摘要
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 effect of laminin on pig urinary bladder matrix（ UBM） on the proliferation of bone marrow mesenchymal stem cells（ BMSCs） and the differentiation into smooth muscle cells. Methods Fresh pig bladder was decellularized by mechanical separation,enzymatic digestion,and freeze drying methods. The morphology of the obtained UBM was examined by hematoxylin and eosin（ HE）staining,hematoxylin / aniline blue staining and scanning electron microscopy. The laminin ＋ UBM material and anti-laminin ＋ UBM material were obtained by incubation of proteins and UBM. The BMSCs were obtained with adherent method,and then seeded on the laminin ＋ UBM material,UBM material,and anti-laminin ＋UBM material after in vitro proliferation. The BMSCs were identified by flow cytometry（ FCM）. The cell proliferation,migration and differentiation in different materials were compared by CCK-8 assay,HE staining and immunohistochemistry methods. Results FCM analysis showed that CD29-positive expression rate of the BMSCs reached 98%, while CD31- and CD34-positive expression rates were 4. 56% and 3. 30%,respectively. CCK-8 analysis showed BMSCs could grow efficiently on the three materials. CCK-8 value was increased significantly in 9-day culture,especially when the BMSCs were seeded on the laminin ＋ UBM material as compared with the other 2 materials. HE staining showed that the 3 materials promoted cell adhesion to the surface. On the 3th day,HE staining showed that the BMSCs formed cell layers on the UBM＋ laminin material,and some cells were migrated into the dense layer. In contrast,no obvious cell migration was observed on the UBM ＋ anti-laminin material and UBM material on the 14 th day. Immunohistochemistry results showed that alpha-smooth muscle actin（ alpha-SMA） expression was detected on the UBM ＋ laminin material,and the expression was negative on the UBM material. Conclusion The laminin on UBM promotes proliferation and migration of the BMSCs and induces differentiation of the BMSCs to smooth muscle cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2015年第8期723-729,共7页
Journal of Third Military Medical University
基金
国家自然科学基金(81270683)~~
