氯化锂及发育期牙胚根端复合体细胞条件培养液对脂肪干细胞增殖分化的影响

发布时间：2018-03-23 18:26

本文选题：条件培养液　切入点：增殖　出处：《安徽医科大学》2014年硕士论文

【摘要】：目的分离培养发育期根端复合体（developing apical complex DAC)并收集上清，制备不同条件培养基用于培养脂肪干细胞，检测发育期根端复合体细胞上清液对脂肪干细胞(adipose tissue-derived stem cells ADSCs)增殖及分化的影响；研究不同浓度经典Wnt信号激活剂氯化锂（lithium chloride，Licl）对ADSCs增殖分化的影响，探索促进ADSCs增殖分化的最佳Licl浓度。方法利用组织块结合酶消化法培养出生后20天（postnatal20days PN20d）的SD大鼠下颌磨牙DAC，并运用形态学、组织学及免疫组化染色方法鉴定DAC组织，收集原代DAC细胞的上清通过过滤及不过滤的方法分别制备成不同的条件培养基（DACCM-滤和DACCM-未滤）；MTT检测不同培养条件培养基对ADSCs增殖活性的影响；碱性磷酸酶活性检测及RT-PCR检测不同培养条件基对碱性磷酸酶（alkaline phosphatase ALP）蛋白活性及基因表达的影响。以不同浓度Licl（0mM、2.5mM、5mM、10mM、20mM、40mM）处理DACCM-未滤培养的ADSCs，MTT检测不同浓度Licl对ADSCs增殖能力的影响；ALP活性检测及RT-PCR检测ALP活性及ALP、BSP基因表达的影响。结果本实验成功分离培养了发育期根端组织复合体，HE染色指出发育期根端组织复合体取自牙根尚未发育完全的大鼠；细胞形态学观察发育期根端组织复合体细胞为长梭形间充质来源细胞和多角形的上皮来源的细胞混合组成；发育期根端组织复合体细胞免疫组化染色结果CK-14，vimentin阳性表达；发育期根端组织复合体上清分别经过滤和未过滤的方法制备了不同条件培养基；MTT分析结果表明发育期根端复合体细胞条件培养基-未滤（DACCM-未滤）培养的脂肪干细胞增殖活性明显高于普通培养基和发育期根端复合体细胞条件培养基-过滤（DACCM-滤）（P0.05）；普通培养基培养的脂肪干细胞增殖活性明显高于发育期根端复合体细胞条件培养液（过滤）组（P0.05）；ALP试剂盒检测ALP活性显示条件培养基（未过滤）培养的脂肪干细胞表达的ALP明显高于普通培养基和DACCM-滤组（P0.05）。 MTT结果表明5mM浓度Licl促进细胞增殖，明显高于其他各浓度组（P0.05），40mM浓度Licl明显抑制细胞增殖（P0.05）；ALP活性检测结果表明5mM浓度Licl促进细胞ALP活性表达，明显高于其他各浓度（P0.05），40mM浓度明显抑制细胞ALP活性表达（P0.05）；5mM浓度Licl明显促进细胞ALP、骨涎蛋白（bone sialoprotein BSP）基因表达（P0.05），40mM浓度明显抑制细胞ALP、BSP基因表达（P0.05）。结论取自牙根尚未发育完全的大鼠的发育期根端复合体，包含牙囊，牙乳头以及上皮根鞘；复合体细胞上清制备的DACCM-未滤对脂肪干细胞增殖分化均有明显促进作用；DACCM-滤对脂肪干细胞增殖具有抑制作用，，但对细胞成骨分化也有一定的促进作用；一定浓度范围的Licl促进细胞增殖分化，其中以5mM作用最明显，浓度过高可对细胞产生细胞毒性作用，并抑制脂肪干细胞增殖分化。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.4

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