中华结核和呼吸杂志
中華結覈和呼吸雜誌
중화결핵화호흡잡지
Chinese Journal of Tuberculosis and Respiratory Diseases
2013年
9期
655-660
,共6页
陈楠%张杰%徐敏%王婷%王玉玲%裴迎华
陳楠%張傑%徐敏%王婷%王玉玲%裴迎華
진남%장걸%서민%왕정%왕옥령%배영화
肉芽组织%成纤维细胞%丝裂霉素C%紫杉醇%药物洗脱气道支架
肉芽組織%成纖維細胞%絲裂黴素C%紫杉醇%藥物洗脫氣道支架
육아조직%성섬유세포%사렬매소C%자삼순%약물세탈기도지가
Granulation tissue%Fibroblasts%Mitomycin C%Paclitaxel%Drug-eluting airway stents
目的 在体外细胞学水平观察丝裂霉素C及紫杉醇抑制人胚肺成纤维细胞增殖的量效及时效关系特点.初步探讨药物抑制细胞增殖的潜在机制,为设定药物洗脱气道支架的药物洗脱浓度提供试验参考.方法 采用噻唑兰(MTT)法分别测定10-11 mol/L至10-4 mol/L(10倍倍比稀释)的丝裂霉素C或紫杉醇持续作用24、48和72 h后对人胚肺成纤维细胞增殖的抑制率.通过AnnexinV-FITC/PI双染及流式细胞术检测5 x 10-6、10-5、5×10-5、10-4、2×10-4 mol/L的丝裂霉素C或紫杉醇持续作用48 h后的细胞凋亡百分比.并采用Hoechst 33342荧光染色法观察细胞凋亡的形态学特征.结果 MTT结果显示,各浓度丝裂霉素C或紫杉醇持续作用24、48及72 h均可在不同程度上抑制人胚肺成纤维细胞增殖.其中,当丝裂霉素C处于10-11 mol/L至10-8 mol/L的较低浓度水平时,药物对细胞增殖的抑制作用较弱.且在此浓度范围内,提高药物浓度或延长作用时间对于改善抑制率无益.而当丝裂霉素C处于10-7 mol/L至10-4 mol/L的较高浓度水平时,随作用时间延长或药物浓度提高,抑制率呈渐进式增高.10-7、10-6、10-5及10-4 mol/L的丝裂霉素C持续作用72 h,抑制率分别为53.52％、60.23％、89.81％及96.47％.紫杉醇对人胚肺成纤维细胞增殖的抑制作用存在明显的“阈浓度效应”.10-5 mol/L的紫杉醇持续作用72 h,抑制率仅为48.22％,但当浓度达到10-4 mol/L后,药物作用24 h时抑制率即可达93.38％,且随作用时间延长,抑制率可进一步升高.细胞凋亡部分的试验结果与MTT部分相吻合,即当药物对细胞增殖的抑制作用较为明显时,采用流式细胞术可检测到大量凋亡细胞,且以早期凋亡为主.此时进行Hoechst 33342荧光染色可观察到典型的凋亡细胞.结论 在体外条件下,一定浓度的丝裂霉素C或紫杉醇持续作用均可对人胚肺成纤维细胞的增殖产生抑制作用.二者在气道药物洗脱支架的制备中具有潜在应用价值,可作为备选涂层药物.为有效抑制成纤维细胞增殖,丝裂霉素C洗脱气道支架的药物洗脱浓度不应低于10-7mol/L,而紫杉醇药物洗脱气道支架的药物洗脱浓度不应低于10-5 mol/L,极限洗脱浓度均为10-4 mol/L,此时丝裂霉素C或紫杉醇持续作用72 h抑制率均可达95％以上.在此基础上进一步提高洗脱浓度对于改善抑制率而言意义不大,反而有增加系统毒性的风险.诱导细胞凋亡是丝裂霉素C及紫杉醇抑制人胚肺成纤维细胞增殖的可能机制之一.
目的 在體外細胞學水平觀察絲裂黴素C及紫杉醇抑製人胚肺成纖維細胞增殖的量效及時效關繫特點.初步探討藥物抑製細胞增殖的潛在機製,為設定藥物洗脫氣道支架的藥物洗脫濃度提供試驗參攷.方法 採用噻唑蘭(MTT)法分彆測定10-11 mol/L至10-4 mol/L(10倍倍比稀釋)的絲裂黴素C或紫杉醇持續作用24、48和72 h後對人胚肺成纖維細胞增殖的抑製率.通過AnnexinV-FITC/PI雙染及流式細胞術檢測5 x 10-6、10-5、5×10-5、10-4、2×10-4 mol/L的絲裂黴素C或紫杉醇持續作用48 h後的細胞凋亡百分比.併採用Hoechst 33342熒光染色法觀察細胞凋亡的形態學特徵.結果 MTT結果顯示,各濃度絲裂黴素C或紫杉醇持續作用24、48及72 h均可在不同程度上抑製人胚肺成纖維細胞增殖.其中,噹絲裂黴素C處于10-11 mol/L至10-8 mol/L的較低濃度水平時,藥物對細胞增殖的抑製作用較弱.且在此濃度範圍內,提高藥物濃度或延長作用時間對于改善抑製率無益.而噹絲裂黴素C處于10-7 mol/L至10-4 mol/L的較高濃度水平時,隨作用時間延長或藥物濃度提高,抑製率呈漸進式增高.10-7、10-6、10-5及10-4 mol/L的絲裂黴素C持續作用72 h,抑製率分彆為53.52％、60.23％、89.81％及96.47％.紫杉醇對人胚肺成纖維細胞增殖的抑製作用存在明顯的“閾濃度效應”.10-5 mol/L的紫杉醇持續作用72 h,抑製率僅為48.22％,但噹濃度達到10-4 mol/L後,藥物作用24 h時抑製率即可達93.38％,且隨作用時間延長,抑製率可進一步升高.細胞凋亡部分的試驗結果與MTT部分相吻閤,即噹藥物對細胞增殖的抑製作用較為明顯時,採用流式細胞術可檢測到大量凋亡細胞,且以早期凋亡為主.此時進行Hoechst 33342熒光染色可觀察到典型的凋亡細胞.結論 在體外條件下,一定濃度的絲裂黴素C或紫杉醇持續作用均可對人胚肺成纖維細胞的增殖產生抑製作用.二者在氣道藥物洗脫支架的製備中具有潛在應用價值,可作為備選塗層藥物.為有效抑製成纖維細胞增殖,絲裂黴素C洗脫氣道支架的藥物洗脫濃度不應低于10-7mol/L,而紫杉醇藥物洗脫氣道支架的藥物洗脫濃度不應低于10-5 mol/L,極限洗脫濃度均為10-4 mol/L,此時絲裂黴素C或紫杉醇持續作用72 h抑製率均可達95％以上.在此基礎上進一步提高洗脫濃度對于改善抑製率而言意義不大,反而有增加繫統毒性的風險.誘導細胞凋亡是絲裂黴素C及紫杉醇抑製人胚肺成纖維細胞增殖的可能機製之一.
목적 재체외세포학수평관찰사렬매소C급자삼순억제인배폐성섬유세포증식적량효급시효관계특점.초보탐토약물억제세포증식적잠재궤제,위설정약물세탈기도지가적약물세탈농도제공시험삼고.방법 채용새서란(MTT)법분별측정10-11 mol/L지10-4 mol/L(10배배비희석)적사렬매소C혹자삼순지속작용24、48화72 h후대인배폐성섬유세포증식적억제솔.통과AnnexinV-FITC/PI쌍염급류식세포술검측5 x 10-6、10-5、5×10-5、10-4、2×10-4 mol/L적사렬매소C혹자삼순지속작용48 h후적세포조망백분비.병채용Hoechst 33342형광염색법관찰세포조망적형태학특정.결과 MTT결과현시,각농도사렬매소C혹자삼순지속작용24、48급72 h균가재불동정도상억제인배폐성섬유세포증식.기중,당사렬매소C처우10-11 mol/L지10-8 mol/L적교저농도수평시,약물대세포증식적억제작용교약.차재차농도범위내,제고약물농도혹연장작용시간대우개선억제솔무익.이당사렬매소C처우10-7 mol/L지10-4 mol/L적교고농도수평시,수작용시간연장혹약물농도제고,억제솔정점진식증고.10-7、10-6、10-5급10-4 mol/L적사렬매소C지속작용72 h,억제솔분별위53.52％、60.23％、89.81％급96.47％.자삼순대인배폐성섬유세포증식적억제작용존재명현적“역농도효응”.10-5 mol/L적자삼순지속작용72 h,억제솔부위48.22％,단당농도체도10-4 mol/L후,약물작용24 h시억제솔즉가체93.38％,차수작용시간연장,억제솔가진일보승고.세포조망부분적시험결과여MTT부분상문합,즉당약물대세포증식적억제작용교위명현시,채용류식세포술가검측도대량조망세포,차이조기조망위주.차시진행Hoechst 33342형광염색가관찰도전형적조망세포.결론 재체외조건하,일정농도적사렬매소C혹자삼순지속작용균가대인배폐성섬유세포적증식산생억제작용.이자재기도약물세탈지가적제비중구유잠재응용개치,가작위비선도층약물.위유효억제성섬유세포증식,사렬매소C세탈기도지가적약물세탈농도불응저우10-7mol/L,이자삼순약물세탈기도지가적약물세탈농도불응저우10-5 mol/L,겁한세탈농도균위10-4 mol/L,차시사렬매소C혹자삼순지속작용72 h억제솔균가체95％이상.재차기출상진일보제고세탈농도대우개선억제솔이언의의불대,반이유증가계통독성적풍험.유도세포조망시사렬매소C급자삼순억제인배폐성섬유세포증식적가능궤제지일.
Objective To observe the inhibitory effect and potential mechanism of mitomycin C and paclitaxel on the proliferation of Human Pulmonary Fibroblast in vitro.So as to providing an experimental reference for the design of drug eluting airway stents.Methods Cell viability was measured by MTT assay after different concentrations of mitomycin C or paclitaxel varying from 10-11 mol/L to 10-4 mol/L had been applied to the fibroblasts for 24,48 or 72 h,respectively.Cell apoptosis was assessed by flow cytometry using dual staining with annexin V-FITC and propidium iodide 48 h after administering mitomycin C or paclitaxet at a concentration of 5 × 10-6,10-5,5 × 10-5,10-4,2 × 10-4 mol/L,respectively.And the morphological character of cell apoptosis was observed by Hoechst 33342 fluorescent staining.Results The results of MTT revealed that cell proliferation were inhibited by mitomycin C and paclitaxel at all concentrations and exposure times.Among them,the inhibitory effect of mitomycin C were weak when the concentrations were between 10-11 mol/L to 10-8 mol/L.And within this context,the inhibitory ratio didn' t correspond to the elevation of the concentration or the prolongation of the exposure times.However,when the concentration were between 10-7 mol/L to 10-4 mol/L,the inhibitory ratio rise progressively as the elevation of the concentration at all exposure times.The inhibitory ratio were 53.52％,60.23％,89.81％ and 96.47％ respectively when cells were treated by 10-7,10-6,10-5 mol/L and 10-4 mol/L mitomycin C for 72 h.An apparent "threshold dose effect" was observed in the paclitaxel treated groups.It' s worth noting that the inhibitory ratio was only 48.22％ when the cells had already been treated by 10-5 mol/L paclitaxel for 72 h.However,when the concentration had reached 10-4 mol/L,the inhibitory ratio sharply climbed to 93.38％ even the cells had only been treated for 24 h.And the inhibitory ratio continued to rise as time prolonged.The results of cell apoptosis were consistent with MTT.When a significant inhibitory effect were detected by MTT,remarkable cell apoptosis could be observed by flow cytometry,and typical apoptotic cell could be identified by Hoechst 33342 fluorescent staining.Conclusions A certain concentration of mitomycin C or paclitaxel can inhibit Human Pulmonary Fibroblast proliferation in vitro.Both of these two drugs have potential value for the preparation of drug eluting airway stents.In order to ensure the inhibitory effect,the eluting concentration of mitomycin C and paclitaxel shouldn't be less than 10-7 mol/L and 10-5 mol/L.But the eluting concentration of these two drugs shouldn' t exceed 10-4 mol/L when both of the inhibitory ratio of these two drugs were higher than 95％.On this basis,elevating the drug concentration has little significance for improving the inhibitory effect,but increase the risk of systemic toxicity.Inducing cell apoptosis is one of the potential mechanisms of mitomycin C and paclitaxel in inhibiting cell proliferation.
