本文選題：條件培養(yǎng)液　切入點：增殖　出處：《安徽醫(yī)科大學》2014年碩士論文

【摘要】：目的分離培養(yǎng)發(fā)育期根端復合體（developing apical complex DAC)并收集上清，制備不同條件培養(yǎng)基用于培養(yǎng)脂肪干細胞，檢測發(fā)育期根端復合體細胞上清液對脂肪干細胞(adipose tissue-derived stem cells ADSCs)增殖及分化的影響；研究不同濃度經(jīng)典Wnt信號激活劑氯化鋰（lithium chloride，Licl）對ADSCs增殖分化的影響，探索促進ADSCs增殖分化的最佳Licl濃度。 方法利用組織塊結(jié)合酶消化法培養(yǎng)出生后20天（postnatal20days PN20d）的SD大鼠下頜磨牙DAC，并運用形態(tài)學、組織學及免疫組化染色方法鑒定DAC組織，收集原代DAC細胞的上清通過過濾及不過濾的方法分別制備成不同的條件培養(yǎng)基（DACCM-濾和DACCM-未濾）；MTT檢測不同培養(yǎng)條件培養(yǎng)基對ADSCs增殖活性的影響；堿性磷酸酶活性檢測及RT-PCR檢測不同培養(yǎng)條件基對堿性磷酸酶（alkaline phosphatase ALP）蛋白活性及基因表達的影響。以不同濃度Licl（0mM、2.5mM、5mM、10mM、20mM、40mM）處理DACCM-未濾培養(yǎng)的ADSCs，MTT檢測不同濃度Licl對ADSCs增殖能力的影響；ALP活性檢測及RT-PCR檢測ALP活性及ALP、BSP基因表達的影響。 結(jié)果本實驗成功分離培養(yǎng)了發(fā)育期根端組織復合體，HE染色指出發(fā)育期根端組織復合體取自牙根尚未發(fā)育完全的大鼠；細胞形態(tài)學觀察發(fā)育期根端組織復合體細胞為長梭形間充質(zhì)來源細胞和多角形的上皮來源的細胞混合組成；發(fā)育期根端組織復合體細胞免疫組化染色結(jié)果CK-14，vimentin陽性表達；發(fā)育期根端組織復合體上清分別經(jīng)過濾和未過濾的方法制備了不同條件培養(yǎng)基；MTT分析結(jié)果表明發(fā)育期根端復合體細胞條件培養(yǎng)基-未濾（DACCM-未濾）培養(yǎng)的脂肪干細胞增殖活性明顯高于普通培養(yǎng)基和發(fā)育期根端復合體細胞條件培養(yǎng)基-過濾（DACCM-濾）（P0.05）；普通培養(yǎng)基培養(yǎng)的脂肪干細胞增殖活性明顯高于發(fā)育期根端復合體細胞條件培養(yǎng)液（過濾）組（P0.05）；ALP試劑盒檢測ALP活性顯示條件培養(yǎng)基（未過濾）培養(yǎng)的脂肪干細胞表達的ALP明顯高于普通培養(yǎng)基和DACCM-濾組（P0.05）。 MTT結(jié)果表明5mM濃度Licl促進細胞增殖，明顯高于其他各濃度組（P0.05），40mM濃度Licl明顯抑制細胞增殖（P0.05）；ALP活性檢測結(jié)果表明5mM濃度Licl促進細胞ALP活性表達，明顯高于其他各濃度（P0.05），40mM濃度明顯抑制細胞ALP活性表達（P0.05）；5mM濃度Licl明顯促進細胞ALP、骨涎蛋白（bone sialoprotein BSP）基因表達（P0.05），40mM濃度明顯抑制細胞ALP、BSP基因表達（P0.05）。 結(jié)論取自牙根尚未發(fā)育完全的大鼠的發(fā)育期根端復合體，包含牙囊，牙乳頭以及上皮根鞘；復合體細胞上清制備的DACCM-未濾對脂肪干細胞增殖分化均有明顯促進作用；DACCM-濾對脂肪干細胞增殖具有抑制作用，，但對細胞成骨分化也有一定的促進作用；一定濃度范圍的Licl促進細胞增殖分化，其中以5mM作用最明顯，濃度過高可對細胞產(chǎn)生細胞毒性作用，并抑制脂肪干細胞增殖分化。
[Abstract]:Objective to isolate and culture the developing apical complex DACin the root tip complex in the developmental stage and collect the supernatants, and to prepare different medium for the culture of adipose stem cells. The effects of supernatant of root end complex cells on the proliferation and differentiation of adipose tissue-derived stem cells ADSCswere detected, and the effects of different concentrations of classical Wnt signal activator Lithium chloride license on the proliferation and differentiation of ADSCs were studied. To explore the best concentration of Licl to promote the proliferation and differentiation of ADSCs. Methods the mandibular molars of SD rats were cultured 20 days after birth by tissue mass combined with enzyme digestion. The DAC tissues were identified by morphological, histological and immunohistochemical staining. The supernatants of primary DAC cells were prepared into different conditioned media by filtration and non-filtration respectively. The effects of different culture conditions on the proliferation of ADSCs were detected by DACCM- and DACCM- unfiltered culture medium. The effect of alkaline phosphatase activity and RT-PCR on the activity and gene expression of alkaline phosphatase alalkaline phosphatase ALP.The proliferation ability of ADSCs treated with different concentration of Licl0 mM2. 5 mM5 mM5 mM5 mM10 mM10 mM10 mM@@. The activity of ALP, the activity of ALP and the expression of ALP gene were detected by RT-PCR. Results in this experiment, we isolated and cultured the root end tissue complex successfully. He staining indicated that the root end tissue complex was taken from the rats whose teeth were not fully developed. CK-14 vimentin positive expression was observed by immunohistochemical staining of the cells from the long fusiform mesenchymal cells and polygonal epithelium. The culture conditions of the supernatant of root end tissue complex were prepared by filtration and unfiltration, respectively. The results of MTT analysis showed that the adipose cells of the root end complex were cultured in conditioned medium-unfiltered DACCM- unfiltered. The proliferative activity of adipose stem cells cultured in ordinary culture medium was significantly higher than that in normal culture medium and in the condition medium of root end complex cells at the developmental stage, and the proliferation activity of adipose stem cells cultured in ordinary culture medium was significantly higher than that in conditioned culture of root end complex cells. The ALP expression of adipose stem cells cultured in conditioned medium (unfiltered) was significantly higher than that in normal culture medium and DACCM- filter group. The results of MTT showed that Licl at 5mM concentration promoted cell proliferation, which was significantly higher than that in other concentration groups, and Licl significantly inhibited the proliferation of cells. The results showed that 5mM concentration Licl promoted the expression of ALP activity in cells. The concentration of P0.05mM significantly inhibited the expression of ALP activity. The concentration of P0.05mM significantly promoted the expression of ALP, and the concentration of bone sialoprotein BSP-P0.05mM significantly inhibited the expression of P0.05mM. Conclusion the root tip complex of the rat with incomplete root development, including dental follicles, dental papillae and epithelial root sheath, was obtained. DACCM- unfiltered from supernatant of complex cells could obviously promote the proliferation and differentiation of adipose stem cells. DACCM- filtration could inhibit the proliferation of adipose stem cells, but it could also promote the osteogenic differentiation of adipose stem cells. Licl at a certain concentration can promote cell proliferation and differentiation, especially 5mM, which can produce cytotoxicity and inhibit the proliferation and differentiation of adipose stem cells when the concentration is too high.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R781.4

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