亚甲蓝对人髓核细胞毒性的CCK-8法检测

doi:10.3969/j.issn.2095-4344.0801

摘要/Abstract

摘要：

文章快速阅读：

文题释义：
CCK-8：CCK-8法使用酶联免疫检测仪测定细胞在450 nm波长处的光吸收值，计算细胞的活力或药物的毒性强度。该方法已被广泛用于一些生物活性因子的活性检测、抗肿瘤药物筛选、细胞增殖试验、细胞毒性试验以及药敏试验等。
MTT法：又称MTT比色法，是一种检测细胞存活和生长的方法。其检测原理为活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT还原为水不溶性的蓝紫色结晶甲瓒(Formazan)并沉积在细胞中，而死细胞无此功能。用酶联免疫检测仪在490 nm波长处测定其光吸收值，可间接反映活细胞数量。在一定细胞数范围内，MTT结晶形成的量与细胞数成正比。

摘要
背景：亚甲蓝在腰椎椎间孔镜手术中具有鉴别及显影退变椎间盘的作用，但是很多专家学者提出亚甲蓝会加速椎间盘的退变，然而国外文献研究发现亚甲蓝具有嗜酸性，推测对具有酸性环境的退变椎间盘具有治疗作用。因此，亚甲蓝对椎间盘是否具有毒性作用一直存在争议。
目的：使用CCK-8法测定亚甲蓝对髓核细胞是否具有毒性作用。
方法：选取2例椎间盘突出患者的废弃髓核组织，消化后提取髓核细胞，培养至细胞长满培养皿80%后消化细胞并制作细胞悬液，分6组接种到96孔板，一组为空白对照(只有培养基、CCK-8溶液而没有细胞)，一组为0加药对照组(只有培养基、细胞和CCK-8溶液)，其余分别加入1%，2%，3%，4%浓度的亚甲蓝干预细胞生长繁殖。在培养24，48，72，96 h后使用CCK-8法测定吸光度，计算细胞活力并观察每一组颜色变化。
结果与结论：0加药对照组颜色最深，亚甲蓝各浓度组随着亚甲蓝浓度升高浓度变浅，细胞活力0加药组活力最强，随着亚甲蓝浓度增高，活力下降。推测亚甲蓝对人髓核细胞具有一定的细胞毒性。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程
ORCID: 0000-0003-2435-0227(何升华)

关键词: 亚甲蓝, 人髓核细胞, 组织构建, 椎间盘突出症, CCK-8, 毒性研究

Abstract:

BACKGROUND: Methylene blue is used as a developer to identify intervertebral disc degeneration in the transforaminal endoscopic surgery. However, many scholars have indicated that methylene blue can accelerate the degeneration process, whilst foreign researches have reported that it may play therapeutic effect on degenerative intervertebral discs under acidic conditions due to its acidophily. Therefore, whether methylene blue holds toxic effect on the disc remains controversial.
OBJECTIVE: To determine whether methylene blue exerts toxic effect on nucleus pulposus cells by cell counting-kit 8 (CCK-8) assay.
METHODS: The discarded nucleus pulposus from two patients with intervertebral disc herniation were selected. After digestion, nucleus pulposus cells were extracted and cultured until proliferated to 80% of the medium. Then, the cells were digested to make cell suspensions, divided into six groups and inoculated into the 96-well plates: blank control (only the medium, CCK-8 solution), control group (only medium, cells and CCK-8 solution), and the other groups were cultured with 1%, 2%, 3%, and 4% methylene blue, respectively. The absorbance values were measured by CCK-8 assay at 24, 48, 72 and 96 hours after incubation to calculate the cell viability, and the color change was observed.
RESULTS AND CONCLUSION: The color in the control group was the deepest, and the color became lighter with the concentration of methylene blue increasing. The cell viability was the highest in the control group, and it was decreased with the concentration of methylene blue increasing. Thus, methylene blue may exert toxic effect on human nucleus pulposus cells.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Intervertebral Disk Displacement, Methylene Blue, Cytotoxicity, Immunologic, Tissue Engineering

中图分类号:

R318

何升华，冯华龙，孙志涛，赖居易，王业广，王建，黄飞强. 亚甲蓝对人髓核细胞毒性的CCK-8法检测[J]. 中国组织工程研究, 2018, 22(12): 1922-1927.

He Sheng-hua1, Feng Hua-long2, Sun Zhi-tao1, Lai Ju-yi2, Wang Ye-guang1, Wang Jian1, Huang Fei-qiang2. Toxicity of methylene blue to human nucleus pulposus cells detected by cell counting-kit 8 assay[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(12): 1922-1927.

图/表（结果） 3

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引言

材料方法

1.1 设计随机对照细胞学观察。

1.2 时间及地点 2016年9月至12月在深圳北京大学香港科技大学医学中心(许可证号：SYXK，粤2010-0106)完成。

1.3 材料

人髓核细胞：髓核组织来源于在深圳市中医院确诊为腰椎间盘突出症患者，其中L_4/5、L₅/S₁椎间盘突出各1例，由同一术者为其进行脊柱内镜手术并取其髓核组织。经患者知情同意，实验由深圳市中医院伦理委员会论证审核通过。

实验用主要试剂及仪器：亚甲蓝，CCK-8试剂盒，培养皿(上海晶安生物科技有限公司)，眼科剪(深圳市瑞沃德生命科技有限公司)，胎牛血清(Hyclone，Cat.No.SH30087.01)，RPMI-1640培养基(Hyclone，Cat.No.SH30809.01B)，青链霉素(Hyclone，Cat.No. SH30010)，PBS磷酸钾缓冲液(Hyclone，胰蛋白酶(Corning)，Cat.No.SH30256.01B)，96孔板细胞培养板(NEST，Cat.No.PPP-001-030)巴氏吸管(JET BIOFIL 3 mL)，苏州安泰洁净工作台(SW-CJ-IFD)，低速离心机(中佳，SC3614)，倒置光学显微镜(OLYMPUS CKX41, U-CTR30-2), 细胞恒温培养箱(Thermo scientific, HERACELL150i)，倒置荧光显微镜(生产商：Leica型号：DMI6000B)。

1.4 方法

1.4.1 实验分组实验设置3个分组，空白组：只有培养基和CCK溶液而没有细胞；0加药组：有细胞、CCK溶液而没有药物溶液；加药组：有细胞、CCK溶液和药物溶液，并将加药组按加药浓度1%，2%，3%，4%分为4个小组，每个小组3个复孔。

1.4.2 人髓核细胞的原代培养术中取出的髓核组织，放入培养基，快速转移至超净台(确保清洁并使用紫外线照射30 min以上)，将髓核组织转移至培养皿，使用含青链霉素的双抗PBS快速冲洗3次，使用眼科剪剪碎至1 mm× 1 mm×1 mm，带溶液转入15 mL离心管，离心，去上清，按体积1∶3加入0.25%胰蛋白酶消化30 min，离心去上清后按体积1∶6加入0.25%Ⅱ型胶原酶，消化2 h至基本消化完成，使用150目滤网过滤，将过滤液加入新的15 mL离心管，离心，去上清，加入含体积分数10%胎牛血清培养基，吹打均匀，转移至培养皿，放入37 ℃，体积分数5%CO₂培养箱中培养，每天倒置显微镜下观察，每2 d换液1次，待细胞贴壁并80%融合后，予以进行实验研究。

1.4.3 实验方法将上述细胞使用0.25%胰蛋白酶在 37 ℃，体积分数5%CO₂培养箱中消化两三分钟，在倒置显微镜下观察，轻拍培养皿壁使大部分细胞脱落，当细胞将要分离而呈现圆粒状时，加入适量含血清之新鲜培养基终止0.25%胰蛋白酶作用。离心，去上清，加入适量10%PBS以吸管上下吸放数次及轻轻吹打培养皿壁以打散细胞团块，混和均匀后，补足3n(n为传代孔数)mL培养基。按实验设计将细胞悬浮液转移至96孔板中(100 μL/孔)并标记组别，加药组按分组加入10 μL不同浓度的亚甲蓝，将96孔细胞培养板四周加入100 μL10%PBS防止细胞培养液挥发，放入37 ℃，体积分数5% CO₂培养箱中培养。在培养24，48，72，96 h后使用CCK-8法测定吸光度，计算细胞活力并观察每一组颜色变化。

1.4.4 CCK-8检测方法将培养板在培养箱孵育一段适当的时间(24，48，72，96 h)，取出后去除原培养液，并用10%PBS冲洗3遍后加入培养基100 μL，向每孔加入10 μL CCK-8溶液(注意不要再孔中生成气泡，它们会影响A值的读数)。将培养板在培养箱内孵育2 h。应用酶联免疫检测仪，在450 nm双波长处测得相应吸光度(A值)。

细胞活力(%)=[加药组-空白组]/[0加药组-空白组] ×100。

1.5 主要观察指标 ①各组不同时间段A值；②加入CCK-8试剂反应后的颜色变化；③细胞活力。

1.6 统计学分析数据以x(_)±s表示，采用SPSS 20.0统计分析，组间比较采用t 检验进行数据分析，检验水准为α=0.05，P < 0.05为差异并非由分组引起，具有统计学意义。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程