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中华细胞与干细胞杂志(电子版) ›› 2019, Vol. 09 ›› Issue (02) : 107 -113. doi: 10.3877/cma.j.issn.2095-1221.2019.02.007

论著

体外诱导骨髓间充质干细胞向甲状腺滤泡细胞分化的实验研究
潘倩1, 章建全1,(), 张传森2,()   
  1. 1. 200003 上海,第二军医大学长征医院超声诊疗科
    2. 200433 上海,第二军医大学再生医学研究中心
  • 收稿日期:2019-02-14 出版日期:2019-04-01
  • 通信作者: 章建全, 张传森
  • 基金资助:
    国家自然科学基金(81171436,81271717)

Committing bone mesenchymal stem cells to differentiate into thyroid follicular cells in vitro

Qian Pan1, Jianquan Zhang1,(), Chuansen Zhang2,()   

  1. 1. Department of Ultrasound in Medicine, Changzheng Hospital, Shanghai 200003, China
    2. Research Center of Regenerative Medcine, Second Military Medical University, Shanghai 200433, China
  • Received:2019-02-14 Published:2019-04-01
  • Corresponding author: Jianquan Zhang, Chuansen Zhang
引用本文:

潘倩, 章建全, 张传森. 体外诱导骨髓间充质干细胞向甲状腺滤泡细胞分化的实验研究[J/OL]. 中华细胞与干细胞杂志(电子版), 2019, 09(02): 107-113.

Qian Pan, Jianquan Zhang, Chuansen Zhang. Committing bone mesenchymal stem cells to differentiate into thyroid follicular cells in vitro[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2019, 09(02): 107-113.

目的

研究体外诱导SD大鼠骨髓间充质干细胞(BMSCs)分化成甲状腺滤泡细胞的培养条件,探讨其对BMSCs向甲状腺滤泡细胞分化的诱导作用。

方法

实验分组:阴性对照组(B组)、阳性对照组(T组)、共培养组(C组)、诱导素组(F组)、共培养+诱导素组(C+F组)。倒置显微镜下观察各组诱导1周后细胞形态的变化;细胞免疫染色法检测甲状腺特有标记物的表达;RT-PCR分析诱导后细胞内甲状腺细胞相关基因表达水平。多组间均数比较使用单因素方差分析ANOVA,两组间资料差异比较采用t检验。

结果

SD大鼠BMSCs (P3)诱导1周:(1)细胞免疫染色结果显示各实验组甲状腺转录因子TTF1和PAX8及钠/碘同向转运体(NIS)、甲状腺过氧化物酶(TPO)与甲状腺球蛋白(Tg)表达情况不同,其中以C+F组染色效果显著加深;(2)RT-PCR分析与C+F组比较,B组、C组、F组PAX8 (6.21±0.04,0.02±0.01,0.54±0.03,3.31±0.30,F = 283.07,P < 0.05)、TTF1 (0.33±0.04,0.03±0.01,0.15±0.03,0.08±0.02,F = 73.36,P < 0.05)、TG (14.90±2.00,0.10±0.05,1.61±0.40,1.91±0.39,F = 134.03,P < 0.05)mRNA水平下降。

结论

在体外BMSCs与FRTL-5间接接触共培养体系中添加TSH、胰岛素、转铁蛋白、生长抑素及氢化可的松可诱导BMSCs分化为表达甲状腺滤泡细胞特异抗原的细胞。

Objective

To induce BMSCs in SD rats to differentiate into thyroid follicular cells by using different culture conditions in vitro, investigate the differentiation of BMSCs into thyroid follicular cells, and to provide a new method and experimental model for differentiation research of adult stem cell-derived thyroid follicular cells.

Methods

The experimental groups: Group C+F (cultured + induced factors) : Group C (cultured) ; Group F (factors) ; and Group B[negative control group (BMSCs) ]and Group T [the positive control group (FRTL-5) ]. The morphological changes of the cells were observed one week after induction by inverted microscopy, the expression of specific thyroid markers was detected by cellular immunostaining, and the expression level of thyroid cell-related genes was analyzed by RT-PCR analysis.

Results

One week after the induction, the findings were as follows: (1) Immunofluorescence analysis showed that the expressions of ttf1 and pax8, sodium/iodide cotransporter (NIS), thyroid peroxidase (TPO) and thyroglobulin (Tg) were different in each experimental group. Among them, the staining effect of Group C+F was significantly deepened. (2) The RT-PCR analysis results were as follows: Compared with Group C+F, PAX8 (6.21±0.04 vs 0.02±0.01, 0.54±0.03, 3.31±0.30; P < 0.05), TTF1 (0.33±0.04 vs 0.03±0.01, 0.15±0.03, 0.08±0.02; P < 0.05) and TG (14.90±2.00 vs 0.10±0.05, 1.61±0.40, 1.91±0.39; P < 0.05) in Group B, Group C and Group F were significantly decreased.

Conclusion

In vitro cultivation system of BMSCs with indirect contact of FRTL-5 adding TSH, insulin, transferrin, somatostatin and hydrocortisone inducing factors can be induced to differentiate into cells expressing specific antigen of thyroid follicular cells.

表1 特异性引物核苷酸序列
图1 倒置相差显微镜下观察培养1周时细胞形态注:a图为C+F组,可见细胞表面凸起略少(×100);b图为C组可见细胞集落形成不明显(×100);c图为F组可见细胞集落形成(×100);d图为BMSCs可见明显细胞集落形成,呈较典型的长梭形(×100);e图为FRTL-5胞体较小,多呈短梭形,边界清晰光整,生长旺盛,易融合成片(×100);f图为C+F组培养孔周边部分细胞形态(×200)
图2 荧光显微镜下观察培养1周后各组细胞TTF1表达情况(免疫细胞荧光染色法)注:a图为FRTL-5阳性对照组(×200) (红色荧光:FTT1蛋白;蓝色荧光:细胞核);b图为BMSCs阴性对照组(×100);c图为共培+诱导素组TTF1阳性表达显示为胞核、胞浆均有着色,以胞核着色为著(×200);d图为共培组TTF1弱阳性表达显示为胞核、胞浆均有着色,以胞核为著(×200);e图为诱导素组TTF1弱阳性表达显示为仅有胞核着色(×400)
图3 免疫细胞荧光法检测培养1周后各组细胞PAX8表达情况注:a图为FRTL-5阳性对照组(×200)(红色荧光:PAX8蛋白;蓝色荧光:细胞核);b图为BMSCs阴性对照组(×100);c图为共培+诱导素组PAX8阳性表达显示为核周及胞浆内均有着色,以核周为著(×200);d图为共培组PAX8阴性(×200);e图为B3诱导素组PAX8阳性表达显示为胞浆内有着色(×200)
图4 免疫细胞荧光法检测培养1周后各组细胞标志性蛋白NIS表达情况注:a图为FRTL-5阳性对照组(×200)(红色荧光:NIS蛋白;蓝色荧光:细胞核);b图为BMSCs阴性对照组(×100);c图为共培+诱导素组NIS阳性表达显示为胞浆内着色(×200);d图为共培组NIS阳性表达显示为胞浆内着色(×200);e图为诱导素组NIS阳性表达显示为胞浆内着色(×200)
图5 免疫细胞荧光法检测培养1周后各组细胞标志性蛋白TPO表达情况注:a图为FRTL-5阳性对照组(×100) (红色荧光:TPO蛋白;蓝色荧光:细胞核);b图为BMSCs阴性对照组(×100);c图为共培+诱导素组TPO阳性表达(×200)显示为胞浆染色;d图为共培组TPO阳性表达(×200)显示为胞核及胞浆染色;e图为诱导素组TPO阳性表达(×200)显示为胞核及胞浆染色
图6 免疫细胞荧光法检测培养1周后各组细胞标志性蛋白Tg表达情况注:a图为FRTL-5阳性对照组(×100)(红色荧光:Tg蛋白;蓝色荧光:细胞核);b图为BMSCs阴性对照组(×100);c图为共培+诱导素组Tg阳性表达显示为胞浆染色(×200);d图为共培组Tg阴性(×200);e图为诱导素组Tg阳性表达显示为胞核染色(×200)
表2 RT-PCR检测TTF1、PAX8、NIS、Tg、TPO在各组细胞中的表达(n = 3, ± s
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