Transwell小室共培养条件下微血管内皮细胞对成骨细胞增殖、凋亡的影响

李高志; 张丽君; 王艺璇; 徐丽群; 胡泽兵; 石菲; 张舒; 曹新生

doi:10.3969/j.issn.2095-5227.2021.01.018

Transwell小室共培养条件下微血管内皮细胞对成骨细胞增殖、凋亡的影响

Effect of microvascular endothelial cells on proliferation and apoptosis of osteoblast under condition of transwell chamber coculture

摘要

摘要:
背景骨微血管在骨稳态维持过程中发挥着重要作用，“血管-成骨耦联”现象已经成为目前临床医学工作者关注的焦点问题，揭示微血管内皮细胞与成骨细胞之间的相互调控关系将为骨相关疾病的临床治疗提供新的思路。
目的探究在共培养条件下，微血管内皮细胞bEnd.3对成骨细胞MC3T3-E1增殖、凋亡的影响。
方法利用Transwell小室建立MC3T3-E1/bEnd.3共培养体系，分别设置对照组(单独培养MC3T3-E1细胞)和共培养组(MC3T3-E1细胞和bEnd.3细胞共同培养)。CCK-8实验检测两组MC3T3-E1细胞增殖活性的变化，PI单染流式细胞术检测两组MC3T3-E1细胞周期的变化，Annexin V-FITC/PI双染流式细胞术检测两组MC3T3-E1细胞凋亡率的变化，Western blotting检测两组MC3T3-E1细胞PCNA、Bax、Cleaved Caspase-3等增殖/凋亡相关蛋白的表达变化情况。
结果与单独培养的MC3T3-E1细胞相比，共培养组的MC3T3-E1细胞增殖活性显著增强(P < 0.05)，S期细胞比例显著增高(P < 0.01)，G₂/M期细胞比例显著增高(P < 0.05)，PCNA表达升高(P < 0.01)，凋亡率下降(P < 0.05)，Bax和Cleaved Caspase-3表达降低(P < 0.05)。
结论在Transwell小室共培养条件下，微血管内皮细胞bEnd.3能够显著促进成骨细胞MC3T3-E1的增殖，并抑制其凋亡。

Abstract:
Background Bone capillaries play important roles in the maintenance of bone homeostasis and the phenomenon of “angiogenesis-osteogenesis coupling” has become the focus of clinicians. Revealing the mutual regulation relationship between microvascular endothelial cells and osteoblasts will provide new ideas for the clinical treatment of bone-related diseases.
Objective To investigate the effect of microvascular endothelial cells bEnd.3 on proliferation and apoptosis of osteoblast MC3T3-E1 cells under coculture conditions.
Methods The MC3T3-E1/bEnd.3 coculture system was established using transwell chambers. The cells were divided into control group (MC3T3-E1 cells were cultured separately) and coculture groups (MC3T3-E1 cells and bEnd.3 cells were co-incubated). CCK-8 assay was used to detect the changes in cell viability of MC3T3-E1 in the two groups. PI single-staining flow cytometry was applied to detect the changes in cell cycle of the two groups in MC3T3-E1, and Annexin V-FITC/PI double-staining flow cytometry for the changes of apoptosis rate of MC3T3-E1 in two groups. Western blotting was used to detect the expression changes of proliferation and apoptosis related-proteins (PCNA, Bax and Cleaved Caspase-3) in two groups of MC3T3-E1.
Results Compared with MC3T3-E1 cells cultured alone, the viability of MC3T3-E1 cells in coculture group enhanced significantly (P < 0.05), the ratio of S phase cells and G₂/M phase cells increased significantly (P < 0.01, P < 0.05 respectively), the expression level of PCNA increased (P < 0.01), while the rate of apoptosis decreased (P < 0.05), Bax and Cleaved Caspase-3 expression were down regulated (P < 0.05).
Conclusion Under the condition of coculture in transwell chamber, bEnd.3 cells can significantly promote the proliferation of MC3T3-E1 cells and inhibit its apoptosis.

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