Chen Chen,Liu Xin,Liu Ge,et al.Comparison of ceiling culture versus insert culture for dedifferentiated fat cells in rats[J].Journal of Clinical Pediatric Surgery,2023,22(01):72-78.[doi:10.3760/cma.j.cn101785-202001032-014]
大鼠去分化脂肪细胞的天花板培养法和滤网培养法比较
- Title:
- Comparison of ceiling culture versus insert culture for dedifferentiated fat cells in rats
- 摘要:
- 目的 探讨大鼠去分化脂肪(dedifferentiated fat,DFAT)细胞天花板培养法和滤网培养法的差异,为DFAT 细胞的原代培养提供研究基础。方法 从8只4~6周SD(Sprague Dawley)大鼠腹股沟脂肪垫中分离出成熟脂肪细胞,应用天花板培养法和滤网培养法体外去分化培养获得DFAT细胞,比较DFAT细胞形态、生长至首次传代需要的时间和完全培养基量,并通过流式细胞分析和细胞免疫荧光比较DFAT细胞纯度和体外诱导分化为平滑肌细胞的潜能。结果 两种方法培养的DFAT细胞均呈成纤维样、梭形、峰谷样排列。天花板培养法和滤网培养法培养DFAT细胞生长至首次传代需要的时间分别为(16.00±1.41)d和(16.25±0.96)d,差异无统计学意义(P>0.05)。两种方法培养的DFAT细胞表面抗原表型均为CD29+CD90+CD31-CD45-,天花板培养法培养的DFAT细胞流式细胞分析结果为CD29+(97.63±3.15)%、CD90+(98.63±0.83)%、CD31+(0.11±0.12)%、CD45+(0.18±0.11)%;滤网培养法培养的DFAT细胞流式细胞分析结果为CD29+(96.93±3.61)%、CD90+(98.65±0.84)%、CD31+(0.16±0.13)%、CD45+(0.16±0.10)%,差异均无统计学意义(P>0.05)。天花板培养法和滤网培养法培养的DFAT细胞在体外经诱导后均可分化为平滑肌细胞,表达α-平滑肌肌动蛋白(alpha smooth muscle actin,αSMA)和平滑肌肌球蛋白重链(smooth muscle myosin heavy chain,SMMHC),αSMA阳性细胞率分别为(73.67±8.04)%、(73.56±8.44)%,差异无统计学意义(P>0.05)。天花板培养法培养DFAT细胞生长至首次传代较滤网培养法需要更多的完全培养基,分别为93.75(90.00,93.75)mL、48.50(48.50,48.50)mL,差异有统计学意义(P<0.05)。结论 天花板培养法和滤网培养法培养DFAT细胞在时间成本、细胞形态、细胞纯度及体外诱导分化为平滑肌细胞的潜能方面均无显著差异,但从经济成本考虑,滤网培养法稍有优势。
- Abstract:
- ObjectiveTo explore the differences between ceiling culture and insert culture for dedifferentiated fat (DFAT) cells in rats to provide research rationales for primary culture of DFAT cells.MethodsMature fat cells were isolated from rat inguinal adipose pad of eight 4-6-week-old Sprague-Dawley (SD) rats and DFAT cells harvested in vitro by dedifferentiation culture using ceiling and insert cultures.Cell morphology,time and amount of complete culture medium required from isolation to first passage were compared.Purity and function of differentiation into smooth muscle cells of DFAT cells were compared by flow cytometry analysis and cell immunofluorescence.ResultsDFAT cells obtained by two methods both were fibroblast-like,spindle-shaped and peak-valley-like.The time required for DFAT cells cultured by ceiling culture and insert culture to grow to the first passage were (16.00±1.41) and (16.25±0.96) days respectively and the difference was not significant (P>0.05).The surface antigen phenotypes of DFAT cells cultured by two methods were positive for CD29,CD90 and negative for CD31,CD45;flow cytometric results of DFAT cells cultured by ceiling culture showed CD29 was expressed in (97.63±3.15)% cells,CD90(98.63±0.83)%,CD31(0.11±0.12)%,CD45(0.18±0.11)%;the results of DFAT cells cultured by insert culture showed CD29 was expressed in (96.93±3.61)% cells,CD90 (98.65±0.84)%,CD31 (0.16±0.13)%,CD45 (0.16±0.10)%.And the differences were not significant (P>0.05).DFAT cells cultured by ceiling and insert cultures could differentiate into smooth muscle cells and express αSMA and SMMHC after induction in vitro.The rates of αSMA positive cells were (73.00±7.88)% and (73.56±8.44)% respectively and the difference was not significant (P>0.05).Ceiling culture for DFAT cell growth to the first passage required more complete medium than insert culture[93.75(90.00,93.75) vs 48.50(48.50,48.50) ml]and the difference was significant (P<0.05).ConclusionNo significant differences exist in time expense,cell morphology,purity or function of induction differentiation into smooth muscle cells in vitro of DFAT cells cultured by ceiling and insert culture.However,from the perspective of economic expense,insert culture offers some advantage.
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备注/Memo
收稿日期:2020-01-14。
基金项目:国家自然科学基金(81701531)
通讯作者:杨屹,Email:yangy2@sj-hospital.com