SHH基因修饰的大鼠鼻黏膜外胚层间充质干细胞向神经样细胞的诱导分化
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摘要
目的:探讨音猬因子(sonic hedgehog,SHH)基因转染对大鼠鼻黏膜外胚层间充质干细胞(ectomesenchymal stem cell,EMSCs)分化为神经样细胞的影响。方法:利用组织贴壁法分离、培养和纯化EMSCs,SHH基因重组腺病毒转染EMSCs(SHH-EMSCs),免疫荧光染色观察转染前后EMSCs的干细胞标志蛋白巢蛋白、波形蛋白和Connexin-43,蛋白质印迹检测SHH蛋白表达。诱导分化实验分为4组:SHH-EMSCs诱导组、SHH-EMSCs正常培养组、EMSCs诱导组、EMSCs正常培养组;倒置相差显微镜观察分化过程中细胞形态变化;免疫荧光染色和蛋白质印迹检测诱导后轴突膜蛋白(GAP-43)、β-3微管蛋白(TUBB3)、胶质纤维酸性蛋白(GFAP)和髓鞘碱性蛋白(MBP)的表达。并将SHH-EMSCs悬液注入大鼠脊髓组织内,观察该细胞在脊髓内存活和分化情况。结果:免疫荧光染色结果显示,经SHH基因转染后的EMSCs巢蛋白、波形蛋白和Connexin-43阳性率达90%;蛋白质印迹结果显示,SHH蛋白表达明显升高。SHH-EMSCs诱导组培养7 d后,细胞呈现双极、多极和锥形的典型神经细胞样形态;免疫荧光染色和蛋白质印迹结果表明SHH-EMSCs诱导组GAP-43、TUBB3、MBP和Smoothen的表达水平明显高于其他3组,差异有统计学意义; GFAP在4组细胞中均为弱表达,无显著差异。SHH-EMSCs在大鼠脊髓内存活情况良好且表达TUBB3。结论:SHH基因转染的EMSCs具有更高的向神经样细胞分化效率,并可在脊髓内存活; EMSCs可望成为移植脊修复髓损伤的一种新的种子细胞。
Objective: To investigate the effect of sonic hedgehog( SHH) gene transfection on differentiation of nasal mucosa-derived ectomesenchymal stem cells( EMSCs) into neural-like cells. Methods:EMSCs were isolated,cultured and purified by tissue adherent method. SHH gene recombinant adenovirus was transfected into EMSCs( SHH-EMSCs). The stem cells markers included Connexin-43,Nestin,and Vimentin in the EMSCs and SHH-EMSCs were stained by immunofluorescence. The expression of SHH was detected by Western blotting. Then these cells were induced to neural cells. The experiments were divided into four groups: SHH-EMSCs inducing group,SHH-EMSCs normal culture group,EMSCs inducinggroup and EMSCs normal culture group. The morphology of the cells were observed by the inverted microscope. The expression of neural markers included GAP-43,TUBB3,MBP and GFAP were detected by immunofluorescence and Western-Blotting. In addition,the SHH-EMSCs suspension was injected into the spinal cord. Two weeks after transplantation,the survived SHH-EMSCs in the rat spinal cord and secreted SHH were examined by immunohistochemistry. Results: Immunofluorescence results showed that the staining positive rates of Connexin-43,Nestin and Vimentin of SHH-EMSCs were all about 90%. Western blotting data showed that SHH-EMSCs highly expressed SHH. Differentiated SHH-EMSCs showed typical bipolar,multipolar and pyramidal neural-like morphology after inducing for 7 days in experimental group.Immunofluorescence and Western-blotting data showed that differentiated SHH-EMSCs expressed higher GAP-43,β3-tubin,MBP and Smoothen compared with the other three groups; GFAP of cells were weakly expressed in the four groups,with no statistical significance. The experimental results in vivo showed that transplanted SHH-EMSCs have higher survival rate and could express TUBB3 and secrete SHH in the spinal cord. Conclusion: SHH gene modified EMSCs are easy to differentiate into neural-like cells and can be used as the excellent seed cells for repairing spinal cord injury.
Objective: To investigate the effect of sonic hedgehog( SHH) gene transfection on differentiation of nasal mucosa-derived ectomesenchymal stem cells( EMSCs) into neural-like cells. Methods:EMSCs were isolated,cultured and purified by tissue adherent method. SHH gene recombinant adenovirus was transfected into EMSCs( SHH-EMSCs). The stem cells markers included Connexin-43,Nestin,and Vimentin in the EMSCs and SHH-EMSCs were stained by immunofluorescence. The expression of SHH was detected by Western blotting. Then these cells were induced to neural cells. The experiments were divided into four groups: SHH-EMSCs inducing group,SHH-EMSCs normal culture group,EMSCs inducinggroup and EMSCs normal culture group. The morphology of the cells were observed by the inverted microscope. The expression of neural markers included GAP-43,TUBB3,MBP and GFAP were detected by immunofluorescence and Western-Blotting. In addition,the SHH-EMSCs suspension was injected into the spinal cord. Two weeks after transplantation,the survived SHH-EMSCs in the rat spinal cord and secreted SHH were examined by immunohistochemistry. Results: Immunofluorescence results showed that the staining positive rates of Connexin-43,Nestin and Vimentin of SHH-EMSCs were all about 90%. Western blotting data showed that SHH-EMSCs highly expressed SHH. Differentiated SHH-EMSCs showed typical bipolar,multipolar and pyramidal neural-like morphology after inducing for 7 days in experimental group.Immunofluorescence and Western-blotting data showed that differentiated SHH-EMSCs expressed higher GAP-43,β3-tubin,MBP and Smoothen compared with the other three groups; GFAP of cells were weakly expressed in the four groups,with no statistical significance. The experimental results in vivo showed that transplanted SHH-EMSCs have higher survival rate and could express TUBB3 and secrete SHH in the spinal cord. Conclusion: SHH gene modified EMSCs are easy to differentiate into neural-like cells and can be used as the excellent seed cells for repairing spinal cord injury.
引文
[1]行勇军.神经嵴来源外胚间充质干细胞在牙向分化中的潜能及相关机制的研究[D].重庆:第三军医大学,2016.
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[3] Sampath K,Rubinstein AL,Cheng AM,et al. Induction of the zebrafish ventral brain and floorplate requires cyclops/nodal signalling[J]. Nature, 1998, 395(6698):185-189.
[4] Castillo-Azofeifa D,Losacco JT,Salcedo E,et al. Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance[J]. Development,2017,144(17):3054-3065.
[5]陈旭东,范文娟,袁科理,等.不同培养条件对小鼠诱导性多能干细胞分化为神经元样细胞的影响[J].细胞与分子免疫学杂志,2015,31(9):1216-1219.
[6]崔学文,张竞新,许艺荠,等. SHH缓释纤维蛋白支架移植促大鼠脊髓损伤的修复[J].神经解剖学杂志,2017,33(2):131-137.
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[10]卓毅,段答,葛丽特,等.人鼻黏膜间充质干细胞生物学特性与自体鼻黏膜间充质干细胞移植治疗脊髓损伤[J].中国组织工程研究,2017,21(29):4666-4672.
[11]贾祎佳.转SHH基因的骨髓间充质干细胞修复大鼠脊髓损伤的实验研究[D].太原:山西医科大学,2014.
[12] Aono H,Choudhury ME,Higaki H,et al. Microglia may compensate for dopaminergic neuron loss in experimental Parkinsonism through selective elimination of glutamatergic synapses from the subthalamic nucleus[J].Glia,2017,65(11):1833-1847.
[13]陈谦,陈静,崔颜宏,等.不同培养代数大鼠骨髓基质细胞向神经元样细胞转分化能力的比较[J].神经解剖学杂志,2009,25(1):1-5.
[14] Faissner A,Reinhard J. The extracellular matrix compartment of neural stem and glial progenitor cells[J].Glia,2015,63(8):1330-1349.
[15] Losada-perez M,Harrison N,Hidalgo A. Molecular mechanism of central nervous system repair by the Drosophila NG2 homologue kon-tiki[J]. J Cell Biol,2016,214(5):587-601.
[16] Buffo A,Rolando C,Ceruti S. Astrocytes in the damaged brain:molecular and cellular insights into their reactive response and healing potential[J]. Biochem Pharmacol,2010,79(2):77-89.
[17] Benowitz LI,Routtenberg A. GAP-43:an intrinsic determinant of neuronal development and plasticity[J].Trends Neurosci,1997,20(2):84-91.
[18] Cao S,Du J,Lv Y,et al. PAX3 inhibitsβ-Tubulin-Ⅲexpression and neuronal differentiation of neural stem cell[J]. Biochem Biophys Res Commun,2017,485(2):307-311.
[19] Deber CM,Reynolds SJ. Central nervous system myelin:structure,function,and pathology[J]. Clin Biochem,1991,24(2):113-134.
[20] Takizawa T,Gudla PR,Guo L,et al. Allele-specific nuclear positioning of the monoallelically expressed astrocyte marker GFAP[J]. Genes Dev,2008,22(4):489-498.
[21] Rauck BM,Novosat TL,Oudega M,et al. Biocompatibility of a coacervate-based controlled release system for protein delivery to the injured spinal cord[J]. Acta Biomaterialia,2015,11(1):204-211.
[22] Jung HY,Jin J,Lee KB,et al. Sonic hedgehog(SHH)and glioblastoma-2(Gli-2)expressions are associated with poor jaundice-free survival in biliary atresia[J]. J Pediatr Surg,2015,50(3):371-376.
[23] Huang Z,Zhen Y,Wei Y,et al. Shh promotes sweat gland cell maturation in three-dimensional culture[J].Cell Tissue Bank,2016,17(2):317-325.
[2] Zhang Z,Li Z,Deng W,et al. Ectoderm mesenchymal stem cells promote differentiation and maturation of oligodendrocyte precursor cells[J]. Biochem Biophys Res Commun,2016,480(4):727-733.
[3] Sampath K,Rubinstein AL,Cheng AM,et al. Induction of the zebrafish ventral brain and floorplate requires cyclops/nodal signalling[J]. Nature, 1998, 395(6698):185-189.
[4] Castillo-Azofeifa D,Losacco JT,Salcedo E,et al. Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance[J]. Development,2017,144(17):3054-3065.
[5]陈旭东,范文娟,袁科理,等.不同培养条件对小鼠诱导性多能干细胞分化为神经元样细胞的影响[J].细胞与分子免疫学杂志,2015,31(9):1216-1219.
[6]崔学文,张竞新,许艺荠,等. SHH缓释纤维蛋白支架移植促大鼠脊髓损伤的修复[J].神经解剖学杂志,2017,33(2):131-137.
[7]崔学文,李正南,张志坚,等.纤维蛋白胶支架搭载外胚间充质源雪旺细胞移植修复大鼠脊髓损伤[J].神经解剖学杂志,2015,31(1):65-72.
[8] Zhang Z,He Q,Deng W,et al. Nasal ectomesenchymal stem cells:multi-lineage differentiation and transformation effects on fibrin gels[J]. Biomaterials,2015,49:57-67.
[9]聂鑫.外胚间充质干细胞的多向分化及在组织工程周围神经再生中的应用[D].西安:第四军医大学,2004.
[10]卓毅,段答,葛丽特,等.人鼻黏膜间充质干细胞生物学特性与自体鼻黏膜间充质干细胞移植治疗脊髓损伤[J].中国组织工程研究,2017,21(29):4666-4672.
[11]贾祎佳.转SHH基因的骨髓间充质干细胞修复大鼠脊髓损伤的实验研究[D].太原:山西医科大学,2014.
[12] Aono H,Choudhury ME,Higaki H,et al. Microglia may compensate for dopaminergic neuron loss in experimental Parkinsonism through selective elimination of glutamatergic synapses from the subthalamic nucleus[J].Glia,2017,65(11):1833-1847.
[13]陈谦,陈静,崔颜宏,等.不同培养代数大鼠骨髓基质细胞向神经元样细胞转分化能力的比较[J].神经解剖学杂志,2009,25(1):1-5.
[14] Faissner A,Reinhard J. The extracellular matrix compartment of neural stem and glial progenitor cells[J].Glia,2015,63(8):1330-1349.
[15] Losada-perez M,Harrison N,Hidalgo A. Molecular mechanism of central nervous system repair by the Drosophila NG2 homologue kon-tiki[J]. J Cell Biol,2016,214(5):587-601.
[16] Buffo A,Rolando C,Ceruti S. Astrocytes in the damaged brain:molecular and cellular insights into their reactive response and healing potential[J]. Biochem Pharmacol,2010,79(2):77-89.
[17] Benowitz LI,Routtenberg A. GAP-43:an intrinsic determinant of neuronal development and plasticity[J].Trends Neurosci,1997,20(2):84-91.
[18] Cao S,Du J,Lv Y,et al. PAX3 inhibitsβ-Tubulin-Ⅲexpression and neuronal differentiation of neural stem cell[J]. Biochem Biophys Res Commun,2017,485(2):307-311.
[19] Deber CM,Reynolds SJ. Central nervous system myelin:structure,function,and pathology[J]. Clin Biochem,1991,24(2):113-134.
[20] Takizawa T,Gudla PR,Guo L,et al. Allele-specific nuclear positioning of the monoallelically expressed astrocyte marker GFAP[J]. Genes Dev,2008,22(4):489-498.
[21] Rauck BM,Novosat TL,Oudega M,et al. Biocompatibility of a coacervate-based controlled release system for protein delivery to the injured spinal cord[J]. Acta Biomaterialia,2015,11(1):204-211.
[22] Jung HY,Jin J,Lee KB,et al. Sonic hedgehog(SHH)and glioblastoma-2(Gli-2)expressions are associated with poor jaundice-free survival in biliary atresia[J]. J Pediatr Surg,2015,50(3):371-376.
[23] Huang Z,Zhen Y,Wei Y,et al. Shh promotes sweat gland cell maturation in three-dimensional culture[J].Cell Tissue Bank,2016,17(2):317-325.