北京生物医学工程

低氧微环境对P（3HB-co-4HB）与小鼠骨髓间充质干细胞共培养形成心肌补片影响的实验研究

Experimental study on the effect of hypoxia microenvironment on the co-culture of P(3HB-co-4HB) and mouse bone marrow mesenchymal stem cells to form myocardial patch

作者：田琨穆军升伯平周帆

单位：首都医科大学附属北京安贞医院心脏外科，北京市心肺血管疾病研究所（北京 100029）解放军总医院第三医学中心超声科(北京 100039)

关键词：低氧微环境； P（3HB-co-4HB）；心肌补片；心肌梗死

分类号：R318.04

出版年·卷·期（页码）：2019·38·3（227-234）

摘要：

目的探究低氧微环境对小鼠骨髓间充质干细胞与P（3HB-co-4HB）材料共培养形成心肌补片的影响，为细胞移植术治疗心肌梗死提供一种更有效的心肌补片。方法全骨髓培养法提取小鼠骨髓间充质干细胞（mMSCs），取5代mMSCs流式细胞术鉴定表面抗原。将聚3羟基丁酸酯-co-4羟基丁酸酯[3-hydroxybutyrate-co-4-hydroxybutyrate，P(3HB-co-4HB)]与小鼠骨髓间充质干细胞（mouse bone marrow mesenchymal stem cells，mMSCs）共培养制作成细胞补片，随机分为常氧组和低氧组,每组各10个样本，0、12、24 h分别用CCK-8法测定细胞增殖情况；扫描电子显微镜（scanning electron microscope，SEM）观察补片存活、黏附、生长情况。加入诱导剂5氮杂胞苷2周后，免疫荧光检测两组心肌肌钙蛋白T(cTnT)的表达情况。结果在共培养24 h后，CCK-8法测定低氧组OD值(0.349±0.038)显著大于常氧组（0.308±0.025）(n=10，P<0.05)，扫描电子显微镜观察到低氧组P(3HB-co-4HB)材料上细胞数量更多，细胞与材料之间的黏附牢固，细胞形态正常。免疫荧光显示低氧组cTnT表达比常氧组更加显著。结论相对于常氧条件，低氧微环境可促进骨髓间充质干细胞在P（3HB-co-4HB）材料上的黏附、存活、增殖、分化，形成一种更有效的心肌补片。

Objective To investigate the effect of hypoxic microenvironment on the co-culture of mouse bone marrow mesenchymal stem cells with P(3HB-co-4HB) material to form a myocardial patch, and to provide a more effective myocardial for cell transplantation in the treatment of myocardial infarction. Methods Mouse bone marrow mesenchymal stem cells (mMSCs) were extracted by whole bone marrow culture. The surface antigens were identified by flow cytometry of 5 generations of mMSCs. 3-hydroxybutyrate-co-4-hydroxybutyrate (P(3HB-co-4HB)) and bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, mMSCs) co-cultured into cell supplements, randomly divided into normoxia group and hypoxia group, each group of 10 samples, 0, 12, 24 hours to determine cell proliferation by CCK-8 method; scanning electron microscope (scanning Electron microscope (SEM) was used to observe the survival, adhesion and growth of the patch. Two weeks after the addition of the inducer 5-azacytidine, the expression of cardiac troponin T (cTnT) was detected by immunofluorescence. Results After co-culture for 24 hours, the OD value of the hypoxic group measured by CCK-8 method (0.349±0.038) was significantly higher than that of the normoxic group (0.308±0.025) (n=10, P<0.05), which was observed by scanning electron microscopy. The number of cells in the oxygen group P(3HB-co-4HB) material is more, the adhesion between the cells and the material is firm, and the cell morphology is normal. Immunofluorescence showed that the expression of cTnT in the hypoxic group was more significant than in the normoxic group. Conclusions Compared with normoxic conditions, hypoxic microenvironment can promote the adhesion, survival, proliferation and differentiation of bone marrow mesenchymal stem cells on P(3HB-co-4HB) materials, and form a more effective myocardial patch.

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