本文選題：滑膜間充質(zhì)干細(xì)胞　切入點(diǎn)：組織塊培養(yǎng)法　出處：《山西醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的:探討應(yīng)用組織塊培養(yǎng)法體外分離、純化、擴(kuò)增兔滑膜間充質(zhì)干細(xì)胞(synovial mesenchymal stem cells,SMSCs)的可行性及其分離細(xì)胞的多向誘導(dǎo)分化潛能;建立增強(qiáng)型綠色熒光蛋白(enhanced Green Fluorescent Protein,e GFP)基因/超順磁性氧化鐵納米顆粒(superparamagnetic iron oxide nanoparticles,SPIO)雙標(biāo)記兔滑膜間充質(zhì)干細(xì)胞的技術(shù),為解決SMSCs對關(guān)節(jié)內(nèi)軟骨損傷修復(fù)提供可靠的體內(nèi)、外示蹤方法。方法:無菌獲取兔膝關(guān)節(jié)脂肪墊滑膜組織,通過組織塊接種法分離原代SMSCs,聯(lián)合有限稀釋法體外純化、擴(kuò)增兔SMSCs;鏡下觀察SMSCs的細(xì)胞形態(tài)學(xué)及其生長特點(diǎn);以最佳感染復(fù)數(shù)(Multiplicity Of Infection,MOI)的e GFP-慢病毒感染處于對數(shù)生長期的兔SMSCs,熒光顯微鏡下及流式細(xì)胞儀觀察e GFP標(biāo)記陽性率;e GFP標(biāo)記SMSCs經(jīng)嘌呤霉素充分篩選后行SPIO標(biāo)記,透射電鏡下及普魯士藍(lán)染色觀察SPIO標(biāo)記結(jié)果。采用Alcian Blue、堿性磷酸酶、油紅 O‖染色檢測雙標(biāo)記SMSCs分別在成軟骨、成骨、成脂誘導(dǎo)液中的定向誘導(dǎo)分化情況。采用CCK8試劑檢測e GFP/SPIO雙標(biāo)記后SMSCs的細(xì)胞增殖能力及其細(xì)胞活性。結(jié)果:1、兔脂肪墊表層滑膜組織經(jīng)組織塊接種法2~3天后,組織塊周圍即可開始長出原代滑膜細(xì)胞,10~12天便可獲得大量滑膜細(xì)胞,經(jīng)有限稀釋法純化擴(kuò)增,SMSCs細(xì)胞形態(tài)更加均一。2、e GFP成功標(biāo)記SMSCs后,熒光顯微鏡下可見細(xì)胞內(nèi)出現(xiàn)綠色熒光,且熒光表達(dá)率隨MOI值的增大而增高;當(dāng)MOI值為100的慢病毒感染SMSCs時,細(xì)胞可獲得(38.4±2.7)%的e GFP轉(zhuǎn)染率,細(xì)胞仍保持良好的狀態(tài),經(jīng)嘌呤霉素篩選后可達(dá)到95%以上的熒光表達(dá)率,標(biāo)記30d之后,標(biāo)記細(xì)胞熒光仍然穩(wěn)定表達(dá),當(dāng)MOI100時,細(xì)胞形態(tài)出現(xiàn)改變。3、以50μg/ml的SPIO標(biāo)記經(jīng)篩選后的e GFP細(xì)胞,經(jīng)普魯士藍(lán)染色,鏡下可見細(xì)胞標(biāo)記率達(dá)(98.4±1.2)%,透射電鏡下觀察顯示SMSCs的細(xì)胞質(zhì)和吞飲小泡內(nèi)可見大量高電子致密度顆粒,而對照組細(xì)胞質(zhì)內(nèi)和吞飲小泡內(nèi)未見明顯高電子致密度顆粒。4、雙標(biāo)記SMSCs經(jīng)成軟骨細(xì)胞定向誘導(dǎo)21天后,誘導(dǎo)細(xì)胞Alcian Blue染色結(jié)果顯示:細(xì)胞外基質(zhì)明顯染成藍(lán)色;雙標(biāo)記SMSCs經(jīng)成骨細(xì)胞定向誘導(dǎo)21天后,誘導(dǎo)細(xì)胞堿性磷酸酶染色顯示:細(xì)胞內(nèi)堿性磷酸酶表達(dá)陽性;雙標(biāo)記SMSCs經(jīng)成脂肪細(xì)胞定向誘導(dǎo)14天后,誘導(dǎo)細(xì)胞油紅 O‖染色結(jié)果顯示:細(xì)胞內(nèi)出現(xiàn)明顯的紅色顆粒狀脂滴。5、CCK-8檢測發(fā)現(xiàn):SMSCs細(xì)胞雙標(biāo)記后仍保持良好的增殖狀態(tài),未標(biāo)記組與標(biāo)記組細(xì)胞比較無顯著差異(P0.05),未產(chǎn)生明顯細(xì)胞毒性。結(jié)論:1、組織塊培養(yǎng)法能高效的獲取原代SMSCs,并且操作簡單,細(xì)胞污染幾率小。2、分離、純化后的SMSCs細(xì)胞具有良好的增殖能力,并具有明顯的間充質(zhì)干細(xì)胞的形態(tài)特性。3、e GFP/SPIO雙標(biāo)記SMSCs效率高,雙標(biāo)記后細(xì)胞仍具有多向誘導(dǎo)分化潛能,未產(chǎn)生明顯細(xì)胞毒性,具有一定可行性,為SMSCs對關(guān)節(jié)內(nèi)軟骨損傷修復(fù)的示蹤研究奠定了實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Objective: to investigate the feasibility of isolating, purifying and amplifying synovial mesenchymal stem cells from rabbit synovial synovial cells by tissue mass culture in vitro and the potential of inducing differentiation of the cells. An enhanced Green Fluorescent protein (GFP) gene / superparamagnetic iron oxide nanoparticles-SPIOO (superparamagnetic iron oxide nanoparticles spIOO) double labeling technique for rabbit synovial mesenchymal stem cells was established to provide a reliable solution for repair of articular cartilage injury by SMSCs in vivo. Methods: the synovial tissue of rabbit knee joint fat pad was obtained by aseptic method. Primary SMSCs were isolated by tissue mass inoculation and purified in vitro with limited dilution method. The morphology and growth characteristics of SMSCs were observed under microscope. The positive rate of e GFP-lentivirus infection in logarithmic growth phase was observed by fluorescence microscope and flow cytometry. The positive rate of e GFP labeled SMSCs was detected by fluorescence microscope and flow cytometry. The SMSCs labeled with e GFP was fully screened by purine mycin and then labeled with SPIO, and the positive rate of e GFP-lentivirus infection was observed by fluorescence microscope and flow cytometry. The results of SPIO labeling were observed under transmission electron microscope and Prussian blue staining. The double labeled SMSCs was detected by Alcian Blue, alkaline phosphatase and oil red O O staining in cartilage formation and osteogenesis, respectively. CCK8 reagent was used to detect the proliferation ability and cell activity of SMSCs labeled with e GFP/SPIO. Results: 1. The synovial tissue of rabbit fat pad was inoculated with tissue mass for 3 days. A large number of synovial cells could be obtained after 10 ~ 12 days of primary synovial cell growth around the tissue block. After purification and amplification with a limited dilution method, the cells were more homogeneous. 2e GFP was successfully labeled with SMSCs, and green fluorescence could be seen in the cells under fluorescence microscope. The fluorescence expression rate increased with the increase of MOI value, when lentivirus with MOI value of 100 was infected with SMSCs, the transfection rate of e GFP was 38.4 鹵2.7%, and the cell remained in good condition, and the fluorescence expression rate was over 95% after purine mycin screening. After 30 days of labeling, the fluorescence of the labeled cells was still stable. When MOI100 was observed, the morphology of the cells was changed. The e GFP cells were labeled with 50 渭 g/ml SPIO and stained with Prussian blue. Under microscope, the cell labeling rate was 98.4 鹵1.20.The results of transmission electron microscopy showed that a large number of high electron density particles could be seen in the cytoplasm of SMSCs and in the vesicles of swallowing and drinking. However, in the control group, no high electron density granules were found in the cytoplasm of the control group and in the vesicles of swallowing drink. The double labeled SMSCs was induced by chondroblastoblast for 21 days. The results of Alcian Blue staining showed that the extracellular matrix was blue obviously. After 21 days of osteoblast induction with double labeled SMSCs, alkaline phosphatase expression in induced cells was positive, while double labeled SMSCs was induced by adipoblast for 14 days. The results showed that the cells were in a good proliferative state after double labeling. The results showed that there were obvious red granular lipid droplets. 5 CCK-8 assay showed that the cells were still in a good proliferative state after double labeling. There was no significant difference between the unlabeled group and the labeled group (P 0.05) and no obvious cytotoxicity. Conclusion: 1: 1, the primary SMSCs can be obtained efficiently by the tissue mass culture method, and the operation is simple, the cell contamination rate is small, and the cell is isolated. The purified SMSCs cells have good proliferative ability, and have obvious morphological characteristics of mesenchymal stem cells. 3e GFP/SPIO double labeled SMSCs has high efficiency. After double labeling, the cells still have multidirectional differentiation potential and no obvious cytotoxicity. It has certain feasibility, which lays an experimental foundation for the tracer study of intraarticular cartilage repair by SMSCs.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R684

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