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中华细胞与干细胞杂志(电子版)

中华细胞与干细胞杂志(电子版) ›› 2019, Vol. 09 ›› Issue (06) : 351 -357. doi: 10.3877/cma.j.issn.2095-1221.2019.06.006

所属专题： 文献；

论著

骨髓间充质干细胞对重症急性胰腺炎胰腺组织修复的促进作用研究
钱道海1, 宋国栋2, 张洲1, 马志龙2, 王冠男1, 于文建1, 陈鹏1, 王小明1,†()   
  1. 1. 241001　芜湖，安徽省芜湖市皖南医学院附属弋矶山医院肝胆外科
    2. 270002　上海，同济大学附属第十人民医院肝胆外科
  • 收稿日期:2019-08-18 出版日期:2019-12-01
  • 通信作者: 王小明
  • 基金资助:
    安徽省教育厅高校自然基金重点项目(KJ2017A271); 安徽省中央引导地方科技发展专项基金(YDZX20183400004899); 弋矶山医院引进人才科研基金(YR201601); 弋矶山医院科研能力"高峰"培育计划项目(GF2019G03)

Positive effect of bone marrow-derived mesenchymal stem cells on the repair of pancreatic tissue in severe acute pancreatitis

Daohai Qian1, Guodong Song2, Zhou Zhang1, Zhilong Ma2, Guannan Wang1, Wenjian Yu1, Peng Chen1, Xiaoming Wang1,†()   

  1. 1. Hepato-biliary Surgery, Yijishan Hospital, Wannan Medical College, Wuhu 241001, China
    2. Hepato-biliary Surgery, the Tenth People's Hospital, Affiliated to Tongji University, Shanghai 270002, China
  • Received:2019-08-18 Published:2019-12-01
  • Corresponding author: Xiaoming Wang
  • About author:
    Corresponding author: Wang Xiaoming, Email:
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引用本文：

钱道海, 宋国栋, 张洲, 马志龙, 王冠男, 于文建, 陈鹏, 王小明. 骨髓间充质干细胞对重症急性胰腺炎胰腺组织修复的促进作用研究[J]. 中华细胞与干细胞杂志(电子版), 2019, 09(06): 351-357.

Daohai Qian, Guodong Song, Zhou Zhang, Zhilong Ma, Guannan Wang, Wenjian Yu, Peng Chen, Xiaoming Wang. Positive effect of bone marrow-derived mesenchymal stem cells on the repair of pancreatic tissue in severe acute pancreatitis[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2019, 09(06): 351-357.

目的

观察骨髓间充质干细胞（BMSCs）抑制坏死性凋亡促进修复重症急性胰腺炎（SAP）的可能机理。

方法

（1）分离、培养和鉴定大鼠BMSCs；（2）构建牛磺胆酸钠（NaT）诱导的SAP大鼠模型，并分成正常组（NC）、假手术组（Sham）、SAP模型组（SAP）、PBS治疗组（PBS）、BMSCs治疗组和Necrostain-1 （Nec-1）治疗组，并检测胰腺病理评分和血淀粉酶水平；（3）运用Western Blot和qRT-PCR方法检测各组受损胰腺组织内RIPK1、RIPK3、Caspase-8、MLKL蛋白及mRNA表达，各组均数间比较采用单因素方差分析，两两比较采用LSD-t检验。

结果

BMSCs可以被诱导分化成骨、软骨、脂肪，并高表达CD44 （99.82﹪）、CD73 （99.87﹪）、CD90 （99.99﹪）、CD105 （99.78﹪），低表达CD11b （0.65﹪）、CD19 （0.85﹪）、CD34 （0.70﹪）和CD45 （1.20﹪）。SAP组胰腺病理评分（12.90±1.79）及血淀粉酶水平（1052.41±183.12） mU/ ml均高于NC组[评分：0.40±0.52，淀粉酶水平：（236.62±33.21） mU/ ml]和Sham组[评分：0.50±0.53，淀粉酶水平：（242.31±27.94） mU/ ml]（F = 200.275，F = 143.245，P均< 0.001），且SAP组受损胰腺组织内RIPK1、RIPK3、MLKL表达升高、Caspase-8表达降低（F = 179.905，P < 0.001）；BMSCs组和Nec-1治疗组胰腺病理评分及血淀粉酶水平均低于PBS治疗组[评分：7.20±1.23、7.00±1.05比12.60±1.65，F = 200.275，P < 0.001；淀粉酶水平：（452.21±101.68）mU/ml、（570.18±148.47） mU/ml比（972.77±204.29） mU/ml，F = 143.245，P < 0.001]，同时受损胰腺组织内RIPK1、RIPK3、MLKL表达下调、Caspase-8表达升高（F = 179.905，P < 0.001）。

结论

BMSCs可能通过抑制坏死性凋亡通路活化来修复SAP。

关键词: 骨髓间充质干细胞, 重症急性胰腺炎, 组织修复, 动物模型
Objective

To investigate the possible mechanism of bone marrow mesenchymal stem cells (BMSCs) promoting the repair of severe acute pancreatitis (SAP) by inhibiting necropotosis.

Methods

(1) the isolation, culture and identification of rat BMSCs; (2) the rat SAP models induced by sodium sulfonycholate (NaT) were constructed and divided into the normal group (NC) , Sham group (Sham) , SAP model group, PBS treatment group, BMSCs transplantation group and the Necrostain-1 (Nec-1) treatment group. (3) The Western-blot and qRT-PCR were used to detect the protein and mRNA expressions of RIPK1, RIPK3, caspase-8 and MLKL in the damaged pancreatic tissues of each group. One-way anova was used for comparison between the mean values of each group, and LSD-t test was used for pair comparison.

Results

BMSCs could be induced to differentiate into bone, cartilage and fat, with high expressions of CD44 (99.82﹪) , CD73 (99.87﹪) , CD90 (99.99﹪) and CD105 (99.78﹪) , low expressions of CD11b (0.65﹪) , CD19 (0.85﹪) , CD34 (0.70﹪) and CD45 (1.20﹪) . Pancreatic pathology scores (12.90±1.79) and blood amylase level (1052.41±183.12) mU/ml in SAP model group were significantly higher than those in NC group [scores: 0.40±0.52, amylase level: (236.62±33.21) mU/ml] and Sham group [scores: 0.50±0.53, amylase level: (242.31±27.94) mU/ml] (F = 200.275, F = 143.245, P < 0.001) . Moreover, the expressions of RIPK1, RIPK3 and MLKL were significantly increased and the expression of caspase-8 was significantly decreased in the SAP model group (F = 179.905, P < 0.001) . Pancreatic pathology scores and blood amylase level in BMSCs transplantation group and Nec-1 treatment group were significantly lower than those in PBS treatment group [score: 7.20±1.23, 7.00±1.05 vs 12.60±1.65, F = 200.275, P < 0.001; Amylase levels: (452.21±101.68) mU/ ml, (570.18±148.47) mU/ml vs (972.77±204.29) mU/ml, F = 143.245, P < 0.001], while the expressions of RIPK1, RIPK3, MLKL were significantly down-regulated and the expression of caspase-8 was significantly increased in the damaged pancreas (F = 179.905, P < 0.001) .

Conclusion

BMSCs may repair SAP by inhibiting the activation of Necroptosispathway .

Key words: Bone marrow mesenchymal stem cells, Severe acute pancreatitis, Tissue repair, Animal model
表1 目的基因引物序列
基因 前向引物（3'-5'） 反向引物（5'-3'）
RIPK1 TCCTCGTTGACCGTGAC GCCTCCCTCTGCTTGTT
RIPK3 CCAGCTCGTGCTCCTTGACT TTGCGGTCCTTGTAGGTTTG
MLKL TCTCCCAACATCCTGCGTAT TCCCGAGTGGTGTAACCTGTA
Caspase-8 GAGTCTTCTGTACCTGTTCC GATAGTCCTCTTCTCCATCTC
GAPDH CGCTAACATCAAATGGGGTG TTGCTGACAATCTTGAGGGAG
图1 倒置显微镜下观察BMSCs形态
图2 流式细胞分析结果
表2 各组血清淀粉酶水平测定（±s）
分组 例数 胰腺病理评分 淀粉酶（mU/ml）
NC组 6 0.40±0.52 236.62± 33.21
Sham组 6 0.50±0.53 242.31± 27.94
SAP组 6 12.90±1.79a 1052.40±183.10a
PBS组 6 12.60±1.65 972.77±204.29
BMSCs组 6 7.20±1.23b 452.21±101.68b
Nec-1治疗组 6 7.00±1.05b 570.18±148.47b
F值 ? 200.275 143.245
P值 ? < 0.001 < 0.001
表3 各组胰腺组织内RIPK1、RIPK3、MLKL、Caspase-8基因表达情况（±s）
分组 例数 RIPK1 RIPK3 MLKL Caspase-8
NC组 6 0.45±0.06 0.37±0.09 0.28±0.10 2.51±0.13
Sham组 6 0.43±0.11 0.40±0.08 0.35±0.16 2.57±0.19
SAP组 6 1.21±0.09a 0.99±0.13a 1.33±0.11a 0.68±0.14a
PBS组 6 1.31±0.17 0.86±0.09 1.34±0.18 0.78±0.08
BMSCs组 6 0.67±0.13b 0.55±0.07b 0.92±0.12b 1.18±0.14b
Nec-1治疗组 6 0.72±0.08b 0.58±0.11b 0.98±0.15b 1.22±0.10b
F值 ? 127.681 47.296 141.026 304.882
P值 ? < 0.001 < 0.001 < 0.001 < 0.001
图3 倒置显微镜下观察胰腺组织HE染色切片（×200）
图4 胰腺组织内RIPK1、RIPK3、MLKL、Caspase-8蛋白表达
图5 间充质干细胞通过抑制RIPK1/RIPK3调控的环死性凋亡发挥治疗作用
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