CN109486749A - 鹿茸细胞无血清培养法 - Google Patents

鹿茸细胞无血清培养法 Download PDF

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CN109486749A
CN109486749A CN201710809881.5A CN201710809881A CN109486749A CN 109486749 A CN109486749 A CN 109486749A CN 201710809881 A CN201710809881 A CN 201710809881A CN 109486749 A CN109486749 A CN 109486749A
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梁国坚
张桂珍
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Guangxi Soft Group Co Ltd
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Abstract

当代研究鹿茸含有近四十种有益物质，特别具有多肽、多胺、多醣，能增加核酸蛋白质合成，修复机体细胞和细胞再生，抑制肿瘤细胞，增加免疫、抗病毒，影响生命全程。但以鹿茸用药功效不显，提取成分困难，成本高，失去活性有药物残留。鹿茸细胞是鹿茸的最小生命单位，研究证明鹿茸细胞中多胺等活性物质多于鹿茸三倍，便于大量生产提取。按生物制药要求研究鹿茸细胞取得成功，获ZL03159604.5号发明专利。进而创新了鹿茸细胞无血清培养法，排除了血清有害成分及耐高温疯牛病毒的风险。建立了鹿茸细胞无血清培养基及取样工具，可从茸体上多点取材培养不同的前体细胞株(系)，组建细胞库，满足工业化生产鹿茸细胞研制新型药物的根本须求。

Description

鹿茸细胞无血清培养法
鹿茸药用始于我国，已有2千余年药用史。在马王堆出土的汉墓皂书中证实鹿茸治疗疾病、蛇咬等。长期视为名贵药材，用于强身健体，益气补阳，为亚洲各国特别在韩国所喜用。
药用鹿茸是梅花鹿、马鹿在额骨上生长的、具有茸毛幼嫩时期的、骨化前的鹿角。每年规律性生长脱落一次，生长快速每日能增长1-2公分超过癌细胞。幼鹿生后第二年开始生角，受日照、紫外线温度、生存环境影响体内性激素消涨，启动骨膜干细胞开始生长鹿茸，由鹿茸干细胞分化为鹿茸前体细胞，形成鹿茸组织神经血管、肋软骨、肋骨组织，成为鹿茸器官。之后，外部皮肤茸毛脱落，钙化后变为鹿的坚硬“武器”，次年春脱落，变成家庭装饰品，完成了一个生长周期。
这种特有的生物学现象从古至今受到关注。我们为了探寻奥秘，研究了鹿茸细胞体外培养取得成功，获得了鹿茸组织细胞培养法ZL03159604.5号发明专利。
鹿茸是鹿的第二性征，在生长过程中进行细胞分化，也是细胞组织功能活性最佳阶段，含有丰富的活性物质。当鹿茸钙化脱皮后形成坚硬的鹿角，活性物质则全部消失。大量试验表明，从鹿茸中提取多肽等活性物质成本高、数量低、有药物残留破坏活性。鹿茸细胞药理药化研究表明，重要的多肽等活性物质含量超鹿茸三倍以上，并且易于从细胞中提取药物单体成分。以大规模细胞培养生产可获得多种活性成分，提供研制生产高浓度、高含量特效新药，进行中药西治。对60岁左右人群补充鹿茸多肽、多胺等活性物质增加全身细胞复制代数，延长生命高龄百岁以上。提供特效新药治疗老年痴呆症、彻底治疗高血糖、高血压、高血脂，治疗预防癌症。
利用鹿茸细胞进行生物制药。鹿茸细胞无血清培养法排除由牛血清所带来的有害成分及损害，防止耐高温疯牛病病毒所能造成的风险。为此我们创立了鹿茸细胞无血清培养法。本培养法为鹿茸细胞在体外培养，在无血清成分的液态环境中能够快速生长，适用鹿茸干细胞以及分化的前体细胞培养。可应用于鹿茸细胞贴壁静止培养、转瓶旋转培养、悬浮培养，适用于工业化鹿茸细胞生产，为分离提取药化单体成分，制备新药奠定了基础。
鹿茸细胞无血清培养法的培养基配方组成及配制：
配制精确称量，依次加入流液，滤过除菌，通过无菌测试、质量检测、低温保存。
鹿茸细胞培养需要不断优化。鹿茸细胞培养实现大规模工业化生产单体药化成分，需要多次培养原代细胞，然后建立细胞株、设立种子库，从天然鹿茸提取选优淘劣，为工业化生产提供优良种细胞。鹿茸是实体组织取材困难，在实行无血清培养技术前，从鹿茸组织上采取原始材料，切割小块组织，施用外科刀对刃切割，手工操作难以均衡取样，取量少，生长速度不一致。现今我们裁外科刀对刀功取法改进，创新为平板细齿锉刀法。能从茸体上任何部位进行平锉，大量锉割组织取样，获得细小2mm立方组织块，用PBS离心清洗，达到全部去除血清成分、红细胞等，种入培养瓶，使均匀分布于生长液中同步大量生长原代细胞，并且缩短了培养时间。可从茸体任何部位除茸皮外，均可取样培养原代细胞。鹿茸上中下段细胞分化程度不同，所能获得的活性生长成分的分子质量也不同，因所含活性因子差异而获得不同的药物。这是使用新工具的技术特征。
鹿茸细胞无血清培养法实例：
进行鹿茸细胞贴壁培养、转瓶培养及悬浮培养为生物制药分离转化鹿茸多肽、多胺等活性物质而行使鹿茸细胞工业化无血清培养。
1、优选临床检查及结核病、布氏杆菌病血清学检疫。从中选择3-7岁阴性个体、产茸量高的健壮公鹿。
2、麻醉割取鹿茸，在半小时内送入实验室取材，茸表灭菌消毒。
3、以专用工具锯锉鹿茸组织加入PBS清洗离心，取组织块种入培养瓶，加入无血清专用培养基。
4、使组织块均匀分布在瓶壁上，在37℃中不可移动静止培养5天，镜检、纪录、照相。
5、约8-10天对大量生长的贴壁细胞进行消化培养。
6、培养8-10天消化收获原代培养细胞，编号冻存。
7、原代细胞传代到15代，之后每7天传代一次，直至形成细胞系。
8、传代达到50次，在正常状态可经过检测符合各种指标即建立细胞株。
9、建立细胞株的工作是长期系统性工作。
10、对种子细胞库进行常态专项管理。鹿茸细胞无血清培养法为鹿茸细胞静止培养、旋转培养、悬浮培养，为工业化生产奠定了坚实基础。
附图说明：附图为本申请单板多齿锉图。
锉刀齿高0.3、宽0.2、长0.2；
锉齿10X24排；
平板厚度0.2公分。

Claims (2)

1.鹿茸细胞无血清培养，其培养基组分：
2.鹿茸取材平板不锈钢锉刀附图：
锉刀齿高0.3、宽0.2、长0.2；
锉齿10X24排；
平板厚度0.2公分。
CN201710809881.5A 2017-09-11 2017-09-11 鹿茸细胞无血清培养法 Pending CN109486749A (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1597934A (zh) * 2003-09-15 2005-03-23 梁国坚 鹿茸组织细胞简易培养方法
KR20060128117A (ko) * 2005-06-09 2006-12-14 김현수 발효녹용추출물을 포함하는 조성물
CN101068556A (zh) * 2006-04-28 2007-11-07 王利忠 富含胰岛素样生长因子的鹿茸纳米化制品及其制备方法
CN102366387A (zh) * 2011-10-24 2012-03-07 汕头市江源化工有限公司 一种鹿茸润肤面霜
CN106148269A (zh) * 2015-04-10 2016-11-23 烟台赛泽生物技术有限公司 一种哺乳动物细胞培养基及其添加剂

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1597934A (zh) * 2003-09-15 2005-03-23 梁国坚 鹿茸组织细胞简易培养方法
KR20060128117A (ko) * 2005-06-09 2006-12-14 김현수 발효녹용추출물을 포함하는 조성물
CN101068556A (zh) * 2006-04-28 2007-11-07 王利忠 富含胰岛素样生长因子的鹿茸纳米化制品及其制备方法
CN102366387A (zh) * 2011-10-24 2012-03-07 汕头市江源化工有限公司 一种鹿茸润肤面霜
CN106148269A (zh) * 2015-04-10 2016-11-23 烟台赛泽生物技术有限公司 一种哺乳动物细胞培养基及其添加剂

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
M SADIGHI 等: "Effects of insulin-like growth factor-I (IGF-I) and IGF-II on the growth of antler cells in vitro", 《JOURNAL OF ENDOCRINOLOGY》 *
储炬 等 主编: "《现代生物工艺学（上册）》", 30 September 2007, 华东理工大学出版社 *
姚文兵 主编: "《生物技术制药概论（第3版）》", 31 August 2015, 中国医药科技出版社 *
朱祖武 等 主编: "《机械加工技术》", 31 January 2017, 吉林大学出版社 *
赵丽红 等: "梅花鹿生茸干细胞的分离培养", 《中国畜牧兽医学会动物解剖学及组织胚胎学分会第十四次学术研讨会论文集》 *

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Application publication date: 20190319