本文選題：骨髓間充質(zhì)干細(xì)胞 + 牙髓細(xì)胞��； 參考：《廣西醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的：體外建立骨髓間充質(zhì)干細(xì)胞(Bone Marrow Mesenchymal Stem Cells, BMMSCs)與牙髓細(xì)胞(Dental Pulp Cells, DPCs)的共培養(yǎng)體系,觀察和評(píng)價(jià)兩種細(xì)胞在共培養(yǎng)后各自細(xì)胞特性的變化和相互影響,探索最佳的兩種細(xì)胞在共培養(yǎng)體系中的細(xì)胞比例和培養(yǎng)時(shí)間,體內(nèi)評(píng)估該共培養(yǎng)體系培養(yǎng)后的細(xì)胞應(yīng)用于牙髓再生中的可行性,為牙髓再生種子細(xì)胞的選擇提供新思路及實(shí)驗(yàn)基礎(chǔ)。方法：1、 體外分離、培養(yǎng)及鑒定BMMSCs和DPCs。將細(xì)胞在0.4 μm的Transwell小室中進(jìn)行共培養(yǎng),分組情況如下：①單一BMMSCs;②單一DPCs; ③BMMSCs和DPCs共培養(yǎng)組。共培養(yǎng)6天后,用MTT法繪制以上各組細(xì)胞的生長曲線并分別提取RNA及蛋白,分別進(jìn)行聚合酶鏈反應(yīng)(PCR)及蛋白免疫印跡(WB)檢測(cè)牙本質(zhì)涎磷蛋白(DSPP)、堿性磷酸酶(ALP)、基質(zhì)細(xì)胞抗原-1(Stro-1)和CD145的表達(dá)情況。2、為探索最佳的共培養(yǎng)細(xì)胞比例和體外共培養(yǎng)時(shí)間,將細(xì)胞在0.4μm的Transwell小室中進(jìn)行共培養(yǎng),BMMSCs與DPCs按照10：I、I：I、1：5和1：10的比例分別共培養(yǎng)3、6、9、12天。提取以上各組細(xì)胞的mRNA,qPCR檢測(cè)DSPP、ALP、Stro-1和CD146的表達(dá)并進(jìn)行統(tǒng)計(jì)學(xué)分析。3、 將人牙牙根段的根管進(jìn)行機(jī)械及化學(xué)預(yù)備,將牙根段隨機(jī)分為4組,每組4個(gè)樣本,將不同的細(xì)胞成分在水凝膠支架中混勻,并注入根管內(nèi),分組情況如下：①BMMSCs組；②DPCs組；③共培養(yǎng)組；④水凝膠支架空白組。將以上制備好的牙根段,移植到裸鼠背部,6周后取出標(biāo)本,制作HE染色切片,并于顯微鏡下觀察組織生長情況。結(jié)果：1、分離培養(yǎng)的SD大鼠骨髓間充質(zhì)細(xì)胞,24小時(shí)后可見細(xì)胞貼壁呈比較均一的圓球狀,3天后呈長梭形、紡錘樣,隨著培養(yǎng)時(shí)間延長,細(xì)胞增殖分裂聚集呈簇似漩渦樣。流式細(xì)胞儀檢測(cè)間充質(zhì)干細(xì)胞表面標(biāo)記物CD29,CD44表達(dá)陽性；造血干細(xì)胞標(biāo)記物CD45,CD34表達(dá)陰性。用成骨誘導(dǎo)培養(yǎng)基、成脂誘導(dǎo)培養(yǎng)基及成軟骨誘導(dǎo)培養(yǎng)基中誘導(dǎo)21天、21天和14天后,分別進(jìn)行茜素紅、油紅0及阿爾新藍(lán)染色陽性。牙髓細(xì)胞傳至第三代后形態(tài)均一,長梭形,呈成纖維樣細(xì)胞形態(tài)。免疫組織化學(xué)染色提示,角蛋白染色陰性,而波形絲蛋白染色陽性,提示培養(yǎng)的牙髓細(xì)胞為間充質(zhì)來源,可用于后續(xù)實(shí)驗(yàn)。2、通過共培養(yǎng)體系,可以提高BMMSCs牙髓細(xì)胞特異標(biāo)志物DSPP和ALP的表達(dá),降低干細(xì)胞標(biāo)志物Stro-1和CD146的表達(dá),顯示有向牙髓細(xì)胞向分化的趨勢(shì)；同時(shí)也可以提高DPCs干細(xì)胞標(biāo)志物的表達(dá),降低牙髓細(xì)胞標(biāo)志物的表達(dá),提示有去分化的趨勢(shì),激活分化為其它細(xì)胞的潛能。3、共培養(yǎng)細(xì)胞比例與時(shí)間改變,會(huì)對(duì)共培養(yǎng)細(xì)胞的分化情況有明顯影響。在BMMSCs與DPCs按照1：1與1：5的比例共培養(yǎng)時(shí),可以較高效的誘導(dǎo)BMMSCs分化及DPCs表達(dá)干細(xì)胞特異標(biāo)記物；體外共培養(yǎng)條件下,BMMSCs牙髓細(xì)胞標(biāo)志物表達(dá)增加、干細(xì)胞標(biāo)志物表達(dá)下降在共培養(yǎng)9-12天增加更為明顯,DPCs干細(xì)胞標(biāo)記物在共培養(yǎng)6-9天增加明顯。4、BMMSCs與DPCs按照1：1比例共培養(yǎng)9天,并裝配至多肽水凝膠支架充盈至經(jīng)機(jī)械化學(xué)預(yù)備后的根管內(nèi),牙根段移植至裸鼠背部6周后,發(fā)現(xiàn)共培養(yǎng)組能再生組織結(jié)構(gòu)與正常牙髓相似的類牙髓組織,而單一細(xì)胞組再生組織量較少且組織結(jié)構(gòu)與正常牙髓差異較大。結(jié)論：1、在共培養(yǎng)體系中,BMMSCs與DPCs相互影響,增殖速率及mRNA、蛋白的表達(dá)發(fā)生變化；BMMSCs受DPCs的影響出現(xiàn)牙髓細(xì)胞向分化的趨勢(shì),DPCs出現(xiàn)去分化表現(xiàn),結(jié)果提示共培養(yǎng)條件在一定程度可調(diào)控共培養(yǎng)細(xì)胞的增殖與分化,用低比例的DPCs獲得更多來源的適合牙髓組織再生的種子細(xì)胞。2、在BMMSCs與DPCs共培養(yǎng)體系中,細(xì)胞共培養(yǎng)比例為1：1、1：5,體外共培養(yǎng)時(shí)間為9天左右時(shí),細(xì)胞增殖與分化互相影響最為明顯。3、體內(nèi)皮下異位實(shí)驗(yàn)的結(jié)果顯示,該共培養(yǎng)體系培養(yǎng)的細(xì)胞可以作為牙髓組織工程再生中的種子細(xì)胞使用。
[Abstract]:Objective: to establish a co culture system of Bone Marrow Mesenchymal Stem Cells (BMMSCs) and dental pulp cells (Dental Pulp Cells, DPCs) in vitro, to observe and evaluate the changes and interaction of the two cells in co culture, and to explore the proportion of the best two cells in the co culture system and the proportion of the cells in the co culture system. Culture time, in vivo evaluation of the viability of the cells used in dental pulp regeneration in vivo, and provide new ideas and experimental basis for the selection of dental pulp regeneration seed cells. Methods: 1, in vitro isolation, culture and identification of BMMSCs and DPCs. cells in the Transwell chamber of 0.4 micron m. The following groups are as follows: 1 Single BMMSCs; (2) single DPCs; (3) BMMSCs and DPCs co culture group. After 6 days of co culture, the growth curves of these cells were plotted by MTT and RNA and protein were extracted respectively. Polymerase chain reaction (PCR) and protein immunoblotting (WB) were used to detect dentin sialosphosin (DSPP), alkaline phosphatase (ALP), matrix cell antigen -1 (Stro-1) and 45.2, in order to explore the best co culture cell ratio and in vitro co culture time, the cells were co cultured in the 0.4 m Transwell chamber, and BMMSCs and DPCs were co cultured for 3,6,9,12 days in proportion to 10:I, I:I, 1:5 and 1:10 respectively. Statistical analysis.3, the root canal of the tooth root canal was mechanically and chemically prepared, and the root segments were randomly divided into 4 groups, each group had 4 samples, and the different cell components were mixed in the hydrogel scaffold and injected into the root canal. The groups were as follows: (1) group BMMSCs; (2) DPCs group; (3) co culture group; (4) the blank group of hydrogel scaffold. The prepared root segments were transplanted into the back of nude mice. After 6 weeks, the specimens were taken out and the HE staining sections were made. The tissue growth was observed under the microscope. Results: 1, the bone marrow mesenchymal cells were separated and cultured in SD rats. After 24 hours, the cells displayed a relatively uniform round ball. After 3 days, the spindle shaped, spindle like, and the culture time prolonged, The cell proliferation and division were clustered like swirling like vortices. Flow cytometry was used to detect the surface markers of mesenchymal stem cells (CD29, CD44), hematopoietic stem cell markers, CD45, CD34 negative. It was induced by osteogenic induction medium, fat induced medium and cartilage induced medium for 21 days, and alizarin red and oil, respectively, after 21 days and 14 days. Red 0 and alnew blue staining were positive. The dental pulp cells were spread to third generations, with long spindle shape and fibroblast like cells. Immunohistochemical staining suggests that keratin is negative, and the coloration of the plasma protein is positive, suggesting that the cultured dental pulp cells are the source of mesenchymal cells, which can be used for subsequent experimental.2 and can be extracted through co culture system. The expression of DSPP and ALP, a specific marker of high BMMSCs dental pulp cells, reduced the expression of stem cell markers Stro-1 and CD146, showing a tendency to differentiate into dental pulp cells, and could also improve the expression of the markers of DPCs stem cells and reduce the expression of the markers of dental pulp cells, suggesting the trend of dedifferentiation and activation to differentiate into other cells. The potential.3, co culture cell proportion and time change, will have a significant influence on the differentiation of co cultured cells. When BMMSCs and DPCs co culture 1:1 and 1:5, it can induce BMMSCs differentiation and DPCs expression of stem cell specific markers more efficiently. Under co culture conditions, the expression of BMMSCs dental pulp cell markers is increased. The decrease of expression of cell markers was more obvious at 9-12 days of co culture. DPCs stem cell markers increased.4 in co culture for 6-9 days, BMMSCs and DPCs were co cultured for 9 days according to 1:1 ratio, and assembled into the root canal of the polypeptide hydrogel scaffold after mechanochemical preparation, and the root segments were transplanted to the back of nude mice for 6 weeks, and the co culture group was found. The regenerated tissue structure is similar to the normal dental pulp, while the single cell group has less tissue and the difference between the tissue structure and the normal pulp. Conclusion: 1, in the co culture system, BMMSCs and DPCs interact with each other, the proliferation rate and the expression of mRNA, and the expression of protein are changed; BMMSCs is affected by DPCs. The trend of the dedifferentiation of DPCs shows that co culture conditions can regulate the proliferation and differentiation of co cultured cells to a certain extent, and use a low proportion of DPCs to obtain more seed cells that are suitable for the regeneration of dental pulp tissue,.2. In the co culture system of BMMSCs and DPCs, the proportion of cell co culture is 1:1,1:5, and in vitro co culture time. At about 9 days, the cell proliferation and differentiation affect the most obvious.3. The result of subcutaneous ectopic experiment in the body shows that the cells cultured in this co culture system can be used as seed cells in the regeneration of dental pulp tissue engineering.
【學(xué)位授予單位】：廣西醫(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)】：R781

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3 劉紅梅;CRABP2基因過表達(dá)逆轉(zhuǎn)卵巢癌鉑類耐藥對(duì)腫瘤微環(huán)境影響探究及卵巢癌3D共培養(yǎng)初探[D];廣西醫(yī)科大學(xué);2016年

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10 王芳兵;腎上腺髓質(zhì)素基因轉(zhuǎn)染的骨髓間充質(zhì)干細(xì)胞缺氧共培養(yǎng)對(duì)新生大鼠心肌細(xì)胞凋亡的影響[D];福建醫(yī)科大學(xué);2012年

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