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
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摘要
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.
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.
引文
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3 Arufe MC,Lu M,Lin RY.Differentiation of murine embryonic stem cells to thyrocytes requires insulin and insulin-like growth factor-1[J].Biochem Biophys Res Commun,2009,381(2):264-270.
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8 Kurmann AA,Serra M,Hawkins F,et al.Regeneration of thyroid function by transplantation of differentiated pluripotent stem cells[J].Cell Stem Cell,2015,17(5):527-542.
9张勤,刘东源.成人骨髓间充质干细胞向甲状腺细胞的诱导分化[J].中国组织工程研究与临床康复,2011,15(6):951-954.
10辛群,刘东源,张勤.骨髓间充质干细胞向甲状腺细胞的分化[J].中国组织工程研究与临床康复,2011,15(32):5909-5912.
11李振想,官立萍,姬明丽,等.人脐带间充质干细胞体外诱导分化为甲状腺细胞的初步探讨[J].实用医学杂志,2013,29(12):1904-1906.
12 Damante G,Tell G,Di Lauro R.A unique combination of transcription factors controls differentiation of thyroid cells[J].Prog Nucleic Acid Res Mol Biol,2001,66:307-356.
13 Nikiforov YE,Nikiforova MN.Molecular genetics and diagnosis of thyroid cancer[J].Nat Rev Endocrinol,2011,7(10):569-580.
14 Altmann A,Schulz RB,Glensch G,et al.Effects of Pax8 and TTF-1thyroid transcription factor gene transfer in hepatoma cells:imaging of functional protein-protein interaction and iodide uptake[J].J Nucl Med,2005,46(5):831-839.
15潘倩,章建全,张传森.甲状腺转录因子在甲状腺器官生发中的作用[J].解剖学杂志,2015,38(3):357-360,361.
16 Nakamura M,Ohtaki S.[Mechanism of thyroid hormone synthesis by thyroid peroxidase][J].Tanpakushitsu Kakusan Koso,1990,35(5):814-824.
17 Nakamura M,Yamazaki I,Nakagawa H,et al.Iodination and oxidation of thyroglobulin catalyzed by thyroid peroxidase[J].J Biol Chem,1984,259(1):359-364.
18 Gerard CM,Lefort A,Christophe D,et al.Control of thyroperoxidase and thyroglobulin transcription by cAMP:evidence for distinct regulatory mechanisms[J].Mol Endocrinol,1989,3(12):2110-2118.
19 Tong Q,Ryu KY,Jhiang SM.Promoter characterization of the rat Na+/I-symporter gene[J].Biochem Biophys Res Commun,1997,239(1):34-41.
20 Akama T,Luo Y,Sellitti DF,et al.Thyroglobulin increases thyroid cell proliferation via the suppression of specific MicroRNAs[J].Mol Endocrinol,2014,28(3):368-379.
2 Arufe MC,Lu M,Kubo A,et al.Directed differentiation of mouse embryonic stem cells into thyroid follicular cells[J].Endocrinology,2006,147(6):3007-3015.
3 Arufe MC,Lu M,Lin RY.Differentiation of murine embryonic stem cells to thyrocytes requires insulin and insulin-like growth factor-1[J].Biochem Biophys Res Commun,2009,381(2):264-270.
4 Ma R,Latif R,Davies TF.Thyrotropin-independent induction of thyroid endoderm from embryonic stem cells by activin A[J].Endocrinology,2009,150(4):1970-1975.
5 Antonica F,Kasprzyk DF,Opitz R,et al.Generation of functional thyroid from embryonic stem cells[J].Nature,2012,491(7422):66-71.
6 Ma R,Latif R,Davies TF.Thyroid follicle formation and thyroglobulin expression in multipotent endodermal stem cells[J].Thyroid,2013,23(4):385-391.
7 Ma R,Latif R,Davies TF.Human embryonic stem cells form functional thyroid follicles[J].Thyroid,2015,25(4):455-461.
8 Kurmann AA,Serra M,Hawkins F,et al.Regeneration of thyroid function by transplantation of differentiated pluripotent stem cells[J].Cell Stem Cell,2015,17(5):527-542.
9张勤,刘东源.成人骨髓间充质干细胞向甲状腺细胞的诱导分化[J].中国组织工程研究与临床康复,2011,15(6):951-954.
10辛群,刘东源,张勤.骨髓间充质干细胞向甲状腺细胞的分化[J].中国组织工程研究与临床康复,2011,15(32):5909-5912.
11李振想,官立萍,姬明丽,等.人脐带间充质干细胞体外诱导分化为甲状腺细胞的初步探讨[J].实用医学杂志,2013,29(12):1904-1906.
12 Damante G,Tell G,Di Lauro R.A unique combination of transcription factors controls differentiation of thyroid cells[J].Prog Nucleic Acid Res Mol Biol,2001,66:307-356.
13 Nikiforov YE,Nikiforova MN.Molecular genetics and diagnosis of thyroid cancer[J].Nat Rev Endocrinol,2011,7(10):569-580.
14 Altmann A,Schulz RB,Glensch G,et al.Effects of Pax8 and TTF-1thyroid transcription factor gene transfer in hepatoma cells:imaging of functional protein-protein interaction and iodide uptake[J].J Nucl Med,2005,46(5):831-839.
15潘倩,章建全,张传森.甲状腺转录因子在甲状腺器官生发中的作用[J].解剖学杂志,2015,38(3):357-360,361.
16 Nakamura M,Ohtaki S.[Mechanism of thyroid hormone synthesis by thyroid peroxidase][J].Tanpakushitsu Kakusan Koso,1990,35(5):814-824.
17 Nakamura M,Yamazaki I,Nakagawa H,et al.Iodination and oxidation of thyroglobulin catalyzed by thyroid peroxidase[J].J Biol Chem,1984,259(1):359-364.
18 Gerard CM,Lefort A,Christophe D,et al.Control of thyroperoxidase and thyroglobulin transcription by cAMP:evidence for distinct regulatory mechanisms[J].Mol Endocrinol,1989,3(12):2110-2118.
19 Tong Q,Ryu KY,Jhiang SM.Promoter characterization of the rat Na+/I-symporter gene[J].Biochem Biophys Res Commun,1997,239(1):34-41.
20 Akama T,Luo Y,Sellitti DF,et al.Thyroglobulin increases thyroid cell proliferation via the suppression of specific MicroRNAs[J].Mol Endocrinol,2014,28(3):368-379.