本文關(guān)鍵詞：低強(qiáng)度脈沖超聲促進(jìn)大鼠牙囊細(xì)胞、骨髓間充質(zhì)干細(xì)胞成骨分化的初步研究　出處：《重慶醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 低強(qiáng)度脈沖超聲 牙囊細(xì)胞 骨髓間充質(zhì)干細(xì)胞 無機(jī)誘導(dǎo)因子復(fù)合性組織工程支架材料 成骨分化

【摘要】：目的:探討低強(qiáng)度脈沖超聲(Low-Intensity Pulsed Ultrasound,LIPUS)對(duì)SD大鼠牙囊細(xì)胞(rat dental follicle cells,rDFCs)、骨髓間充質(zhì)干細(xì)胞(rat bone marrow mesenchymal stem cells,rBMSCs)成骨分化能力的影響。方法:體外分離培養(yǎng)rDFCs、rBMSCs,流式細(xì)胞術(shù)檢測(cè)細(xì)胞表面標(biāo)記物表達(dá),茜素紅染色、油紅染色鑒定細(xì)胞多向分化能力;分別于第7d、21d采用定量PCR及茜素紅染色檢測(cè)LIPUS(30m W/cm2,20min/d)對(duì)rDFCs、rBMSCs體外成骨分化的影響;然后,將rDFCs、rBMSCs分別接種無機(jī)誘導(dǎo)因子復(fù)合性組織工程支架材料進(jìn)行培養(yǎng),于第3、5、7、9天采用掃描電鏡觀察細(xì)胞生長(zhǎng)情況;最后,分別構(gòu)建rDFCs-支架材料及rBMSCs-支架材料的細(xì)胞三維培養(yǎng)復(fù)合體,分組進(jìn)行裸鼠皮下移植:(1)支架材料組(空白對(duì)照組);(2)rDFCs+支架材料組;(3)rBMSCs+支架材料組;(4)rDFCs+支架材料組+LIPUS組;(5)rBMSCs+支架材料+LIPUS組;LIPUS組(30m W/cm2,20min/d)對(duì)植入部位進(jìn)行輻照,8周后收集樣本,制作組織切片,HE和Masson染色觀察組織修復(fù)狀況。結(jié)果:本研究成功分離培養(yǎng)rDFCs、rBMSCs,流式細(xì)胞儀檢測(cè)提示兩種細(xì)胞均具有間充質(zhì)干細(xì)胞特征,茜素紅及油紅染色實(shí)驗(yàn)提示兩種細(xì)胞具備多向分化潛能;定量PCR檢測(cè)發(fā)現(xiàn)LIPUS處理組ALP、Runx2、OSX、COL-1相對(duì)表達(dá)量較空白對(duì)照組更高;茜素紅染色結(jié)果顯示LIPUS處理組較對(duì)照組染色更明顯,鈣結(jié)節(jié)數(shù)量更多;掃描電鏡觀察結(jié)果顯示rDFCs、rBMSCs可順利粘附在材料上,隨培養(yǎng)時(shí)間的增加,支架材料表面及孔隙中可見大量細(xì)胞生長(zhǎng),呈梭形,可見細(xì)胞分泌細(xì)胞外基質(zhì);HE染色結(jié)果顯示空白對(duì)照組未見明顯細(xì)胞及新生組織存在。rDFCs+支架材料組及rBMSCs+支架材料組可見少量細(xì)胞及新生類骨組織;rDFCs+支架材料組+LIPUS組及rBMSCs+支架材料+LIPUS組支架材料間可見大量細(xì)胞及類骨組織;Masson染色結(jié)果顯示空白對(duì)照組未見明顯細(xì)胞及新生纖維組織,rDFCs+支架材料組及rBMSCs+支架材料組可見少量細(xì)胞、新生纖維組織及血管組織存在;rDFCs+支架材料組+LIPUS組及rBMSCs+支架材料+LIPUS組細(xì)胞數(shù)量明顯增多,可見大量新生纖維組織及血管組織。結(jié)論:體外聯(lián)合LIPUS輻照可在一定程度上增強(qiáng)rDFCs、rBMSCs成骨分化能力;電鏡掃描結(jié)果證明rDFCs、rBMSCs接種無機(jī)誘導(dǎo)因子復(fù)合性組織工程支架材料后生長(zhǎng)良好;構(gòu)建rDFCs-支架材料及rBMSCs-支架材料的細(xì)胞三維培養(yǎng)復(fù)合體并植入裸鼠皮下,LIPUS輻照后能有效提高rDFCs、rBMSCs骨組織再生能力。以上結(jié)果初步提示LIPUS可在一定程度上促進(jìn)rDFCs、rBMSCs成骨分化,為L(zhǎng)IPUS物理刺激應(yīng)用于牙周組織工程提供了一定的參考依據(jù)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R454.3;R781

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