Isolation of bone marrow mesenchymal stem cells in transgenic pigs and co-culture with porcine islets
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摘要:
目的 探讨α-1, 3-半乳糖基转移酶（GGTA1）基因敲除（GTKO）、GTKO/人补体调节蛋白hCD46插入、单磷酸胞嘧啶-N-乙酰神经氨酸羟化酶（CMAH）/GGTA1双基因敲除（Neu5GC/Gal）猪骨髓间充质干细胞（BMSC）的分离培养，以及与猪胰岛共培养对胰岛的保护作用。 方法 从不同转基因修饰GTKO、GTKO/hCD46及Neu5GC/Gal猪中提取骨髓，采用全骨髓法分离猪BMSC后进行培养。对BMSC进行形态学观察，并使用流式细胞术鉴定BMSC表面标志物。同时，观察BMSC诱导的多向分化，通过绿色荧光蛋白（GFP）转染标记猪BMSC来实现对BMSC的标记和示踪。将GFP转染标记的猪BMSC与猪胰岛细胞共培养，观察猪胰岛形态变化，与单纯猪胰岛细胞培养组进行比较。 结果 猪来源的BMSC在体外培养时呈梭形，表达标志物CD29、CD44、CD73、CD90、CD105及CD166，不表达CD34、CD45，具有向脂肪细胞、成骨细胞、软骨细胞分化的能力；通过GFP转染标记的猪BMSC能够实现BMSC的标记和示踪，且在细胞分裂后的子代细胞中也能够稳定表达。猪BMSC对胰岛细胞有一定保护能力。 结论 成功建立了GFP标记的GTKO、GTKO/hCD46及Neu5GC/Gal猪来源的BMSC，其对胰岛细胞具有一定的保护能力。 Abstract:Objective To investigate the isolation and culture of porcine bone marrow mesenchymal stem cell (BMSC) with α-1, 3-galactosyltransferase (GGTA1) gene knockout (GTKO), GTKO/ human CD46 (hCD46) insertion and cytidine monopho-N-acetylneuraminic acid hydroxylase (CMAH)/GGTA1 gene knockout (Neu5GC/Gal), and the protective effect of co-culture with porcine islets on islet cells. Methods Bone marrow was extracted from different transgenic pigs modified with GTKO, GTKO/hCD46 and Neu5GC/Gal. Porcine BMSC were isolated by the whole bone marrow adherent method and then cultured. The morphology of BMSC was observed and the surface markers of BMSC were identified by flow cytometry. Meantime, the multi-directional differentiation induced by BMSC was observed, and the labeling and tracing of BMSC were realized by green fluorescent protein (GFP) transfection. The porcine BMSC transfected with GFP were co-cultured with porcine islet cells. Morphological changes of porcine islet cells were observed, and compared with those in the porcine islet cell alone culture group. Results BMSC derived from pigs were spindle-shaped in vitro, expressing biomarkers of CD29, CD44, CD73, CD90, CD105 and CD166 rather than CD34 and CD45. These cells were able to differentiate into adipocytes, osteoblasts and chondrocytes. Porcine BMSC with GFP transfection could be labeled and traced, which could be stably expressed in the daughter cells after cell division. Porcine BMSC exerted certain protective effect on islet cells. Conclusions GFP-labeled porcine BMSC modified with GTKO, GTKO/hCD46 and Neu5GC/Gal are successfully established, which exert certain protective effect upon islet cells. -
图 1 猪BMSC形态学表现（×100）
注：A图为野生型外三元猪BMSC；B图为野生型巴马猪BMSC；C图为野生型五指山猪BMSC；D图为GTKO/hCD46五指山猪BMSC；E图为GTKO巴马猪BMSC；F图为GTKO五指山猪BMSC；G图为野生型藏猪BMSC；H图为Neu5GC/Gal五指山猪BMSC。
Figure 1. Morphological findings of BMSC of pig
图 2 GTKO五指山猪BMSC的三系分化结果（×100）
注：A图为原位茜素红染色；B图为油红-O染色；C图为阿利新蓝染色。
Figure 2. The trilineage differentiation results of BMSC of GTKO Wuzhishan pig
图 3 GTKO五指山猪BMSC表面标志物表达情况
Figure 3. Expression of cell surface markers of BMSC of GTKO Wuzhishan pig
图 4 GTKO五指山猪BMSC标记示踪结果（×100）
注：A图为BMSC培养36 h后GFP表达情况；B图为BMSC培养48 h后GFP表达情况。
Figure 4. Labeling and tracing results of BMSC of GTKO Wuzhishan pig
图 5 猪胰岛与GTKO五指山猪BMSC的共培养结果（×100）
注：A图为猪胰岛单独培养；B图为GFP转染标记的BMSC单独培养；C图为猪胰岛与GFP转染标记的BMSC共培养。
Figure 5. Co-culture results of porcine pancreatic islets and BMSC of GTKO Wuzhishan pig
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