灌注脱细胞系统制备全膀胱脱细胞基质并联合脂肪干细胞构建组织工程膀胱的研究  

The study of the whole bladder acellular matrix prepared by the perfusion decellularization system combined with the adipose-derived stem cells to construct the tissue engineering bladder

作　　者：肖树伟[1] 符伟军[1] 王鹏超[1] 赵健[1] 凌争云 安子彦 符舟洋 张旭[1] Xiao Shuwei;Fu Weijun;Wang Pengchao;Zhao Jian;Ling Zhengyun;An Ziyan;Fu Zhouyang;Zhang Xu

机构地区：[1]解放军总医院泌尿外科,北京100853

出　　处：《中华泌尿外科杂志》2023年第3期211-217,共7页Chinese Journal of Urology

基　　金：国家自然科学基金(81873600);北京市自然科学基金(7182154)。

摘　　要：目的应用自行设计的灌注脱细胞系统制备全膀胱脱细胞基质(BAM),并探讨采用脂肪干细胞(ADSCs)构建组织工程膀胱的可行性。方法本研究于2020年10月至2021年4月进行。采用自行设计的膀胱灌注脱细胞系统,按照灌注液流动方向和不同脱细胞液作用时间的不同,制订4种不同的脱细胞方案(分别为A、B、C、D组)。其中A组和B组均以膀胱组织的尿道口作为灌注液的流出道,C组和D组剪去膀胱顶部部分组织以膀胱顶部开口作为灌注液的流出道。A组和C组采用1%Triton X-100作用6 h,1%十二烷基硫酸钠(SDS)作用2 h;B组和D组采用1%Triton X-100作用7 h,1%SDS作用1 h。另设正常膀胱组作为对照,其组织为直接取材的天然膀胱组织,不需要灌注和冷冻保存。通过HE、DAPI、Masson三色染色和阿尔新蓝染色,以及试剂盒定量分析以筛选出快速且高效的脱细胞方案,制备全膀胱支架。以制备的BAM为支架材料,ADSCs为种子细胞构建组织工程膀胱,HE和DAPI染色观察ADSCs在BAM上的分布情况。结果HE和DAPI染色结果显示C组未见明显的细胞核残留,Masson三色染色和阿尔新蓝染色结果表明C组的胶原结构和糖胺聚糖得到较好保留。C组膀胱壁厚度与正常膀胱组[(975.44±158.62)μm与(1064.49±168.52)μm]差异无统计学意义(P>0.05)。C组DNA含量[(43.59±4.59)ng/mg]明显低于正常膀胱组、A组、B组和D组[分别为(532.50±26.69)、(135.17±6.99)、(182.49±13.69)、(84.00±4.38)ng/mg],差异均有统计学意义(P<0.05)。C组胶原含量[(10.98±0.29)μg/mg]和糖胺聚糖含量[(2.30±0.18)μg/mg]与正常膀胱组[(11.69±0.49)μg/mg和(2.36±0.09)μg/mg]比较,差异均无统计学意义(P>0.05)。扫描电镜检查结果显示A~D组制备的BAM表面可见大量的孔隙结构,利于细胞黏附。分离培养ADSCs,流式细胞术鉴定证实CD90和CD29阳性表达,CD45和CD106阴性表达。活/死细胞双染色法和CCK-8检测证实C组制备的BAM无细胞毒性�Objective To prepare the whole bladder acellular matrix(BAM)using the self-designed perfusion decellularization system,and evaluate the feasibility of constructing the tissue engineering bladder with the adipose-derived stem cells(ADSCs).Methods This study was conducted from October 2020 to April 2021.The self-designed perfusion decellularization system was used,and four different decellularization protocols(group A,group B,group C and group D)were formulated,according to the flow direction of the perfusate and the action time of different decellularization solutions.Among them,the urethral orifice of the bladder tissue was used as the outflow tract of the perfusion fluid in groups A and B.The top of the bladder was cut off and used as the outflow tract of the perfusion fluid in groups C and D.In groups A and C,1%Triton X-100 was treated for 6 h,and 1%sodium dodecyl sulfate(SDS)was treated for 2 h.In groups B and D,1%Triton X-100 was treated for 7 h,and 1%sodium dodecyl sulfate(SDS)was treated for 1 h.In addition,the tissue in the normal bladder group was directly obtained from the natural bladder tissue,which did not require perfusion,cryopreservation and thawing.The fast and efficient decellularization protocol was screened out through HE,DAPI,Masson trichrome and Alcian Blue staining and quantitative analyses to prepare the whole bladder scaffold.The prepared BAM was used as the scaffold material,and the ADSCs were used as the seeding cells to construct the tissue engineering bladder.HE and DAPI staining were used to observe the distribution of ADSCs on the BAM.Results HE and DAPI staining showed that there was no obvious nuclear residue in the group C.Masson trichrome and Alcian Blue staining showed that the collagen structure and glycosaminoglycan were well preserved in the group C.There was no significant difference in bladder wall thickness between the group C and the normal bladder group[(975.44±158.62)μm vs.(1064.49±168.52)μm,P>0.05].The DNA content in the group C[(43.59±4.59)ng/mg]was lower than th

关 键 词：膀胱 灌注脱细胞系统 膀胱脱细胞基质 脂肪干细胞 组织工程 

分 类 号：R694[医药卫生—泌尿科学]