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目的探讨猪膀胱细胞外基质(urinary bladder matrix,UBM)上的Laminin蛋白对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖和向平滑肌分化的影响。方法采用机械分离法、酶消化法及冷冻干燥法等方法对新鲜猪膀胱进行脱细胞处理,采用蛋白与支架共孵育,制备出UBM+Laminin及UBM+Anti-Laminin复合支架。采用贴壁法培养BMSCs,流式细胞仪鉴定BMSCs。采用P3代BMSCs,分别接种于UBM+Laminin、UBM及UBM+Anti-Laminin上。采用CCK-8检测、HE染色观察及免疫组化鉴定等方法检测Laminin蛋白对BMSCs增殖、迁移和分化的影响。结果流式细胞仪鉴定BMSCs CD29阳性表达率为98%,CD31阳性表达率为4.56%,CD34阳性率为3.30%。3种材料均能支持BMSCs生长,且随共孵育时间的延长,D(450)值逐渐升高,其中UBM+Laminin复合材料组细胞D(450)最高(P<0.01)。HE染色结果显示UBM+Laminin组细胞接种第3天形成黏附层,并向深部迁移,第14天可达材料致密层;UBM组第7天仅少许细胞向深层迁移,第14天未达基底层;UBM+Anti-Laminin组仅在支架表层生长。免疫组化检测显示UBM+Laminin组、UBM组、UBM+Anti-Laminin组细胞α-SMA表达分别呈阳性、弱阳性和阴性。结论 UBM材料上Laminin蛋白能促进负载BMSCs增殖、迁移、并有诱导其向平滑肌方向分化的作用。
Objective To investigate the effects of Laminin on the proliferation and differentiation of rat bone marrow mesenchymal stem cells (BMSCs) induced by urinary bladder matrix (UBM). Methods The fresh porcine bladder was decellularized by mechanical separation, enzymatic digestion and freeze-drying. UBM + Laminin and UBM + Anti-Laminin composite scaffolds were prepared by co-incubation of protein and scaffolds. BMSCs were cultured by adherent method and BMSCs were identified by flow cytometry. P3 generation BMSCs were inoculated on UBM + Laminin, UBM and UBM + Anti-Laminin respectively. The effects of Laminin on the proliferation, migration and differentiation of BMSCs were detected by CCK-8 assay, HE staining and immunohistochemistry. Results The positive expression rate of CD29 in BMSCs was 98%, the positive rate of CD31 was 4.56% and the positive rate of CD34 in BMSCs was 3.30% by flow cytometry. All three kinds of materials could support the growth of BMSCs. With the extension of incubation time, D (450) increased gradually, and the highest value of D (450) was found in UBM + Laminin composite group (P <0.01). The results of HE staining showed that UBM + Laminin cells formed adhesion layer on day 3 and migrated deeply, reaching the dense layer on the 14th day. In the UBM group, only a few cells migrated to the deeper layer on the 7th day and reached the basal layer on the 14th day UBM + Anti-Laminin group only grew on the surface of scaffolds. Immunohistochemistry showed that the expression of α-SMA in UBM + Laminin group, UBM group and UBM + Anti-Laminin group were positive, weakly positive and negative respectively. Conclusion The Laminin protein on UBM can promote the proliferation and migration of BMSCs and induce its differentiation into smooth muscle.