去垢剂浓度对灌流法制备脱细胞肺支架效果的影响

Effect of detergent concentration on preparation of acellular pulmonary scaffold by perfusion

目的比较不同浓度的十二烷基硫酸钠(SDS)和3-[3-(胆酰胺丙基)二甲氨基]丙磺酸内盐(CHAPS)对制备脱细胞肺支架效果的影响。方法选用SD大鼠21只,鼠龄8~9周,体质量200~300 g。采用蠕动泵灌洗系统,分别用磷酸盐缓冲溶液(对照组)及3种不同浓度的两种去垢剂(0.1%SDS、0.2%SDS、0.3%SDS、4 mmol/L CHAPS、8 mmol/L CHAPS、12 mmol/L CHAPS 6个实验组)对大鼠肺脏进行灌洗制备脱细胞肺支架,并且在此过程中每隔0.5 h拍照截取肺组织图片;再分别测定各组脱细胞肺支架残留DNA的量及观察各组胶原质3及纤维连接蛋白的免疫荧光染色。结果随着灌洗时间的延长,各实验组大鼠肺脏透明度均逐渐增高。且脱细胞后肺都保持着肺泡、支气管等基本结构。不同浓度的同一种试剂灌洗后脱细胞肺支架大体形态观无明显差别。随着去垢剂浓度的增加,各SDS组及各CHAPS组DNA残留量分别为(20.41±4.85)ng/mg、(24.90±3.73)ng/mg、(27.92±5.11)ng/mg、(114.14±7.82)ng/mg、(118.93±10.84)ng/mg、(121.04±6.46)ng/mg,对照组为(177.77±9.37)ng/mg,各实验组与对照组之间差异均有统计学意义(P<0.05),各实验组之间差异无明显统计学意义(P>0.05)。标记胶原质3和标记纤维连接蛋白的免疫荧光染色结果显示,各实验组4’,6-二脒基-2-苯基吲哚(DAPI)蓝染的细胞核均模糊、分散,对照组DAPI蓝染的细胞核较完整。实验组及对照组红染的阳性标记物胶原质3及纤维连接蛋白分布较分散。结论不同浓度下的SDS和CHAPS对制备脱细胞肺支架有着一定的影响。

Objective To compare the effect of different concentrations of detergents sodium dodecyl sulfate(SDS)and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate(CHAPS)on preparation of acellular pulmonary scaffold.Methods A total of 21 SD rats were selected,aged 8-9 weeks old with body mass of 200-300 g.The decellularized pulmonary scaffold was prepared by lavaged rat lung in peristaltic pump lavage system which contained phosphate buffer solution(control group)and 3 different concentrations of 2 detergents(0.1%SDS,0.2%SDS,0.3%SDS,4 mmol/L CHAPS,8 mmol/L CHAPS,12 mmol/L CHAPS experimental groups).The lung tissue images were taken every 0.5-hour,the residual DNA in each group of decellularized pulmonary scaffold were measured and observed.The immunofluorescence staining of collagen 3 and fibronectin was observed for each group.Results As the lavage time was prolonged,the lung transparency of the rats in experimental group gradually increased.After decellularization,the lungs maintain basic structures such as alveoli and bronchi.There was no significant difference in the general morphology of decellularized pulmonary scaffold after lavage of different concentrations of the same reagent.With increased detergent concentration,the DNA residues in each SDS group and each CHAPS group were(20.41±4.85)ng/mg,(24.90±3.73)ng/mg,(27.92±5.11)ng/mg,(114.14±7.82)ng/mg,(118.93±10.84)ng/mg,(121.04±6.46)ng/mg,and control group was(177.77±9.37)ng/mg.The difference was statistically significant between experimental group and control group(P<0.05),while no statistically significant difference between experimental groups(P>0.05).The immunofluorescence staining of labeled collagen 3 and fibronectin showed that the 4’,6-diamidino-2-phenylindole(DAPI)blue-stained nuclei of each experimental group were blurred and dispersed,while the nuclei in control group was complete.The red-stained positive marker collagen 3 and fibronectin were more dispersed in experimental group and control group.Conclusion It is demonstrated that different concentration of detergents have certain effect on the preparation of acellular pulmonary scaffold.