蔡振陸 馮韻然 李星星 李牧 呂海俠
[摘要] 目的 探討綠茶水浸出物對(duì)發(fā)育期神經(jīng)細(xì)胞存活和增殖的影響。 方法 選取PC12細(xì)胞作為毒理學(xué)研究的模型,將綠茶于去離子水中100℃連續(xù)煮沸3次,留取各次煮沸后的水溶液進(jìn)行過濾及冷凍干燥,繼而以40倍體積無菌純水溶解,制備綠茶水浸出物。以DMEM培養(yǎng)基對(duì)綠茶水浸出物進(jìn)行100、200、400、800和1600倍稀釋,用于處理PC12細(xì)胞。通過觀察細(xì)胞形態(tài)、繪制細(xì)胞生長(zhǎng)曲線、CCK-8試劑盒檢測(cè)細(xì)胞活力、流式細(xì)胞儀檢測(cè)分析細(xì)胞凋亡,Ki67免疫細(xì)胞化學(xué)染色觀察細(xì)胞增殖,分析綠茶水浸出物對(duì)PC12細(xì)胞的影響。 結(jié)果 與低濃度(800倍及1600倍稀釋)綠茶比較,較高濃度(100稀釋)綠茶處理后,PC12細(xì)胞貼壁能力下降,死細(xì)胞碎屑明顯增多,活細(xì)胞數(shù)量及細(xì)胞活力較對(duì)照組明顯下降(P < 0.05),Ki67陽性細(xì)胞比例顯著降低(P < 0.05),但細(xì)胞凋亡比率與對(duì)照組比較,差異無統(tǒng)計(jì)學(xué)意義(P > 0.05)。 結(jié)論 高濃度綠茶對(duì)PC12細(xì)胞的存活和增殖有顯著抑制作用,建議孕期盡量減少飲用濃茶。
[關(guān)鍵詞] 綠茶水浸出物;PC12細(xì)胞;存活;凋亡;增殖
[中圖分類號(hào)] R715.3 [文獻(xiàn)標(biāo)識(shí)碼] A [文章編號(hào)] 1673-7210(2018)05(c)-0004-04
Effect of water extraction of green tea on survival and proliferation of PC12 cells
CAI Zhenlu1 FENG Yunran2 LI Xingxing1 LI Mu3▲ LV Haixia1
1.Institute of Neurobiology, Xi′an Jiaotong University Health Science Center, Shaanxi Province, Xi′an 710061, China;2.the Affiliated Middle School of Shaanxi Normal University, Shaanxi Province, Xi′an 710061, China;3.Department of Obstetrics and Gynecology, the Second Hospital of Xi′an Jiaotong University, Shaanxi Province, Xi′an 710004, China
[Abstract] Objective To investigate the effect of green tea on the survival and proliferation of neural cells during development. Methods PC12 cells were selected as the cell model for toxicological analysis, green tea leaves were boiled in distill water of 100℃ for three times, tea solution was then filtered and freeze dried. The powder was dissolved with sterilized pure water. DMEM medium was applied to make different concentrations of tea extraction (diluer for 100, 200 400, 800, and 1600 times) before use. The morphology of PC12 cells was observed, cell viability and apoptosis were detected by CCK-8 assay and flow cytometry assay after treatment. The proliferation of PC12 was detected by Ki67 immunostaining. Results Compared with low concentration (diluer 800 and 1600 times), after treated with green tea extraction in high concentration, PC12 cell morphology changed dramatically and cell viability was decreased significantly (P < 0.05). Proliferation of PC12 also declined dramatically, as showed by the reduced number of Ki67+ cells (P < 0.05). While the apoptosis of PC12 had no statistically significant difference compared with control group (P > 0.05). Conclusion Green tea in high concentration, so it is recommended to reduce the drinking of strong green teadurn pregancy could reduce the survival and proliferation of PC12 cells
[Key words] Water extraction of green tea; PC12 cells; Survival; Apoptosis; Proliferation
中國(guó)傳統(tǒng)的茶文化源遠(yuǎn)流長(zhǎng),茶水也已成為中國(guó)人群普遍接受的飲品。茶葉中含有茶多酚、茶多糖、茶蛋白、生物堿等成分,已被證實(shí)具有抗氧化、抗癌等功效[1-6],備受食品界和醫(yī)學(xué)界的重視。然而妊娠期婦女是否可以飲茶?茶水對(duì)胚胎發(fā)育尤其是神經(jīng)系統(tǒng)發(fā)育會(huì)產(chǎn)生怎樣的影響?一直是困擾婦產(chǎn)科醫(yī)生和妊娠婦女的難題。有實(shí)驗(yàn)證實(shí)綠茶有對(duì)抗神經(jīng)損傷的作用[7-8],然而Ye團(tuán)隊(duì)[9]對(duì)北方人群飲茶和胎兒神經(jīng)管畸形的發(fā)生率之間的關(guān)系調(diào)查結(jié)果證實(shí),圍妊娠期間持續(xù)每日飲茶者其胎兒發(fā)生神經(jīng)管缺陷的風(fēng)險(xiǎn)明顯增高。究竟飲用茶水是否會(huì)影響神經(jīng)系統(tǒng)的發(fā)育?茶水會(huì)對(duì)神經(jīng)細(xì)胞產(chǎn)生怎樣的影響仍值得探討。
綠茶屬于不發(fā)酵茶葉,已經(jīng)被證實(shí)在抗氧化應(yīng)激損傷以及抗腫瘤等方面發(fā)揮重要的作用[1-4,6-8]。本研究選取綠茶作為研究對(duì)象,以開水煮沸的方式模擬日常生活中的茶葉沖泡過程。選取大鼠嗜鉻細(xì)胞瘤系PC12細(xì)胞為神經(jīng)毒理研究模型,觀察不同濃度的綠茶處理后細(xì)胞的存活、凋亡以及增殖情況,分析飲茶對(duì)發(fā)育期神經(jīng)系統(tǒng)的可能影響,以期為指導(dǎo)臨床實(shí)踐提供實(shí)驗(yàn)依據(jù)。
1 材料與方法
1.1 實(shí)驗(yàn)材料與實(shí)驗(yàn)試劑
PC12細(xì)胞系夠自美國(guó)ATCC細(xì)胞庫,由西安交通大學(xué)醫(yī)學(xué)部神經(jīng)生物研究所保存。綠茶為市售流行品牌。胰蛋白酶、DMEM(高糖)培養(yǎng)基、胎牛血清((FBS))購(gòu)自Gibco公司;CCK-8試劑盒購(gòu)自七海復(fù)泰生物科技有限公司;小鼠抗大鼠Ki67單克隆抗體購(gòu)自Abcam公司(1∶500)、TRITC標(biāo)記二抗購(gòu)自北京中杉金橋生物科技有限公司;Hochest33258染液及DAPI購(gòu)自中國(guó)碧云天生物科技有限公司。磷酸鹽緩沖液(PBS)以及4%多聚甲醛由本實(shí)驗(yàn)室配制。
1.2 實(shí)驗(yàn)方法
1.2.1 不同濃度茶葉水浸出物制備 稱取綠茶干茶葉5.0 g,加入200 mL去離子水,100℃條件下煮沸30 min,每5分鐘晃動(dòng)1次。連續(xù)提取3次后靜置,待顆粒物沉淀,取上清液進(jìn)行過濾,并于40℃條件下真空干燥。使用前以50 mL滅菌去離子水復(fù)溶,取200 μL原液稀釋至1 mL,以0.22 μm微孔濾膜過濾,以DMEM培養(yǎng)基進(jìn)行100、200、400、800和1600倍稀釋,用于處理PC12細(xì)胞。
1.2.2 PC12細(xì)胞培養(yǎng)及處理 按常規(guī)方法將PC12細(xì)胞培養(yǎng)于含10% FBS的DMEM培養(yǎng)液中,5% CO2、37℃條件下培養(yǎng),待細(xì)胞生長(zhǎng)至80%~90%匯合時(shí)用0.25%胰蛋白酶消化傳代。傳代細(xì)胞進(jìn)入對(duì)數(shù)生長(zhǎng)期后,于培養(yǎng)環(huán)境中加入不同濃度的綠茶水浸出物,對(duì)照組添加等量培養(yǎng)基。
1.3 PC12細(xì)胞活力檢測(cè)
PC12細(xì)胞傳代接種于96孔培養(yǎng)板,每孔3000個(gè)細(xì)胞,對(duì)進(jìn)入對(duì)數(shù)生長(zhǎng)期的PC12細(xì)胞給予不同濃度的綠茶水浸出物處理。倒置相差顯微鏡下觀察細(xì)胞形態(tài),連續(xù)進(jìn)行細(xì)胞計(jì)數(shù)并繪制生長(zhǎng)曲線;與此同時(shí),于處理后不同時(shí)間點(diǎn),每孔加入10 μL的CCK-8溶液,37℃孵育50 min,用BIO-RAD680型酶標(biāo)儀(美國(guó))測(cè)定波長(zhǎng)450 nm處的吸光度(OD值)。參考以下公式計(jì)算細(xì)胞存活率:細(xì)胞存活率=(實(shí)驗(yàn)組OD-空白組OD)/(對(duì)照組OD-空白組OD)×100%。每組實(shí)驗(yàn)至少重復(fù)3次。
1.4 PC12細(xì)胞凋亡及增殖能力檢測(cè)
同上處理PC12細(xì)胞,以流式細(xì)胞儀檢測(cè)細(xì)胞凋亡情況;部分細(xì)胞傳代于預(yù)先經(jīng)過多聚賴氨酸包被的玻片,置96孔培養(yǎng)板繼續(xù)培養(yǎng)3 d后進(jìn)行Ki76免疫細(xì)胞化學(xué)染色,觀察細(xì)胞增殖情況。
1.5 免疫細(xì)胞化學(xué)染色
PC12細(xì)胞以4%多聚甲醛室溫固定20 min,固定后細(xì)胞經(jīng)PBS洗滌3次,以5%正常山羊血清封閉,甩掉不洗,繼而與1∶500稀釋的單克隆Ki67抗體室溫孵育30 min后于4℃過夜,次日經(jīng)室溫復(fù)溫2 h、PBS洗滌3次后滴加TRITC標(biāo)記二抗,室溫避光孵育2 h,以含有DAPI的封片劑進(jìn)行封片,熒光顯微鏡下觀察并計(jì)數(shù)陽性細(xì)胞。陰性對(duì)照組以含5%正常山羊血清的PBS代替一抗,分別選取3張染色后玻片,每張玻片計(jì)數(shù)3個(gè)視野,計(jì)算陽性細(xì)胞占細(xì)胞總數(shù)百分比。每組實(shí)驗(yàn)至少重復(fù)3次。
1.6 統(tǒng)計(jì)學(xué)方法
采用SPSS 13.0統(tǒng)計(jì)學(xué)軟件進(jìn)行數(shù)據(jù)分析,計(jì)量資料用均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,兩組間比較采用t檢驗(yàn),以P < 0.05為差異有統(tǒng)計(jì)學(xué)意義。
2 結(jié)果
2.1 綠茶水浸出物對(duì)PC12細(xì)胞生長(zhǎng)的影響
正常培養(yǎng)條件下,PC12以貼壁狀態(tài)生長(zhǎng),細(xì)胞輪廓清晰,胞體和突起折光性強(qiáng);低濃度(800倍稀釋)綠茶水浸出物處理后細(xì)胞形態(tài)變化不大,而高濃度(100倍稀釋)綠茶水浸出物處理后細(xì)胞貼壁能力下降,死細(xì)胞碎屑明顯增多,與對(duì)照組比較細(xì)胞數(shù)量顯著減少(圖1);生長(zhǎng)曲線顯示高濃度綠茶水浸出物處理后PC12細(xì)胞數(shù)量隨培養(yǎng)時(shí)間的延長(zhǎng)沒有明顯增加(圖2),與對(duì)照組比較,差異有統(tǒng)計(jì)學(xué)意義(P < 0.01)。
2.2 綠茶水浸出物對(duì)PC12細(xì)胞活力的影響
以不同濃度綠茶水浸出物處理PC12細(xì)胞,CCK-8檢測(cè)結(jié)果顯示:高濃度綠茶(0~100倍稀釋)浸出物處理細(xì)胞后,細(xì)胞活力[OD值:0.30±0.01)]與對(duì)照組(0.50±0.08)比較顯著下降,差異有統(tǒng)計(jì)學(xué)意義(P < 0.05)。相對(duì)而言,以中、低濃度綠茶(200~800倍稀釋)處理PC12細(xì)胞,細(xì)胞活力(0.46±0.08、0.45±0.07、0.48±0.06)與對(duì)照組比較無明顯變化,差異無統(tǒng)計(jì)學(xué)意義(P > 0.05)(圖3)。
2.3 綠茶水浸出物對(duì)PC12細(xì)胞凋亡的影響
流式細(xì)胞儀檢測(cè)不同濃度的綠茶水浸出物處理后PC12細(xì)胞凋亡情況,結(jié)果顯示:對(duì)照組細(xì)胞凋亡比率為(3.7±0.59)%,高濃度綠茶水浸出物處理后PC12細(xì)胞凋亡率為(5.24±1.05)%,兩組比較差異無統(tǒng)計(jì)學(xué)意義(P > 0.05)。見圖4。
2.4 綠茶水浸出物對(duì)PC12細(xì)胞增殖的影響
免疫細(xì)胞化學(xué)染色的方法觀察茶葉水浸出物處理后細(xì)胞增殖的變化,結(jié)果顯示:高濃度綠茶水處理組(圖5B,封四),Ki67陽性細(xì)胞(正在增殖的細(xì)胞)比率為(33.5±2.89)%,與對(duì)照組[(79.8±3.95)%,圖5A,封四]比較顯著下降,差異有統(tǒng)計(jì)學(xué)意義(P < 0.05)。
3 討論
茶葉中富含多種營(yíng)養(yǎng)成份,本實(shí)驗(yàn)選用未發(fā)酵的綠茶,以煮沸方式獲取其浸出物,能更好地模擬日常生活中的茶葉浸泡。已經(jīng)證實(shí)未發(fā)酵茶葉經(jīng)過熱水浸泡后其浸出物以茶多酚和嘌呤類的生物堿為主,而茶多酚有明確的神經(jīng)保護(hù)作用,可以顯著減輕谷氨酸對(duì)PC12細(xì)胞的興奮性神經(jīng)毒性[10-13],對(duì)于母親患糖尿病引起的神經(jīng)管畸形有緩解作用[8]。本實(shí)驗(yàn)為了更好地模擬正常妊娠婦女飲茶,未對(duì)細(xì)胞做損傷性處理,即重點(diǎn)觀察綠茶水浸出物對(duì)正常培養(yǎng)的PC12細(xì)胞的影響,具有較高的指導(dǎo)意義。
PC12細(xì)胞系源于大鼠嗜鉻細(xì)胞瘤,已被廣泛用于神經(jīng)系統(tǒng)細(xì)胞模型[12-20]。本研究觀察不同濃度綠茶水浸出物對(duì)PC12細(xì)胞存活、凋亡以及增殖等生物學(xué)行為的影響,用于分析綠茶對(duì)發(fā)育中神經(jīng)系統(tǒng)細(xì)胞產(chǎn)生的可能影響。Menezes等[7]的研究證明飲茶可以緩解動(dòng)物因母嬰分離而引起的認(rèn)知功能障礙,發(fā)揮積極的作用,而Ye等[9]的研究認(rèn)為茶葉中的茶酸成分可能通過抑制二氫葉酸還原酶的活性而干擾葉酸的代謝,并抑制葉酸在腸道的吸收,從而引起神經(jīng)管發(fā)育異常。發(fā)育期大腦神經(jīng)細(xì)胞對(duì)外界因素的影響極為敏感,綠茶中的多種成分可能通過不同的途徑影響神經(jīng)細(xì)胞的行為[1,4,12,16],從而對(duì)神經(jīng)發(fā)育產(chǎn)生影響。本研究結(jié)果顯示當(dāng)高濃度綠茶水浸出物處理PC12細(xì)胞后,細(xì)胞活力以及增殖能力顯著下降,而凋亡細(xì)胞比率無顯著性差異,相對(duì)來講,淡茶水對(duì)PC12細(xì)胞行為的影響和對(duì)照組比較,差異無統(tǒng)計(jì)學(xué)意義。上述結(jié)果提示孕期婦女如果飲用高濃度綠茶,茶多酚可能通過影響神經(jīng)細(xì)胞的存活和增殖從而影響神經(jīng)系統(tǒng)的正常發(fā)育,必須謹(jǐn)慎。相對(duì)而言,低濃度綠茶對(duì)細(xì)胞的存活和增殖無明顯的影響,相對(duì)安全。因此,我們建議妊娠婦女在孕期盡量減少茶水的飲用,如果需要,盡可能以淡茶為主。
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(收稿日期:2018-01-29 本文編輯:任 念)