大鼠子宫去细胞化支架的制备和评价

The preparation for decellularized scaffolds in the rat uterine and its evaluation

目的:通过去细胞化灌注技术,探讨成功制备天然子宫去细胞化生物支架的理想灌注策略,并通过系统的评价体系,以鉴定其能否作为子宫组织再生工程研究的良好应用载体。方法:选择健康成年雌性SD大鼠,采用子宫动脉入路途径,通过冻融、酶解及曲亚通(Triton X-100)联合十二烷基硫酸钠(SDS)的洗脱灌注等流程,获取子宫去细胞化支架。并通过大体形态观察、美兰染色、HE染色观察、基因组DNA定量分析、EGF、b FGF、TGF-β含量测定、电镜观察、胶原蛋白总量检测及主要胶原成分鉴定等对去细胞化后的子宫支架进行全面的评价。结果:成功建立动脉灌注入路并获得了肉眼透明的子宫去细胞化支架,HE染色和透射电镜观察均表明去细胞化子宫支架内无细胞残留,DNA含量检测表明支架内DNA残留量为(45.6±7.3)ng/mg,不足正常子宫的5%(P<0.01),美兰染色和扫描电镜显示子宫支架的脉管网络和空间结构保存完整;而胶原蛋白含量由新鲜子宫组织的(0.57±0.12)μg/mg增加到去细胞化子宫支架的(0.88±0.10)μg/mg,差异有统计学意义(P<0.05),结合各型胶原的免疫组织化学定性分析,说明去细胞化后子宫支架的细胞外基质成分保留得较为完整;ELISA结果显示去细胞化子宫支架中细胞因子EGF、b FGF和TGF-β分别保留了66%、85%和54%,表明其仍具备一定的生物活性。结论:本研究方法能够成功制备理想的去细胞化子宫支架,并且支架的基质成分和理化特性完整保留,能够作为子宫组织工程研究的良好载体。

Objective:To explore the feasible strategy for the preparation of natural uterine decellularized scaffolds and to evaluate it systematically.Methods:Female SD rats were used,detergents including TritonX-100 and sodium dodecyl sulfate were perfused into the uterus through the uterine artery after physical freezing and thawing and enzymatic hydrolysis.After decellularization,scaffolds were evaluated by general observation,methylene blue staining,HE staining,quantitative analysis of genomic DNA,cytokine content determination,transmission and scanning electron microscopy(TEM and SEM),collagen detection and identification.Results:After the perfusion,the transparent uterine scaffold was obtained,HE staining and TEM showed that there was no cell residual in it.DNA content was confirmed to be(45.6±7.3)ng/mg,less than 5%of the normal uterus(P<0.01).Methylene blue staining and SEM showed that vascular network and spatial structure were intact.Collagen content increased from(0.57±0.12)μg/mg in fresh uterine tissue to(0.88±0.10)μg/mg in scaffolds(P<0.05),combined with IHC study,indicating that extracellular matrix components of the scaffolds were completely preserved.In addition,ELISA results showed that the cytokines EGF,bFGF and TGF-βwere still retained in the decellularized scaffolds(66%,85%and 54%,respectively),indicating that they still had some biological activity.Conclusion:The decellularized uterine scaffold could be successfully prepared with its physical and chemical properties preserved,and it may be used as an alternative platform for uterine tissue engineering study.