4种肺脏全器官脱细胞支架制备方法的比较

Comparison of four preparation methods of lung whole-organ extracellular scaffold

目的分别采用4种不同的方法灌注大鼠肺脏,采集灌注后肺组织大体形态;采取5种不同的染色方法比较脱细胞后肺组织的变化,筛选出较优的脱细胞方法。方法选择SD大鼠12只,雌雄不限,鼠龄8～9周,体质量200～300 g。随机分成4组[3-[(3-胆酰胺丙基)二甲氨基]丙磺酸内盐(CHAPS)组、乙二胺四乙酸二钠盐(EDTA-2Na)组、十二烷基硫酸钠(SDS)组、冻/融组],每组3只。取出整个大鼠肺,用设定的灌注系统采用0.9%氯化钠溶液(生理盐水)适当冲洗其上血液。采用下述脱细胞方法制备:CHAPS组用浓度8 mmol/L CHAPS灌注,EDTA-2Na组用浓度25 mmol/L EDTA-2Na灌注,SDS组用0.1%SDS灌注,冻/融组先冻融后再用0.1%SDS灌注,灌注完毕后均采用0.9%氯化钠溶液200 m L灌注冲洗,最后对脱细胞肺基质分别进行大体形态拍照、苏木精-伊红(HE)染色、弹性纤维(EVG)染色、masson染色、COL-Ⅰ免疫组织化学染色、COL-Ⅲ免疫组织化学染色。结果 CHAPS组肺组织大体形态最为透明;HE染色可见EDTA-2Na组仍残留较多细胞核成分,其余组未见明显的细胞核成分;各组脱细胞后,肺基质构成的肺泡、细支气管等支架结构基本完整。EVG染色可见SDS组和CHAPS组染色较淡,但是SDS组对微结构破坏较大,CHAPS组肺基质保留较完整;COL-Ⅰ免疫组织化学染色可见CHAPS组肺基质保留较其他组完好;COL-Ⅲ免疫组织化学染色可见:SDS组和冻/融组较CHAPS组染色较深,COL-Ⅲ保留较多,且SDS组同冻/融组深浅无明显差别。masson染色可见:CHAPS组和冻/融组肺胶原纤维保留较完整,SDS组肺胶原纤维破坏较大。结论浓度8 mmol/L CHAPS灌注方法是一种较好的制备肺脏脱细胞支架的方法。

Objective To collect gross tissue appearance after perfusion by 4 different methods in rat lung and compare the changes in lung tissue after decellularization with 5 staining methods,and select the better method of decellularization.Methods Twelve SD rats,male or female,aged 8-9 weeks old with body weight of 200-300 g,were selected and sacrificed.All of them were randomly divided into 4 groups,3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS)group,ethylenediaminetetraacetic acid disodium salt(EDTA-2 Na)group,sodium dodecyl sulfate(SDS)group and frozen/melted group,n=3 in each group.The whole lung was removed and washed out blood with 0.9%sodium chloride solution(normal saline)by set in the perfusion system.The following decellularization methods were used to prepare 8 mmol/L CHAPS perfusion(CHAPS group),25 mmol/L EDTA-2 Na perfusion(EDTA-2 Na group),0.1%SDS perfusion(SDS group)and 0.1%SDS perfusion after freezing and thawing(frozen/melted group),then 200 mL of 0.9%sodium chloride solution was used to continue perfusion.Finally,the acellular lung matrix was taken morphological photograph,hematoxylin and ecosin(HE)staining,elastic Van Gieson(EVG)staining,masson staining,collagenⅠ(COL-Ⅰ)immunohistochemical staining and collagenⅢ(COL-Ⅲ)immunohistochemical staining.Results The lung tissue obtained in CHAPS group was the most transparent.HE staining showed many nuclear in EDTA-2 Na group,while no obvious nuclear were observed in other groups.After decellularization in each group,the lung matrix formed by alveolar and bronchial scaffolds was basically complete.EVG staining showed that SDS group and CHAPS group were less staining,but SDS group had great damage in microstructure,and lung matrix was retained intact in CHAPS group.Immunohistochemical staining of COL-Ⅰshowed that matrix in CHAPS group remained more intact than in other groups;Immunohistochemical staining of COL-Ⅲshowed that SDS group and frozen/melted group had deeper staining than in CHAPS group,and COL-Ⅲretained more,and there was no significant difference in depth of staining between SDS group and frozen/melted group.Masson staining showed that CHAPS group and frozen/melted group retained more intact collagen fibers,and the collagen fibers in SDS group were significantly damaged.Conclusion It is demonstrated that the concentration of 8 mmol/L CHAPS perfusion method is the better method for preparation of lung acelluar scaffold.