CN116574683B - 一种细胞生长促进剂及其应用 - Google Patents
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Abstract
本发明涉及一种细胞生长促进剂及其应用,采用Fe2+与卵白蛋白形成Fe2+‑卵白蛋白复合物促进卵白蛋白细胞内吞,该细胞生长促进剂能够在无氨基酸的环境中维持细胞生长,在有氨基酸的环境中促进细胞增殖,并且,Fe2+‑卵白蛋白复合物能够在细胞培养中替代昂贵的动物血清白蛋白,该细胞生长促进剂对于细胞活力的促进效果显著,且成本低廉。
Description
技术领域
本发明属于生物技术领域,具体涉及一种细胞生长促进剂及其应用。
背景技术
蛋白质是机体生长所必需的营养物质,科研工作者们经常通过添加胎牛血清来促进细胞的增殖或维持细胞活性。胎牛血清中包含丰富的血清白蛋白、球蛋白、纤连蛋白、转铁蛋白等蛋白质,其中牛血清白蛋白占总蛋白的比例超过50%。血清白蛋白也是机体内重要的营养物质,机体每天消耗14 g/70 kg的血清白蛋白,同时肝脏合成等量的血清白蛋白以维持平衡。在健康人体内,脂肪与蛋白提供能量比例为15:1,癌症患者中脂肪与蛋白提供能量比例为3:1-1:1。因此,血清蛋白质是细胞或机体生长的重要营养物质来源。
卵白蛋白(OVA)是一种单体磷酸糖蛋白,由385个氨基酸残基组成,其氨基酸组成营养均衡,是优异的膳食补充剂。因此,OVA是重要的营养物质来源。此外,每个OVA分子有一个内部二硫键和四个游离巯基,其能与自由基直接反应,从而具有较强的抗氧化活性。卵白蛋白相比于血清白蛋白纯化方法更加简单,更易得到,其成本更低,因此寻找一种简单的方法来替代动物血清白蛋白(BSA)来促进细胞增殖或维持细胞活力将具有重要的应用价值。
胞吞是维持生物体生命活动的基础。胞吞是调节营养物质内化的关键过程,调节蛋白质的胞吞效率有利于提高蛋白质被机体或者细胞更多的被利用,从而有利于维持细胞的正常生长。提高白蛋白的胞吞效率,将有利于提高其营养物质的生物利用率。Noah等人和Gustafsson等人通过管腔给药后,在肠道中观察到卵白蛋白,表明OVA可以在肠道发生细胞内吞,这为OVA发挥生物学功能和营养价值提供了良好的条件。这些研究结果表明了细胞可以通过胞吞作用内化OVA,从而发挥OVA的营养价值。增加OVA的胞吞效率可能将有助于增强其生物学功能以及其营养价值,将为OVA在细胞培养基中的应用以及提高OVA的营养价值提供一定的理论支持。现有技术没有合适的方法去增加细胞对OVA的胞吞效率,亟需开发一种促进细胞增殖和维持细胞活力的新方法。
发明内容
为了解决上述问题,本发明提供一种提高细胞活力的方法及其应用,本发明的目的是通过以下技术方案实现的:
本发明第一方面提供一种细胞生长促进剂,所述细胞生长促进剂为Fe2+-卵白蛋白复合物。
本发明第二方面提供一种能够细胞培养基,所述细胞培养基包含本发明第一方面所述的细胞生长促进剂;所述细胞培养基中生长促进剂的浓度为1μg/ml ~1000 μg/ml,包括但不限于100μg/ml、200μg/ml、250μg/ml、500μg/ml、1000μg/ml。
进一步的,所述细胞培养基中不含氨基酸;
和/或,所述细胞培养基中不含动物血清白蛋白;
和/或,所述细胞培养基中还包含细胞培养基辅料。
优选的,所述细胞培养基辅料还包括:NaCl、KCl、NaHCO3、KH2PO4、Na2HPO4、葡萄糖和/或水。
更优选的,所述细胞培养基辅料还包括:137.93mM NaCl, 5.33mM KCl, 4.17mMNaHCO3, 0.441mM KH2PO4, 0.338mM Na2HPO4, 5.56mM 葡萄糖和/或水,pH值为7.1-7.5;
优选的,所述动物血清白蛋白包括人血清白蛋白、α血清白蛋白或牛血清白蛋白。
本发明第三方面提供一种细胞培养方法,在含有本发明第一方面所述的细胞生长促进剂的细胞培养基或本发明第二方面所述的细胞培养基中培养细胞。
本发明第四方面提供一种含有本发明第一方面所述的细胞生长促进剂药物、食品、保健食品或化妆品。
本发明第五方面提供一种提升细胞活力的方法,所述方法中添加本发明第一方面所述的细胞生长促进剂。
进一步的,所述方法在不含氨基酸或氨基酸缺乏的环境下进行。
进一步的,所述方法中采用本发明第一方面所述的细胞生长促进剂替代动物血清蛋白。
进一步的,细胞培养体系中所述Fe2+的终浓度为10~100µM,添加所述卵白蛋白的终浓度为10~100 µM。
进一步的,所述细胞为哺乳动物细胞,优选的,所述细胞为鼠细胞、猪细胞、牛细胞、水牛细胞、绵羊细胞、山羊细胞、鹿细胞、野牛细胞、骆驼细胞、马鹿细胞、兔细胞和/或人细胞,更优选为人细胞;
和/或,所述细胞为上皮细胞、内皮细胞、巨噬细胞、肾小管细胞、肝实质细胞、肝细胞、心肌细胞、乳腺癌细胞、胃癌细胞、肺癌细胞、肝癌细胞、宫颈癌细胞、淋巴癌细胞、甲状腺癌细胞、食道癌细胞、肾癌细胞、胰腺癌细胞、胶质瘤细胞、黑色素瘤细胞、膀胱癌细胞或前列腺癌细胞。
本发明的有益效果
(1)本发明发现Fe2+可以促进卵白蛋白的细胞内吞效率,从而提升细胞活力,促进细胞增殖,增加OVA的利用度;
(2)本发明发现添加结合Fe2+的卵白蛋白,当Fe2+与卵白蛋白形成复合物时,能够在无氨基酸培养基中维持细胞活力;同时,在有氨基酸培养基中,能够促进细胞增殖。
(3)本发明发现在细胞培养基中,Fe2+与卵白蛋白形成复合物时能够替代动物血清白蛋白的作用。
附图说明
图1所示为在氨基酸饥饿的条件下比较Fe2+-OVA和OVA对Caco-2细胞活力的影响;
图2所示为富含氨基酸的条件下的培养基中HSA、BSA、OVA、Fe2+-OVA对Caco-2细胞活力的影响;
图3所示为Fe2+对OVA细胞内吞效率的影响;
图4所示为Fe2+-OVA的细胞内吞过程的照片;
图5所示为Fe2+-OVA在IEC-6细胞中的胞吞的荧光图;
图6所示为Fe2+-OVA在小鼠单核巨噬细胞(RAW264.7)中的胞吞的荧光图;
图7所示为Mg2+对OVA的细胞内吞效率的影响的荧光图;
图8所示为Ca2+对OVA的细胞内吞效率的影响的荧光图;
图9所示为实施例3 Fe2+加入顺序对OVA胞吞效率的影响;
图10所示为实施例4添加不同浓度Fe2+的OVA荧光淬灭结果图,图例代表了OVA和Fe2+的摩尔比。
具体实施方式
除非另有定义,本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
本发明中,术语“内吞”是指细胞内吞作用或胞吞作用(endocytosis)是细胞从胞外获取大分子和颗粒状物质的一种重要方式,它是真核细胞中普遍存在的一种生理现象。胞外物质通过质膜包裹,质膜内陷并形成膜包被的囊泡,囊泡与质膜脱离进入胞内并在胞内产生一系列的生理活动和生理功能。胞吞作用与多种生命活动有着密切关系,如免疫应答、神经递质运输、细胞信号转导、细胞和组织代谢平衡等;
本发明中,术语“Caco-2细胞”是指一种人克隆结肠腺癌细胞,结构和功能类似于分化的小肠上皮细胞,具有微绒毛等结构,并含有与小肠刷状缘上皮相关的酶系,可以用来进行模拟体内肠转运的实验。在细胞培养条件下,生长在多孔的可渗透聚碳酸酯膜上的细胞可融合并分化为肠上皮细胞,形成连续的单层,这与正常的成熟小肠上皮细胞在体外培育过程中出现反分化的情况不同。细胞亚显微结构研究表明,Caco-2细胞与人小肠上皮细胞在形态学上相似,具有相同的细胞极性和紧密连接。胞饮功能的检测也表明,Caco-2细胞与人小肠上皮细胞类似;
本发明中,术语“IEC-6细胞”是指一种从大鼠小肠上皮分离出的细胞系,常被用作研究肠道生理和疾病机制的模型。这些细胞具有上皮细胞的形态特征和功能,包括涂层肠上皮的表面区域和表达多种消化酶的细胞刷缘。IEC-6细胞不仅可以用于研究肠道的吸收、转运、分泌和黏附功能等方面,还可以用于模拟炎症、肿瘤和肠道感染等疾病。
本发明中,术语“Western blot”是指蛋白质印迹法(免疫印迹试验),其基本原理是通过特异性抗体对凝胶电泳处理过的细胞或生物组织样品进行着色。通过分析着色的位置和着色深度获得特定蛋白质在所分析的细胞或组织中表达情况的信息。
本发明中,术语“OVA(ovalbumin)”是指卵白蛋白。
本发明中,术语“氨基酸饥饿”(amino acid starvation)是指细胞培养时候除去培养基中的所有氨基酸,以此模拟肿瘤病人体内的营养缺乏状态环境。
本发明中,术语“HBSS”是指Hank's平衡盐溶液。
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限制本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实验方法
1.1 Western blot分析
1.1.1、提取蛋白
采用含有不同浓度的Fe2+的OVA处理Caco-2细胞和IEC-6细胞,处理4h后,弃上清,用PBS洗3次后,每孔加入100 μL细胞裂解液(含1 mM PMSF),将孔板置于冰上静置30 min,以提取蛋白用于OVA胞吞含量的测定。每组设置3个平行处理。用刮刀按顺时针方向刮下细胞于离心管中,然后离心(4℃, 12000×g, 20 min),吸取上层蛋白提取液,作为待测液,用于测量提取液中的总蛋白浓度及目标蛋白的水平。
1.1.2、提取液中总蛋白浓度的测定
1.1.2.1试剂的配制:
1.1.2.1.1、30%凝胶配制溶液(A 液) :称取29.2 g丙烯酰胺和0.8 g N,N-亚甲基双丙烯酰胺于80 mL超纯水中,待充分溶解后用超纯水定容至100 mL。4℃保存备用。
1.1.2.1.2、SDS 溶液(10%,w/v):称取10 g SDS 粉末充分溶解并用超纯水定容至100 mL。
1.1.2.1.3、4×分离胶缓冲液(B 液):量取75 mL Tris-HCl(pH 8.8)和4 mL 10%SDS,用超纯水定容至100 mL。
1.1.2.1.4、4×浓缩胶缓冲液(C 液):量取50 mL Tris-HCl(pH 6.8)和4 mL 10%SDS,用超纯水定容至100 mL
1.1.2.1.5、1×电泳缓冲液储备液:称取3 g Tris、14.4 g 甘氨酸和1 g SDS,用加超纯水定容至1 L,室温保存备用。
1.1.2.1.6、1×转膜缓冲液储备液:称取3 g Tris和14.4 g甘氨酸,加超纯水定容至1 L,室温保存备用。
1.1.2.1.7、1×TBS(pH 7.5)储备液:称取2.42 g Tris-base 和8 g NaCl 溶解于800 mL 超纯水中,待充分溶解后加盐酸调pH 至7.5,随后加超纯水定容至1 L,最后加入0.5 mL 吐温20,充分混匀后室温保存备用。
1.1.2.1.8、封闭液:称取5 g脱脂奶粉于100 mL TBST中,振荡混匀,4℃暂存,现配现用。
1.1.2.2采用BCA 蛋白检测试剂盒(therom,23227)测定提取的蛋白样品中总蛋白质的浓度:
1.1.2.2.1用裂解液稀释标准品蛋白(2mg/ml的牛血清白蛋白溶液,购自therom,货号为23227),依次得到 0mg/mL,0.5mg/mL,0.75mg/mL,1mg/mL,1.5mg/mL,2 mg/mL的蛋白标准溶液。
1.1.2.2.2工作液:20mL试剂1(1%二辛可宁酸二钠盐,2%无水碳酸钠,0.16%酒石酸钠,0.4%氢氧化钠,0.95%碳酸氢钠. 混合调pH值至11.25)与400μL试剂2(4%硫酸铜)混匀。
1.1.2.2.3加样检测:每孔加入10 µL蛋白标准溶液或待测样),再加入200 µL工作液,37℃孵育30 min。孵育结束后测定570 nm处的OD 值。
1.1.2.2.4绘制蛋白浓度-OD 值标准曲线,计算提取的蛋白样品中的总蛋白浓度。
1.1.2.3、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)
将所有样本的蛋白提取液调至统一的浓度,然后分别取80 μL稀释后的蛋白提取液(1、提取蛋白步骤中的待测液经由超纯水稀释而成),加入5×上样缓冲溶液20 μL,95℃金属浴10 min。冷却后保存于-80℃待用。
根据目标蛋白的分子量大小本实施例采用10%分离胶以及4%浓缩胶进行分离蛋白样品。按照表1、2中的试剂体积配制分离胶和浓缩胶。然后灌入制胶玻璃板中,室温静置30min,充分凝固。样品孔两边各加入3 µL蛋白Marker。电泳条件为:先以80 V恒压使样品浓缩为同一高度的细线,然后以120 V 恒压将样品在分离胶部分进行分离。
表 1 SDS-PAGE分离胶配制方法
表 2 SDS-PAGE浓缩胶配制方法
1.1.2.4、转膜和抗体孵育
将孔径为0.45 μm的聚偏二氟乙烯(polyvinylidene difluoride, PVDF)膜浸泡于甲醇中活化30 s,然后放入转膜缓冲液。转膜夹黑色面朝下,从下往上依次放置转印海绵-转印滤纸-分离胶-PVDF膜-转印滤纸-转印海绵。转膜条件为:恒流200 mA,2 h。
转膜完成后,将PVDF膜截留蛋白的一面朝上,4℃封闭过夜。封闭结束后,将膜浸入稀释至合适浓度的一抗溶液中,4℃孵育14 h,使一抗与目标蛋白结合。用甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)或β-actin作为内参,均以 1:1000 的比例稀释。孵育结束后将一抗回收,用TBST洗膜液,每隔10min换洗膜液,重复清洗步骤3次,以除去非特异性结合的抗体。加入二抗(稀释比例为1:4000)稀释液,室温孵育1h。然后用 TBST 重复相同的洗涤步骤将残留二抗洗去。
1.1.2.5、显影和计算目标蛋白相对含量
将发光液按A液和B液等体积混合配制成显影液,将显影液(北京华兴博创基因技术有限公司,HX1868)均匀覆于膜上,然后采用凝胶成像仪进行显影。使用 Image J软件对目标蛋白的相对含量进行计算,根据每个泳道目标蛋白条带得到的灰度值与相对应的内参蛋白条带灰度值的比值来计算目标蛋白的相对含量。
1.2 免疫荧光染色
具体步骤如下:
1.2.1、采用含有不同含量Fe2+的HSA处理细胞4 h后,用PBS浸洗3次,每次3 min。
1.2.2、采用4%的多聚甲醛固定细胞15 min后,用PBS洗涤3次,每次3 min。
1.2.3、采用0.5% Triton X-100对细胞通透5 min后,用PBS洗涤3次,每次5 min,
1.2.4、采用免疫荧光封闭液对细胞封闭30 min后,去除封闭液。
1.2.5、加入稀释至合适浓度的一抗(albumin抗体稀释比例为 1:50),4℃孵育过夜。去除一抗稀释液后,用PBST洗涤3次,每次5 min。
1.2.6、加入稀释好的荧光二抗(稀释比例为1:500),室温孵育1 h,去除二抗稀释液后,用PBST洗涤3次,每次5 min。
1.2.7、加入DAPI对细胞核进行染色,避光孵育5 min,去除DAPI后,用PBST洗涤4次,每次5 min,
1.2.8、用吸水纸吸干爬片上的液体,用含抗荧光淬灭剂的封片液滴入至载玻片上,再倒置爬片于载玻片上,最后通过激光共聚焦显微镜(CLSM)采集图像。
1.3 透射电子显微镜
将细胞培养基去除,然后用PBS浸洗干净。加入2.5%戊二醛溶液,4℃固定过夜,然后转移至1%锇酸中,在4℃条件下振荡3 h,使细胞再次固定。然后用不同浓度的乙醇对样品进行脱水处理,采用环氧丙烷置换乙醇后,再置于环氧树脂中4℃条件下浸透包埋;最后置于70℃条件下18 h,使树脂聚合形成样品包埋块。将包埋块切成60 nm薄片,染色后通过透射电子显微镜观察HSA的细胞内吞过程。
实施例1 Fe2+对细胞活力和内吞效率的影响
(1)Caco-2细胞培养
含有结合Fe2+的OVA溶液(Fe2+-OVA)的培养基配制:添加一定浓度的Fe2+于OVA中,添加至离心管中,然后在37℃孵育15 min,分成两组,用Hanks细胞培养基稀释,稀释至Fe2+的终浓度为22 µM,OVA的终浓度为22 µM、另一组仅添加同等浓度的OVA于离心管中(即不添加Fe2+),然后在37℃孵育15 min,用Hanks细胞培养基稀释,稀释至OVA的终浓度为22 µM,并于不同的时间观察其细胞状态。
(2)测定OVA的Caco-2细胞内吞效率
含有结合不同浓度Fe2+的OVA溶液(Fe2+-OVA)的培养基配制:添加一定浓度的OVA和分别加入不同浓度的Fe2+于离心管中,然后在37℃孵育15min,最后用Hanks培养基将复合物稀释至Fe2+的终浓度分别为0µM,22µM,44µM,88µM,OVA的终浓度均为22µM。将配制好的培养基分别加入12孔板各孔中,37℃处理4h后,弃上清,用冰PBS洗3次,孔板置于冰上,提取蛋白用于Western blot分析(分析方法参见前述“实验方法”)
(3)测定细胞活性
分别采用含有不同浓度的牛血清白蛋白(BSA),人血清白蛋白(HSA),OVA,Fe2+-OVA的无氨基酸培养基(Hanks培养基)或富含氨基酸的培养基(DNEM培养基)培养Caco-2细胞24h后,弃去孔内培养基,每孔重新加入DMEM培养基与CCK-8的混液100 µL,37℃ 孵育1 h后,测定450 nm和650 nm处的OD值,来表示其细胞活性。
实验结果
如图1所示,在无氨基酸的培养基中(Hanks培养基),只添加卵白蛋白的培养基不能维持Caco-2细胞的生长,而添加结合Fe2+的OVA的培养基可以维持Caco-2细胞活力,Fe2+-OVA组的OD值远大于OVA组,说明Fe2+的结合促进了细胞对OVA的利用,从而增加OVA的营养价值,维持了细胞活力。
如图2所示,以BSA和HSA组为阳性对照,以OVA组和Fe2+-OVA组为实验组,在富含氨基酸的培养基(DMEM)环境中,Fe2+-OVA组的OD值高于OVA、HSA、BSA组,即Fe2+-OVA组的细胞活力高于OVA、HSA、BSA组,说明单纯的卵白蛋白(OVA)没有促进细胞的增殖,而结合Fe2+的OVA(Fe2+-OVA)在富含氨基酸的培养基中仍然可以促进细胞的增殖。结果表明Fe2+-OVA可以在氨基酸缺乏环境中仍然维持细胞活力以及可以正常生理条件下显著促进细胞增殖。为了探究Fe2+提高OVA细胞活力的原因,本实施例进一步检测了Fe2+对卵白蛋白胞吞效率的影响,采用Western blot技术测定了添加不同剂量Fe2+对OVA在Caco-2细胞和IEC-6细胞内吞效率的影响。
如图3所示,考察Fe2+对OVA细胞内吞效率的影响,添加一定浓度的OVA和不同浓度的Fe2+于离心管中,然后在37℃孵育15min,最后用DMEM培养基稀释Fe2+-OVA,稀释至每组培养基中Fe2+终浓度分别为0µM,22µM,44µM,88µM,每组培养基中OVA的终浓度为22µM。将配制好的各组Fe2+-OVA培养基分别加入12孔板各孔中,37℃处理4h后,弃上清,用冰PBS洗3次,孔板置于冰上,提取蛋白用于Western blot分析。
如图3,随着Fe2+终浓度的增加,OVA的细胞内吞效率显著增加(P<0.05),表明Fe2+增加了OVA的细胞内吞效率。
为了进一步明确OVA的细胞内吞过程,本实施例采用TEM技术对其进行拍摄,如图4所示,Fe2+-OVA的细胞内吞过程为:Fe2+-OVA首先结合到细胞膜上后,细胞膜向内凹陷形成内吞小泡,然后小泡脱落进入细胞,Fe2+-OVA位于细胞内体中。
实施例2 Fe2+对OVA在不同种类细胞中的胞吞效率的影响
本发明前述实施例表明了Fe2+促进OVA在Caco-2细胞中的胞吞效率,为了研究Fe2+促进OVA胞吞是否在不同种类的细胞中仍具有促进效果,本发明采用了免疫荧光的方法测定OVA和Fe2+结合物对大鼠肠上皮细胞(IEC-6)胞吞效率的影响。如图5所示,图5的OVA、1:0.5Fe2+、1:1Fe2+、1:2Fe2+分别代表单独添加OVA、OVA:Fe2+的摩尔比为1:0.5、OVA:Fe2+的摩尔比为1:1、OVA:Fe2+的摩尔比为1:2,采用DAPI染料对细胞核进行荧光标记,并对细胞中的OVA进行免疫荧光染色。细胞核形态行浅色圆圈表示细胞核,OVA在细胞分布行的亮色表示OVA在细胞内的含量。从图5中可以看出,OVA的荧光信号(即OVA在细胞分布行的亮色部分)随着Fe2+剂量的增加而逐渐越强,说明Fe2+促进OVA在IEC-6细胞中的胞吞效率。
本发明还考察了OVA和Fe2+结合物对小鼠单核巨噬细胞(RAW264.7)细胞胞吞效率的影响,采用DAPI染料对细胞核进行荧光标记,并对细胞中的OVA进行免疫荧光染色。圆形中等亮色部分表示细胞核,亮度最高的部分表示OVA在细胞内的含量(如右1右2图中最亮的呈白色的点状部分表示OVA)。如图6所示,图6的横轴表示在37℃条件下,浓度为22μM的OVA的组、含有浓度11μM Fe2+的OVA(OVA浓度为22μM)的组、含有浓度22μM Fe2+的OVA(OVA浓度为22μM)的组、含有44μM Fe2+的OVA(浓度为22μM)的组、含有88μM Fe2+的OVA(浓度为22μM)的组,其中,如含有44μM Fe2+的OVA、含有88μM Fe2+的OVA两组所示,表征OVA的荧光信号(亮度最高的点状部分)随着Fe2+剂量的增加而逐渐越强,说明高浓度的Fe2+促进OVA在RAW264.7细胞中的胞吞效率。这些结果再次证明了Fe2+促进OVA发生细胞内吞。且在不同种类的细胞中均存在促进作用。
实施例3 Fe2+和OVA的结合形态
实验步骤:
为了验证Fe2+和OVA是否是以结合态促进胞吞,本发明对比了Fe2+加入顺序对OVA胞吞效率的影响。组别为“培养基+Fe2++OVA”的培养基的配制方法为:在富含氨基酸培养基中加入不同浓度Fe2+后,再加入OVA,体系中Fe2+的终浓度分别为5.5µM、11µM、22µM、44 µM,OVA的终浓度为22µM。
组别为“培养基+Fe2+-OVA”的培养基的配制方法为:首先配制结合Fe2+的OVA溶液(Fe2+-OVA):添加不同浓度的Fe2+于OVA中离心管中,然后在37℃孵育15 min。最后分别用富含氨基酸的培养基将复合物稀释至Fe2+的终浓度分别为5.5µM、11µM、22µM、44 µM,OVA的终浓度为22 µM。并采用western blot测定其胞吞效率。
实验结果:
若Fe2+先加入至富含氨基酸的培养基中,则Fe2+会先和培养基中氨基酸结合,从而降低了Fe2+和OVA的结合效率。而先配制结合Fe2+的OVA溶液,则可以保证Fe2+和OVA先发生结合从而提高其结合效率。因此本发明对比了在富含氨基酸培养基中加入不同浓度Fe2+后,再加入OVA(浓度为22 μM),以及先配制结合Fe2+的OVA溶液后,再加入至培养基中这两种方式对OVA的细胞胞吞效率的影响。通过图9可以发现,“培养基+Fe2+-OVA”组别中的条带比组别“培养基+Fe2++OVA”更深,表明“培养基+Fe2+-OVA”处理的细胞比“培养基+Fe2++OVA”处理的细胞内吞OVA的含量更多,证明了Fe2+和OVA发生结合后,才可以更加高效的促进OVA的细胞内吞效率。
实施例4 进一步验证Fe2+和OVA的结合形态
实验步骤:
称取一定量的OVA溶解到10 mM Na2HPO4/KH2PO4缓冲液中,OVA终浓度为22µM,取一定量含有Fe2+的溶液于OVA中至Fe2+终浓度分别为11µM、22µM、44µM、和88 µM,37℃反应15min,现用现配。图10的1:0.5、1:1、1:2、1:4分别表示体系中OVA和Fe2+的摩尔比,OVA表示,不含有Fe2+。
实验结果:
本发明采用荧光分光光谱法测定了添加不同浓度Fe2+的OVA荧光淬灭图谱。如图10所示,随Fe2+的浓度升高OVA的荧光猝灭程度逐渐增加,可能是由于Fe2+和HSA形成了非荧光配合物导致OVA发生荧光猝灭效应,表明Fe2+可以与OVA发生相互作用。
对比例1 不同种类的二价阳离子对OVA细胞内吞的影响
Mg2+对细胞内吞OVA的效率
细胞培养:含有结合不同浓度Mg2+的OVA溶液(Mg2+-OVA)的培养基配制:添加一定浓度的OVA和分别加入不同浓度的Mg2+于离心管中,OVA蛋白与Mg2+离子的摩尔比分别为1:1、1:2、1:4、1:8、1:10、1:20,然后在37℃孵育15 min,最后用DMEM培养基将复合物稀释至Mg2+的终浓度分别为0 µM,22 µM,44 µM,88 µM,176 µM、220 µM、440 µM;OVA的终浓度为22µM。将配制好的培养基分别加入12孔板各孔中,37℃处理4 h后,弃上清,用冰PBS洗3次,孔板置于冰上,提取蛋白用于Western blot分析。
结果如图7所示,图7的横坐标从左到右分别表示:Control组(仅添加DMEM培养基)、OVA组(OVA的终浓度为22 µM)、OVA与Mg2+摩尔比分别为1:1(即OVA的终浓度为22 µM+Mg2+的终浓度为22 µM)、OVA与Mg2+摩尔比分别为1:2(即OVA的终浓度为22 µM+Mg2+的终浓度为44 µM)、OVA与Mg2+摩尔比分别为1:4(即OVA的终浓度为22 µM+Mg2+的终浓度为88 µM)、OVA与Mg2+摩尔比分别为1:8(即OVA的终浓度为22 µM+Mg2+的终浓度为176 µM)、OVA与Mg2+摩尔比分别为1:10(即OVA的终浓度为22 µM+Mg2+的终浓度为226 µM)、OVA与Mg2+摩尔比分别为1:20(即OVA的终浓度为22 µM+Mg2+的终浓度为440 µM);其中,采用DAPI染料对细胞核进行荧光标记,并对细胞中的OVA进行免疫荧光染色。DAPI-细胞核行圆形亮色部分表示细胞核,OVA行的亮色部分表示OVA在细胞内的分布,叠加列是指将两种荧光信号叠加到一起的结果。随着Mg2+添加比例的增加,表征OVA的荧光信号无明显变化,表明了Mg2+对细胞内吞OVA的效率无明显影响。
Ca2+对细胞内吞OVA的效率
实验方法参照实施例1,测定添加不同剂量Ca2+对Caco-2细胞内吞OVA的影响,如图8所示,图8的横坐标从左到右分别表示:Control组(仅添加DMEM培养基)、OVA组(OVA的终浓度为22 µM)和OVA与Ca2+摩尔比分别为1:1(即OVA的终浓度为22 µM+Ca2+的终浓度为22 µM)、1:2(即OVA的终浓度为22 µM+Ca2+的终浓度为44 µM)、1:4(即OVA的终浓度为22 µM+Ca2+的终浓度为88 µM)、1:8(即OVA的终浓度为22 µM+Ca2+的终浓度为176 µM)、1:10(即OVA的终浓度为22 µM+Ca2+的终浓度为226 µM)、1:20(即OVA的终浓度为22 µM+Ca2+的终浓度为440µM)组,其中,采用DAPI染料对细胞核进行荧光标记,并对细胞中的OVA进行免疫荧光染色。DAPI-细胞核行圆形亮色部分表示细胞核,OVA行的亮色部分表示OVA在细胞内的分布,叠加列是指将两种荧光信号叠加到一起的结果。随着Ca2+剂量的增加,表征OVA的荧光信号强度无明显变化,Ca2+对细胞内吞OVA的效率无影响。
综上所述,本发明发现了结合Fe2+的卵白蛋白相比于单独卵白蛋白或者牛血清白蛋白可以更多的被细胞利用,从而可以在不含氨基酸的培养基中仍可以维持细胞的生长,在富含氨基酸的培养基中仍可以促进细胞增殖。进一步为了探究其促进细胞增殖的机制,本发明又测定了Fe2+对OVA胞吞效率的影响,发现Fe2+可以促进OVA的细胞内吞效率,这可能是结合Fe2+的OVA促进细胞增殖的主要原因,这将为细胞培养基的研发提供更多的选择。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,但本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (5)
1.一种细胞培养基,其特征在于,所述细胞培养基包含Fe2+-卵白蛋白复合物;
所述细胞培养基中Fe2+-卵白蛋白复合物的浓度为1μg/ml~1000μg/ml;
所述细胞培养基中不含氨基酸;
所述细胞培养基中不含动物血清白蛋白。
2.一种细胞培养方法,其特征在于,在权利要求1所述的细胞培养基中培养细胞。
3.一种提升细胞活力的方法,其特征在于,添加Fe2+-卵白蛋白复合物; Fe2+的终浓度为10~100µM,添加所述卵白蛋白的终浓度为10~100µM;所述细胞为哺乳动物细胞,所述方法为非诊断或治疗目的的。
4.如权利要求3所述的提升细胞活力的方法,其特征在于,所述方法在不含氨基酸或氨基酸缺乏的环境下进行。
5.如权利要求3所述的提升细胞活力的方法,其特征在于,所述方法中采用Fe2+-卵白蛋白复合物替代动物血清白蛋白。
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