CN114480275A - 一种外周血淋巴细胞培养基 - Google Patents
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Abstract
本发明涉及外周血淋巴细胞的增殖培养领域,具体来说是一种外周血淋巴细胞培养基,培养基包含:10‑20g/L的RPMI Medium 1640,1‑3g/L的碳酸氢钠,10‑15mg/L的植物血凝素,体积比为15%‑25%的胎牛血清,0.1‑0.5g/L的庆大和两性霉素,20‑50mg/L的肝素钠,pH值为7.2±0.2。本发明的人体外周血淋巴细胞培养基稳定性好,淋巴细胞转化率与分裂指数高,经过培养的淋巴细胞转换率大于65%,分裂指数大于7%,培养基的pH在密闭情况下依然可以保持7.2左右,培养环境更加稳定。
Description
技术领域
本发明涉及外周血淋巴细胞的增殖培养领域,具体来说是一种外周血淋巴细胞培养基。
背景技术
人体外周血小淋巴细胞,通常都处在G1期(或G0期),一般情况下不进行分裂。如在培养液中加入植物血凝素(PHA),这种小淋巴细胞受到刺激可转化为淋巴母细胞,进入有丝分裂。短期培养后,经秋水仙素处理,低渗和固定,即可得到大量的有丝分裂细胞。这种方法广泛用于临床医学、病理学、药理学、遗传病毒性学等方面的广泛应用。
细胞培养基是培养细胞中供给细胞营养和促使细胞生殖增殖的基础物质,也是培养细胞生长和繁殖的生存环境。培养基的种类很多,按其物质状态分为半固体培养基和液体培养基两类;按其来源分为合成培养基和天然培养基。将合成培养基能使细胞生存的特性和天然培养基能使细胞增殖生长的特性相结合,使两种培养基的特性都能获得发挥。
授权公告号为CN101550408B的中国发明专利公开了一种人体外周血淋巴细胞培养基,其原料组成的质量百分比为:RPMI 1640液体培养基77-81.9%,牛血清10-14.9%,植物凝血素4-6%,透明质酸2-5%,酪蛋白磷酸肽2-5%,青霉素和链霉素0.05-0.1%。授权公告号为CN101550408B的培养基已经将淋巴细胞转化率提高至60%,分裂指数提高至6%,但是本发明改进培养及配方后,能够将转化率提高至65%,分裂指数提高至7%。
发明内容
本发明的目的在于解决现有技术的不足,为培养淋巴细胞提供更好的条件,有效提高淋巴细胞转换率和淋巴细胞分裂指数,提供一种改良后的外周血淋巴细胞培养基。
为了实现上述目的,设计一种外周血淋巴细胞培养基,培养基包含:10-20g/L的RPMI Medium 1640,1-3g/L的碳酸氢钠,10-15mg/L的植物血凝素,体积比为15%-25%的胎牛血清,0.1-0.5g/L的庆大和两性霉素,20-50mg/L的肝素钠,pH值为7.2±0.2。
本发明同现有技术相比,其优点在于:
本发明的人体外周血淋巴细胞培养基稳定性好,淋巴细胞转化率与分裂指数高,经过培养的淋巴细胞转换率大于65%,分裂指数大于7%,培养基的pH在密闭情况下依然可以保持7.2左右,培养环境更加稳定。
具体实施方式
下面结合具体实施例对本发明作进一步说明,本发明的结构和原理对本专业的人来说是非常清楚的。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1(采用胎牛血清):
1.配置培养基:在培养管中按照以下比例配置培养基,10-20g/L的RPMI Medium1640,1-3g/L的碳酸氢钠,10-15mg/L的植物血凝素,体积比为15%-25%的胎牛血清,0.1-0.5g/L的庆大和两性霉素,20-50mg/L的肝素钠,pH值为7.2±0.2。
2.采血:采血0.5mL至培养管中,颠倒混匀5次。
3.培养:将培养管水平放置在培养箱内,37℃恒温培养72小时。
实施例2:(采用小牛血清)
1.配置培养基:在培养管中按照以下比例配置培养基,10-20g/L的RPMI Medium1640,1-3g/L的碳酸氢钠,10-15mg/L的植物血凝素,体积比为15%-25%的小牛血清,0.1-0.5g/L的庆大和两性霉素,20-50mg/L的肝素钠,pH值为7.2±0.2。
2.采血:采血0.5ml至培养管中,颠倒混匀5次。
3.培养:将培养管水平放置在培养箱内,37度恒温培养72小时。
通过以下步骤分别得到两种不同培养基培养后的淋巴细胞的转换率和细胞分裂指数。
1.加秋水仙素:向培养72小时后的培养基内加秋水仙素,使用浓度为0.02-0.05μg/mL。
2.继续培养1小时~2小时。
3.低渗处理:离心力200g,离心时间10分钟,吸出上清液5mL;加入预温至37℃的0.075M氯化钾低渗溶液5mL,振荡混匀1分钟,保温10分钟。
4.预固定:离心200g,10分钟,吸出上清液5mL。加入新鲜配制并预冷至2~8℃的卡诺氏固定液0.5mL预固定,静置10分钟,然后再加入固定液1mL混合后静置5分钟。
5.固定:半固定结束后,离心200g,5分钟。吸取上清液,管内保留0.5mL液体,然后加入5mL固定液,振荡混匀后静置5分钟。
6.再固定:200g离心力,5分钟,吸取上清液5mL,管内保留约0.5mL细胞沉淀物。
7.收获:加入0.5mL固定液,振荡混匀。此细胞沉淀物计约1mL即为收获的淋巴细胞染色体。
8.滴片:将免洗载玻片2-8℃预冷,吸取上述细胞沉淀物50μL滴片。
9.热固定:将滴片以后的载玻片60~80度固定2-5分钟。
10.胰酶处理:使用0.025~0.1%乙二胺四乙酸-胰蛋白酶溶液处理30秒左右。
11.染色:用2~5%姬姆萨染色液染色5~7分钟,用纯化水冲去多余的染色液,冷风吹干载玻片即可。
淋巴细胞转换率的检测与计算方法:转变换率=淋巴细胞转换数/1000*100%。即每1000个细胞中转换为淋巴母细胞的百分数。
淋巴细胞分裂指数检测与计算方法:分裂指数=分裂细胞书/(1000-分裂细胞数)*100%。即每1000个细胞中的分裂细胞数比上为分裂细胞数的百分数。
检测结果:
用小牛血清容易出现溶血现象,胎牛血清可以有效避免溶血现象,而且胎牛血清培养后的淋巴细胞转换率更高,分裂指数液更高。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (1)
1. 一种外周血淋巴细胞培养基,其特征在于,培养基包含:10-20g/L的RPMI Medium1640,1-3g/L的碳酸氢钠,10-15mg/L的植物血凝素,体积比为15%-25%的胎牛血清,0.1-0.5g/L的庆大和两性霉素,20-50mg/L的肝素钠,pH值为7.2±0.2。
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