CN115109739B - 一种快速高密度培养沙漠蔷薇悬浮细胞高产绿原酸的方法 - Google Patents
一种快速高密度培养沙漠蔷薇悬浮细胞高产绿原酸的方法 Download PDFInfo
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Abstract
本发明属于植物组织培养类的化妆品原料领域,公开了一种快速高密度培养沙漠蔷薇悬浮细胞高产绿原酸的方法。该方法包括无菌叶片的获得、愈伤组织诱导、愈伤组织的继代增殖、悬浮细胞的培养和筛选,规模性放大培养,高产绿原酸诱导。该方法以沙漠蔷薇植株新鲜幼嫩叶片为外植体,可快速诱导细胞脱分化形成愈伤组织,愈伤组织经过固体继代及液体震荡培养,筛选获得可进行高密度悬浮培养细胞系,其可以在一个培养周期内,约5~7天沙漠蔷薇悬浮细胞鲜重就达到600‑800g/L;本发明悬浮细胞生长速度快、可耐受高转速、可实现高密度培养,使得该技术具有耗时短、成本低,获得的悬浮细胞密度高等特点。
Description
技术领域
本发明属于植物组织培养类的化妆品原料领域,特别涉及一种快速高密度培养沙漠蔷薇悬浮细胞高产绿原酸的方法。
背景技术
沙漠蔷薇(Adenium Obesum.),又名沙漠玫瑰,天宝花,是夹竹桃科沙漠玫瑰属,为多年生落叶肉质植物,原产东非至阿拉伯半岛南部,该地区地处赤道附近,常年干旱少雨,光照充足。特殊的地理环境造成沙漠蔷薇喜高温干燥和阳光充足的环境,耐酷暑,耐干旱,能在恶劣的环境中生长,能够从干燥的沙漠环境中获取水分,有很强的保水能力。
沙漠蔷薇提取物有很好的药用价值和美容功效,沙漠蔷薇中可提取出30种强心苷和2种孕烷,可用于治疗心脏病和妇科病。沙漠蔷薇叶提取物具有保湿润肤,抗衰老等美容功效。沙漠蔷薇花提取物含高浓度海藻糖,有减少晒黑和皮肤老化、有效提高肌肤的储水功能。
沙漠蔷薇自花结实率低,不容易长果荚,种子少,收集难;且人工授粉较难,操作成功率低,一般是用半成熟枝扦插来栽培,但繁殖速度慢,易出现根腐病。因此,依靠种植实生苗或扦插苗提取功效成分,存在资源不足的问题。
专利CN 104622759 A中指出原料沙漠蔷薇叶细胞提取物的常规制备方法是利用新鲜、无杂质的沙漠蔷薇叶经水蒸气蒸馏法获得,但得率非常低,仅有万分之三。
专利CN 111280063 A利用沙漠蔷薇成熟种子萌发为无菌苗,以无菌苗的茎、叶片和根为外植体获得愈伤组织,得到沙漠蔷薇悬浮细胞系,进行规模化培养,解决了沙漠蔷薇细胞产量不足的问题。但是,该专利是利用种子作为外植体,而沙漠蔷薇存在种子少,收集难问题,同时,需要种子进行萌发获得无菌苗,再进行愈伤组织诱导,操作繁琐,周期长。并且放大生产需要利用到CN103224882A或CN204385208U的中国专利文献所披露的昂贵生物反应器,致使其进一步应用受到制约。
发明内容
为了克服现有技术中存在的缺点和不足,本发明的目的在于提供一种快速高密度培养沙漠蔷薇悬浮细胞高产绿原酸的方法;该方法以沙漠蔷薇植株新鲜幼嫩叶片为外植体,可快速诱导细胞脱分化形成愈伤组织,愈伤组织经过固体继代及液体震荡培养,筛选获得可进行高密度悬浮培养细胞系,其可以在一个培养周期内,约5~7天沙漠蔷薇悬浮细胞鲜重就达到600-800g/L;本发明悬浮细胞生长速度快、可耐受高转速、可实现高密度培养,使得该技术具有耗时短、成本低,获得的悬浮细胞密度高等特点。
本发明目的通过以下技术方案实现:
一种快速高密度培养沙漠蔷薇悬浮细胞高产绿原酸的方法,包括无菌叶片的获得、愈伤组织诱导、愈伤组织的继代增殖、悬浮细胞的培养和筛选,规模性放大培养,高产绿原酸诱导。
一种快速高密度培养沙漠蔷薇悬浮细胞高产绿原酸的方法,具体包括以下操作步骤:
(1)取沙漠蔷薇的幼嫩叶片,按照常规的消毒方法对其进行消毒处理;
(2)将步骤(1)灭菌处理过的叶片切除叶边缘,横切过叶中脉,切成2~3cm见方小块接入诱导培养基中进行诱导培养,温度24℃,湿度70~80%,避光暗培养;
(3)将步骤(2)诱导出来的愈伤组织,选取色泽鲜黄的疏松愈伤组织进行继代培养,继代周期为10-15天/次,培养条件光照强度1000~1500LX,16h光照/8h黑暗,温度24℃,湿度70%~80%;所述继代培养的培养基配方为:MS+2,4-D(2,4-二氯苯氧乙酸)1~4mg/L+6-BA(6-苄氨基嘌呤)0.5~2mg/L+TDZ(噻苯隆)0.005~0.05mg/L+蔗糖10~30g/L+琼脂6g/L,pH5.8;
(4)取步骤(3)继代培养2~3次的愈伤组织,选择分散性好、增殖速度快的鲜黄色愈伤组织,用镊子夹碎后转入液体培养基震荡培养,接种量10-25%,每250mL三角瓶接种80ml悬浮细胞液,培养条件为避光或暗光,温度28℃,震荡培养5~7天后进行悬浮细胞继代培养,继代次数3~5次,得到高分散悬浮细胞;
所述震荡培养和悬浮细胞继代培养使用的培养基均为液体培养基,具体由以下组分组成:MS+2,4-D 1~4mg/L+6-BA 0.1~1mg/L+TDZ 0.01~0.1mg/L+蔗糖30g/L+胰蛋白胨100~500mg/L+苯丙氨酸50~500mg/L,pH5.8;
所述震荡培养的第1天摇床转速为80~120rpm,自培养第3天开始摇床速度在150~220rpm的范围内逐步提高搅拌速度;
(5)将步骤(4)筛选获得的高分散悬浮细胞,按10%接种量接种至带有曝气装置10L波浪式生物反应器进行扩大培养,设定参数:温度25~28℃,转速80~120rpm,pH5.5~6.0,DO值为3%~10%,培养期间,每天取细胞培养液样品,考察细胞密度和细胞活率变化情况,以及上清液中绿原酸含量变化。
步骤(1)中所述消毒方法具体按照以下步骤:取沙漠蔷薇的幼嫩叶片,清洗表面污渍,流水冲洗30~50min,超净工作台内无菌室清洗2~3遍,用体积百分浓度70%的酒精浸泡30s,再用无菌水清洗3~4遍,然后用含5%有效率的次氯酸钠溶液处理8~15min,无菌水清洗3~4遍,无菌滤纸吸干叶片表面水分。采用此方法消毒后的组织,染菌几率小于1‰。
步骤(2)中所述诱导培养基的配方为MS+NAA(α-萘乙酸)0.1~1mg/L+6-BA 1~2mg/L,附加蔗糖30g/L,琼脂6.5g/L,pH5.8;所述诱导培养的时间为7-10天,叶片边缘会诱导产生愈伤组织,诱导率>90%。附加蔗糖和琼脂可以显著促进愈伤组织产生。
步骤(3)中所述继代培养的培养基中的蔗糖浓度优选为30g/L,其利于获得颗粒细小,疏松易碎的愈伤组织用于悬浮培养。
步骤(4)中所述悬浮细胞继代培养时,高分散悬浮细胞的筛选方法为:首先将震荡培养所得悬浮细胞培养液摇匀后静置1min,使用大口吸管吸取上层细胞,去除较大颗粒细胞团,收集细胞液通过300~500g离心5min,去除细胞碎片,获得的细胞液与新鲜液体培养液按照1:1~3接种量进行继代,经过3~5次继代培养后,悬浮培养液中主要由单细胞和小细胞团组成,即得到高分散悬浮细胞。所得高分散悬浮细胞分散均匀,生长活力旺盛。
将步骤(4)所得高分散悬浮细胞进行高密度培养,细胞培养密度>5*104个/ml,生长曲线呈S型,培养5~7天后,悬浮细胞湿重量达到500-800g/L。
步骤(5)所述扩大培养方式为半连续培养,接种后培养至5~7天时,停止通气和摇动,静置1~2min从反应器底部放出培养液,保留上清液,按照10%接种量补加新鲜液体培养基。
本发明相对于现有技术具有如下的优点及有益效果:
(1)本发明方法筛选得到可以耐受较高转速悬浮细胞,一般植物细胞培养的转速为80~120rpm(转速过高会对细胞产生冲击力,使细胞破裂),而本发明做出来的悬浮细胞可以在180rpm条件下进行增殖生长,从而实现高密度。
(2)本发明提供了一种快速高密度培养沙漠蔷薇悬浮细胞高产绿原酸的方法,操作简单,细胞增殖率高,生长周期短,易于进行放大生产,可以大量提供沙漠蔷薇悬浮细胞原料,解决了沙漠蔷薇繁殖速度慢问题;
(3)本发明筛选获得的沙漠蔷薇悬浮细胞系抗机械搅拌力强,增殖率高,可进行高密度悬浮培养,最高培养密度可达800g/L,细胞培养液接近呈膏体状。克服了传统植物悬浮细胞培养存在培养密度低,周期长、细胞抗剪切能力弱等问题。
(4)本发明悬浮培养基含有高浓度的2,4-D、高活性细胞分裂素TDZ,同时复配天然物质,为细胞增殖提供充足的营养环境,使得增殖率高细胞可以快速达到较高培养密度;
(5)本发明悬浮细胞生长曲线呈“S”型,摇瓶筛选阶段,第0天至第2天细胞处于适应期,在120rpm和180rpm条件下,细胞的增殖速率基本一致,因此摇床转速选择在剪切力较小的80~120rpm范围内,自培养第3天开始悬浮细胞进入快速增殖期,提高转速可加快悬浮细胞的增殖,因此,摇床在150~220rpm的范围内逐步提高转速,通过此方法可使得增殖率高、抗机械搅拌力强悬浮细胞迅速分散至培养液,保持细胞增殖力,从而实现悬浮细胞高密度培养。
(6)本发明悬浮细胞筛选阶段,通过短时间沉降去除增殖力弱的大细胞团,以及低速离心去除细胞碎片,保留分散性好,增殖率高的悬浮细胞系,通过3~5次继代培养筛选后,获得的悬浮细胞系分散性好,培养液密度高。
(7)本发明利用苯丙氨酸作为前体物质促进绿原酸合成,获得沙漠蔷薇悬浮细胞提取液中绿原酸含量为31.35ug/ml,特征吸收峰位置5.879min,
(8)本发明获得的沙漠蔷薇悬浮细胞的水提液(1:10)具有抗氧化、促修护作用,其对DPPH自由基的抗氧化能力为50.48%,对鸡胚无刺激性,而对细胞的黏附、增殖有促进作用,同时可抑制炎症因子TNF-α释放。
附图说明
图1为诱导的沙漠蔷薇愈伤组织、悬浮细胞和悬浮细胞微观图;
图2为考察不同转速和TDZ对悬浮细胞增殖影响;
图3为醇提液的绿原酸高效液相图;
图4为水提液对DPPH自由基的清除能力;
图5为水提液对鸡胚刺激性试验;
图6为水提液对细胞黏附的促进作用;
图7为水提液对细胞增殖促进作用;
图8为水提液对炎症因子TNF-α释放抑制作用。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例1沙漠蔷薇愈伤组织的诱导
步骤一:取沙漠蔷薇的幼嫩叶片,清洗表面污渍,流水冲洗30min,超净工作台内无菌室清洗3遍,体积百分浓度70%的酒精浸泡30s,无菌水清洗4遍,利用含5%有效率的次氯酸钠溶液处理8min,无菌水清洗3遍,滤纸吸干叶片表面水分。
步骤二:将经过步骤一灭菌处理过的叶片切除叶边缘,横切过叶中脉,切成约2-3cm见方小块接入培养基进行诱导培养,配方为MS+NAA0.3mg/L+6-BA1mg/L,附加蔗糖30g/L,琼脂6.5g/L,pH5.8,温度24℃,湿度70%~80%,暗光培养;在诱导培养基上生长7-10天后,叶片边缘会诱导产生淡黄色愈伤组织,诱导率100%(如图1的a所示)。
步骤三:将诱导出来的愈伤组织,选取色泽鲜黄,较疏松愈伤组织进行继代培养,继代周期为10-15天/次,培养基配方为MS+2,4-D 2mg/L+6-BA0.5mg/L+TDZ 0.01mg/L+蔗糖30g/L+琼脂6g/L,pH5.8,培养条件,光照强度1500LX,16h光照/8h黑暗,温度24℃,湿度70%,继代2~3次后得选择分散性好、增殖速度快的鲜黄色愈伤组织(如图1的b所示)。
实施例2考察震荡培养转速和激素TDZ对沙漠蔷薇悬浮细胞增殖影响
将实施例1所得分散性好、增殖速度快的鲜黄色愈伤组织,用镊子夹碎后转入液体培养基震荡培养,接种量15%,以MS+2,4-D 2mg/L+6-BA 0.5mg/L+100mg/L苯丙氨酸作为悬浮培养的基础培养基,培养条件为避光,温度28℃,摇床转速120rpm,每5~7天进行一次悬浮细胞更新继代,继代方法:将三角瓶内悬浮液静置后倒去,使用大口吸管吸取上层细胞,去除较大颗粒细胞团,收集细胞液通过300~500g离心5min,去除细胞碎片,获得的细胞液与新鲜液体培养液按照1:1~3接种量进行继代,继代培养条件不变。经过3~5次继代培养后,悬浮培养液主要由单细胞和小细胞团(不多于20个细胞)组成,如图1中的d所示沙漠蔷薇悬浮细胞微观结构图。
利用筛选获得的悬浮细胞系,分别考察细胞分裂素TDZ和摇床震荡培养转速对悬浮细胞增殖影响,设计四个不同实验组方案如下:
①震荡培养转速120rpm;
②震荡培养转速120rpm,悬浮培养基补加TDZ至0.01mg/L;
③震荡培养转速180rpm;
④摇床转速180rpm,悬浮培养基补加TDZ至0.01mg/L;
每天取样称其鲜重量,每个实验组设置3个重复,取鲜重的平均值作为统计结果。悬浮细胞增殖测试结果如图2,由图可以看出悬浮细胞生长曲线呈“S”型,第0天至第2天细胞处于适应期,震荡培养转速在120rpm和180rpm条件下,细胞的增殖速率基本一致,因此,沙漠蔷薇悬浮细胞接种后第0天至第2天摇床转速可选择在常规的剪切力较小的120rpm范围内,自培养第3天开始悬浮细胞进入快速增殖期,可耐受180rpm高转速,并且通过提高转速可以加快悬浮细胞的增殖,因此,增殖期震荡培养转速可以选择在180rpm左右。同时,通过考察悬浮培养基中添加TDZ对细胞增殖影响,结果显示通过添加TDZ可促进悬浮细胞增殖,其与转速具有协同性,影响因素的主次顺序为:T(转速)>D(分裂素TDZ)。
实施例3沙漠蔷薇悬浮细胞摇瓶震荡培养
根据实施例2中对沙漠蔷薇悬浮细胞震荡培养条件探索,将悬浮培养条件设定为:第0~2天悬浮细胞处于适应期,选择常规悬浮细胞培养条件:摇床转速80~120rpm,自培养第3天开始悬浮细胞进入快速增殖期,摇床转速选择在150~220rpm的范围内,悬浮培养基组成:MS+2,4-D 1~42mg/L+6-BA 0.5mg/L+TDZ 0.01mg/L+蔗糖30g/L+胰蛋白胨500mg/L+苯丙氨酸500mg/L,pH5.8,按照1:1~3接种量进行继代,培养条件为避光,培养温度28℃,每250mL三角瓶接种80ml悬浮细胞液,每5~7天进行一次悬浮细胞继代,每天取样进行细胞湿重量测试,结果显示:在一个培养周期7天内,悬浮培养的沙漠蔷薇细胞的生长曲线呈“S”型,培养至第7天,鲜重可达最大值680g/L(如图2),悬浮细胞培养液呈膏体状(如图1中的c所示)。
实施例4沙漠蔷薇悬浮细胞提取物的抗氧化活性研究
1、沙漠蔷薇悬浮细胞醇提液的制备:收集实施例2中得到的沙漠蔷薇悬浮细胞,纯水清洗2遍后减压过滤,采用80%乙醇按照料液比1:3(M(g):V(ml))混合后进行粉碎,40℃超声浸提30min后,4000rpm进行离心分离,收集上清用0.45um有机滤膜过滤备用;
2、将上述得到的醇提液用于以下实验:
HPLC检测绿原酸:分析柱:C18(150mm×4.6mm×5μm);流动相:A相:乙腈,B相:0.5%磷酸-水;梯度洗脱如下:0~9.00min,15%A;9.00~20min,30%A;20.00min~25.00min,15%A;流速:1mL/min;进样量:10μL,柱温:30℃,紫外检测波长:327nm
精密称取绿原酸对照品,利用80%乙醇溶液配制成100ug/ml溶液作为母液,分别稀释2、4、8、16倍。
结果:
(1)绿原酸标准曲线图如图3的(a)所示,回归方程为y=29241x-14367,r2值为1,浓度在6.25~100ug/ml的浓度范围内与峰面积呈良好的线性关系。
如图3的(b)所示,为绿原酸标准品,其在5.871min附近处存在特征吸收峰,图3的(c)为沙漠蔷薇悬浮细胞醇提液的液相图,其在5.879min处存在强吸收峰,峰面积为1207754,与绿原酸标准对照品出峰时间相一致,说明沙漠蔷薇悬浮细胞提取液中有绿原酸存在,浓度为31.35ug/ml。其中,图3(d)为其他步骤同实施例2,只是愈伤组织悬浮培养的培养基由以下组分组成:MS+2,4-D 2mg/L+6-BA 0.5mg/L+TDZ 0.01mg/L+蔗糖30g/L+胰蛋白胨500mg/L(不含苯丙氨酸),制备得到的悬浮液细胞,然后按照上述醇提的方法得到的醇提液,其绿原酸出峰时间为5.863min,绿原酸浓度为21.12ug/ml,因此,通过在沙漠蔷薇悬浮细胞培养液中添加前体物苯丙氨酸,绿原酸的获得量可增加48.44%。
(2)讨论:通过本实施例说明苯丙氨酸作为绿原酸重要的中间代谢物质,能够起到调节沙漠蔷薇悬浮细胞绿原酸积累;同时,绿原酸具有抗氧化作用,其是一种酚型抗氧化剂,能够清除体内的自由基,可以保护组织不受氧化反应伤害,延缓衰老,使得获得的沙漠蔷薇悬浮培养物具有一定美容功效。
实施例5沙漠蔷薇水提液功效测试
1、悬浮细胞水提液制备:收集实施例2中得到的沙漠蔷薇悬浮细胞,纯水清洗2遍后减压抽干,采用纯水按照料液比1:3(M(g):V(ml))混合后进行粉碎,40℃超声浸提30min后,5000rpm进行离心分离,收集上清用0.45um有机滤膜过滤备用;
2、将上述得到的水提液用于以下试验:
(1)悬浮细胞水提液对DPPH自由基清除能力:称取1,1-二苯基-2-三硝基苯肼(DPPH)12mg,溶解于100ml的95%乙醇溶液,配制浓度为0.12mg/L的DPPH溶液,冻存于-20℃冰箱。按照表1的实验体系进行添加,充分混合后,避光条件下室温反应30min,取反应液移入比色皿中,在517nm处测定溶液的吸光度值。
表1DPPH自由基清除实验反应体系
上述测得的吸光度按照以下公式进行计算:
清除率(%)=(1-(T-T0)/(C-C0))×100%
T:试样溶液与DPPH溶液混合后OD517
T0:试样溶液与95%乙醇溶液混合后OD517
C:95%乙醇与DPPH溶液混合后OD517
CO:95%溶剂OD517
分别选用200ug/ml Vc溶液、沙漠蔷薇悬浮细胞水提液,测量其反应后的吸光度值OD517,分别计算出它们的自由基清除率,并比较其清除能力。
(2)鸡胚试验:取出9日龄鸡胚,将鸡胚随机分组,每组设置3个平行试验,以0.9%的生理盐水为阴性对照(3个平行),质量分数0.288%的SDS溶液为阳性对照(3个平行),样品溶液为测试组。剥去已标记气室位置的蛋壳部分,加入一定量0.9%的生理盐水浸湿鸡胚外膜,用镊子缓慢的揭掉鸡胚外膜,拍照记录原始CAM血管情况,放置受试环(在小血管密集处),分别加入100μL生理盐水、SDS溶液、样品溶液于受试环内,观察环内血管情况,并进行拍照。
(3)促粘附试验:1)取阳性对照品(动物胶原)、阴性对照品(PBS溶液)、样品溶液100μL加入96孔板中,密封于4℃过夜。实验时,弃掉液体,PBS洗一遍,备用。2)胰酶消化处于对数期的HaCat细胞,10%FBS的培养基重悬,添加100μL稀释好的细胞悬液于上述96孔板中,使细胞密度为1.5万/孔。3)接种3小时后,显微镜下观察细胞贴壁状况。4)弃去细胞培养液,每孔中加入100μL的10%CCK8溶液,混匀后继续培养1小时,然后从培养箱中取出,放在酶标仪上,检测波长为450nm处的吸光度,记录测定结果。
A0:PBS溶液处理组OD450
An:样品溶液处理组OD450
(4)促细胞迁移试验:1)胰酶消化处于对数期的NIH3T3细胞,10%FBS的培养基重悬,以10万/mL,1mL/孔接种细胞至12孔板中;2)第二天观察长至90%后,用无血清培养基配制样品所需的稀释浓度,每个浓度需2ml;3)吸弃12孔板中的上清,用200μL枪头沿着直尺垂直划线,每孔划两条互相垂直,交点过培养板中心。用PBS洗涤细胞1-2次,去除脱落下的细胞。4)加入配置好的标准品(1ug/ml bFGF)、空白对照和样品溶液,900ul/孔,做好标记;5)放入37℃,5%CO2培养箱培养;按0,6h,24h,48h选取位置拍照:
(5)抑制炎症因子TNF-α释放:1)Raw264.7细胞株用完全培养液于37℃、5%CO2条件下培养,取对数生长期的细胞,将细胞轻轻吹打至脱落。2)将脱落的细胞收集至离心管中后500rpm、离心5min。3)离心结束后,弃掉上清液,向离心管中加入新鲜含1%FBS(胎牛血清)的DMEM培养液,吸管吹打重悬细胞,血球计数板在显微镜下进行计数。3)用细胞培养基稀释细胞至接种密度3×105/mL,接种至96孔板中,每孔加入100μL细胞悬液。4)接种结束后,放置于CO2培养箱中培养24±2h。5)实验中设置空白对照组、模型组(LPS)、阳性对照组(LPS+甘草酸二钾)及样品组,每个浓度梯度下设置3个复孔。6)给药:待96孔板中细胞融合率达到60%左右时进行给药。空白对照组和模型组每孔加入100μL的无血清培养基;样品组每孔加入100μL含有相应浓度的样品工作液;阳性对照组加入100μL含有终浓度为100μg/mL的甘草酸二钾。给药完成后,将96孔板放置于CO2培养箱中培养4h。7)LPS刺激:孵育4h后,根据实验设计,分别向已给药的孔板中加入120μL终浓度为1μg/mL的LPS的无血清培养基,空白对照组中加入无血清培养基120μL,混匀后将96孔板放置于CO2培养箱中培养20h;7)收样:孵育培养结束后,收集110μL细胞上清液于1.5mL无菌离心管中,4℃条件下1000rpm离心15分钟取上清,立即用于后续ELISA实验。8)根据TNF-ɑELISA检测试剂盒的操作说明书进行检测;
结果:
(1)如图4所示:悬浮细胞水提液对DPPH自由基的清除率为50.48%,阳性对照组200ug/ml的Vc溶液对羟基自由基清除率为54.34%,二者清除作用效果相当。
(2)如图5所示:鸡胚刺激试验结果,阳性试验组(受试环内添加0.228%SDS)出现了明显溶血现象,阴性对照组(0.9%生理盐水)受试环内血管保持完好,沙漠蔷薇水提液样品受试环内未出现溶血,测试时间内,血管形态保持完好,与阴性对照组相当,说明沙漠蔷薇水提液对鸡胚无刺激性;
(3)如图6所示:沙漠蔷薇悬浮细胞提取液对HaCaT细胞迁移具有促进作用,与1ug/ml bFGF溶液促迁移作用效果相当,明细好于空白对照组。说明,沙漠蔷薇悬浮细胞提取液具有促细胞迁移作用,可用于促修护产品;
(4)如图7所示:沙漠蔷薇悬浮细胞提取液对HaCaT细胞的粘附具有促进作用,同时在考察的浓度范围内,其作用效果好于阳性对照组(动物胶原);如图8所示:沙漠蔷薇悬浮细胞提取液可以抑制炎症TNF-α释放,可以用于肌肤修护类产品。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (7)
1.一种快速高密度培养沙漠蔷薇悬浮细胞高产绿原酸的方法,其特征在于具体包括以下操作步骤:
(1)取沙漠蔷薇的幼嫩叶片,按照常规的消毒方法对其进行消毒处理;
(2)将步骤(1)灭菌处理过的叶片切除叶边缘,横切过叶中脉,切成2~3cm见方小块接入诱导培养基中进行诱导培养,温度24℃,湿度70~80%,避光暗培养;
(3)将步骤(2)诱导出来的愈伤组织,选取色泽鲜黄的疏松愈伤组织进行继代培养,继代周期为10-15天/次,培养条件光照强度1000~1500LX,16h光照/8h黑暗,温度24℃,湿度70%~80%;所述继代培养的培养基配方为:MS+2,4-D 1~4mg/L+6-BA 0.5~2mg/L+TDZ0.005~0.05mg/L+蔗糖10~30g/L+琼脂6g/L,pH5.8;
(4)取步骤(3)继代培养2~3次的愈伤组织,选择分散性好、增殖速度快的鲜黄色愈伤组织,用镊子夹碎后转入液体培养基震荡培养,接种量10-25%,每250mL三角瓶接种80ml悬浮细胞液,培养条件为避光或暗光,温度28℃,震荡培养5~7天后进行悬浮细胞继代培养,继代次数3~5次,得到高分散悬浮细胞;
所述震荡培养和悬浮细胞继代培养使用的培养基均为液体培养基,具体由以下组分组成:MS+2,4-D 1~4mg/L+6-BA 0.1~1mg/L+TDZ 0.01~0.1mg/L+蔗糖30g/L+胰蛋白胨100~500mg/L+苯丙氨酸50~500mg/L,pH5.8;
所述震荡培养的第1天摇床转速为80~120rpm,自培养第3天开始摇床速度在150~220rpm的范围内逐步提高搅拌速度;
(5)将步骤(4)筛选获得的高分散悬浮细胞,按10%接种量接种至带有曝气装置10L波浪式生物反应器进行扩大培养,设定参数:温度25~28℃,转速80~120rpm,pH5.5~6.0,DO值为3%~10%,培养期间,每天取细胞培养液样品,考察细胞密度和细胞活率变化情况,以及上清液中绿原酸含量变化。
2.根据权利要求1所述的一种快速高密度培养沙漠蔷薇悬浮细胞高产绿原酸的方法,其特征在于:步骤(1)中所述消毒方法具体按照以下步骤:取沙漠蔷薇的幼嫩叶片,清洗表面污渍,流水冲洗30~50min,超净工作台内无菌室清洗2~3遍,用体积百分浓度70%的酒精浸泡30s,再用无菌水清洗3~4遍,然后用含5%有效率的次氯酸钠溶液处理8~15min,无菌水清洗3~4遍,无菌滤纸吸干叶片表面水分。
3.根据权利要求1所述的一种快速高密度培养沙漠蔷薇悬浮细胞高产绿原酸的方法,其特征在于:步骤(2)中所述诱导培养基的配方为MS+NAA0.1~1mg/L+6-BA 1~2mg/L,附加蔗糖30g/L,琼脂6.5g/L,pH5.8;所述诱导培养的时间为7-10天,叶片边缘会诱导产生愈伤组织,诱导率>90%。
4.根据权利要求1所述的一种快速高密度培养沙漠蔷薇悬浮细胞高产绿原酸的方法,其特征在于:步骤(3)中所述继代培养的培养基中的蔗糖浓度为30g/L。
5.根据权利要求1所述的一种快速高密度培养沙漠蔷薇悬浮细胞高产绿原酸的方法,其特征在于:步骤(4)中所述悬浮细胞继代培养时,高分散悬浮细胞的筛选方法为:首先将震荡培养所得悬浮细胞培养液摇匀后静置1min,使用大口吸管吸取上层细胞,去除较大颗粒细胞团,收集细胞液通过300~500g离心5min,去除细胞碎片,获得的细胞液与新鲜液体培养液按照1:1~3接种量进行继代,经过3~5次继代培养后,悬浮培养液中主要由单细胞和小细胞团组成,即得到高分散悬浮细胞。
6.根据权利要求1所述的一种快速高密度培养沙漠蔷薇悬浮细胞高产绿原酸的方法,其特征在于:将步骤(4)所得高分散悬浮细胞继续进行高密度培养,细胞培养密度>5*104个/ml,生长曲线呈S型,培养5~7天后,悬浮细胞的湿重量达到500-800g/L。
7.根据权利要求1所述的一种快速高密度培养沙漠蔷薇悬浮细胞高产绿原酸的方法,其特征在于:步骤(5)所述扩大培养方式为半连续培养,接种后培养至5~7天时,停止通气和摇动,静置1~2min从反应器底部放出培养液,保留上清液,按照10%接种量补加新鲜液体培养基。
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