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转基因猪骨髓间充质干细胞的分离及与猪胰岛的共培养研究

朱淑芳, 曲泽澎, 陆赢, 等. 转基因猪骨髓间充质干细胞的分离及与猪胰岛的共培养研究[J]. 器官移植, 2024, 15(1): 55-62. doi: 10.3969/j.issn.1674-7445.2023205
引用本文: 朱淑芳, 曲泽澎, 陆赢, 等. 转基因猪骨髓间充质干细胞的分离及与猪胰岛的共培养研究[J]. 器官移植, 2024, 15(1): 55-62. doi: 10.3969/j.issn.1674-7445.2023205
Zhu Shufang, Qu Zepeng, Lu Ying, et al. Isolation of bone marrow mesenchymal stem cells in transgenic pigs and co-culture with porcine islets[J]. ORGAN TRANSPLANTATION, 2024, 15(1): 55-62. doi: 10.3969/j.issn.1674-7445.2023205
Citation: Zhu Shufang, Qu Zepeng, Lu Ying, et al. Isolation of bone marrow mesenchymal stem cells in transgenic pigs and co-culture with porcine islets[J]. ORGAN TRANSPLANTATION, 2024, 15(1): 55-62. doi: 10.3969/j.issn.1674-7445.2023205

转基因猪骨髓间充质干细胞的分离及与猪胰岛的共培养研究

doi: 10.3969/j.issn.1674-7445.2023205
基金项目: 国家重点研发计划(2017YFC1103704);国家自然科学基金重大项目(32095504);深圳市科技计划(GJHZ20200731095207021)
详细信息
    作者简介:
    通讯作者:

    牟丽莎(ORCID 0000-0001-6232-8341),博士,副研究员,研究方向为胰岛移植,Email:lishamou@gmail.com

  • 中图分类号: R617, Q78

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。

     

  • 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

    图  6  不同培养条件下猪胰岛细胞活力

    注:A图为Ham培养猪胰岛细胞;B图为HamCDM培养猪胰岛细胞;C图为Ham培养猪胰岛细胞48 h后细胞染色;D图为HamCDM培养猪胰岛48 h细胞染色(绿色为活细胞染色,红色为死细胞染色)。

    Figure  6.  Viability of porcine pancreatic islet cells under different culture conditions

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出版历程
  • 收稿日期:  2023-10-09
  • 网络出版日期:  2023-11-29
  • 刊出日期:  2024-01-11

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