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目的比较同一供者及不同供者的人胎盘MSCs(human placenta-derived MSCs,HPMSCs)与人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)三维复合培养效果,为体外构建三维血管化组织工程骨提供实验依据。方法取健康足月产妇自愿捐赠的胎盘及脐带组织,采用Ⅳ型胶原酶消化和人淋巴细胞分层液密度梯度离心法分离培养HPMSCs,通过流式细胞学检测细胞表型,取第2代细胞向成骨细胞诱导培养鉴定;以Ⅰ型胶原酶消化分离培养HUVECs,通过Ⅷ因子相关抗原因子免疫组织化学染色进行鉴定。实验分为两组,A组取不同供者的第3代HUVECs和成骨诱导培养3周的第3代HPMSCs以1∶1比例复合种植于胶原水凝胶,制备细胞-胶原水凝胶复合物;B组将同一供者的以上两种细胞同法复合制备复合物。复合培养第1、3、5、7天取材,行CD31免疫荧光染色,于激光共聚焦显微镜下观察其成血管倾向,测量复合物中的索道长度和节点数,并进行统计学分析。结果经鉴定,成功分离培养HPMSCs及HUVECs。CD31免疫荧光染色示,与A组相比,B组细胞-胶原水凝胶复合物中的HUVECs可在三维空间上较早、较好地伸展,在水凝胶内部相互交织能力较强,形成的索道更长,节点更多,网络更密集。第7天时B组索道长度和节点数分别为(8.11±0.62)mm/mm2及(21.30±1.41)个/mm2,显著优于A组的(6.68±0.35)mm/mm2及(17.10±1.10)个/mm2,差异均有统计学意义(t=0.894,P=0.000;t=0.732,P=0.000)。结论将同一供者的HPMSCs与HUVECs体外复合种植于胶原水凝胶支架上,比不同供者来源的细胞形成的网络成血管倾向更明显,交织连续性好,层次丰富。
Objective To compare the effects of three-dimensional (3D) culture of human placenta-derived MSCs (HPMSCs) and human umbilical vein endothelial cells (HUVECs) on the same donor and donor donors, Bone provide experimental basis. Methods Placenta and umbilical cord tissues from healthy full-term pregnant women were collected. HPMSCs were isolated and cultured by collagenase digestion and gradient centrifugation of human lymphocytes. Flow cytometry was used to detect the phenotype of the second generation of cells The osteoblasts were induced to culture. HUVECs were isolated and digested with type Ⅰ collagenase and identified by Ⅷ factor-related antigen immunohistochemical staining. The experiment was divided into two groups. The third generation HUVECs from different donors in group A and the third generation HPMSCs which were induced by osteogenic induction for three weeks were planted in collagen hydrogel at the ratio of 1: 1 to prepare cell-collagen hydrogel composite Group B will be the same donor of the above two cells with the law complex preparation of complexes. Cultures were harvested on day 1, 3, 5, and 7 for CD31 immunofluorescence staining. The angiogenic tendency was observed under laser scanning confocal microscopy. The length of the cableway and the number of nodes in the composite were measured and statistically analyzed. Results After identification, HPMSCs and HUVECs were successfully isolated and cultured. CD31 immunofluorescence staining showed that compared with group A, HUVECs in group B cell-collagen hydrogel composite could be stretched earlier and better in three-dimensional space, and could interweave strongly in hydrogel to form Longer cable, more nodes, more dense network. On the 7th day, the cable length and node numbers of group B were (8.11 ± 0.62) mm / mm2 and (21.30 ± 1.41) / mm2, respectively, which were significantly superior to those of group A (6.68 ± 0.35) mm / mm2 and (17.10 ± 1.10) A / mm2, the differences were statistically significant (t = 0.894, P = 0.000; t = 0.732, P = 0.000). Conclusion The HPMSCs and HUVECs of the same donor were implanted in vitro on collagen hydrogel scaffolds in vitro. Compared with the networks formed by cells from different donors, HPMSCs and HUVECs were more likely to become blood vessels with better interweave continuity and richness.