热休克汗腺细胞诱导人骨髓间充质干细胞的表型转化

背景：未休克汗腺细胞与骨髓间充质干细胞的共培养和休克汗腺细胞与骨髓间充质干细胞的直接共培养均对骨髓间充质干细胞的表型改变无影响。 目的：在体外建立热休克汗腺细胞模型和骨髓间充质干细胞与汗腺细胞间接共培养体系;分析共培养前后骨髓间充质干细胞的形态学、细胞表型的变化情况,探讨骨髓间充质干细胞向汗腺细胞分化的可行性。 方法：体外分别分离培养、扩增并鉴定骨髓间充质干细胞和汗腺细胞,建立汗腺细胞体外热休克模型,继续孵育3-5 d,收集上清液作为条件培养基对骨髓间充质干细胞分化诱导,对比共培养5,10 d骨髓间充质干细胞形态学变化;应用免疫组织化学和流式细胞仪法检测对比共培养10 d后骨髓间充质干细胞细胞表型的改变。 结果与结论：①骨髓间充质干细胞表面标志 CD29、CD44、CD105阳性,汗腺细胞表面标志CK7、CK8、CK18、CK19、CEA阳性。②骨髓间充质干细胞诱导分化后细胞表型的改变：被诱导的细胞中CEA、CK7、CK8和CK19 染色阳性,而对照组没有检测到CEA、CK7、CK8和CK19染色阳性的细胞;流式细胞仪检测CEA、CK7、CK8和CK19的阳性率分别是60.67%,53.34%,54.11%和58.62%。说明实验成功诱导骨髓间充质干细胞,并获得汗腺细胞表型;通过建立适当的体外汗腺诱导培养体系,中胚层的骨髓间充质干细胞可以通过跨胚层分化途径转变为汗腺细胞。

Heat-shocked sweat gland cells induce the phenotype transformation of human bone marrow mesenchymal stem cells

BACKGROUND:Co-culture of bone marrow mesenchymal stem cells with heat-shocked sweat gland cells or non-shocked sweat gland cells does not influence the phenotype transformation of human bone marrow mesenchymal stem cells. OBJECTIVE:To establish heat-shocked sweat gland cell models in vitro and a co-culture system of bone marrow mesenchymal stem cells and sweat gland cells. Morphological and phenotypic changes in bone marrow mesenchymal stem cells before and after co-culture were analyzed. The feasibility of bone marrow mesenchymal stem cells differentiating into sweat gland cells was investigated. METHODS:Bone marrow mesenchymal stem cells and sweat gland cells were isolated, cultured, amplified and identified in vitro. In vitro heat-shocked sweat gland cell models were established and further incubated for 3-5 days. The supernatants were collected as conditioned medium to induce the differentiation of bone marrow mesenchymal stem cells. Morphology of bone marrow mesenchymal stem cells was compared between 5 and 10 days of co-culture. Phenotypic changes of bone marrow mesenchymal stem cells after 10 days of co-culture were detected by immunohistochemical staining and flow cytometry. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells were positive for surface markers CD29, CD44 and CD105, and sweat gland cells were positive for surface markers CK7, CK8, CK18, CK19 and CEA. After induction, the differentiated cells were positive for CEA, CK7, CK8 and CK19. However, positive expression of CEA, CK7, CK8 and CK19 was not detected in the differentiated cells after co-cultured with non-heat-shocked sweat gland cells. Through the flow cytometry, the positive expression rate of CEA, CK7, CK8 and CK19 was 60.67%, 53.34%, 54.11% and 58.62%, respectively in the differentiated cells. These findings suggest that bone marrow mesenchymal stem cells were successfully induced and phenotype of sweat gland cells was acquired. By the cross-mesoderm way, bone marrow mesenchymal stem cells from the mesoderm can convert into sweat gland-like cells through establishing a proper in vitro culture system.