RFP标记及bFGF/BMP-2信号诱导的牙组织工程的体内实验研究
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摘要
目的 RFP标记的牙胚细胞和b FGF/BMP-2转染的骨髓间充质干细胞混合培养,复合3D打印聚乙烯醇(PVA)/双相陶瓷骨支架(DCCP)材料构建牙组织工程的体内孵育,探究其分化能力。方法取健康SD大鼠128只,随机分为4组:细胞团块组;PVA/DCCP支架材料组;细胞团块+PVA/DCCP支架材料组;空白对照组,不做任何干预,均在全麻下植入大鼠肾被膜下。术后按4个时间点(5、10、14、28 d各8只)分期取材,进行免疫组化染色和激光共聚焦切片观察。结果免疫组化:析因分析显示细胞团块+PVA/DCCP支架材料组在5、10 d DMP-1、BMP-4表达量最高且均高于其余各组,随着时间推移表达量逐渐有减小趋势,差异有统计学意义P<0.05;DSP在各组各时间点均为阴性表达,差异无统计学意义P>0.05。激光共聚焦显微镜显示:细胞团块+PVA/DCCP支架材料组各时间点切片组织中都可观测到荧光标记的牙胚细胞。结论 RFP标记的牙胚细胞和b FGF/BMP-2转染骨髓间充质干细胞混合培养,复合PVA/DCCP支架材料,构建牙组织工程成活并分化,其分化机制有待进一步研究。
Objective Mixed culture of RFP markers of tooth germ cells and b FGF/BMP-2 transfecting the bone marrow stem cells( BMSCs) was conducted and polyvinyl alcohol( PVA)/biphasic ceramic bone scaffold( DCCP) stand was painted threedimensionally to construct heterogeneous tooth tissue engineering( HTTE). In order to investigate their ability to differentiate in vivo.Methods 128 healthy SD rats were randomly divided into four groups: the cell pellet group; PVA/DCCP scaffold group; cell clumps+ PVA/DCCP scaffold group; control group which was without any intervention. The HTTE was implanted into the renal subcapsular of rats under general anesthesia. Four time points after surgery were chosen to collect samples for testing,Immunohistochemical staining and confocal slice observation. At the 5 th,10 th,14 th,28 th day,8 samples were collected from each group. Results Immunohistochemistry: Factorial analysis showed that cell clumps + PVA/DCCP scaffold group in 5,10 days DMP-1,BMP-4 expression were the highest and were higher than the rest groups,the expression level gradually decreased over time,and the difference was statistically significant P<0.05; DSP in each group at each time point was negative,the difference was not statistically significant P> 0.05. Confocal microscopy showed: cell clumps +PVA/DCCP scaffolds group at each time point biopsy tissue could be observed in tooth germ cell fluorescently marker. Conclusion RFP labeled tooth germ cells and b FGF/BMP-2 transfecting BMSCs mixed culture,composite PVA/DCCP scaffold successfully construct tooth tissue engineering and tooth differentiation,but its differentiation mechanism needs further study.
Objective Mixed culture of RFP markers of tooth germ cells and b FGF/BMP-2 transfecting the bone marrow stem cells( BMSCs) was conducted and polyvinyl alcohol( PVA)/biphasic ceramic bone scaffold( DCCP) stand was painted threedimensionally to construct heterogeneous tooth tissue engineering( HTTE). In order to investigate their ability to differentiate in vivo.Methods 128 healthy SD rats were randomly divided into four groups: the cell pellet group; PVA/DCCP scaffold group; cell clumps+ PVA/DCCP scaffold group; control group which was without any intervention. The HTTE was implanted into the renal subcapsular of rats under general anesthesia. Four time points after surgery were chosen to collect samples for testing,Immunohistochemical staining and confocal slice observation. At the 5 th,10 th,14 th,28 th day,8 samples were collected from each group. Results Immunohistochemistry: Factorial analysis showed that cell clumps + PVA/DCCP scaffold group in 5,10 days DMP-1,BMP-4 expression were the highest and were higher than the rest groups,the expression level gradually decreased over time,and the difference was statistically significant P<0.05; DSP in each group at each time point was negative,the difference was not statistically significant P> 0.05. Confocal microscopy showed: cell clumps +PVA/DCCP scaffolds group at each time point biopsy tissue could be observed in tooth germ cell fluorescently marker. Conclusion RFP labeled tooth germ cells and b FGF/BMP-2 transfecting BMSCs mixed culture,composite PVA/DCCP scaffold successfully construct tooth tissue engineering and tooth differentiation,but its differentiation mechanism needs further study.
引文
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[18]张健,田卫东,聂鑫,等.纤维粘连蛋白及骨形成蛋白-2、4在牙胚发育中的表达研究[J].四川大学学报(医学版),2007,38(5):826-828.
[19]占柳,谢淑娟,潘卫红.牙本质基质蛋白-1及其特异性表达的研究进展[J].现代口腔医学杂志,2013,27(1):55-58.
[20]张菁,梅陵宣.牙本质涎蛋白在牙齿发育、牙本质疾病中的表达和作用[J].牙体牙髓牙周病学杂志,2009,19(5):294-297.
[21]陈慧鲜,裴路.牙本质基质蛋白-1的研究近况[J].口腔医学,2014,34(2):141-143.
[22]王细文,梁平.激光扫描共聚焦显微镜在人胆管癌组织免疫荧光染色切片观察中的应用[J].激光杂志,2001,22(3):40-42.
[2]吴丽娜,温秀杰,刘鲁川,等.外胚间充质干细胞在牙组织工程的应用与基因调控[J].现代生物医学进展,2014,14(8):1595-1597.
[3]邬薇薇,胡杨,何惠宇,等.BMP-2、b FGF基因转染的大鼠骨髓干细胞与牙胚细胞在体外混合培养对成牙基因的影响[J].口腔医学研究,2015,31(1):18-22.
[4]马文渊,何惠宇,杨楠,等.PVA水凝胶、海藻酸钠凝胶与羊椎骨支架粘合植入动物体内观察[J].口腔医学研究,2013,29(6):501-504.
[5]杨楠,何惠宇,胡杨,等.复合骨髓间充质干细胞同种异体支架骨修复羊髂骨极限缺损[J].中国组织工程研究,2013,17(16):2859-2868.
[6]胡杨,盛磊,马莹,等.碱性成纤维细胞生长因子慢病毒真核表达载体的构建及转染[J].中国组织工程研究与临床康复,2011,15(6):1009-1014.
[7]韩祥祯,何惠宇,等.人骨形态发生蛋白-2重组慢病毒载体转染羊骨髓间充质干细胞及其成骨调节作用[J].中华实用诊断与治疗杂志,2014,28(6):540-542.
[8]张嘉宇,米雪.三维打印组织工程骨支架计算机辅助建模及快速成型技术[J].口腔医学研究,2013,29(12):1097-1101.
[9]巴娇娇,何惠宇,胡杨,等.牙胚细胞体外培养过程中成牙基因的表达[J].中国组织工程研究,2014,18(2):193-198.
[10]唐小雪.b FGF基因转染对羊骨髓间充质干细胞成骨调节作用的研究[D].乌鲁木齐:新疆医科大学,2012.
[11]胡杨,胡建江,王锋,等.海藻酸钙或聚乙烯醇双相陶瓷骨支架与牙胚细胞相容性的研究[J].中华临床医师杂志(电子版),2015,9(7):1168-1173.
[12]米雪,张嘉宇,何惠宇.三维打印组织工程骨支架的生物相容性实验研究[J].中华实用诊断与治疗杂志,2017,31(7):640-644.
[13]Butler WT,Brunn JC,Qin C,et al.Extracellular matrix proteins and the dynamics of dentin formation[J].Connect Tissue Res,2002,43(2-3):301-307.
[14]Fen JQ,Zhang J,Dallas SL,et al.Dentin matrix protein 1,a target molecule for Cbfa1 in bone,is a unique bone marker gene[J].J Bone Miner Res,2002,17(10):1822-1831.
[15]Qin C,Brunn JC,Cook RG,et al.Evidence for the Proteolytic Processing of Dentin Matrix Protein 1[J].J Biol Chem,2003,278(36):34700-34708.
[16]Ye L,Mac Dougall M,Zhang S,et al.Deletion of dentin matrix protein-1 leads to a partial failure of maturation ofpredentin into dentin,hypomineralization,and expanded cavities of pulp and root canal during postnatal tooth development[J].J Biol Chem,2004,279(18):19141-19148.
[17]Jacobson MD,Weil M,Raff MC.Programmed cell death in animaldevelopment[J].Cell,1997,88(3):347-354.
[18]张健,田卫东,聂鑫,等.纤维粘连蛋白及骨形成蛋白-2、4在牙胚发育中的表达研究[J].四川大学学报(医学版),2007,38(5):826-828.
[19]占柳,谢淑娟,潘卫红.牙本质基质蛋白-1及其特异性表达的研究进展[J].现代口腔医学杂志,2013,27(1):55-58.
[20]张菁,梅陵宣.牙本质涎蛋白在牙齿发育、牙本质疾病中的表达和作用[J].牙体牙髓牙周病学杂志,2009,19(5):294-297.
[21]陈慧鲜,裴路.牙本质基质蛋白-1的研究近况[J].口腔医学,2014,34(2):141-143.
[22]王细文,梁平.激光扫描共聚焦显微镜在人胆管癌组织免疫荧光染色切片观察中的应用[J].激光杂志,2001,22(3):40-42.