BrdU, CFSE和GFP標(biāo)記大鼠間充質(zhì)干細(xì)胞的比較

1、BrdU, CFSE和GFP標(biāo)記大鼠間充質(zhì)干細(xì)胞的比較         08-10-11 14:02:00     編輯：studa20           作者：付霞霏，何援利，楊芳，彭冬先，劉木彪   【摘要】  目的: 研究BrdU, CFSE和GFP作為大鼠間充質(zhì)干細(xì)胞標(biāo)記物的可行性. 方法: 分別用上述三種標(biāo)記物標(biāo)記大鼠間充質(zhì)干細(xì)胞，檢測(cè)

2、即時(shí)、15和30 d的標(biāo)記率，通過檢測(cè)細(xì)胞周期、凋亡率和描繪生長(zhǎng)曲線，來(lái)明確標(biāo)記物對(duì)體外培養(yǎng)大鼠間充質(zhì)干細(xì)胞生長(zhǎng)特性的影響；通過流式細(xì)胞儀檢測(cè)標(biāo)記物對(duì)細(xì)胞表面標(biāo)志（CD29, CD34, CD44, CD45）表達(dá)的影響. 結(jié)果: BrdU, CFSE, GFP的即時(shí)標(biāo)記率為77.0%, 99.4%, 89.7%，1 mo后分別下降至51.9%, 77.5%, 82.9%. 三者對(duì)體外培養(yǎng)的大鼠間充質(zhì)干細(xì)胞的生長(zhǎng)特性和表面標(biāo)志的表達(dá)均無(wú)明顯影響. 結(jié)論: GFP, BrdU和CFSE均可以作為大鼠間充質(zhì)干細(xì)胞的標(biāo)記物. 【關(guān)鍵詞】  綠色熒光蛋白；溴脫氧尿嘧啶核苷；熒光染料CFSE；

3、間充質(zhì)干細(xì)胞【Abstract】 AIM: To  investigate the feasibility of labeling of BrdU, CFSE and GFP as labels of rat mesenchymal stem cells (MSCs) and to investigate the influence of the 3 markers on the growth characteristics and expressions of surface markers of rat MSCs. METHODS: Wistar rats bone marrow

4、 derived MSCs were isolated and purified in vitro by Percoll density gradient centrifugation combined with adherent method and were labeled with BrdU, CFSE and GFP, respectively. The immediate, 15day and 30day labeling efficiency of the 3 markers was quantified. The labeling efficiency of BrdU was d

5、etected with immunocytochemical method, and that of CFSE and GFP was detected via flow cytometry (FCM). The labeled MSCs were observed under microscope. Cell cycle and apoptosis rate of labeled MSCs were revealed and the growth curves were delineated, so as to identify whether or not the markers cas

6、ted an influence on the cell growth characteristics in vitro. Furthermore, the surface markers (CD29, CD34, CD44, CD45)  of the labeled MSCs were detected through flow cytometry and were compared with those of nonlabeled MSCs. RESULTS: The immediate labeling efficiency of BrdU, CFSE, and GFP wa

7、s 77.0%, 99.4%,  89.7% respectively, and the efficiency decreased to 51.9%, 77.5%, and 82.9% respectively after 30 d. There was no statistical difference in cell cycle and apoptosis ratio between labeled MSCs and nonlabeled MSCs. The growth curves of labeled MSCs were similar to that of nonlabe

8、led MSCs. The 3 markers didnt influence the expressions of surface markers (CD29, CD34, CD44, CD45) of rat MSCs. CONCLUSION: The 3 markers have no effects on the growth characteristics and expressions of surface markers of rat MSCs cultured in vitro. BrdU, CFSE and GFP can serve as labeling markers

9、of rat MSCs. BrdU and CFSE labeling can be used for short time tracing, and GFP labeling for long time tracing.【Keywords】 GFP; BrdU; CFSE; MSCs0引言骨髓間充質(zhì)干細(xì)胞（mesenchymal stem cells, MSCs）是定居于骨髓微環(huán)境中的一種成體干細(xì)胞. MSCs不僅能分泌多種細(xì)胞因子調(diào)節(jié)造血細(xì)胞的增生與分化，還具有自我增殖、多向分化的潛能1. 因其采集方法簡(jiǎn)單、能在體外大量擴(kuò)增、可自體移植避免免疫排斥和倫理問題，已成為組織修復(fù)的理想種子細(xì)胞和

10、基因治療的靶細(xì)胞2. 而尋找合適的標(biāo)記方法用于追蹤觀察MSCs在實(shí)驗(yàn)研究中是很重要的. 目前常用的標(biāo)記方法包括：溴脫氧尿嘧啶（5bromodeoxyuridine, BrdU）、熒光染料（如二醋酸鹽琥珀酰亞胺酯，5,6carboxyfluorescein diacetate succinimidyl ester，CFSE）和綠色熒光蛋白（green fluorescent protein，GFP）標(biāo)記. 我們從標(biāo)記率、MSCs的生物學(xué)特性和表面標(biāo)志的表達(dá)等方面對(duì)這三種標(biāo)記方法進(jìn)行較全面的比較.1材料和方法1.1材料近交系SPF級(jí)68周齡Wistar大鼠20只，雌雄不拘，體質(zhì)量120150 g，

11、由廣東省動(dòng)物中心提供. DMEMF12培養(yǎng)基（Gibco公司），胎牛血清（Hyclone公司），CFSE（Sigma公司），BrdU（Sigma公司），小鼠抗BrdU抗體（Chemicon公司），山羊抗小鼠FITC（Santa Crutz公司），AdGFP（珠江醫(yī)院婦產(chǎn)科構(gòu)建），CCK8試劑盒（Dojindo公司），磷酸鹽緩沖液（PBS，武漢博士德公司）. CO2培養(yǎng)箱（Shell公司），熒光顯微鏡（Leica公司），流式細(xì)胞儀（Becton Dickinson公司）.1.2方法1.2.1大鼠MSCs的分離、培養(yǎng)及擴(kuò)增用100 g/L水合氯醛麻醉大鼠后，無(wú)菌條件下取出脛骨和股骨，制備單細(xì)胞懸液

12、，小心加入預(yù)先有密度為1.083的Percoll分離液的離心管內(nèi)，兩種液體體積比為11. 離心后，小心吸取中間界面乳白色的單個(gè)核細(xì)胞層，用PBS洗2次后加完全培養(yǎng)基（含100 mL/L胎牛血清的DMEM/F12培養(yǎng)基）重懸，接種培養(yǎng). 待細(xì)胞鋪滿瓶底的80%90%時(shí)，按12傳代. 本實(shí)驗(yàn)中選用生長(zhǎng)良好的第3代大鼠MSCs.1.2.2BrdU標(biāo)記方法及標(biāo)記率的檢測(cè)待細(xì)胞長(zhǎng)至約50%融合時(shí)，吸棄培養(yǎng)基，加入BrdU濃度為10 mol/L的培養(yǎng)基，置培養(yǎng)箱中孵育48 h. 棄去培養(yǎng)基，PBS清洗，以40 g/L多聚甲醛固定30 min. 再用PBS清洗3次后，加入10 g/L牛血清白蛋白，甩凈余液后

13、，加入小鼠的抗BrdU抗體，于濕盒中37孵育4 h，PBS清洗3次后，避光加入山羊抗小鼠FITC熒光抗體，避光孵育1 h，PBS清洗3次后，熒光顯微鏡下觀察細(xì)胞的即時(shí)標(biāo)記情況. 隨機(jī)選取5個(gè)視野，平均標(biāo)記率，即為BrdU的即時(shí)標(biāo)記率. 同批BrdU標(biāo)記的大鼠MSCs繼續(xù)培養(yǎng)、傳代，分別于15，30 d后再檢測(cè)標(biāo)記率.1.2.3CFSE標(biāo)記方法和標(biāo)記率檢測(cè)待大鼠MSCs長(zhǎng)至約鋪滿瓶底的80%時(shí)，加入CFSE，并使其終濃度為5 mol/L，置培養(yǎng)箱中繼續(xù)孵育40 min. PBS洗3次后，流式細(xì)胞儀檢測(cè)熒光標(biāo)記率，以未標(biāo)記的MSCs作為對(duì)照. 同批CFSE標(biāo)記的大鼠MSCs繼續(xù)培養(yǎng)、傳代，分別于15，30 d后再檢測(cè)標(biāo)記率.1.2.4AdGFP標(biāo)記方法和標(biāo)記率的檢測(cè)取病毒儲(chǔ)存液在HEK293細(xì)胞中反復(fù)擴(kuò)增，收集培養(yǎng)上清及細(xì)胞，反復(fù)凍溶破壞細(xì)胞離心取上清、過濾除菌，氯化銫密度梯度離心法進(jìn)行純化，空斑實(shí)驗(yàn)法測(cè)定轉(zhuǎn)染液病毒滴度為2×1013 pfuL. 待大鼠MSCs長(zhǎng)至約鋪滿瓶底的80%時(shí)，加入感染復(fù)數(shù)（multiplicity of infection，MOI）為100的AdGFP，并于12，24，48和72 h，于熒光顯微鏡下觀察標(biāo)記情況. GFP標(biāo)記72 h后，以未標(biāo)記的MSCs做對(duì)照，用流式細(xì)胞儀進(jìn)行熒光標(biāo)記率檢測(cè). 同批GFP標(biāo)記