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目的通过分离扩增版纳微型猪骨髓间充质干细胞(BMSCs)观察传代对慢病毒转染的绿色荧光蛋白(GFP)表达的影响。方法采集4月龄版纳微型猪的骨髓,在DMEM/F12培养液中分离间充质干细胞(MSCs),并根据其形态学、抗原标志表达和分化潜能给予鉴定。MSCs与GFP慢病毒载体共培养,荧光显微镜观察GFP的表达,流式细胞仪检测传代后MSCs GFP表达率的变化。标记后传代至1、2、3、4代细胞间比较用非参数检验。结果采用直接培养骨髓,可以分离到高表达CD13、CD29、CD90、CD105的MSCs,并可诱导分化为脂肪、骨和软骨细胞。MSCs与携带GFP的慢病毒载体共培养3天即可观察到GFP的表达,最佳转染复数(MOI)值为50,最佳共培养时间为6 d。传代后MSCs GFP表达率呈下降趋势[1代转染率(42.3±2.25)%、2代转染率(41.6±2.65)%、3代转染率(41.4±3.75)%和4代转染率(38.2±4.75)%],但传至4代GFP表达率的变化差异无统计学意义(P>0.05)。结论采用含有10%胎牛血清的DMEM/F12培养液可以从版纳微型猪骨髓中分离到MSCs,GFP慢病毒转染是标记MSCs的有效方法,连续传代4代不会显著影响GFP的表达。
Objective To observe the effect of passage on the expression of green fluorescent protein (GFP) transfected with lentivirus by amplifying Banna miniature bone marrow mesenchymal stem cells (BMSCs). Methods Bone marrow of 4-month-old Banna miniature pigs were collected and mesenchymal stem cells (MSCs) were isolated from DMEM / F12 medium and identified according to their morphology, antigenic marker expression and differentiation potential. MSCs were co-cultured with GFP lentivirus vector. The expression of GFP was observed by fluorescence microscopy. The expression of GFP in MSCs was detected by flow cytometry. Passage to 1, 2, 3, 4 generations of cells after comparison with non-parametric test. Results Directly cultured bone marrow, MSCs with high expression of CD13, CD29, CD90 and CD105 were isolated and differentiated into adipose, bone and chondrocytes. MSCs were co-cultured with lentiviral vector carrying GFP for 3 days to observe the expression of GFP. The optimal MOI was 50, and the optimal co-culture time was 6 days. After passage, the GFP expression rate of MSCs showed a decreasing trend (42.3 ± 2.25%), 2 (41.6 ± 2.65)%, 3 (41.4 ± 3.75)% and 4 passages (38.2 ± 4.75)%], but there was no significant difference in the expression rate of GFP between the 4th passage and the 4th passage (P> 0.05). Conclusion MSCs can be isolated from Banna miniature pig bone marrow by DMEM / F12 medium containing 10% fetal bovine serum. GFP lentivirus transfection is an effective method for labeling MSCs. Continuous passage 4 generations will not significantly affect the expression of GFP.