构建新型细胞增殖评价方法的实验研究

华　琨 , 聂　宇

(1. 首都医科大学附属北京安贞医院 心外科, 北京, 100029;

2. 国家心血管病中心 心血管病国家重点实验室, 北京, 100037)



构建新型细胞增殖评价方法的实验研究

华琨1, 聂宇2

(1. 首都医科大学附属北京安贞医院 心外科, 北京, 100029;

2. 国家心血管病中心 心血管病国家重点实验室, 北京, 100037)

摘要:目的建立胞质分裂基因评分系统(CC-Score)。方法初步选择10个胞质分裂相关基因: Anln, Aurkb, Cenpa, Kif4, Kif23, Prc1, RhoA, Spin1, TACC2和CDC42。计算CC-Score评分。H9C2细胞分别接受GHRH、FGF-1、DMSO、IFN-α、AngII和Endothelin I处理，模拟细胞增殖促进、抑制和细胞肥大的状态。与传统心肌细胞增殖评价方法(如MTT法、Brdu法、免疫荧光染色和流式细胞分析法)进行比较。结果GHRH组、FGF-1组、DMSO组、IFN-α组、AngII处理组、Endothelin I处理组的CC-Score分值依次为+3.39 (P<0.01)、+3.22 (P<0.01)、-1.47 (P<0.01)、-0.93 (P<0.05)、+0.15 (P>0.05)和+0.09 (P>0.05)。结论CC-Score不仅能够有效反映心肌细胞不同增殖状态，而且能够有效区分细胞增殖和肥大两种不同的状态。

关键词:基因评分; 细胞增殖; 胞质分裂; 细胞肥大

细胞周期相关基因评分(CCP-Score)是目前肿瘤学研究的热点之一[1-5]。CCP-Score能够很好地评估乳腺癌、肺癌和前列腺癌中肿瘤细胞的增殖情况，对于肿瘤术后复发和预后具有良好的预测作用。本研究建立胞质分裂相关基因的评分(CC-Score)体系[6-10]，并将CC-Score与传统评价心肌细胞增殖方法进行比较，现报告如下。

1资料与方法

1.1实验细胞

H9C2细胞系，购于中国医学科学院北京协和医学院基础所细胞库，细胞培养条件为37 ℃、5% CO2无菌恒温培养箱，培养基成分: DMEM+10%FBS+1%青霉素+1%链霉素, pH值7.2～7.4。

1.2主要实验试剂

促生长激素释放激素(GHRH)细胞因子、成纤维生长(FGF-1)细胞因子、干扰素-α(INF-α)、内皮素I (endothelin I)、血管紧张素II(angiotensin II)、MTT试剂(Sigma)、DMEM、胎牛血清、Trizo(Invitrogen), 胰蛋白酶, Power SYBR Green PCR Master Mix(ABI), CELLBANKER ( ZENOAQ公司), PBS缓冲液(碧云天), Brdu ELISA试剂盒 (Roche), 辣根过氧化物酶标记的二抗(CST), Ki-67 免疫荧光抗体(Abcam), TNT免疫荧光抗体(Abcam), H3P免疫荧光抗体(Millipore), DAPI染料(VECTOR)。

1.3主要实验设备

实时荧光定量 PCR仪(ABI-7300)(ABI), PCR扩增仪(Bio-Rad公司), 紫外可见分光光度计(UV-1206)(SHIMADZU公司), 微量移液器(Eppendorf公司), 流式细胞检测仪及 CellQuest 软件( BD公司), 倒置相差/荧光显微镜(BX31)(Olympus公司), 恒温水浴箱(SANYO 公司)。

1.4胞质分裂基因的选取和胞质分裂基因评分的制定方法

根据NCBI数据库提供的信息[11-15], 选取10个与细胞胞质分裂密切相关的基因: Prc1, RhoA, Cenpa, Kif4, Anln, Spin1, Aurkb, Kif23, CDC42和TACC2基因。

1.5统计学分析

应用 SPSS 15.0软件进行统计学分析，组内比较采用单因素方差分析(One-Way ANOVA), 均数间的两两比较采用LSD检验，结果中的数据以均值±标准差表示。P<0.05为差异有统计学意义。

2结果

2.1Real-Time PCR

Anln, GHRH组上调>7倍(P<0.05)和FGF-1组上调3倍(P<0.05)。DMSO组和IFN-α组均表达下调(P<0.05)，AngII和Endothelin I组未见显著变化(P>0.05)。Aurkb在GHRH组和FGF-1组表达上调>4倍(P<0.05)，DMSO组和IFN-α组下调(P<0.05)，在AngII和Endothelin I组表达量轻微上调(P>0.05)。Cenpa在GHRH组表达上调4倍(P<0.05)，FGF-1组表达上调2倍(P<0.05)，DMSO组和IFN-α组表达量下调，在AngII和Endothelin I表达量未见显著改变(P>0.05)。Kif4，GHRH组上调5倍(P<0.05)，FGF-1组表达量上调4倍(P<0.05), DMSO组和IFN-α组表达量均显著下调(P<0.05)，同时在AngII和Endothelin I组表达量未见显著改变(P>0.05)。同样的表达量变化趋势在KIF23基因的表达中也发现(P<0.05)。Prc1基因的表达在GHRH组>2倍的上升(P<0.05)，在FGF-1组表达量呈2倍增长(P<0.05), 在DMSO组和IFN-α组均表现为表达量显著下降(P<0.05), 在AngII和Endothelin I表达量未见显著改变(P>0.05)。RhoA和Spin1在GHRH组和FGF-1组表达量均出现显著上调(P<0.05), 在DMSO组和IFN-α组表达量均出现显著下降(P<0.05)。 TACC2在GHRH组表现为稍有上调，约1.5倍(P<0.05)。在DMSO组和IFN-α组表达量呈现显著下调(P<0.05)。CDC42在GHRH组和FGF-1组显著上调(P<0.05)，DMSO和IFN-α组表达量均显著下调(P<0.05)，在AngII和Endothelin I表达量未见显著改变(P>0.05)。

2.2胞质分裂相关基因评分

GHRH组、FGF-1组、DMSO组、IFN-α组、AngII处理组、Endothelin I处理的CC-Score分值依次为+3.39 (P<0.01)、+3.22 (P<0.01)、-1.47(P<0.01)、-0.93 (P<0.05)、+0.15 (P>0.05)和+0.09 (P>0.05)。

2.3MTT法和Brdu法

MTT结果提示: GHRH组增殖率为145%, FGF增殖率为170%。DMSO组增殖率为50%, IFN-α组增殖率为40%。AngII组增殖率为103%, Endothelin I组增殖率为102%，均无显著差异(P>0.05)。Brdu提示: GHRH组增殖率为160%, FGF增殖率为190%。DMSO组增殖率为80%, IFN-α组增殖率为50%。AngII组增殖率为102%, Endothelin I组增殖率为101%，均无显著差异(P>0.05)。细胞免疫荧光染色见图1，流式细胞周期检测见表1。

3讨论

本研究选取了10个与心肌细胞胞质分裂密切相关的基因，包括 Aurkb, Kif23, Prc1, TACC2, Spin1, RhoA, Cenpa, Kif4, Anln和CDC42基因。所有选取的基因都能调控心肌细胞胞质分裂进程，表达胞质分裂需要的结构蛋白，促进完整细胞分裂正常进行[16-19]。通过对这10个基因的整合制定出CC-Score评分，这种方法与以往单个增殖指标相比，能够更全面、综合、准确地反映细胞增殖的情况。CC-Score根据已有的公式计算简单易行，Real-Time PCR检测各基因的相对表达量变化也是相对简单可行[20-21]。本研究首次将CC-Score评分应用到细胞增殖评价，CC-Score也需要不断调整和优化，最终形成一种简单、稳定、准确的细胞增殖评价方法。

图1 免疫荧光染色细胞增殖评价

表1　流式细胞周期检测结果

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Construction of a new evaluation method for cell proliferation

HUA Kun1, NIE Yu2

(1.DepartmentofCardiacSurgery,BeijingAnzhenHospitalAffiliatedtoCapitalMedicalUniversity,Beijing, 100029; 2.StateKeyLaboratoryofCardiovascularDiseases,NationalCenterforCardiovascularDiseases,Beijing, 100037)

ABSTRACT:ObjectiveTo construct cytokinesis gene scoring system (CC-Score). MethodsTen cytokinesis-correlated genes (Anln, Aurkb, Cenpa, Kif4, Kif23, Prc1, RhoA, Spin1, TACC2, and CDC42) were selected to establish the CC-Score. The H9C2 cells were processed by GHRH, FGF-1, DMSO, IFN-α, AngII and Endothelin I respectively to imitate state of cell proliferation, inhibition and hypertrophy. The results of the CC-Score were compared with those of the conventional methods such as MTT assay, Brdu assay, immunofluorescence staining and flow cytometry. ResultsThe results of CC-Score in the GHRH group, FGF-1 group, DMSO group, IFN-α group, Ang II group and Endothelin I group were +3.39 (P<0.01), +3.22 (P<0.01), -1.47 (P<0.01), -0.93(P<0.05), +0.15 (P>0.05) and +0.09 (P>0.05) respectively. ConclusionThe CC-Score can not only effectively reflect the different proliferations of cardiac muscle cells, but also can effectively distinguish different states between cell proliferations and hypertrophy.

KEYWORDS:gene scoring; proliferation; cytokinesis; hypertrophy

中图分类号:R 329.2

文献标志码:A

文章编号:1672-2353(2016)07-018-03

DOI:10.7619/jcmp.201607005

收稿日期:2016-01-06