低强度脉冲超声促进大鼠牙囊细胞、骨髓间充质干细胞成骨分化的初步研究

发布时间：2017-12-26 20:37

本文关键词：低强度脉冲超声促进大鼠牙囊细胞、骨髓间充质干细胞成骨分化的初步研究　出处：《重庆医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:探讨低强度脉冲超声(Low-Intensity Pulsed Ultrasound,LIPUS)对SD大鼠牙囊细胞(rat dental follicle cells,rDFCs)、骨髓间充质干细胞(rat bone marrow mesenchymal stem cells,rBMSCs)成骨分化能力的影响。方法:体外分离培养rDFCs、rBMSCs,流式细胞术检测细胞表面标记物表达,茜素红染色、油红染色鉴定细胞多向分化能力;分别于第7d、21d采用定量PCR及茜素红染色检测LIPUS(30m W/cm2,20min/d)对rDFCs、rBMSCs体外成骨分化的影响;然后,将rDFCs、rBMSCs分别接种无机诱导因子复合性组织工程支架材料进行培养,于第3、5、7、9天采用扫描电镜观察细胞生长情况;最后,分别构建rDFCs-支架材料及rBMSCs-支架材料的细胞三维培养复合体,分组进行裸鼠皮下移植:(1)支架材料组(空白对照组);(2)rDFCs+支架材料组;(3)rBMSCs+支架材料组;(4)rDFCs+支架材料组+LIPUS组;(5)rBMSCs+支架材料+LIPUS组;LIPUS组(30m W/cm2,20min/d)对植入部位进行辐照,8周后收集样本,制作组织切片,HE和Masson染色观察组织修复状况。结果:本研究成功分离培养rDFCs、rBMSCs,流式细胞仪检测提示两种细胞均具有间充质干细胞特征,茜素红及油红染色实验提示两种细胞具备多向分化潜能;定量PCR检测发现LIPUS处理组ALP、Runx2、OSX、COL-1相对表达量较空白对照组更高;茜素红染色结果显示LIPUS处理组较对照组染色更明显,钙结节数量更多;扫描电镜观察结果显示rDFCs、rBMSCs可顺利粘附在材料上,随培养时间的增加,支架材料表面及孔隙中可见大量细胞生长,呈梭形,可见细胞分泌细胞外基质;HE染色结果显示空白对照组未见明显细胞及新生组织存在。rDFCs+支架材料组及rBMSCs+支架材料组可见少量细胞及新生类骨组织;rDFCs+支架材料组+LIPUS组及rBMSCs+支架材料+LIPUS组支架材料间可见大量细胞及类骨组织;Masson染色结果显示空白对照组未见明显细胞及新生纤维组织,rDFCs+支架材料组及rBMSCs+支架材料组可见少量细胞、新生纤维组织及血管组织存在;rDFCs+支架材料组+LIPUS组及rBMSCs+支架材料+LIPUS组细胞数量明显增多,可见大量新生纤维组织及血管组织。结论:体外联合LIPUS辐照可在一定程度上增强rDFCs、rBMSCs成骨分化能力;电镜扫描结果证明rDFCs、rBMSCs接种无机诱导因子复合性组织工程支架材料后生长良好;构建rDFCs-支架材料及rBMSCs-支架材料的细胞三维培养复合体并植入裸鼠皮下,LIPUS辐照后能有效提高rDFCs、rBMSCs骨组织再生能力。以上结果初步提示LIPUS可在一定程度上促进rDFCs、rBMSCs成骨分化,为LIPUS物理刺激应用于牙周组织工程提供了一定的参考依据。
[Abstract]:Objective: To investigate the effect of Low-Intensity Pulsed Ultrasound (LIPUS) on the osteogenic differentiation ability of SD rat dental follicle cells (rat dental follicle cells, rDFCs) and bone marrow mesenchymal stem cells (rat cells). Methods: the cultured rDFCs and rBMSCs in vitro, detect the expression of cell surface markers by flow cytometry, alizarin red staining, oil red staining to identify the cell differentiation; respectively at 7d, 21d PCR and alizarin red staining using quantitative detection of LIPUS (30M W/cm2,20min/d) of rDFCs and rBMSCs in vitro osteogenic differentiation; and rDFCs, rBMSCs, were inoculated with inorganic scaffolds induced factor composite tissue engineering were cultured in third, fifth, seventh and 9 days by using scanning electron microscope to observe cell growth; finally, three-dimensional cell culture scaffold complex rDFCs- and rBMSCs- scaffolds were constructed, were transplanted subcutaneously in nude mice: (1) the scaffold group (blank control group); (2) rDFCs+ scaffold group; (3) rBMSCs+ scaffold group; (4) rDFCs+ scaffold group +LIPUS group; (5) rBMSCs+ scaffold group +LIPUS; group LIPUS (30M of W/cm2,20min/d) The implanted sites were irradiated, and samples were collected 8 weeks later. Tissue sections were made. HE and Masson staining were used to observe the status of tissue repair. Results: the successful isolation of rDFCs and rBMSCs in this study, flow cytometry showed that two kinds of cells have the characteristics of mesenchymal stem cells, alizarin red and oil red staining experiments indicated that two kinds of cells have the potential of multi-directional differentiation; quantitative PCR assay showed that LIPUS treatment group ALP, Runx2, OSX, COL-1 relative expression compared with the blank higher in the control group; alizarin red staining showed that the LIPUS treatment group than in the control group were more obvious, more amount of calcium nodules; scanning electron microscopy showed that rDFCs and rBMSCs can be well adhered to the material, with the increase of culture time, scaffold material surface and pore can be seen in a large number of cell growth, spindle shaped cells the secretion of extracellular matrix; HE staining showed the presence of blank control group had no obvious cell and tissue. RDFCs+ scaffold group and rBMSCs+ scaffold group showed a few of cells and new bone tissue scaffolds; rDFCs+ group +LIPUS group and rBMSCs+ group +LIPUS scaffold scaffold can be seen between a large number of cells and bone tissue; Masson staining showed that the control group had no obvious cell and new fibrous tissue, rDFCs+ scaffold group and rBMSCs+ scaffold group showed a few of cells, fibrous tissue and vascular tissue; the number of rDFCs+ scaffold group +LIPUS group and +LIPUS group of rBMSCs+ scaffold cells increased obviously and showed large amount of new fibrous tissue and vascular tissue. Conclusion: the in vitro combined with LIPUS irradiation in a certain extent, rBMSCs enhanced rDFCs osteogenic differentiation; scanning electron microscopy results show that rDFCs and rBMSCs were induced by inorganic composite tissue engineering scaffold material factor after good growth; construction of rDFCs- scaffolds and rBMSCs- scaffold complex three-dimensional cell culture and transplanted subcutaneously in nude mice, can effectively improve the rDFCs rBMSCs bone tissue regeneration ability after LIPUS irradiation. These results suggest that LIPUS can promote osteogenic differentiation of rDFCs and rBMSCs to some extent, providing a reference for LIPUS physical stimulation in periodontal tissue engineering.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R454.3;R781

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