丝裂霉素C及紫杉醇对人胚肺成纤维细胞增殖及凋亡的影响

The influence of mitomycin C and paclitaxel on the proliferation and apoptosis of human pulmonary fibroblast

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10-11mol／L至10-8moL／L（10倍倍比稀释）的丝裂霉素C或紫杉醇持续作用24、48和72h后对人胚肺戍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5×10-6、10-5、5×10-5、10-4、2×10-4mol／L的丝裂霉素C或紫杉醇持续作用48h后的细胞凋亡百分比。并采用Hoechst33342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72h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10-11mol／L至10-8mol／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10-7mol／L至10-4mol／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10-4mol／L的丝裂霉素C持续作用72h，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”。10-5mol／L的紫杉醇持续作用72h，抑制率仅为48．22％，但当浓度达到10-4mol／L后，药物作用24h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Mr丌部分相吻合，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Hoechst33342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10-5mol／L，极限洗脱浓度均为10-4mol／L，此时丝裂霉素C或紫杉醇持续作用72h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。

Objective To observe the inhibitory effect and potential mechanism of mitomycin C and paclitaxel on the proliferation of Human Puhnonary 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 b,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 paclitaxel 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 10-5 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 tool/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 nfitomycin 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 tooL/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 effeet,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.