Chinese Journal of Tissue Engineering Research ›› 2019, Vol. 23 ›› Issue (13): 2016-2021.doi: 10.3969/j.issn.2095-4344.1676
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Guo Bo1, Liu Jia1, Cui Xiaolan2, Shi Han1, Zhang Sheyi1, Wang Jia1, Shan Xia1, Wang Yizhong1
Revised:
2018-12-24
Online:
2019-05-08
Published:
2019-05-08
Contact:
Wang Yizhong, MD, Chief physician, Department of Hematology and Endocrinology, Aerospace Clinical Medical College of Peking University, Beijing 100049, China
About author:
Guo Bo, Master candidate, Department of Hematology and Endocrinology, Aerospace Clinical Medical College of Peking University, Beijing 100049, China
Supported by:
the Scientific Research Fund Project of the Aerospace Clinical Medical College of Peking University, No. YN201806 (to WYZ)
CLC Number:
Guo Bo, Liu Jia, Cui Xiaolan, Shi Han, Zhang Sheyi, Wang Jia, Shan Xia, Wang Yizhong. Human umbilical cord mesenchymal stem cells combined with immunotherapy for the treatment of type 1 diabetic mice[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(13): 2016-2021.
2.1 人脐带间充质干细胞的表型鉴定结果 如图1所示,流式细胞仪检测结果:CD44-FITC(99.9%)、CD90-APC (98.9%)、CD105-PE(99.9%)高表达,CD14-FITC (0.081%)、CD34-PE(0.43%)、HLA-DR-APC (0.024%)低表达,细胞表型符合人脐带间充质干细胞的表型特征。 2.2 BALB/c Foxp3-DTR-EGFP阳性小鼠的流式鉴定结果 取四五周龄小鼠,经眼眶采血进行流式鉴定,共鉴定115只小鼠,其中阳性小鼠59只,阳性率51.3%,图2A为阳性结果,图2B为阴性结果。 2.3 BALB/c Foxp3-DTR-EGFP阳性小鼠Treg细胞敲除 BALB/c Foxp3-DTR-EGFP阳性小鼠经腹腔注射白喉毒素后24 h,再次通过流式细胞仪对小鼠进行鉴定,结果如图2C所示,Q2象限细胞群消失,Treg细胞被成功敲除。"
2.4.2 体质量变化 治疗后4周,正常对照组小鼠体质量明显增加,模型组小鼠体质量下降,两组对比差异有统计学意义(P < 0.01);免疫干预组、人脐带间充质干细胞组、联合治疗组小鼠体质量均有不同程度的增加,但与模型组对比,免疫干预组小鼠体质量变化不显著(P > 0.05);人脐带间充质干细胞组、联合治疗组小鼠体质量明显升高,与模型组对比差异有统计学意义(P < 0.05),其中联合治疗组体质量升高更加显著(P < 0.01),人脐带间充质干细胞组和联合治疗组小鼠体质量增量接近正常对照组水平。见表1。 2.4.3 C肽变化 治疗后4周,正常对照组小鼠C肽水平在合适范围内,模型组小鼠C肽水平明显下降,两组对比差异有统计学意义(P < 0.01);与模型组对比,免疫干预组小鼠血清C肽水平变化不显著(P > 0.05);人脐带间充质干细胞组及联合治疗组小鼠血清C肽水平显著升高,与模型组对比差异有统计学意义(P < 0.01);其中联合治疗组小鼠血清C肽水平改善最明显,接近正常对照组水平。见表2。"
2.5 苏木精-伊红染色观察胰腺组织形态变化 正常对照组小鼠胰岛形态规则,呈圆形或者椭圆形细胞团,边界清楚,胰岛数及胰岛内细胞数量多,排列致密,胰岛细胞大小一致,细胞胞体饱满,胞质丰富,呈嗜酸性,核染色清晰呈卵圆形;模型组小鼠胰岛明显萎缩,轮廓不规则、扭曲、变形,边界模糊,胰岛数及胰岛内细胞数量明显减少,排列紊乱,分布稀疏;免疫干预组、人脐带间充质干细胞组、联合治疗组小鼠胰岛细胞受损程度较轻,胰岛轻度萎缩,胰岛形态较规则,界限清晰,胰岛内细胞数量较多,尤其是联合治疗组,部分小鼠胰岛形态完整接近正常组小鼠胰岛。见图3。 2.6 小鼠胰岛素免疫组织化学染色 正常对照组小鼠胰岛素染色阳性面积占满整个胰岛,分布均匀;模型组小鼠胰岛素染色阳性面积仅占胰岛小部分,且分布紊乱;免疫干预组、人脐带间充质干细胞组、联合治疗组小鼠胰岛素染色阳性面积占胰岛的大部分,且分布较均匀;与模型组对比,人脐带间充质干细胞组及联合治疗组小鼠胰岛素染色阳性面积变化显著,其中部分人脐带间充质干细胞组及联合治疗组小鼠胰岛素染色阳性面积接近正常组。见图4。"
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