CN113913370A - N-乙酰半胱氨酸在绵羊卵巢颗粒细胞体外培养中的应用 - Google Patents
N-乙酰半胱氨酸在绵羊卵巢颗粒细胞体外培养中的应用 Download PDFInfo
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Abstract
本发明提供了N‑乙酰半胱氨酸的一种新用途,具体地是N‑乙酰半胱氨酸用于在绵羊卵巢颗粒细胞体外培养的培养基配方中的应用,实验结果显示:含有N‑乙酰半胱氨酸的细胞体外培养基可用于促进绵羊颗粒细胞原代培养初期时的体外增殖与贴壁,并有效降低细胞内活性氧(ROS)水平,有利于细胞活力的提升。
Description
技术领域
本发明涉及家畜养殖技术领域,具体涉及N-乙酰半胱氨酸在绵羊卵巢颗粒细胞体外培养中的应用。
背景技术
卵泡是卵巢的基本功能单位,每一个卵泡都由一层或者多层卵巢颗粒细胞细胞包裹的卵母细胞组成。卵巢上表现出来的各级卵泡发育,其卵泡内体现着卵子成熟的过程。卵子成熟是由卵巢内外的多种因素的综合调节结果。在卵泡发育过程中,卵母细胞减数分裂和成熟的能力是逐步获得的,并且取决于发育卵母细胞和其附近卵巢颗粒细胞的相互作用关系的结果。卵母细胞与其周围的颗粒细胞之间的信号交流,对于卵母细胞发育和颗粒细胞分化、增殖都是至关重要的。卵巢颗粒细胞可为卵母细胞核和细胞质成熟提供必需的营养和调节信号,从而促使卵母细胞获得发育能力。
N-乙酰半胱氨酸是合成半胱氨酸和还原型谷胱甘肽的前体,是一种含巯基的化合物,能干扰自由基生成,清除活性氧自由基。NAC还能通过激活细胞信号通路调节激酶阻止细胞凋亡、促进细胞存活,常用来治疗某些退化性疾病。与半管氨酸相比,N-乙酰半胱氨酸分子量较小,更容易进入细胞,通过脱乙酰基形成半胱氨酸为细胞内还原型谷胱甘肽的合成提供原料。NAC分子中的乙酰基使其抗氧化性更稳定,临床上可通过口服、肌肉注射、静脉注射等多种方式给药。N-乙酰半胱氨酸因其还原性可直接调节多种蛋白,修饰DNA和多种分子模式;而被广泛用于治疗呼吸道疾病、癌症、心脏病、HIV感染、以及重金属中毒等疾病。
N-乙酰半胱氨酸可作为巯基供给体直接清除ROS,如超氧自由基(O2-)和过氧化氢等,减轻其对脂质和膜蛋白的损害,而维持其正常功能。同时,N-乙酰半胱氨酸可通过PI3K/Akt信号途径上调FOXO1、FOXO3、FOXO4基因的表达降低ROS生成、上调细胞内Bcl-2的相对表达来实现抗细胞凋亡。迄今为止,N-乙酰半胱氨酸在绵羊卵巢颗粒细胞体外培养中作用和影响报道较少。
发明内容
本发明的目的在于提供N-乙酰半胱氨酸的一种新用途,用于在制备绵羊卵巢颗粒细胞体外培养基中的应用。
为实现上述目的,本发明采用如下技术方案。
N-乙酰半胱氨酸在用于绵羊卵巢颗粒细胞体外培养基中的应用,所述的应用指的是提升细胞的活力;所述绵羊卵巢颗粒细胞体外培养基指的是:N-乙酰半胱氨酸16.3μg/mL、胎牛血清体积比为15%,高糖培养基体积比为84%,双抗体积比为1%。其中所述双抗为青霉素和链霉素溶液,其中青霉素和链霉素浓度均为10000单位/mL。所述提升细胞的活力指的是:体外培养基为促进绵羊卵巢颗粒细胞原代培养初期的体外增殖和贴壁促进剂,以及作为细胞活性氧的清除剂。所述绵羊体外卵巢颗粒细胞培养基,用于原代细胞铺板后48小时内的培养,在此期间,有利于其体外增殖和贴壁;超过48小时后,后续换液时可使用不含N-乙酰半胱氨酸的培养基进行正常传代培养。实验结果显示,含有N-乙酰半胱氨酸的细胞体外培养基可在绵羊颗粒细胞原代培养初期时促进体外增殖与贴壁,并有效降低细胞内活性氧(ROS)水平,有利于细胞活力的提升。
本发明更加详细的描述如下:
N-乙酰半胱氨酸用于绵羊卵巢颗粒细胞体外培养基中的应用。优选地,所述N-乙酰半胱氨酸用于制备促进原代绵羊卵巢颗粒细胞体外培养初期的贴壁与增殖促进剂;所述N-乙酰半胱氨酸在绵羊卵巢颗粒细胞培养基中的用量为16.3μg/mL。其培养基中的胎牛血清所占体积比为15%,高糖培养基(HyClone,美国,货号SH30022.01)所占体积比为84%,双抗溶液所占体积比为1%。所述的双抗溶液,由青霉素和链霉素溶液组成,其中青霉素和链霉素浓度均为10000单位/mL。所述绵羊卵巢颗粒细胞体外培养基,用于原代细胞铺板后48小时内的培养,在此期间,有利于其体外增殖和贴壁以及细胞内活性氧的降低和细胞活力的提升;超过48小时后,后续换液时可使用不含N-乙酰半胱氨酸的培养基进行正常传代培养。
本发明主要解决了绵羊卵巢颗粒细胞原代培养贴壁困难以及增殖速度慢的问题,重点考察了含有16.3μg/mL的N-乙酰半胱氨酸的培养基对绵羊颗粒细胞的贴壁、增殖和细胞活性氧和细胞活力的影响,主要的难点在于培养基中N-乙酰半胱氨酸适宜浓度的摸索研究。
本发明公开的N-乙酰半胱氨酸在绵羊卵巢颗粒细胞体外培养中的应用的有益效果在于:
本发明提高N-乙酰半胱氨酸用于在制备绵羊卵巢颗粒细胞体外培养基中的应用,具体地说,N-乙酰半胱氨酸可用于绵羊卵巢颗粒细胞前期的原代培养过程,该培养基在48小时内的体外培养过程中,有利于绵羊卵巢颗粒细胞前期的快速增殖和细胞贴壁;还可用于制备临床研究相关的用于促进绵羊卵巢颗粒细胞增殖,细胞内活性氧(ROS)清除以及细胞活力的提升研究。
附图说明
图1为倒置显微镜下的贴壁颗粒细胞(10倍镜);
图2为两组培养基培养48小的卵巢颗粒细胞贴壁情况(4倍镜)注:左图培养基A组,右图培养基B组;
图3为培养基A(NAC)和培养基B(对照组)培养颗粒细胞24小时和48小时的细胞活力比较。
图4为培养基A(NAC)和培养基B(对照组)培养颗粒细胞48小时的细胞内活性氧(ROS)水平比较。
具体实施方式
下面通过具体的实施方案叙述本发明。除非特别说明,本发明中所用的技术手段均为本领域技术人员所公知的方法。另外,实施方案应理解为说明性的,而非限制本发明的范围,本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言,在不背离本发明实质和范围的前提下,对这些实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。N-乙酰半胱氨酸、胎牛血清、高糖培养基、青霉素和链霉素等原料及试剂均有市售。
实施例1
卵巢颗粒细胞培养:
1. 在屠宰场收集空怀母羊的新鲜卵巢,用含有双抗体积比为1%的300mL的PBS保存,并低温运输至实验室。
2. 运输至实验室后,利用75%的乙醇溶液进行快速清洗卵巢,利用灭菌后的生理盐水进行卵巢多次清洗后,小心剪去卵巢外的结缔组织。
3. 处理好的卵巢换上含有1%双抗的生理盐水,置于超净台中等待颗粒细胞的抽取。
4. 配制两种颗粒细胞培养基:
培养基A:7.5mL胎牛血清+42mL高糖培养基+0.5mL双抗溶液+815μg N-乙酰半胱氨酸;
培养基B:5mL胎牛血清+44.5mL高糖培养基+0.5mL双抗溶液。
5. 使用10mL的一次性注射器先吸入12mL的培养基A,并对准绵羊卵巢卵泡抽取颗粒细胞,转移至装有8mL高糖培养基的15mL离心管中。
6. 抽完颗粒细胞后,15mL离心管至于离心机中2000rpm离心5分钟。
7. 弃掉上清液,利用6mL的高糖培养基重悬,之后在1500rpm离心5min,重复两次后,加入2mL培养基A,经过细胞计数后于10cm培养皿中铺板,置于CO2培养箱中37℃培养48小时。
8. 培养48小时后,吸走培养基A,加入6mL培养基B继续培养48小时,可择机收取贴壁颗粒细胞用于后续试验(图1)。
实施例2
培养基A和B对颗粒细胞培养增殖的影响:
1. 按照实施例1中的细胞培养步骤,以培养基A为试验组,以培养基B为对照组,调整细胞浓度为5×104个/mL(24孔板内),分别对绵羊颗粒细胞进行原代培养铺板。
2. 培养48h小时后,利用胰酶进行消化后,可通过全自动细胞计数仪进行计数。
3. 如图2所示,相比于对照组(培养基B),利用培养基A进行绵羊卵巢颗粒细胞体外培养时,其48小时后的细胞数量明显高于对照组。
实施例3
培养基A和B对颗粒细胞培养48小时后的细胞活力影响:
1. 按照实施例1中的细胞培养步骤,以培养基A为试验组,以培养基B为对照组,调整细胞浓度为5×104个/mL(96孔板),分别对绵羊颗粒细胞进行原代培养铺板。
2. 培养48h小时后(24h时,分别将旧培养基吸出,PBS清洗两遍),每孔加入100μL含10%(体积比)CCK8的对应培养基,且另加入一组(6个重复)没有细胞的孔做阴性对照。
3. 继续置于饱和湿度、37℃、5% CO2浓度的培养箱中培养4个小时后,用酶标仪测定各孔在450nm波长下的吸光值。
4. 根据细胞活力计算公式:细胞活力(%)=(试验组吸光值-阴性对照组吸光值)/(对照组吸光值-阴性对照组吸光值);如图3所示,相比于对照组(培养基B),利用培养基A进行绵羊卵巢颗粒细胞体外培养时,其24和48小时的细胞活力均显著高于对照组。
实施例4
培养基A和B对颗粒细胞培养48小时后的细胞活性氧清除比较:
1. 按照实施例1中的细胞培养步骤,以培养基A为试验组,以培养基B为对照组,调整细胞浓度为5×104个/mL(96孔板),分别对绵羊颗粒细胞进行原代培养铺板。
2. 培养48h小时后(24h时,分别将旧培养基吸出,PBS清洗两遍),避光操作,用培养基稀释ROS探针,终浓度10μM。每孔加入100μl探针工作液,37℃培养箱孵育20min。弃掉探针孵育工作液,PBS温和地洗1次,然后各孔加入100μl培养基。在荧光酶标仪上检测荧光强度。
3. ROS探针标记后,移除多余培养基,经过多聚甲醛固定后,适当PBS清洗。加入100μL的10μg/ml的Hochest染细胞核,并同样在荧光酶标仪上检测荧光强度。
4. 针对每一个培养孔,利用ROS探针荧光强度值/对应孔Hochest荧光强度值,计算每个细胞培养孔的细胞内活性氧(ROS)相对定量值。如图4所示,相比于对照组(培养基B),利用培养基A进行绵羊卵巢颗粒细胞体外培养时,其48小时的细胞内活性氧(ROS)水平显著低于对照组。
结论:综合以上试验结果,含有N-乙酰半胱氨酸的细胞体外培养基可用于促进绵羊颗粒细胞原代培养初期时的体外增殖与贴壁,并有效降低细胞内活性氧(ROS)水平,有利于细胞活力的提升。
Claims (4)
1. N-乙酰半胱氨酸在用于绵羊卵巢颗粒细胞体外培养基中的应用,所述的应用指的是提升细胞的活力;所述绵羊卵巢颗粒细胞体外培养基指的是:N-乙酰半胱氨酸16.3μg/mL、胎牛血清体积比为15%,高糖培养基体积比为84%,双抗体积比为1%。
2.权利要求1所述的应用,其中所述双抗为青霉素和链霉素溶液,其中青霉素和链霉素浓度均为10000单位/mL。
3.权利要求1所述的应用,其中所述提升细胞的活力指的是:体外培养基为促进绵羊卵巢颗粒细胞原代培养初期的体外增殖和贴壁促进剂,以及作为细胞活性氧的清除剂。
4.权利要求3所述的应用,其中所述绵羊体外卵巢颗粒细胞培养基,用于原代细胞铺板后48小时内的培养,在此期间,有利于其体外增殖和贴壁;超过48小时后,后续换液时可使用不含N-乙酰半胱氨酸的培养基进行正常传代培养。
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