CN116270413B - 一种脂肪间充质干细胞冻干粉的制备方法 - Google Patents
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Abstract
本发明提供了一种含脂肪间充质干细胞冻干粉的制备方法。本发明首次提供了针对Caspase‑3的单克隆抗体,该抗体具备良好的结合活性和特异性,能有效抑制细胞中的Caspase‑3蛋白的活性,抑制细胞凋亡,延长细胞的冻存周期。经实验验证,以含有Caspase‑3单克隆抗体的冻存保护剂可以有效提升冻存细胞的复苏活性,减少因冻存过程中因为环境变化而激发的凋亡刺激。延长细胞的冻存时间。采用本发明实验组冻存保护剂冻存1个月后复苏的脂肪间充质干细胞可以高效率地促进成纤维细胞进行增殖和促进成纤维细胞分泌I型和III型胶原,且其效果明显优于对照组(P<0.05),差异显著,具有统计学意义。
Description
技术领域
本发明涉及干细胞保存技术领域,特别涉及一种脂肪间充质干细胞冻干粉的制备方法。
背景技术
人脂肪间充质干细胞是一种富含于人体脂肪组织中的成体间充质干细胞。这些干细胞具有多向分化潜能,在体外可以向脂肪,骨,软骨,肌肉和神经细胞分化。并且人脂肪间充质干细胞能够分泌多种生物活性物质,包括蛋白质、多肽及细胞因子等,比如干细胞生长因子(SCGF)、血管内皮生长因子(VEGF)、胰岛素样生长因子(IGF)、肝细胞生长因子(HGF)和转化生长因子等,这些细胞生长因子能够控制或者维持受伤组织细胞,引导其自身修复。与胚胎干细胞相比,人脂肪间充质干细胞作为成体干细胞可以避免相关的伦理问题,与骨髓干细胞相比,人脂肪间充质干细胞获取方式微创。综合以上优点,人脂肪间充质干细胞在细胞治疗及再生医学中具有广泛的应用前景。
目前临床使用的MSCs大多就是经体外培养、低温保存后的。冻存过程中导致的损伤一般是由细胞在冷冻过程中脱水、氧自由基产生和细胞凋亡酶如caspase的激活所致。caspase-3在细胞凋亡中起着不可替代的作用,caspase-3基因转染昆虫Sf9细胞后引起细胞凋亡,这个过程可以被Bcl-2阻断;在发生凋亡的细胞提取液中去除caspase-3后,这些提取液就失去了诱导细胞凋亡的能力;再加入纯化的caspase-3它又能恢复致凋亡的功能。有研究显示用早期代数的MSCs的治疗效果要比晚期的效果好。因此,通过抑制细胞冻存过程中的凋亡可以有效维持MSCs移植时细胞状态。
发明内容
针对现有技术所存在的技术问题,本发明提供了一种脂肪间充质干细胞冻干粉的制备方法。所述冻干粉能够促进成纤维细胞的活性,延缓细胞衰老。
具体而言,本发明首先提供了一种脂肪间充质干细胞冻干粉,所述冻干粉是将脂肪间充质干细胞与冻存保护剂混合后冻干制备到。
优选地,所述冻存保护剂包括Caspase-3单克隆抗体、牛血清白蛋白、海藻糖、蔗糖和PEG。
优选地,所述冻存保护剂是采用DMEM完全培养基配置。
优选地,所述冻存保护剂包括5-15%Caspase-3单克隆抗体、2-20%牛血清白蛋白、2-20%海藻糖、2-20%蔗糖和2-20%PEG。
进一步优选地,所述冻存保护剂包括5%Caspase-3单克隆抗体、10%牛血清白蛋白、10%海藻糖、10%蔗糖、10%PEG。
进一步优选地,所述冻存保护剂包括10%Caspase-3单克隆抗体、15%牛血清白蛋白、15%海藻糖、15%蔗糖、15%PEG。
进一步优选地,所述冻存保护剂包括15%Caspase-3单克隆抗体、20%牛血清白蛋白、20%海藻糖、20%蔗糖、20%PEG。
优选地,所述Caspase-3单克隆抗体的重链可变区序列如SEQ ID NO:2所示,轻链可变区序列如SEQ ID NO:3所示。
进一步优选地,所述Caspase-3单克隆抗体的重链可变区包括CDR-H1、CDR-H2、CDR-H3,所述CDR-H1-3的序列如SEQ ID NO.4-6所示,轻链可变区包括CDR-L1、CDR-L2、CDR-L3,所述CDR-L1-3的序列如SEQ ID NO.7-9所示。
另一方面,本发明还提供了所述冻存保护剂在制备间充质干细胞冻干粉中的用途。
进一步,所述冻干粉能够促进成纤维细胞的活性,延缓细胞衰老。
另一方面,本发明还提供了一种间充质干细胞冻干粉的制备方法,其特征在于,所述方法是将脂肪间充质干细胞与冻干保护剂混合后冻干制备到。
另一方面,本发明还提供了一种Caspase-3单克隆抗体,其特征在于重链可变区序列如SEQ ID NO:2所示,轻链可变区序列如SEQ ID NO:3所示。
优选地,本发明还提供了所述Caspase-3单克隆抗体在制备冻存保护剂中的用途。
本发明的优点如下:
1)本发明首次提供了针对Caspase-3的单克隆抗体,该抗体具备良好的结合活性和特异性,能有效抑制细胞中的Caspase-3蛋白的活性,抑制细胞凋亡,延长细胞的冻存周期。经实验验证,以含有Caspase-3单克隆抗体的冻存保护剂可以有效提升冻存细胞的复苏活性,减少因冻存过程中因为环境变化而激发的凋亡刺激。延长细胞的冻存时间。
2)采用本发明实验组冻存保护剂冻存1个月后复苏的脂肪间充质干细胞可以高效率地促进成纤维细胞进行增殖和促进成纤维细胞分泌I型和III型胶原,且其效果明显优于对照组(P<0.05),差异显著,具有统计学意义。
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明,以使本领域的技术人员更加清楚地理解本发明。
以下各实施例,仅用于说明本发明,并不用来限制本发明的范围。基于本发明中的具体实施例,本领域普通技术人员在没有做出创造性劳动的情况下,所获得的其他所有实施例,都属于本发明的保护范围。
在本发明实施例中,若无特殊说明,所有原料组分均为本领域技术人员熟知的市售产品;在本发明实施例中,若未具体指明,所用的技术手段均为本领域技术人员所熟知的常规手段。
实施例1Caspase-3单克隆抗体的制备
根据Caspase-3的氨基酸序列(GenPept:CAC88866.1),分析其相应的主要活性结合表位,综合筛选获高活性的抗原表位作为免疫原制备单克隆抗体。经筛选获得的抗原表位序列为ngpvdlkkit nffrgdrcrs ltgkpklfii qacrgteldc(SEQ ID NO.1)。
人工合成SEQ ID NO.1的编码序列,并将其用于构建原核表达质粒pET-N-GST-PreScission(上海碧云天生物技术有限公司,产品编号D2916),在大肠杆菌DH5α中表达N端含有GST标签的Caspase-3重组蛋白。重组蛋白经谷胱甘肽琼脂糖凝胶亲和层析纯化,再经PreScission进行酶切除去GST标签,并收集Caspase-3短肽。经SDS-PAGE电泳检测和Western blot鉴定,确认为目标蛋白Caspase-3截短肽表达正确,将蛋白浓度调整为5mg/mL备用。
按照50μg重组蛋白/只小鼠的剂量对6周龄雌性BALB/c小鼠进行免疫;首次免疫后第14天采用同样方法加强免疫一次;二免后第14天进行第三次免疫注射,方法与剂量同二免。三免后第10天对小鼠断尾采血,用间接ELISA方法监测血清抗体效价。当抗体水平达到要求后,于三免第14天后,选择效价最高的免疫小鼠,用纯的抗原液尾静脉加强免疫,3天后无菌采取小鼠脾细胞与SP2/0细胞融合制备得到杂交瘤细胞。取杂交瘤细胞株的培养上清,采用IsoStrip Mouse Monoclonal Antibody Isotyping试剂盒(Roche公司)测定抗体重链、轻链类型。
经过三轮免疫后,最终获得了Caspase-3单克隆抗体高表达单克隆细胞株,命名为4F-6。将单克隆细胞扩增后进行腹水制备纯化抗体,经试剂盒鉴定,所述Caspase-3单克隆抗体重链可变区序列如SEQ ID NO.2所示,轻链可变区序列如SEQ ID NO.3所示,所述重链可变区包括CDR-H1、CDR-H2、CDR-H3,所述CDR-H1-3的序列如SEQ ID NO.4-6所示,所述轻链可变区包括CDR-L1、CDR-L2、CDR-L3,所述CDR-L1-3的序列如SEQ ID NO.7-9所示。
采用非竞争ELISA方法检测Caspase-3单克隆抗体对SEQ ID NO.1多肽的亲和力。将SEQ ID NO.1多肽进行10倍稀释至0.01μg/mL,使用酶标仪读取OD450数值,检测结果显示,Caspase-3单克隆抗体对SEQ ID NO.1多肽的EC50可达到0.09956μg/mL,证明其具备较好的结合活性。
采用Caspase-7、Caspase-8、GST和BSA作为特异性抗原,通过western blot检测发现,Caspase-3单克隆抗体只与Caspase-3形成特异性条带,与其他均无条带产生,表明Caspase-3单克隆抗体具有较好的特异性。
实施例2脂肪间充质干细胞的分离制备
在无菌条件下,通过抽脂获取腹部脂肪组织20mL,经500rpm离心5min,去除上层油脂后,将底部脂肪颗粒沉淀转移至0.25% I型胶原酶溶液中,置于37℃持续振荡摇匀,消化60min后,加入等体积DMEM完全培养基进行终止消化。经500rpm离心5min,PBS清洗两次后加入2倍体积的红细胞裂解液,混匀后冰上放置20min,期间间隔5min涡旋一次,待红细胞渗透涨破。4500rpm离心5min,弃上清,PBS重悬细胞沉淀。将细胞悬液经100μm滤网过滤,分离出作为原代的脂肪间充质干细胞;经血球计数板计数后接种于T225培养瓶,加入DMEM完全培养液进行培养;首次传代后每3天更换一次培养基,传至第4代做免疫表型鉴定,通过流式细胞仪测定表面标记物CD13、CD29、CD31、CD34、CD45、CD44、CD73、CD90、CD105和HLA-DR的表达情况。结果显示脂肪间充质干细胞的CD73、CD90、CD105表达呈阳性,阳性率>99%;CD34、CD45和HLA-DR几乎不表达。
实施例3脂肪间充质干细胞的冷冻实验
实验组冻存保护剂:5%Caspase-3单克隆抗体、10%牛血清白蛋白、10%海藻糖、10%蔗糖、10%PEG,DMEM完全培养液中溶解混匀。
对照组冻存保护剂:10%牛血清白蛋白、10%海藻糖、10%蔗糖、10%PEG,DMEM完全培养液中溶解混匀。
收集第5代脂肪间充质干细胞,待其细胞贴壁生长融合率达到70-80%时,经胰蛋白酶消化,PBS清洗2遍后,在细胞沉淀中加入两倍体积的冻存保护剂。并采用分段降温法对脂肪间充质干细胞进行冻存(4℃下平衡20min,-20℃下平衡30min,-80℃下平衡12h,-196℃下存储)。
冻存细胞的复苏:分别将冻存1个月、5个月和10个月的样品复苏,每组3个重复,采用快速复温法,将冻存管至于37℃水浴箱中快速解冻,用DMEM完全培养基复苏,复苏率=复苏后活细胞的总数/冻存前活细胞总数×100%。细胞数量总计方法如下:取50μL细胞培养液和等体积的0.4%台盼蓝混合均匀后统计活细胞的数量。
表1冻存复苏率
组别 | 实验组 | 对照组 |
冻存前(%) | 98.63±1.21 | 98.63±1.21 |
1个月复苏率(%) | 97.89±1.65 | 93.45±1.53 |
5个月复苏(%) | 96.78±2.13 | 86.78±2.91 |
10个月复苏(%) | 94.59±2.45 | 79.65±2.69 |
结果如表1所示:实验组中经冻存1个月后的脂肪间充质干细胞的平均复苏率为97.89±1.65%,而对照组中的复苏率仅达到了93.45±1.53%,相比于对照组,实验组具有显著的提升效果,这充分说明本发明的Caspase-3单克隆抗体能够有效的抑制细胞在冻存过程中因为环境变化而激发的凋亡刺激,实现了细胞存活率提高的效果。且伴随着冻存试验的延长,本发明实验组的复苏效果逐渐显示其优势,相比于对照组的复苏率具有明显的提升,且差异显著,P<0.05。
实施例4冻存前后脂肪干细胞对成纤维细胞增殖的影响
采用MTT细胞增殖检测试剂盒(型号M1020,购自北京百奥创新科技有限公司)检测冻存前后脂肪干细胞对成纤维细胞HFF-1(货号ZB063,购自上海致备生物科技有限公司)的增殖影响。具体检测步骤可根据试剂盒说明书进行常规调整,其中,空白组为DMEM完全培养基,实验组含有经本发明实验组冻存保护剂冻存1个月后复苏的脂肪间充质干细胞的DMEM完全培养基,对照组含有经对照组冻存保护剂冻存1个月后复苏的脂肪间充质干细胞的DMEM完全培养基,实验组和对照组中的细胞用量均在5×103个。
表2成纤维细胞增殖分析
检测结果如下表2所示:空白组不能促进成纤维细胞的增殖,而采用本发明实验组冻存保护剂冻存1个月后复苏的脂肪间充质干细胞可以高效率地促进成纤维细胞进行增殖,且其促增殖效果明显优于对照组(P<0.05),差异显著,具有统计学意义。
实施例5冻存前后脂肪干细胞对成纤维细胞胶原蛋白分泌的影响
根据实施例4的分组情况,分别处理后的成纤维细胞HFF-1进行培养,培养24h后,收集细胞,通过ELISA试剂盒(货号EH7533/EH2866,购自武汉菲恩生物科技有限公司)检测I型和III型胶原分泌情况。结果如表3所示。
表3 I型和III型胶原分泌
组别 | I型胶原(ng/mL) | III型胶原(ng/mL) |
空白组 | 104±2.98 | 1.46±0.056 |
实验组 | 260±3.15 | 5.67±0.098 |
对照组 | 159±2.16 | 3.45±0.015 |
从表3的结果可以看出,采用本发明实验组冻存保护剂冻存1个月后复苏的脂肪间充质干细胞可以高效率地促进成纤维细胞分泌I型和III型胶原,该效果明显优于对照组(P<0.05),差异显著,具有统计学意义。
在此有必要指出的是,以上实施例仅限于对本发明的技术方案做进一步的阐述和说明,并不是对本发明的技术方案的进一步的限制,本发明的方法仅为较佳的实施方案,并非用于限定本发明的保护范围。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 (6)
1.一种脂肪间充质干细胞冻干粉,其特征在于,所述冻干粉是将脂肪间充质干细胞与冻存保护剂混合后冻干制备得到,其中所述冻存保护剂包括Caspase-3单克隆抗体、牛血清白蛋白、海藻糖、蔗糖和PEG;所述Caspase-3单克隆抗体的重链可变区包括CDR-H1、CDR-H2、CDR-H3,所述CDR-H1-3的序列如SEQ ID NO.4-6所示,轻链可变区包括CDR-L1、CDR-L2、CDR-L3,所述CDR-L1-3的序列如SEQ ID NO.7-9所示。
2.如权利要求1所述的冻干粉,其特征在于,所述Caspase-3单克隆抗体的重链可变区序列如SEQ ID NO:2所示,轻链可变区序列如SEQ ID NO:3所示。
3.如权利要求1所述的冻干粉,其特征在于,所述冻存保护剂是采用DMEM完全培养液溶解混匀。
4.如权利要求1所述的冻干粉,其特征在于,所述冻存保护剂包括5-15%Caspase-3单克隆抗体、2-20%牛血清白蛋白、2-20%海藻糖、2-20%蔗糖和2-20%PEG。
5.一种间充质干细胞冻干粉的制备方法,其特征在于,所述方法是将脂肪间充质干细胞与权利要求1-4任一项所述的冻存保护剂混合后冻干制备得到。
6.一种Caspase-3单克隆抗体,其特征在于,所述Caspase-3单克隆抗体的重链可变区包括CDR-H1、CDR-H2、CDR-H3,所述CDR-H1-3的序列如SEQ ID NO.4-6所示,轻链可变区包括CDR-L1、CDR-L2、CDR-L3,所述CDR-L1-3的序列如SEQ ID NO.7-9所示。
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