脂牛奶稀释后的二抗(1：10000)，室温摇床孵育1小时；结束后用TBST清洗3次，每次10分钟，然后用TBS洗1次，洗10分钟；之后滴加显色液在BIO-RAD成像系统中进行曝光成像。
如图5显示，相较于未经体外培养水牛卵巢皮质空白对照组，以及由基础培养基培养7天的对照组，利用150μM bpv(hopic)激活剂处理体外培养的水牛卵巢皮质的激活组中AKT、mTOR等原始卵泡激活相关蛋白表达量都显著增加。(P<0.05)
需要说明的是，以上列举的仅是本发明的若干个具体实施例，显然本发明不仅仅限于以上实施例，还可以有其他变形。本领域的技术人员从本发明公开内容直接导出或间接引申的所有变形，均应认为是本发明的保护范围。
Claims (4)
1.一种水牛卵巢皮质体外培养用激活培养基，其特征在于，所述激活培养基是在体外基础培养基中外加bpv(hopic)，其中所述bpv(hopic)的浓度为150μM。
2.根据权利要求1所述水牛卵巢皮质体外培养用激活培养基，其特征在于，50mL所述体外基础培养基含有以下成分：
所述液体培养基补充剂中含有浓度为1.0mg/mL的人重组胰岛素，浓度为0.55mg/mL的人转铁蛋白以及浓度为0.5μg/mL的亚硒酸钠。
3.一种体外培养水牛卵巢皮质的方法，其特征在于，包括以下步骤：
(1)水牛卵巢的选择及准备：从屠宰场刚屠宰的雌性水牛腹腔中采集卵巢，生理盐水清洗表面后立即放入37℃的高压灭菌处理后的生理盐水中，1小时内运回实验室；
(2)分离卵巢皮质，将水牛卵巢的周边修整干净后从韧带部分沿纵轴一分为二，去除掉髓质、白膜后，然后将卵巢皮质分离出来,将分离出的卵巢皮质切成4mm×3mm×1mm的窄条并置于分离培养基中，备用；
(3)水牛卵巢皮质的体外培养
首先将细胞培养小室放入六孔板中，加入权利要求1或权利要求2所述激活培养基，液面以正好没过培养小室底部的交换滤膜为宜；然后将细胞培养小室放入温度为39℃、5％CO2、相对湿度95％的培养箱中，备用；
取出置于分离培养基中的水牛卵巢皮质，并置于细胞培养小室的交换滤膜上，并置于上述培养箱中进行激活培养24小时；
激活培养后将细胞培养小室中的激活培养基换成体外基础培养基，然后放入上述培养箱中继续培养6天，每隔两天换一次体外培养基础液。
4.根据权利要求1所述体外培养水牛卵巢皮质的方法，其特征在于，50mL所述分离培养基中含有以下成分：
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