華　琨 , 聶　宇

(1. 首都醫(yī)科大學(xué)附屬北京安貞醫(yī)院 心外科, 北京, 100029;

2. 國家心血管病中心 心血管病國家重點(diǎn)實(shí)驗(yàn)室, 北京, 100037)

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構(gòu)建新型細(xì)胞增殖評價方法的實(shí)驗(yàn)研究

華琨1, 聶宇2

(1. 首都醫(yī)科大學(xué)附屬北京安貞醫(yī)院 心外科, 北京, 100029;

2. 國家心血管病中心 心血管病國家重點(diǎn)實(shí)驗(yàn)室, 北京, 100037)

摘要:目的建立胞質(zhì)分裂基因評分系統(tǒng)(CC-Score)。方法初步選擇10個胞質(zhì)分裂相關(guān)基因: Anln, Aurkb, Cenpa, Kif4, Kif23, Prc1, RhoA, Spin1, TACC2和CDC42。計(jì)算CC-Score評分。H9C2細(xì)胞分別接受GHRH、FGF-1、DMSO、IFN-α、AngII和Endothelin I處理，模擬細(xì)胞增殖促進(jìn)、抑制和細(xì)胞肥大的狀態(tài)。與傳統(tǒng)心肌細(xì)胞增殖評價方法(如MTT法、Brdu法、免疫熒光染色和流式細(xì)胞分析法)進(jìn)行比較。結(jié)果GHRH組、FGF-1組、DMSO組、IFN-α組、AngII處理組、Endothelin I處理組的CC-Score分值依次為+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)。結(jié)論CC-Score不僅能夠有效反映心肌細(xì)胞不同增殖狀態(tài)，而且能夠有效區(qū)分細(xì)胞增殖和肥大兩種不同的狀態(tài)。

關(guān)鍵詞:基因評分; 細(xì)胞增殖; 胞質(zhì)分裂; 細(xì)胞肥大

細(xì)胞周期相關(guān)基因評分(CCP-Score)是目前腫瘤學(xué)研究的熱點(diǎn)之一[1-5]。CCP-Score能夠很好地評估乳腺癌、肺癌和前列腺癌中腫瘤細(xì)胞的增殖情況，對于腫瘤術(shù)后復(fù)發(fā)和預(yù)后具有良好的預(yù)測作用。本研究建立胞質(zhì)分裂相關(guān)基因的評分(CC-Score)體系[6-10]，并將CC-Score與傳統(tǒng)評價心肌細(xì)胞增殖方法進(jìn)行比較，現(xiàn)報告如下。

1資料與方法

1.1實(shí)驗(yàn)細(xì)胞

H9C2細(xì)胞系，購于中國醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院基礎(chǔ)所細(xì)胞庫，細(xì)胞培養(yǎng)條件為37 ℃、5% CO2無菌恒溫培養(yǎng)箱，培養(yǎng)基成分: DMEM+10%FBS+1%青霉素+1%鏈霉素, pH值7.2～7.4。

1.2主要實(shí)驗(yàn)試劑

促生長激素釋放激素(GHRH)細(xì)胞因子、成纖維生長(FGF-1)細(xì)胞因子、干擾素-α(INF-α)、內(nèi)皮素I (endothelin I)、血管緊張素II(angiotensin II)、MTT試劑(Sigma)、DMEM、胎牛血清、Trizo(Invitrogen), 胰蛋白酶, Power SYBR Green PCR Master Mix(ABI), CELLBANKER ( ZENOAQ公司), PBS緩沖液(碧云天), Brdu ELISA試劑盒 (Roche), 辣根過氧化物酶標(biāo)記的二抗(CST), Ki-67 免疫熒光抗體(Abcam), TNT免疫熒光抗體(Abcam), H3P免疫熒光抗體(Millipore), DAPI染料(VECTOR)。

1.3主要實(shí)驗(yàn)設(shè)備

實(shí)時熒光定量 PCR儀(ABI-7300)(ABI), PCR擴(kuò)增儀(Bio-Rad公司), 紫外可見分光光度計(jì)(UV-1206)(SHIMADZU公司), 微量移液器(Eppendorf公司), 流式細(xì)胞檢測儀及 CellQuest 軟件( BD公司), 倒置相差/熒光顯微鏡(BX31)(Olympus公司), 恒溫水浴箱(SANYO 公司)。

1.4胞質(zhì)分裂基因的選取和胞質(zhì)分裂基因評分的制定方法

根據(jù)NCBI數(shù)據(jù)庫提供的信息[11-15], 選取10個與細(xì)胞胞質(zhì)分裂密切相關(guān)的基因: Prc1, RhoA, Cenpa, Kif4, Anln, Spin1, Aurkb, Kif23, CDC42和TACC2基因。

1.5統(tǒng)計(jì)學(xué)分析

應(yīng)用 SPSS 15.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析，組內(nèi)比較采用單因素方差分析(One-Way ANOVA), 均數(shù)間的兩兩比較采用LSD檢驗(yàn)，結(jié)果中的數(shù)據(jù)以均值±標(biāo)準(zhǔn)差表示。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2結(jié)果

2.1Real-Time PCR

Anln, GHRH組上調(diào)>7倍(P<0.05)和FGF-1組上調(diào)3倍(P<0.05)。DMSO組和IFN-α組均表達(dá)下調(diào)(P<0.05)，AngII和Endothelin I組未見顯著變化(P>0.05)。Aurkb在GHRH組和FGF-1組表達(dá)上調(diào)>4倍(P<0.05)，DMSO組和IFN-α組下調(diào)(P<0.05)，在AngII和Endothelin I組表達(dá)量輕微上調(diào)(P>0.05)。Cenpa在GHRH組表達(dá)上調(diào)4倍(P<0.05)，F(xiàn)GF-1組表達(dá)上調(diào)2倍(P<0.05)，DMSO組和IFN-α組表達(dá)量下調(diào)，在AngII和Endothelin I表達(dá)量未見顯著改變(P>0.05)。Kif4，GHRH組上調(diào)5倍(P<0.05)，F(xiàn)GF-1組表達(dá)量上調(diào)4倍(P<0.05), DMSO組和IFN-α組表達(dá)量均顯著下調(diào)(P<0.05)，同時在AngII和Endothelin I組表達(dá)量未見顯著改變(P>0.05)。同樣的表達(dá)量變化趨勢在KIF23基因的表達(dá)中也發(fā)現(xiàn)(P<0.05)。Prc1基因的表達(dá)在GHRH組>2倍的上升(P<0.05)，在FGF-1組表達(dá)量呈2倍增長(P<0.05), 在DMSO組和IFN-α組均表現(xiàn)為表達(dá)量顯著下降(P<0.05), 在AngII和Endothelin I表達(dá)量未見顯著改變(P>0.05)。RhoA和Spin1在GHRH組和FGF-1組表達(dá)量均出現(xiàn)顯著上調(diào)(P<0.05), 在DMSO組和IFN-α組表達(dá)量均出現(xiàn)顯著下降(P<0.05)。 TACC2在GHRH組表現(xiàn)為稍有上調(diào)，約1.5倍(P<0.05)。在DMSO組和IFN-α組表達(dá)量呈現(xiàn)顯著下調(diào)(P<0.05)。CDC42在GHRH組和FGF-1組顯著上調(diào)(P<0.05)，DMSO和IFN-α組表達(dá)量均顯著下調(diào)(P<0.05)，在AngII和Endothelin I表達(dá)量未見顯著改變(P>0.05)。

2.2胞質(zhì)分裂相關(guān)基因評分

GHRH組、FGF-1組、DMSO組、IFN-α組、AngII處理組、Endothelin I處理的CC-Score分值依次為+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結(jié)果提示: GHRH組增殖率為145%, FGF增殖率為170%。DMSO組增殖率為50%, IFN-α組增殖率為40%。AngII組增殖率為103%, Endothelin I組增殖率為102%，均無顯著差異(P>0.05)。Brdu提示: GHRH組增殖率為160%, FGF增殖率為190%。DMSO組增殖率為80%, IFN-α組增殖率為50%。AngII組增殖率為102%, Endothelin I組增殖率為101%，均無顯著差異(P>0.05)。細(xì)胞免疫熒光染色見圖1，流式細(xì)胞周期檢測見表1。

3討論

本研究選取了10個與心肌細(xì)胞胞質(zhì)分裂密切相關(guān)的基因，包括 Aurkb, Kif23, Prc1, TACC2, Spin1, RhoA, Cenpa, Kif4, Anln和CDC42基因。所有選取的基因都能調(diào)控心肌細(xì)胞胞質(zhì)分裂進(jìn)程，表達(dá)胞質(zhì)分裂需要的結(jié)構(gòu)蛋白，促進(jìn)完整細(xì)胞分裂正常進(jìn)行[16-19]。通過對這10個基因的整合制定出CC-Score評分，這種方法與以往單個增殖指標(biāo)相比，能夠更全面、綜合、準(zhǔn)確地反映細(xì)胞增殖的情況。CC-Score根據(jù)已有的公式計(jì)算簡單易行，Real-Time PCR檢測各基因的相對表達(dá)量變化也是相對簡單可行[20-21]。本研究首次將CC-Score評分應(yīng)用到細(xì)胞增殖評價，CC-Score也需要不斷調(diào)整和優(yōu)化，最終形成一種簡單、穩(wěn)定、準(zhǔn)確的細(xì)胞增殖評價方法。

圖1 免疫熒光染色細(xì)胞增殖評價

表1　流式細(xì)胞周期檢測結(jié)果

參考文獻(xiàn)

[1]Adler C P, Friedburg H. Myocardial DNA content, ploidy level and cell number in geriatric hearts: post-mortem examinations of human myocardium in old age[J].J Mol Cell Cardiol, 1986, 18(1): 39.

[2]Adler C P. Relationship between deoxyribonucleic acid content and nucleoli in human heart muscle cells and estimation of cell number during cardiac growth and hyperfunction[J]. Recent Adv Stud Cardiac Struct Metab, 1975, 8: 373.

[3]Laflamme M A, Murry C E. Heart regeneration[J]. Nature, 2011, 473(7347): 326.

[4]Bergmann O, Bhardwaj R D, Bernard S, et al. Evidence for cardiomyocyte renewal in humans[J]. Science, 2009, 324(5923): 98.

[5]Olivetti G, Cigola E, Maestri R, et al. Aging, cardiac hypertrophy and ischemic cardiomyopathy do not affect the proportion of mononucleated and multinucleated myocytes in the human heart[J]. J Mol Cell Cardiol, 1996, 28(7): 1463.

[6]van de Vijver M J, He Y D, van′t Veer L J, et al. A gene-expression signature as a predictor of survival in breast cancer[J]. N Engl J Med, 2002, 347(25): 1999.

[7]Pasumarthi K B, Field L J. Cardiomyocyte cell cycle regulation[J]. Circ Res, 2002, 90(10): 1044.

[8]Porrello E R, Mahmoud A I, Simpson E, et al. Transient regenerative potential of the neonatal mouse heart[J]. Science, 2011, 331(6020): 1078.

[9]Cuzick J, Swanson G P, Fisher G, et al. Prognostic value of an RNA expression signature derived from cell cycle proliferation genes in patients with prostate cancer: a retrospective study[J]. Lancet Oncol, 2011, 12(3): 245.

[10]Whitfield M L, Sherlock G, Saldanha A J, et al. Identification of genes periodically expressed in the human cell cycle and their expression in tumors[J]. Mol Biol Cell, 2002, 13(6): 1977.

[11]Zhang J, Liu B, Jiang X, et al. A systems biology-based gene expression classifier of glioblastoma predicts survival with solid tumors[J].PLoS ONE, 2009, 4(7): e6274.

[12]Lau S L, Yuen M L, Kou C Y, et al. Interferons induce the expression of IFITM1 and IFITM3 and suppress the proliferation of rat neonatal cardiomyocytes[J]. J Cell Biochem, 2012, 113(3): 841.

[13]Glotzer M. The molecular requirements for cytokinesis[J]. Science, 2005, 307(5716): 1735.

[14]Speir E, Tanner V, Gonzalez A M, et al. Acidic and basic fibroblast growth factors in adult rat heart myocytes.Localization, regulation in culture, and effects on DNA synthesis[J]. Circ Res, 1992, 71(2): 251.

[15]Walsh S, Pontén A, Fleischmann B K, et al.Cardiomyocyte cell cycle control and growth estimation in vivo-an analysis based on cardiomyocyte nuclei[J]. Cardiovasc Res, 2010, 86(3): 365.

[16]Murry C E, Reinecke H, Pabon L M. Regeneration gaps: observations on stem cells and cardiac repair[J]. J Am Coll Cardiol, 2006, 47(9): 1777.

[17]Watkins S J, Borthwick G M, Arthur H M. The H9C2 cell line and primary neonatal cardiomyocyte cells show similar hypertrophic responses in vitro[J]. In Vitro Cell Dev Biol Anim, 2011, 47(2): 125.

[18]Wei X, Song L, Jiang L, et al. Overexpression of MIP2, a novel WD-repeat protein, promotes proliferation of H9c2 cells[J]. Biochem Biophys Res Commun, 2010, 393(4): 860.

[19]Carvalho A B, de Carvalho A C. Heart regeneration: Past, present and future[J]. World J Cardiol, 2010, 2(5): 107.

[20]Olivetti G, Melissari M, Capasso J M, et al. Cardiomyopathy of the aging human heart.Myocyte loss and reactive cellular hypertrophy[J].Circ Res, 1991, 68(6): 1560.

[21]Whelan R S, Kaplinskiy V, Kitsis R N. Cell death in the pathogenesis of heart disease: mechanisms and significance[J]. Annu Rev Physiol, 2010, 72: 19.

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

中圖分類號:R 329.2

文獻(xiàn)標(biāo)志碼:A

文章編號:1672-2353(2016)07-018-03

DOI:10.7619/jcmp.201607005

收稿日期:2016-01-06