CN114107191B - 一种89Zr标记人脐带间充质干细胞的方法 - Google Patents
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Abstract
本发明公开了一种89Zr标记人脐带间充质干细胞hMSCs的方法，所述方法包含的步骤为：将待标记hMSCs细胞与前体89Zr‑oxine混合，孵育，得到89Zr‑hMSCs细胞。本发明的同位素细胞标记方法标记的效率更高，同位素流出率低，投入产出比更高，并降低DMSO了对细胞的影响。移植放射性同位素89Zr标记的hMSCs在体内分布定量检测时，灵敏度高，检测限低、可以微量检测。
Description
技术领域
本发明涉及核医学技术领域，具体涉及一种89Zr标记人脐带间充质干细胞的方法。
背景技术
人脐带间充质干细胞(hMSCs)是一种能分化成骨细胞、脂肪细胞和软骨细胞的多能干细胞。其具有强大的增殖能力，且参与构成造血微环境，因此被广泛应用于组织工程，细胞治疗和基因治疗。目前，人脐带间充质干细胞作为一个研究热点被广泛应用于再生医学和组织工程，特别是在骨、心血管和神经系统等疾病的领域。众所周知，干细胞治疗需要将干细胞移植到患者体内，因此确认干细胞在体内的迁移路径对判断干细胞移植的治疗效果至关重要。
目前常规的用于hMSCs体内分布定量研究的方法是解剖动物取样后用q-PCR的方式检测。但是该方法不够灵敏，检测限比较高，做不到微量检测。
放射性同位素标记技术能够在活体中对干细胞移植后组织分布和定量进行监测，该技术主要原理是移植携带放射性同位素的干细胞后用单光子发射计算机断层成像术(SPECT)或正电子发射体层扫描(PET)在体扫描，根据各组织器官的放射活性比例可以确定干细胞在体内的数量分布。
目前有111铟(111In)、99m锝(99mTc)和89锆(89Zr)等核素用于细胞标记研究。其中，89Zr作为常用的PET核素，半衰期为78.4小时，可以连续追踪细胞至少14天，非常适合用来标记细胞。
发明内容
本发明要解决的技术问题之一是，针对现有技术中hMSCs体内分布定量的测定方法存在灵敏度低、检测限高、需要解剖动物取样之后才能检测等缺点，提供了一种通过放射性同位素89Zr标记hMSCs，借助PET对hMSCs在体内的分布和迁移示踪，从而对其进行定量检测的方法。该方法检测灵敏度高，检测限低，可以活体成像。
本发明要解决的技术问题之二是，现有技术中使用的细胞同位素标记的方式存在标记效率低、同位素流出率高、DMSO对细胞的影响大等缺点。而且，现有技术中的细胞同位素标记的方式以实验员的手工操作为主，实验场景涉及化学合成、细胞培养等多种场景切换，时间漫长且难以做到方方面面的放射性防护。
针对上述技术问题，本发明提供的技术方案为：一种89Zr标记人脐带间充质干细胞hMSCs的方法，所述方法包含的步骤为：将待标记hMSCs细胞与前体89Zr-oxine混合，孵育，得到89Zr-hMSCs细胞。
在本发明一优选实施方案中，所述待标记hMSCs细胞与所述前体89Zr-oxine混合后，所得到的混合液中DMSO的体积百分比不超过总体积的0.5％。
如技术方案所述的方法，所述待标记hMSCs细胞的重悬方式可为本领域常规，例如，使用DPBS重悬，或者可用含蛋白的HEPES重悬。
在本发明一具体实施方案中，使用含1～5％(v/v)人血白蛋白的HEPES重悬。
本发明中所用1～5％(v/v)的人血白蛋白为使用10～20mM HEPES将20％(v/v)的人血白蛋白(本领域常规)稀释后得到。
优选地，使用10mM HEPES将20％(v/v)的人血白蛋白(本领域常规)稀释至1％(v/v)。
如技术方案所述的方法，所述孵育优选室温振荡孵育；所述振荡孵育的转速优选650rpm；所述振荡孵育的时间优选30min。
如本发明技术方案所述的方法，所述前体89Zr-oxine可为本领域常规，在本发明一优选实施方案中，所述前体89Zr-oxine的制备包括以下步骤：
(1)将pH 7.0～8.0的89Zr草酸溶液与溶于HEPES的8-羟基喹啉混匀；
(2)加入氯仿，混匀；
(3)分离出下层氯仿相，蒸发氯仿；
(4)用DMSO复溶，得到所述前体89Zr-oxine。
在步骤(1)中，所述89Zr草酸溶液的pH可为7.4。
在步骤(1)中，所述8-羟基喹啉的浓度可为1mg/mL。
在步骤(1)中，所述混匀为室温振荡混匀；所述振荡混匀的转速优选3000rpm；所述振荡混匀的时间优选30min。
在步骤(2)中，加入的氯仿与步骤(1)中的8-羟基喹啉优选为等体积。
在步骤(2)中，所述混匀可为室温振荡混匀；所述振荡混匀的转速优选3000rpm；所述振荡混匀的时间优选15min。
在步骤(3)中，所述蒸发氯仿的方式可为本领域常规，优选使用真空离心浓缩仪来蒸发氯仿。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明比现有技术主要的改进点在于：
①用HEPES缓冲液而不是氯仿来溶解8-羟基喹啉，一则控制了pH条件的细微差异对标记结果的影响，二则大大提高了89Zr和8-羟基喹啉的结合效率。
②用真空离心浓缩仪来蒸发氯仿，比起用氮气吹干的方法，缩小了最终干燥的89Zr-oxine的面积，使DMSO的用量减少。本发明DMSO的含量不超过0.5％，远低于现有技术中认为的DMSO含量不超过2％。
③在细胞标记时保持低速振荡，相比不振荡的标记率要高，且对细胞无损伤。
本发明的积极进步效果在于：
1.本发明的同位素细胞标记方法标记的效率更高，同位素流出率低，投入产出比更高，并降低DMSO了对细胞的影响。
2.本发明的同位素细胞标记方法由于更换了8-羟基喹啉的溶媒、并且在细胞标记时低速振荡孵育，加快了细胞结合前体的速度。
3.移植放射性同位素89Zr标记的hMSCs在体内分布定量检测时，灵敏度高，检测限低、可以微量检测。
4.移植放射性同位素89Zr标记的hMSCs在体内分布定量检测时，可以活体成像，大大节省动物，更符合动物伦理。
附图说明
图1为89Zr-oxine合成的示意图。
图2为89Zr-oxine标记人脐带间充质干细胞的示意图。
图3为SD大鼠静脉单次注射89Zr-hMSCs的PET图谱。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1 89Zr-oxine合成
1.取250μCi 89Zr-oxalate(PerkinElmer)，用1M Na2CO3调节pH至7.4左右，然后加入500μL 8-羟基喹啉(1mg/mL，溶于HEPES)，室温3000rpm振荡30min，其原理如图1所示。
2.加入500μL氯仿进行萃取，室温3000rpm振荡15min左右。
3.将下层的氯仿相分离出来，得到89Zr-oxine，用真空离心浓缩仪干燥氯仿，再用100μL DMSO复溶，完全溶解后加入DPBS稀释至200μL。
实施例2 89Zr-oxine标记人脐带间充质干细胞
4.收集2×106个人脐带间充质干细胞(常规来源)，用1mL含10mM HEPES的1％(v/v)人血白蛋白重悬，加入上述10μL 89Zr-oxine(DMSO含量不超过总体积的0.5％)，室温650rpm孵育30min，其原理如图2所示。
5.离心去除上清，然后用DPBS清洗标记细胞3次，得到89Zr标记的人脐带间充质干细胞。
6.标记前后用台盼蓝拒染法分别计算细胞存活率，标记前细胞存活率91％，标记后细胞存活率93.8％。说明标记对细胞存活率没有影响。
7.标记细胞以2×104/孔的密度接种于24孔板中，置于37℃、5％CO2的培养箱中培养，分别在24h、48h、96h的时间点收集上清，然后将细胞消化下来，分别检测上清和细胞的放射性。计算放射性的流出率分别为：36.6％(上清5339.72CPM，细胞9235.33CPM)、46.1％(上清5082.87CPM，细胞5944.59CPM)、56.4％(上清3917.39CPM，细胞3030.28CPM)，在接受范围内。
实施例3 SD大鼠单次静脉注射89Zr-hMSCs注射液后Micro-PET/MR显像
2只SD大鼠单次心肌注射89Zr-人脐带间充质干细胞注射液。给药剂量为1×106cells/只，分别于给药后24h、72h、120h、168h、216h和264h六个时间点进行Micro-PET/MR扫描，并用Pmod软件对Micro-PET/MR扫描数据进行处理。图3为该动物各时间点Micro-PET/MR扫描结果(Micro-PET/MR型号：BRUKER PET/MR 3T；Pmod版本号：4.104)。
对比例1 8-羟基喹啉溶媒的筛选
在实施例1的步骤1中，将8-羟基喹啉溶于氯仿，其余操作步骤与实施例1完全相同。
使用氯仿溶解的8-羟基喹啉，与89Zr结合需要持续反应2小时以上，且最终标记率30-70％。对比使用HEPES溶解8-羟基喹啉，反应只需30min，标记率就可以达到70％。
对比例2 DMSO的含量对于细胞生长的影响
分别用含0.12％、0.48％、1.2％DMSO的DPBS对细胞进行模拟标记，并将三组细胞分别孵育24h后用CCK-8法检测其增殖活力。用0.12％DMSO处理的细胞24h相对增殖活力100％，用0.48％DMSO处理的细胞24h相对增殖活力93.4％，用1.2％DMSO处理的细胞24h相对增殖活力44.5％。
对比例3低速振荡对细胞标记率的影响
在实施例2的步骤1中，细胞加入89Zr-oxine后室温不振荡孵育，其余操作步骤与实施例2完全相同。
不振荡1小时标记率10％，振荡30min标记率70％，在标记过程中加入振荡，可以使标记效率最高提高60％。
Claims (1)
1.一种89Zr标记人脐带间充质干细胞hMSCs的方法，其特征在于，所述方法包含的步骤为：
（I）收集2×106个人脐带间充质干细胞，用1 mL含10 mM HEPES的1%v/v人血白蛋白重悬，加入DMSO含量不超过总体积0.5%的10 μL的前体89Zr-oxine，室温650 rpm孵育30 min；
（II）离心去除上清，然后用DPBS清洗标记细胞3次，得到89Zr标记的人脐带间充质干细胞；
所述前体89Zr-oxine的制备包括以下步骤：
（1）取250 μCi 89Zr-oxalate，用1M Na2CO3调节pH至7.4，然后加入500 μL溶于HEPES的1mg/mL的8-羟基喹啉，室温3000 rpm振荡30 min；
（2）加入500 μL氯仿进行萃取，室温3000 rpm振荡15 min；
（3）将下层的氯仿相分离出来，得到89Zr-oxine，用真空离心浓缩仪干燥氯仿，再用100μL DMSO复溶，完全溶解后加入DPBS稀释至200 μL。
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