不同浓度利福喷丁对兔脂肪干细胞增殖活性的影响

发布时间：2018-10-08 16:55

【摘要】：目的：探讨在体外条件下不同浓度的利福喷丁溶液对兔脂肪干细胞增殖活性的影响。方法：本试验取健康新西兰大白兔(雌雄不限)，体重2.5-3.0kg，由新疆医科大学实验动物中心提供。用3％戊巴比妥钠(1ml/kg)或速眠新(0.3ml/kg)麻醉后，8％硫化钠水溶液术区脱毛备皮，消毒，取肩胛间区皮下脂肪，无菌切取皮下脂肪。放入装有10ml PBS的培养皿中，带回实验室。用I型胶原酶将细胞消化后，接种在DMEM生长液中进行培养。在倒置显微镜下观察细胞生长态势以及形状。待细胞增殖到一定程度时进行传代，取第三代细胞接种后加入成骨诱导剂诱导成骨，于成骨诱导第17天镜下观察有钙结节形成，茜素红染色结果阳性，，证实所取细胞为兔脂肪干细胞，然后再取第三代兔脂肪干细胞分别设3组不同利福喷丁浓度(根据每组利福喷丁浓度不同，分为10μg/ml组、20μg/ml组、40μg/ml组)及空白对照组进行细胞培养，采用CCK-8比色法进行增殖分化活性比较。用碱性磷酸酶及矿化结节染色鉴定成骨细胞分化能力。结果：碱性磷酸酶及矿化结节染色均为阳性。与空白对照组相比：利福喷丁溶液浓度为10μg/ml、20μg/ml、40μg/ml时细胞增殖活性在统计学上无差异(P＞0.05)；在显微镜下可观察到利福喷丁溶液浓度为40μg/ml时兔脂肪干细胞形态发生明显变化。结论：从兔脂肪组织中分离、培养出的脂肪干细胞(ADSCs)在具有在体外诱导成为成骨细胞(OB)的分化潜能。利福喷丁溶液浓度为10μg/ml、20μg/ml、40μg/ml时细胞增殖活性在统计学上无差异(P＞0.05)；在显微镜下可观察到利福喷丁溶液浓度为40μg/ml时兔脂肪干细胞形态发生明显变化。
[Abstract]:Aim: to investigate the effect of rifapentine solution at different concentrations on the proliferation of rabbit adipose stem cells in vitro. Methods: the healthy New Zealand white rabbits (male and female), weighing 2.5-3.0 kg, were provided by Experimental Animal Center of Xinjiang Medical University. After anaesthesia with 3% pentobarbital sodium (1ml/kg) or 0.3ml/kg, 8% sodium sulphide solution was used to remove hair and skin, sterilize, take subcutaneous fat in interscapular area, and cut subcutaneous fat aseptically. Put it in a petri dish containing 10ml PBS and bring it back to the lab. Cells were digested with type I collagenase and cultured in DMEM growth liquid. Cell growth and shape were observed under inverted microscope. After the third generation cells were inoculated with osteogenic inducer to induce osteogenesis, calcium nodules were observed under the microscope on the 17th day of osteogenesis induction, and alizarin red staining was positive. The cells were confirmed to be rabbit adipose stem cells, and then the third generation of rabbit adipose stem cells were divided into three groups with different concentrations of rifapentine (10 渭 g/ml group, 20 渭 g/ml group, 40 渭 g/ml group) and blank control group, respectively. The activity of proliferation and differentiation was compared by CCK-8 colorimetry. The differentiation ability of osteoblasts was evaluated by alkaline phosphatase and mineralized nodule staining. Results: alkaline phosphatase and mineralized nodules were positive. When the concentration of rifapentine solution was 10 渭 g / ml or 20 渭 g / ml or 40 渭 g/ml, the proliferative activity of rabbit adipose stem cells had no statistical difference (P > 0. 05), and the morphological changes of adipose stem cells were observed under microscope when the concentration of rifapentine solution was 40 渭 g/ml. Conclusion: adipose stem cell (ADSCs) isolated from rabbit adipose tissue has the potential to differentiate into osteoblast (OB) in vitro. When the concentration of rifapentine solution was 10 渭 g / ml ~ (20 渭 g / ml), the proliferative activity of rabbit adipose stem cells had no statistical difference (P > 0. 05), and the morphological changes of adipose stem cells were observed under microscope when the concentration of rifapentine solution was 40 渭 g/ml.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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