本文關(guān)鍵詞：納米材料體外細胞毒性試驗標準的關(guān)鍵項目研究，，由筆耕文化傳播整理發(fā)布。

納米材料體外細胞毒性試驗標準的關(guān)鍵項目研究

Study on key parameters of the in vitro cytotoxicity test in nanomaterials assessment

[1] [2] [3] [4] [5] [6] [7]

FU Hai-yang,SHAO An-liang,FANG Yu,WANG Han,ZHANG Na,XU Li-ming（National Institutes for Food and Drug Control,Beijing 100050,China）

中國食品藥品檢定研究院,北京100050

文章摘要：目的：參考ASTM標準,研究納米材料體外細胞毒性檢測方法（MTT試驗和LDH試驗）中的關(guān)鍵項目指標,為制定納米材料體外細胞毒性試驗標準提供實驗依據(jù)。方法：通過全波長掃描獲得MTT試驗和LDH試驗的檢測波長;測試陽性對照品APAP的使用劑量和保存條件;通過改變MTT試劑的配制介質(zhì),以及DMSO溶解后加入和不加入甘氨酸緩沖液來測試MTT試驗條件;考察含不同濃度血清（10%,5%,2.5%）的細胞培養(yǎng)液對LDH試驗的背景影響;在確定優(yōu)化的實驗條件下用MTT試驗和LDH試驗,對某納米銀凝膠產(chǎn)品進行細胞毒性評價。結(jié)果：MTT試驗和LDH試驗在測定吸光度時雙波長可分別設(shè)定為540～570/680 nm和490/680 nm。APAP作為陽性對照的合適濃度為20～30 mmol.L-1,該溶液保存條件為-20℃,14 d內(nèi)有效。血清對LDH試驗的干擾與濃度呈正相關(guān),為確保細胞正常生長,該方法中血清濃度確定為10%,血清造成的背景值干擾可從空白組扣除。納米銀凝膠產(chǎn)品MTT試驗和LDH試驗結(jié)果（IC50）分別為26.8 mg.mL-1和61.8 mg.mL-1。結(jié)論：MTT試驗和LDH試驗相結(jié)合可以在1個實驗體系內(nèi)獲得2個檢測結(jié)果,能夠更全面地評價納米材料可能引起的細胞毒性。本課題確定了納米材料體外細胞毒性檢測方法（MTT試驗和LDH試驗）的關(guān)鍵項目指標,使該方法具有良好的操作性與可行性。

Abstr：Objective： To investigate the key factors and parameters of the in vitro cytotoxicity test（MTT assay and LDH assay） for assessing nanomaterials referring to ASTM,so that to provide evidences for establishing standards of nanomaterial’s cytotoxicity test.Methods： The detection wavelength of MTT assay and LDH assay were obtained by full-wavelength scanning;the concentration and storage condition of the positive control APAP were tested;the experimental factors of MTT assay were tested by changing the MTT media with or without glycine buffer after adding DMSO;the experimental conditions of LDH assay were measured by changing the concentration of serum（10%,5%,2.5%） in cell culture media.The cytotoxicity of the silver nanoparticle containing hydrogel was assessed by refined MTT assay and LDH assay.Results： The double-wavelength was found at 540-570 /680 nm and 490 /680 nm,which is suitable for MTT assay and LDH assay respectively,in this experimental system.The proper concentration of positive control APAP was 20-30 mmol.L-1,which should be stored at-20 ℃ and will be stable for 14 days.The serum contained in the cell culture media could affect the LDH test with a serum concentration-depended manner,but this kind of interference could be deducted by the blank control.The IC50 of silver nanoparticle-containing hydrogel tested by refined MTT assay and LDH assay were 26.8 mg.mL-1 and 61.8 mg.mL-1,respectively.Conclusions： Two assessment points can be obtained in one experiment by combining re-fined MTT assay and LDH assay,so that the cytotoxicity of nanomaterials can be evaluated more comprehensively.In addition,this study defines the key factors and parameters of the cytotoxicity test（MTT assay and LDH assay） for assessing nanom

文章關(guān)鍵詞：

Keyword：:nanomaterials cytotoxicity MTT assay LDH assay standard study medical devices

課題項目：國家科技支撐計劃：支撐認證認可的評價分析/檢測驗證與有效性保障技術(shù)研究與示范;（2012BAK26B00）,子課題：快速檢測關(guān)鍵材料評價技術(shù)研究與示范; 東南大學(xué)生物電子學(xué)國家重點實驗室開放研究基金：納米醫(yī)療器械的免疫學(xué)研究（2011E04）; 北京市自然基金：納米銀透過體外血腦屏障模型引起腦神經(jīng)毒性的機理研究（311204）; 中國食品藥品檢定研究院學(xué)科帶頭人基金：金銀敷料中銀存在形式和釋放特性的表征方法研究（2012X2）; 中央財政支持國家局醫(yī)療器械行業(yè)標準（N2012099－T－zjs）

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  本文關(guān)鍵詞：納米材料體外細胞毒性試驗標準的關(guān)鍵項目研究，由筆耕文化傳播整理發(fā)布。