CN111307775A - 一种应用刃天青试剂检测体外3d培养条件下pbmc活性的方法 - Google Patents
一种应用刃天青试剂检测体外3d培养条件下pbmc活性的方法 Download PDFInfo
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Abstract
本发明公开了一种应用刃天青试剂检测体外3D培养条件下PBMC活性的方法,具体步骤如下:步骤一、刃天青母液配制;步骤二、刃天青检测试剂配制;步骤三、体外3D培养条件下PBMC活性测定。本发明建立的标准化、可大规模化生产的用以体外检测人外周血单个核细胞在体外3D培养体系培养后活性的刃天青溶液,对细胞活性检测灵敏度高,操作便捷,对细胞无不良影响,对环境无污染,且经过实际测试证明该试剂适合于检测3D培养体系内PBMC的活性。
Description
技术领域
本发明涉及体外诊断技术领域,具体涉及一种应用刃天青试剂检测体外3D培养条件下PBMC活性的方法。
背景技术
体外细胞培养是指通过模拟机体内的生理条件,将从生物机体内取出的器官、组织或细胞等,在体外进行培养,并使其继续生存、生长和繁殖的过程。体外细胞培养已经成为现代生物研究的基本技术之一,广泛应用于现代生物医学和生物科学研究的各个方面。
目前,体外细胞水平的研究很大程度上是在二维培养条件下完成的。当细胞在二维条件下生长时,由于细胞与其基质之间复杂的细胞信号无法再现,细胞逐渐丧失了其体内原有的性状,在形态、结构和功能方面都与其在体内自然生长的状态相去甚远,因此体外实验数据无法完全转化为临床试验。近年来发展的3D(三维,three-dimensional)细胞培养技术则是应对这一挑战,作为一个更好的模型而更准确地体现体内生理条件,因此3D体外细胞培养方法获得的细胞在形态结构、增殖分化、基因表达及细胞的功能活动等方面均与二维培养存在显著不同。3D细胞培养能更好的模拟体内细胞生长的微环境,克服二维细胞培养方式的缺陷,为细胞水平的研究提供了一种更简单、更安全、更可靠的方法。
细胞在体外培养主要呈现悬浮型和贴壁型两种状态。其中贴附型细胞包括1、成纤维细胞型;2、上皮型细胞;3、游走细胞型;4、多型细胞型。悬浮型细胞见于少数特殊的细胞,如PBMC(外周血单个核细胞)、某些类型的癌细胞及白血病细胞。悬浮型细胞胞体呈圆形,不贴于支持物上,呈悬浮生长。与之对应,细胞体外培养也分为两大类如下:
一、贴壁培养法:指细胞贴附在一定的固相表面进行的培养。需要附着于带适量电荷的固体或半固体表面才能生长,大多数动物细胞,包括非淋巴组织细胞和许多异倍体细胞均属于这一类。其基本操作过程是:先将采集到的活体动物组织在无菌条件下采用物理(机械分散法)或化学(酶消化法)的方法分散成细胞悬液,经过滤、离心、纯化、漂洗后接种到加有适宜培养液的培养皿(瓶、板)中,再放入二氧化碳培养箱进行培养。用此法培养的细胞生长良好且易于观察,适于实验室研究。但贴壁生长的细胞有接触抑制的特性,一旦细胞形成单层,生长就会受到抑制,细胞产量有限。如要继续培养,还需将已形成单层的细胞再分散,稀释后重新接种,然后进行传代培养。
二、悬浮培养法:指细胞在反应器中自由悬浮生长的过程,使细胞始终处于分散悬浮于培养液内的培养方法。悬浮培养法是在微生物发酵的基础上发展起来的,主要用于非贴壁依赖型细胞培养,如人外周血单个核细胞(PBMC)、杂交瘤细胞等。该法将采集到的活体动物组织分散、过滤、离心、纯化、漂洗后接种到适宜的培养液中,并置于特定条件下进行自由悬浮培养。便于进行定量研究。适于悬浮培养的动物细胞种类很少,大多数动物细胞属于贴壁依赖性的,因此不能悬浮培养。
刃天青是一种呈靛青蓝色的染料,本身微弱荧光,在还原条件下可以不可逆地转化成一种粉红色荧光终产物试卤灵。刃天青具有Kreft二色性指数中已知的最高值之一,这意味着当观察到的样品的厚度或浓度增加或减少时,其感知色调的变化很大,其最大吸光度为573nm,荧光为激发光579nm/发射光584nm。刃天青检测原理为在细胞增殖过程中,细胞内NADPH/NADP、FADH/FAD、FMNH/FMN和NADH/NAD的比值升高,处于还原环境。摄入细胞内的刃天青被这些代谢中间体及细胞色素类还原成试卤灵后释放到细胞外并溶于培养基中,使培养基从无荧光的靛青蓝变成有荧光的粉红色。最后用普通分光光度计或荧光光度计进行检测,吸光度和荧光强度与活性细胞数成正比。
发明内容
鉴于PBMC作为悬浮型细胞,更适合于使用3D培养体系进行体外培养,但目前缺乏相应的检测PBMC在体外3D培养体系下活性的方法。本发明提供了一种应用刃天青试剂检测体外3D培养条件下PBMC活性的方法,能够针对性检测PBMC在3D培养基条件下活化、增殖情况。具体技术方案如下:
一种应用刃天青试剂检测体外3D培养条件下PBMC活性的方法,具体步骤如下:
步骤一、刃天青母液配制:取刃天青钠1g加200mL三蒸水,配制成终浓度为0.5%的溶液,使用孔径为0.22μm的一次性过滤器对刃天青溶液进行除菌,然后将除菌后的刃天青溶液放入4℃冰箱保存备用;
步骤二、刃天青检测试剂配制:根据实验需要,取适量的刃天青母溶液,加入一定量的三蒸水,配制成终浓度为0.15%-0.2%的刃天青溶液,即为刃天青检测试剂;
步骤三、体外3D培养条件下PBMC活性测定:按照3D培养基体积与刃天青检测试剂容积的比例为9:1,计算所需要的刃天青检测试剂使用量,向3D培养体系中加入刃天青检测试剂后,在不同时间段用酶标仪检测。
进一步地,步骤三中酶标仪检测采用荧光检测模式,激发光550nm/发射光590nm。
一种用于检测体外3D培养条件下PBMC活性的刃天青检测试剂,该试剂是浓度为0.15%-0.2%的刃天青溶液。
本发明首先配制刃天青检测试剂,然后将刃天青检测试剂定量加入含有PBMC的3D细胞培养体系中,通过不同时间点的检测,确定适合的检测时间。本发明所用刃天青检测试剂检测灵敏高,吸光度或荧光强度与细胞的活性和损伤成比例;操作便捷,刃天青钠盐为水溶性,因此配制和使用方便、快捷,只需均质的“加样-孵育-检测”即可,最短10min即可观察到结果;安全性高,刃天青细胞活性检测染料试剂对细胞不具毒性,可根据需要延长细胞孵育时间。
本发明提供的应用刃天青试剂检测体外3D培养条件下PBMC活性的方法,与现有技术相比具有以下有益效果:
1、与经典的[3H]-胸腺嘧啶核苷掺入法相比:刃天青对细胞和环境无不良影响,尤其是不需要处理放射性废物;与常用的基于MTT四唑盐检测方法相比:刃天青钠盐为水溶性,可以即时使用,短至10分钟即可检测结果;而MTT四唑盐为非水溶性,需要DMSO等有机溶剂助溶,而这些有机溶剂对细胞有一定的毒性,因此会影响检测结果,另一方面导致操作繁琐而增加检验过程时间,同时容易出错;
2、本发明建立的标准化、可大规模化生产的用以体外检测人外周血单个核细胞在体外3D培养体系培养后活性的刃天青溶液,对细胞活性检测灵敏度高,操作便捷,对细胞无不良影响,对环境无污染,且经过实际测试证明该试剂适合于检测3D培养体系内PBMC的活性。
附图说明
图1为不同时间点的PBMC在体外3D琼脂糖水凝胶细胞培养体系的检测结果。
图2为不同浓度的PBMC在体外3D琼脂糖水凝胶细胞培养体系的检测结果。
图3为不同时间点的PBMC在体外3D琼脂糖水凝胶细胞培养体系的检测结果(扣除空白对照)。
图4为不同浓度的PBMC在体外3D琼脂糖水凝胶细胞培养体系的检测结果(扣除空白对照)。
具体实施方式
为更进一步阐述本发明所采取的技术手段及其效果,以下结合本发明附图及优选实施例进行详细描述。
实施例1
一种应用刃天青试剂检测体外3D培养条件下PBMC活性的方法,具体步骤如下:
步骤一、刃天青母液配制:取刃天青钠1g加200mL三蒸水,配制成终浓度为0.5%的溶液,使用孔径为0.22μm的一次性过滤器对刃天青溶液进行除菌,然后将除菌后的刃天青溶液放入4℃冰箱保存备用;
步骤二、刃天青检测试剂配制:根据实验需要,取适量的刃天青母溶液,加入一定量的三蒸水,配制成终浓度为0.15%的刃天青溶液,即为刃天青检测试剂;
步骤三、体外3D培养条件下PBMC活性测定:按照3D琼脂糖水凝胶培养基体积与刃天青检测试剂容积的比例为9:1,计算所需要的刃天青检测试剂使用量,向3D琼脂糖水凝胶培养体系中加入刃天青检测试剂后,在不同时间段用酶标仪检测,酶标仪采用荧光检测模式,激发光550nm/发射光590nm。
进一步地,所述3D琼脂糖水凝胶培养基各组分及其含量如下:1640培养基液10mg/mL,胎牛血清10-20v/v%,琼脂糖1-1.5wt%。
实施例2
一种应用刃天青试剂检测体外3D培养条件下PBMC活性的方法,具体步骤如下:
步骤一、刃天青母液配制:取刃天青钠1g加200mL三蒸水,配制成终浓度为0.5%的溶液,使用孔径为0.22μm的一次性过滤器对刃天青溶液进行除菌,然后将除菌后的刃天青溶液放入4℃冰箱保存备用;
步骤二、刃天青检测试剂配制:根据实验需要,取适量的刃天青母溶液,加入一定量的三蒸水,配制成终浓度为0.17%的刃天青溶液,即为刃天青检测试剂;
步骤三、体外3D培养条件下PBMC活性测定:按照3D甲基纤维素水凝胶培养基体积与刃天青检测试剂容积的比例为9:1,计算所需要的刃天青检测试剂使用量,向3D甲基纤维素水凝胶培养体系中加入刃天青检测试剂后,在不同时间段用酶标仪检测,酶标仪检测采用荧光检测模式,激发光550nm/发射光590nm。
进一步地,所述3D甲基纤维素水凝胶培养基各组分及其含量如下:1640培养基液10mg/mL,胎牛血清10-20v/v%,甲基纤维素1-1.5wt%。
实施例3
一种应用刃天青试剂检测体外3D培养条件下PBMC活性的方法,具体步骤如下:
步骤一、刃天青母液配制:取刃天青钠1g加200mL三蒸水,配制成终浓度为0.5%的溶液,使用孔径为0.22μm的一次性过滤器对刃天青溶液进行除菌,然后将除菌后的刃天青溶液放入4℃冰箱保存备用;
步骤二、刃天青检测试剂配制:根据实验需要,取适量的刃天青母溶液,加入一定量的三蒸水,配制成终浓度为0.2%的刃天青溶液,即为刃天青检测试剂;
步骤三、体外3D培养条件下PBMC活性测定:按照3D胶原蛋白水凝胶培养基体积与刃天青检测试剂容积的比例为9:1,计算所需要的刃天青检测试剂使用量,向3D胶原蛋白水凝胶培养体系中加入刃天青检测试剂后,在不同时间段用酶标仪检测,酶标仪检测采用荧光检测模式,激发光550nm/发射光590nm。
进一步地,所述3D胶原蛋白水凝胶培养基各组分及其含量如下:1640培养液10mg/ml,胎牛血清10-20v/v%,甲基纤维素1-1.5wt%,胶原蛋白1.5-2.5mg/mL。
实施例1采用本发明提供的应用刃天青试剂检测方法对体外琼脂糖浓度为0.5wt%的3D琼脂糖水凝胶培养条件下PBMC活性进行检测。图1、图2分别为不同时间点、不同浓度的PBMC在体外3D琼脂糖水凝胶细胞培养体系的检测结果;图3、图4分别为扣除空白对照后不同时间点、不同浓度的PBMC在体外3D琼脂糖水凝胶细胞培养体系的检测结果。
结果表明本发明涉及的刃天青检测试剂能够在3D体外培养基检测到PBMC活性,而且检测的荧光值与细胞浓度呈正相关;由于刃天青为水溶性物质,在3D体外培养基中扩散和渗透入细胞并发生还原反应,需要的时间较水溶液体系时间长,因此检测适宜时间确定在4-6小时。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,本领域普通技术人员对本发明的技术方案所做的其他修改或者等同替换,只要不脱离本发明技术方案的精神和范围,均应涵盖在本发明的权利要求范围当中。
Claims (3)
1.一种应用刃天青试剂检测体外3D培养条件下PBMC活性的方法,其特征在于,具体步骤如下:
步骤一、刃天青母液配制:取刃天青钠1g加200mL三蒸水,配制成终浓度为0.5%的溶液,使用孔径为0.22μm的一次性过滤器对刃天青溶液进行除菌,然后将除菌后的刃天青溶液放入4℃冰箱保存备用;
步骤二、刃天青检测试剂配制:根据实验需要,取适量的刃天青母溶液,加入一定量的三蒸水,配制成终浓度为0.15%-0.2%的刃天青溶液,即为刃天青检测试剂;
步骤三、体外3D培养条件下PBMC活性测定:按照3D培养基体积与刃天青检测试剂容积的比例为9:1,计算所需要的刃天青检测试剂使用量,向3D培养体系中加入刃天青检测试剂后,在不同时间段用酶标仪检测。
2.根据权利要求1所述的应用刃天青试剂检测体外3D培养条件下PBMC活性的方法,其特征在于,步骤三中酶标仪检测采用荧光检测模式,激发光550nm/发射光590nm。
3.一种用于检测体外3D培养条件下PBMC活性的刃天青检测试剂,其特征在于,该试剂是浓度为0.15%-0.2%的刃天青溶液。
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