CN109652369B - 利用外周血体外制备成熟红细胞的方法及制剂 - Google Patents
利用外周血体外制备成熟红细胞的方法及制剂 Download PDFInfo
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Abstract
本发明涉及一种利用外周血体外制备成熟红细胞的方法及制剂,特别涉及一种利用外周血中的Lin‑CD34‑细胞制备成熟红细胞的方法,包括富集Lin‑CD34‑细胞,往红系细胞分化Lin‑CD34‑细胞以及促进红细胞脱核成熟的步骤;另外还涉及利用所述方法制备得到的成熟红细胞以及其中应用的分化培养基以及细胞因子组合,该制备方法具有以下优点:原材料易得,红细胞得率高,得到的红细胞性能良好。
Description
技术领域
本发明涉及干细胞与细胞再生领域,特别涉及一种利用外周血体外制备成熟红细胞的方法及直接,还涉及利用所述方法制备得到的成熟红细胞,以及单个核细胞用于制备成熟红细胞的用途。
背景技术
众多周知,输血是治疗严重缺血患者(例如:急性贫血、严重的血小板减少症、血友病)的救命手段,但是目前在临床医学中存在很多的问题,主要体现在血液短缺以及输血风险两个方面。临床用血短缺一直是全国乃至全球医疗健康领域存在的问题,据研究显示,医疗临床用血量每年以两位数的比例逐年呈增长趋势,而与此同时,合格献血者数量在减少,再加上我国部分地方陆续取消互助献血,导致了严重的血液供需矛盾-供不应求;并且不容忽视的是,通过同种异体输血的方式还存在血源传播性疾病传播的风险,由于血液存在“窗口期”,很多新型病原体在检测期间是不会被检测出来的,从而带来了一定程度的输血风险。另外输血医学还面临着抗原/抗体不匹配导致患者发生免疫反抗以及稀有血型患者难以及时得到合适的血源的问题,以上种种用血问题引发了人们对临床用血问题的关注,如何解决用血紧张的问题,成为了迫在眉睫的难题。
为了解决以上的用血问题,红细胞再生技术迅猛地发展,目前所用的红细胞再生方法主要是基于CD34+骨髓造血干细胞或CD34+脐带血造血干细胞进行的,该方案虽然也可以实现体外的红系造血分化,但是提取CD34+骨髓造血干细胞会给细胞供应者造成极大的身体痛苦和伤害,而CD34+脐带血造血干细胞只有在新生儿出生的特定时间段能够获取,细胞取材困难,因此以上两种方法在临床应用上存在极大的困难。
授权号为CN 103667188B的中国专利“一种制备成熟红细胞的方法”中提到的了利用脐带血或外周血中的单个核细胞经过体外培养制备成熟红细胞的方法,虽然该方法初步提到了从外周血中利用单个核细胞作为起始材料直接制备红细胞的方法,但是该方法中存在的问题:其依旧是通过扩增CD34+细胞的方式来获取红细胞,细胞获取难度非常大,大规模临床应用可能性小。
发明内容
本发明的目的在于提供一利用外周血体外制备成熟红细胞的方法及制剂,具体而言,是利用外周血中少量的Lin-CD34-细胞体外分化培养大量红细胞的方法,并建立稳定的针对该细胞的红系分化培养体系及条件,解决红细胞体外分化这一重要难题。
本发明的第一方面提供一利用外周血体外制备成熟红细胞的方法及制剂,所述方法从外周血中分离得到单个核细胞,将单个核细胞和红细胞及血小板等不同的细胞进行初步分离,获取并富集单个核细胞中的Lin-CD34-细胞,在红系相关细胞因子充足的条件下诱导Lin-CD34-细胞向红系分化并实现大量扩增,通过红细胞脱核得到成熟的红细胞。
本发明的第二方面提供一利用外周血体外制备成熟红细胞的方法,包括以下步骤:
步骤S1:从外周血中提取Lin-CD34-细胞;
步骤S2:富集Lin-CD34-细胞;
步骤S3:诱导所述Lin-CD34-细胞向红系分化并进行扩增;以及
步骤S4:通过红细胞脱核得到成熟的红细胞。
更具体而言,所述利用外周血体外制备成熟红细胞的方法包括以下步骤:
S1.获取外周血,分离得到外周血单个核细胞;离心步骤S1得到的单个核细胞,利用添加了牛血清白蛋白和EDTA的磷酸盐缓冲液的细胞分离缓冲液重悬细胞,添加生物素标记的抗体,加入链霉亲和素标记的磁珠,在磁力作用下分离去除带有特殊表面标记物的细胞,收集未被吸附分离的分离缓冲液,离心得到Lin-CD34-细胞;
S2.在第0天,收集步骤S1得到的Lin-CD34-细胞,接种到造血干细胞扩增培养基中,并添加细胞因子组合以及青链霉素,培养3-5天,富集Lin-CD34-细胞;
S3.在第3-5天,收集步骤S2得到的细胞,接种到分化第一阶段培养基,其中所述分化第一阶段培养基内只提供向红系发育相关的细胞因子,继续培养至第12-14天,诱导Lin-CD34-细胞向红系分化并实现大量扩增;
S4.在第13-15天,收集步骤S3得到的细胞,接种到分化第二阶段培养基,其中所述分化第二阶段培养基相对于所述分化第一阶段培养基缺少部分细胞因子,继续培养至第21-23天,促进红细胞脱核成熟。
根据本发明的第二方面的方法,所述步骤S1当中,单个核细胞利用外周血的全血分离得到,分离过程是使用磷酸盐缓冲液1:1稀释全血后,使用淋巴细胞分离液(LymphoprepTM,STEMCELL Technologies)以及淋巴细胞分离管,在1200×g离心15分钟,分离得到外周血单个核细胞;在该步骤当中是通过淋巴细胞分离液实现对细胞成分的密度梯度离心分离,将外周血单个核细胞与红细胞及血小板等不同的细胞进行初步分离,保证后续对Lin-CD34-细胞富集。
根据本发明的第二方面的方法,所述步骤S1当中,所述生物素标记的抗体包括20-40μg/ml生物素标记的鼠抗人CD3抗体(Biotin Mouse Anti-human CD3)、20-40μg/ml生物素标记的鼠抗人CD14抗体(Biotin Mouse Anti-human CD14)、20-40μg/ml生物素标记的鼠抗人CD16抗体(Biotin Mouse Anti-human CD16)、20-40μg/ml生物素标记的鼠抗人CD19抗体(Biotin Mouse Anti-human CD19)、20-40μg/ml生物素标记的鼠抗人CD41a抗体(BiotinMouse Anti-human CD41a)、20-40μg/ml生物素标记的鼠抗人CD56抗体(Mouse Anti-humanCD56)和20-40μg/ml生物素标记的鼠抗人CD235a抗体(Biotin Mouse Anti-humanCD235a),其中所述生物素标记的鼠抗人CD3抗体用以分离富集T细胞,所述生物素标记的鼠抗人CD14抗体用以分离富集单核细胞、巨噬细胞以及树突状细胞,所述生物素标记的鼠抗人CD16抗体用以分离富集自然杀伤细胞,所述生物素标记的鼠抗人CD19抗体用以分离富集B细胞,所述生物素标记的鼠抗人CD41a抗体用以分离富集巨核细胞及血小板,所述生物素标记的鼠抗人CD56抗体用以分离富集自然杀伤T细胞,所述生物素标记的鼠抗人CD235a抗体用以分离富集红系细胞。
所述步骤S1进一步包括以下步骤:
S11:将单个核细胞300×g离心10分钟后,去除上清液,利用添加了2%牛血清白蛋白和1mM EDTA的磷酸盐缓冲液的细胞分离缓冲液重悬细胞,按照0.5-2×106个/毫升重悬细胞,得到细胞悬液;
S12:使用细胞悬液10倍体积的细胞分离缓冲液对生物素标记的抗体进行稀释,混合所述生物素标记的抗体和所述细胞悬液,并添加所述链霉亲和素标记的磁珠,在4℃孵育30分钟;以及
S13:随后将步骤S12得到的溶液置于磁力架内6-8分钟,进行谱系细胞分离,收集未被吸附分离的分离缓冲液,300×g离心10分钟,得到Lin-CD34-细胞。
步骤S1采用的原理是通过细胞表面的抗原会与相应的生物素标记的抗体结合,并通过生物素与链霉亲和素标记的磁珠结合,在磁力作用下可将细胞表面表达各谱系分化后带有特殊表面标志物的细胞分离后去除,从而得到未被磁珠分离的没有谱系特异性表面标志物的Lin-CD34-细胞。
根据本发明的第二方面的方法,所述步骤S2当中,所述细胞因子组合包括但不限于含有50-100ng/ml重组人类fms样酪氨酸激酶3配体(Flt3L)、50-100ng/ml重组人干细胞因子(SCF)、50-100ng/ml重组人白细胞介素三(IL-3)、200-800pg/ml重组人白细胞介素六(IL-6);所述造血干细胞扩增培养基为StemSpanTMSFEM无血清扩增培养基,通过添加细胞因子组合对Lin-CD34-细胞进行扩展培养,用以获取更多的Lin-CD34-细胞。
所述步骤S2当中,扩增培养条件为:37℃,5%CO2。
根据本发明的第二方面的方法,所述步骤S3当中,所述分化第一阶段培养基包括IMDM(Iscove's Modified Dulbecco's Medium,Sigma-Aldrich),胎牛血清(FBS,Gibco),人血浆(Plasma),谷氨酰胺,牛血清白蛋白(BSA,Albumin from bovine serum),人转铁蛋白(holo human transferrin,Sigma-Aldrich),重组人胰岛素(recombinant humaninsulin,Sigma-Aldrich),青链霉素(Penicillin-Streptomycin,Gibco),重组人白细胞介素三(rhIL-3,Peprotech),重组人促红细胞生成素(rhEpo,Amgen),重组人干细胞因子(rhSCF,Peprotech),该培养基中只提供向红系发育相关的细胞因子,保证了Lin-CD34-细胞向红系的增殖分化。
其中所述分化第一阶段培养基中,胎牛血清选自10-15%,例如11、12、13、14%;人血浆的成为为5-10%,例如6、7、8、9等;谷氨酰胺的成分为1-4mM,例如:2、3nM;牛血清蛋白选自1-2%;人转铁蛋白的成分为300-600μg/ml,例如400/500μg/ml;重组人胰岛素的成分为8-13μg/ml,例如9、10、11、12μg/ml;青链霉素的比例为2%;重组人白细胞介素三的成分为3-5ng/ml,例如:4ng/m;重组人促红细胞生成素的成分为4-7U/ml,例如5、6U/ml;重组人干细胞因子的成分为100ng/ml;该步骤当中,扩增培养条件依旧为:37℃,5%CO2。
根据本发明的第二方面的方法,所述步骤S4当中,所述分化第二阶段培养基的成分为IMDM,胎牛血清,人血浆,谷氨酰胺,牛血清白蛋白,人转铁蛋白,重组人胰岛素,2%青链霉素,重组人促红细胞生成素,其中所述分化第二阶段培养基相较所分化第一阶段培养基少了重组人白细胞介素三和重组人干细胞因子,促进红细胞脱核成熟。
其中所述分化第二阶段培养基中,胎牛血清选自10-15%,例如11、12、13、14%;人血浆的成为为5-10%,例如6、7、8、9等;谷氨酰胺的成分为1-4mM,例如:2、3nM;牛血清蛋白选自1-2%;人转铁蛋白的成分为300-600μg/ml,例如400/500μg/ml;重组人胰岛素的成分为8-13μg/ml,例如9、10、11、12μg/ml;青链霉素的比例为2%;重组人促红细胞生成素的成分为1-5U/ml,例如2、3、4U/ml;该步骤当中,扩增培养条件依旧为:37℃,5%CO2
值得一提的是,根据本发明的第二方面,在红细胞脱核阶段,每隔2-4天对培养基进行一次更换,并且在期间通过流式细胞仪测定体外培养分化期间的细胞的CD235a,CD117,CD71,Hoechst 33342的变化情况。
本发明的第三方面提供根据本发明的第一至第二方面任一项所述方法制备得到的成熟红细胞。
得到的成熟红细胞的性能参数:细胞大小6-8微米,每个细胞血红蛋白含量30±5pg,其中成人血红蛋白含量94.23%,胎儿血红蛋白含量2.82%,成人血红蛋白α2含量3.04%。
本发明的第四方面提供涉及外周血中Lin-CD34-细胞在制备成熟红细胞中的用途。
本发明的第五方面提供涉及细胞因子组合物,其含有重组人类fms样酪氨酸激酶3配体(Flt3L)、重组人干细胞因子(SCF)、重组人白细胞介素三(IL-3)、重组人白细胞介素六(IL-6)。
本发明的第六方面提供细胞因子组合物在由外周血单个核细胞中富集Lin-CD34-细胞的用途。
本发明的第七方面提供分化阶段培养基及其在由Lin-CD34-细胞制备成熟红细胞中的用途,其中所述分化阶段培养基包括分化第一阶段培养基和分化第二阶段培养基。
以上对本方案中的专业术语的介绍:
外周血:外周血是除骨髓之外的血液,临床上常用一些方法把骨髓中的造血干细胞释放到血液中,再从血液中提取分离得到造血干细胞,我们把这样得到的干细胞称为外周血干细胞。
外周血单个核细胞:是外周血中具有单个核的细胞,包括淋巴细胞和单核细胞,目前外周血单个核细胞主要的分离方法是Ficoll-hypaque(聚蔗糖-泛影葡胺)密度梯度离心法。
Lin-CD34-细胞:Lin-细胞是指并未进入各个造血谱系分化的细胞,CD34-是指细胞表面不表达CD34表面标志物,Lin-CD34-细胞是并未进入造血谱系分化且不表达CD34表面标志物的细胞。
生物素标记的抗体:是在抗体上连接生物素(biotin),生物素标记反应简单、温和且很少抑制抗体活性,将生物素与抗体共价结合是一种非常简便、直接的标记方法。
造血干细胞扩增培养基:StemSpanTMSFEM无血清扩增培养基,是一款造血干细胞扩增培养基,不含血清,在加入造血生长因子和/或用户选择的其他刺激因子后,可促进人造血干/祖细胞(HSPC)的扩增或向特定谱系的分化。
重组人类fms样酪氨酸激酶3配体(rhFlt3L):FMS样酪氨酸激酶3配体(Flt-3配体)也称为FL,Flt3L和FLT3LG,是促进多种造血细胞谱系分化的α-螺旋细胞因子。FLT3LG在结构上与干细胞因子(SCF)和集落刺激因子1(CSF-1)同源。FLT3LG作为生长因子,通过激活造血祖细胞来增加细胞的数量。
重组人干细胞因子(rhSCF):Kit配体(KITLG)也称为干细胞因子(SCF),属于SCF家族的I型跨膜糖蛋白。KITLG是受体型蛋白酪氨酸激酶KIT的配体。SCF在调节细胞存活和增殖,造血,干细胞维持,细胞发育,迁移和功能中起重要作用。
重组人白细胞介素三(rhIL-3):是属于造血生长因子家族的糖蛋白,其在临床前体外和体内研究中表现出多谱系活性。造血祖细胞在IL-3蛋白的帮助下增殖和分化成成熟的红细胞,肥大细胞,巨核细胞和粒细胞。
重组人白细胞介素六(rhIL-6):是一种调节免疫反应,造血功能,急性期反应和炎症反应的多功能细胞因子。与IL-3协同促进细胞造血细胞增殖。
重组人促红细胞生成素(rhEpo):是主要的红细胞生成因子,其与从多能祖细胞发育红细胞谱系的各种其他生长因子(例如,IL-3,IL-6,糖皮质激素和SCF)协同作用。爆式形成单位-红细胞(BFU-E)细胞开始促红细胞生成素受体表达并且对促红细胞生成素敏感。是重要的红系造血细胞因子。
人转铁蛋白(holo human transferrin):是血浆中主要含铁蛋白,可以与铁离子形成复合物,供红细胞中血红蛋白生成。
链霉亲和素:是Streptomyces avicllrdi菌培养过程中的分泌产物,主要通过2一亚氨基生物素亲和层析法提纯,1L培养液中含蛋白10~60mg.
CD235a:血型糖蛋白A,是一种单跨膜糖蛋白,表达于成熟红细胞和红系前体细胞,为红细胞表面特殊的标记蛋白。CD235a表达说明该细胞向红系细胞分化,从流式细胞术结果分析表明,在SFEM阶段,细胞不表达CD235a,说明该细胞并未进入红系分化,而在将细胞换液为分化1阶段培养基后,培养基中的细胞因子诱导细胞向红系分化,细胞开始表达CD235a,且比例随分化时间不断增加,在更换为分化2阶段培养基后,由于细胞已经完全进入红系,故几乎所有细胞均表达CD235a,表明几乎所有细胞均为红系细胞。该细胞表明标志物说明我们的分化体系在体外培养红系细胞的诱导过程中是成功的。
CD117:又称c-kit,是SCF干细胞生长因子受体,表达于造血干细胞及其他细胞表面。SCF在调节细胞存活和增殖,造血,干细胞维持,细胞发育,迁移和功能中起重要作用。其受体的表达量变化反应出细胞利用SCF能力的变化,在SFEM条件下CD117并无表达,表明Lin-细胞在SFEM中培养阶段并未利用SCF。当细胞进入分化1阶段培养基后细胞进入红系分化,CD117表达快速上升,达到顶峰,此时培养基中加入SCF以调节细胞存活和增殖,造血,干细胞维持,细胞发育。而当细胞进入分化2阶段培养基后,由于细胞进入成熟脱核阶段,故CD117表达又逐渐下降。
CD71:转铁蛋白受体1,是一种跨膜糖蛋白,由两个通过两个二硫键连接的二硫键连接的单体组成。每个单体结合一个全转铁蛋白分子,产生铁-转铁蛋白-转铁蛋白受体复合物,其通过胞吞作用进入细胞,供细胞向红系发育过程中血红蛋白生成。在SFEM条件下CD71并无表达,表明Lin-细胞在SFEM中培养阶段并未利用转铁蛋白,细胞并未进入红系开始摄取铁合成血红蛋白。当细胞进入分化1阶段培养基后,由于细胞进入红系分化,需要摄取大量的转铁蛋白,故CD71表达迅速上升,满足细胞转铁蛋白的摄取。当细胞进入分化2阶段培养基后,由于红系细胞已经合成了足够多的血红蛋白,故CD71表达逐渐下降,红细胞走向成熟。
Hoechst33342:是用于染色DNA的荧光染料。该染料可以穿过细胞膜与DNA相结合,如果细胞未脱核,则该染料与DNA结合可以通过流式细胞术检测到信号为阳性,如果该细胞脱核,则通过流失细胞术检测到信号为阴性。
同时由于CD235a是红细胞的表面标志物,通过流式细胞术同时检测检测两种标志物共表达的情况,CD235a阳性Hoechst 33342阳性为红系未脱核细胞,CD235a阳性Hoechst33342阴性为成熟红细胞。在SFEM和分化1阶段培养基中,细胞由Lin-细胞进入红系分化,CD235a表达升高,但并未脱核,故该细胞为Hoechet 33342阳性。当细胞进入分化2阶段培养基后,细胞开始成熟脱核,出现Hoechst 33342阴性细胞,表明该红细胞成熟,本发明可以对成熟红细胞进行形态、结构和功能鉴定。
本发明利用外周血中少量的Lin-CD34-细胞制备成熟红细胞,具有以下有益效果:
1、创新型的使用外周血中的Lin-CD34-细胞进行体外造血,避免了传统CD34+细胞造血方法中对供体采集者进行药物干预骨髓细胞动员或骨髓穿刺等伤害供体的身体的做法;由于CD34+脐带血来源的细胞只有在新生儿出生时能够采集,本方法可以随时利用外周血进行造血,也有明显优势。
2、红细胞的增值率高,在22天左右就能实现约1000倍增殖分化,为未来的临床应用提供了可能。
附图说明
图1是本发明的一实施例的利用外周血体外制备成熟红细胞的细胞增殖曲线,本发明中以两个捐献者的数据作为比对。
图2和图3是在富集Lin-CD34-细胞阶段,两个捐献者的CD235a以及CD117表达水平的示意图以及CD71以及hoechst33342/CD235a共染后鉴定红细胞脱核水平。
图4和图5是在Lin-CD34-细胞分化阶段,两个捐献者的CD235a以及CD117表达水平的示意图以及CD71以及hoechst33342/CD235a共染后鉴定红细胞脱核水平。
图6和图7是在红细胞脱核成熟阶段,两个捐献者的CD235a以及CD117表达水平的示意图以及CD71以及hoechst33342/CD235a共染后鉴定红细胞脱核水平。
图8是分化培养阶段细胞CD235a表达变化曲线。
图9是分化培养阶段细胞CD71表达变化曲线。
图10是分化培养阶段细胞CD117表达变化曲线。
图11是制备过程中的红细胞脱核情况。
图12和图13分别是细胞分化完成后对细胞进行涂片染色,镜下检测红细胞形态。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员所获得的所有其他实施例,都属于本发明保护的范围。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。
实施例1:
一、成分准备:
1.1生物素标记的抗体
配置1ml生物素标记的抗体需要如下成分:
成分 | 体积 |
生物素标记的鼠抗人CD3抗体 | 20ul |
生物素标记的鼠抗人CD14抗体 | 20ul |
生物素标记的鼠抗人CD16抗体 | 20ul |
生物素标记的鼠抗人CD19抗体 | 20ul |
生物素标记的鼠抗人CD41a抗体 | 20ul |
生物素标记的鼠抗人CD56抗体 | 20ul |
生物素标记的鼠抗人CD235a抗体 | 20ul |
1.2、富集细胞培养基(包括造血干细胞扩增培养基中,细胞因子组合以及青链霉素)
配置1L富集细胞培养基需要如下成分:
1.3、分化第一阶段培养基的配置:
配置1L分化第一阶段培养基需要如下成分:
成分 | 终浓度 | 体积 |
IMDM | 85% | 850ml |
胎牛血清(FBS,Gibco) | 10% | 100ml |
人血浆(Plasma) | 5% | 50ml |
谷氨酰胺 | 2nM | 10 |
牛血清白蛋白 | 2% | 20ml |
人转铁蛋白 | 500μg/ml | 100 |
重组人胰岛素 | 10μg/ml | 100μl |
青链霉素 | 2% | 20ml |
重组人白细胞介素3 | 100ng/ml | 1ml |
重组人促红细胞生成素 | 100ng/ml | 1ml |
重组人干细胞因子 | 100ng/ml | 1ml |
1.4、分化第二阶段培养基的配置:
配置1L分化第二阶段培养基需要如下成分:
二、外周血单个核细胞的分离:
取用全血,使用磷酸盐缓冲液1:1稀释全血后,使用淋巴细胞分离液(LymphoprepTM,STEMCELL Technologies)以及淋巴细胞分离管,在1200×g离心15分钟,用毛细血管吸取单个核细胞。
三、外周血中Lin-CD34-细胞的获取:
将步骤二获取的外周血单个核细胞在300×g离心10分钟后,使用添加了2%牛血清白蛋白,1mM EDTA的磷酸盐缓冲液的细胞分离缓冲液,按1×106个/毫升的比例重悬细胞,得到细胞悬浮液。
使用细胞悬液10倍体积的细胞分离缓冲液对生物素标记的抗体进行稀释,往细胞悬浮液中以20-40μg/ml添加生物素标记的抗体,加入300μl的链霉亲和素标记的磁珠,在4℃孵育30分钟;以及随后将溶液置于磁力架内6-8分钟,进行谱系细胞分离,收集未被吸附分离的分离缓冲液,在300×g离心10分钟,得到Lin-CD34-细胞。
四、制备成熟红细胞:
在第0天,将0.1-0.5×105的Lin-CD34-细胞,接种到造血干细胞扩增培养基中,并添加细胞因子组合以及青链霉素,其中造血干细胞扩增培养基中,细胞因子组合以及青链霉素组成富集细胞培养基,所述富集细胞培养基的成分如前所述,培养到第四天,该阶段被定义为富集Lin-CD34-细胞阶段;在第5天更换培养体系,将培养基更换为分化第一阶段培养基,该培养基的成分如前介绍所示,在置于37℃,5%CO2条件下培养至第13天,该阶段被定义为Lin-CD34-细胞分化阶段;在第14天更换培养体系,将培养基更换为分化第二阶段培养基,该培养基的成分如前介绍所示,在置于37℃,5%CO2条件下培养至第22天,该阶段被定义为红细胞脱核成熟阶段,从0-22天全期间每2-4天更换相应阶段培养基。
五、细胞扩增数量分析:
在第0-22天分别对细胞数量进行检测,每3-4天,将培养体系中的细胞充分重悬,取10μl细胞悬液与10μl台盼蓝染液混合,通过细胞计数仪计数。
具体地,细胞增殖情况如下所述,此时将起始细胞数量标准化到1×106:
天数 | 0 | 4 | 7 | 11 | 13 | 17 | 19 | 22 |
捐献者1 | 1 | 2.69 | 21.53 | 89.20 | 209.17 | 738.24 | 996.62 | 1011.38 |
捐献者2 | 1 | 2.33 | 13.84 | 36.91 | 126.73 | 406.03 | 501.92 | 517.22 |
将得到的细胞数量绘制细胞增殖曲线,如图1所示,可见,在该培养条件下红细胞能够正常的增殖分化,且能够实现1000倍增殖。
六、细胞表型分析:
分别取用富集Lin-CD34-细胞阶段、Lin-CD34-细胞分化阶段以及红细胞脱核成熟阶段的细胞进行分析,具体的操作过程。
将培养体系中的细胞充分重悬,取50-100μl细胞悬液加入500μl磷酸盐缓冲液中。分别加入鼠抗人CD235a-APC抗体(mouse Anti-human CD235a APC)0.5μl,鼠抗人CD71-PerCP Cy5.5抗体(mouse Anti-human CD71-PerCP Cy5.5)2μl,鼠抗人CD117-PE Cy7抗体(mouse Anti-human CD117-PE Cy7)2μl,Hoechst33342染料0.5μl,充分混匀后避光孵育20分钟,流式细胞仪上机检测。对目的细胞进行特定的表面标记物表达水平进行分析。通过不同时间点的分析绘制变化曲线,得到如图2到如10所示的示意图。
七、红细胞脱核情况的分析:
根据流式细胞术分析CD235a阳性/Hoechst 33342阴性细胞的比例,该比例可以反映细胞脱核情况。
得到如图11所示的示意图,由图可知,细胞从第13天开始脱核水平明显提高。
八、细胞形态分析:
取0.5-1×106细胞,使用血细胞离心涂片机进行离心涂片。
使用-20℃预冷的甲醇室温固定2分钟。
将固定后的血涂片水洗3次,每次5分钟,并置于室温风干。
使用10ml磷酸盐缓冲液溶解对二氨基联苯片剂(Benzidine,Sigma),添加10μl过氧化氢溶液,过滤。使用300-500μl该溶液对血涂片进行染色,室温1小时。
将染色后的血涂片水洗3次,每次5分钟,并置于室温风干。
使用吉姆萨染液对血涂片二次染色,室温35-40分钟。
将染色后的血涂片水洗3次,每次5分钟,并置于室温风干。
使用封片剂封片,镜下观察拍照。
得到如图12所示的示意图,由图可知,在第22天细胞分化完成后,明显出现成熟的红细胞。
本发明不局限于上述最佳实施方式,任何人在本发明的启示下都可得出其他各种形式的产品,但不论在其形状或结构上作任何变化,凡是具有与本申请相同或相近似的技术方案,均落在本发明的保护范围之内。
Claims (1)
1.利用外周血体外制备成熟红细胞的方法,其特征在于,包括以下步骤:
步骤S1:从外周血中提取得到Lin-CD34-细胞:
利用外周血的全血分离得到外周血单个核细胞:使用磷酸盐缓冲液1:1稀释全血后,使用淋巴细胞分离液以及淋巴细胞分离管,在1200×g离心15分钟,分离得到外周血单个核细胞;
离心上述步骤得到的外周血单个核细胞得到Lin-CD34-细胞:利用添加了牛血清白蛋白和EDTA的磷酸盐缓冲液重悬上述步骤得到的外周血单个核细胞,添加生物素标记的抗体,加入链霉亲和素标记的磁珠,在磁力作用下分离去除表面带有会与相应生物素标记的抗体结合的抗原的细胞,收集未被磁珠吸附的分离缓冲液,离心得到Lin-CD34-细胞;其中所述生物素标记的抗体为生物素标记的鼠抗人CD3抗体、生物素标记的鼠抗人CD14抗体、生物素标记的鼠抗人CD16抗体、生物素标记的鼠抗人CD19抗体、生物素标记的鼠抗人CD41a抗体、生物素标记的鼠抗人CD56抗体和生物素标记的鼠抗人CD235a抗体;
步骤S2:富集Lin-CD34-细胞:
在步骤S2中,在第0-5天,将步骤S1得到的Lin-CD34-细胞接种到造血干细胞扩增培养基中,并添加细胞因子组合以及青链霉素,富集Lin-CD34-细胞;其中所述细胞因子组合为50-100ng/ml重组人类fms样酪氨酸激酶3配体、50-100ng/ml重组人干细胞因子、50-100ng/ml重组人白细胞介素三、200-800pg/ml重组人白细胞介素六;
步骤S3:诱导Lin-CD34-细胞向红系分化并进行扩增:
将步骤S2得到的Lin-CD34-细胞接种到分化第一阶段培养基,继续培养至第9-11天;所述分化第一阶段培养基为IMDM,10-15%胎牛血清,5-10%人血浆,1-4mM谷氨酰胺,1-2%牛血清白蛋白,300-600μg/ml人转铁蛋白,8-13μg/ml重组人胰岛素,2%青链霉素,100ng/ml重组人白细胞介素三,100ng/ml重组人促红细胞生成素以及100ng/ml重组人干细胞因子;以及
步骤S4:通过红细胞脱核得到成熟的红细胞:
将步骤S3得到的细胞接种到分化第二阶段培养基,继续培养7-9天;其中所述分化第二阶段培养基为IMDM,15%胎牛血清,5-10%人血浆,1-4mM谷氨酰胺,1-2%牛血清白蛋白,300-600μg/ml人转铁蛋白,8-13μg/ml重组人胰岛,2%青链霉素以及1-5U/ml重组人促红细胞生成素。
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