论文标题:柚皮苷对大鼠骨髓间充质干细胞增殖和骨向分化的影响
The Effects on Proliferation and Differentiation to Osteoblast of SD Rat Bone Marrow Stromal Cells in Vitro of Naringin
论文作者
论文导师 张荣华,论文学位 硕士,论文专业 中西医结合临床
论文单位 暨南大学,点击次数 61,论文页数 78页File Size4913K
2006-05-01论文网 http://www.lw23.com/lunwen_589195362/
Naringin; bone marrow mesenchymal stem cell; osteoporosis; Osteoblast proliferation; induce differentiation
目的：研究骨碎补的有效成分柚皮苷对体外培养的BMSCs增殖和骨向分化的影响，确定诱导的最佳添加浓度和添加时间，以及在诱导过程与向OB分化相关的重要蛋白BMP-2和Cbfa1mRNA的影响，从不同角度探讨中药防治骨质疏松的机理。 方法： 1．运用全骨髓贴壁法分离、培养病鉴定SD大鼠的BMSCs； 2．将柚皮苷配成高中低三大组浓度，每大组中再设置不同的浓度梯度，用CFU-F和MTT法检测柚皮苷对BMSCs增殖的影响，了解量效关系(CFU-F：2μg/ml～500μg/ml；MTT：0.06μg／ml～500μg／ml)，并分别设置对照组； 3．用改良钙钴法对加入柚皮苷诱导后的细胞进行ALP染色，确定柚皮苷是否具有成骨诱导作用，并通过ALP阳性细胞计数，确定柚皮苷的添加浓度(2μg/ml～500μg/ml)和添加时间(3～15d)，空白组为对照组； 4．用最佳浓度的柚皮苷诱导BMSCs向OB分化，分柚皮苷组、空白组、经典诱导组、柚皮苷和经典诱导液协同作用组共四组，检测ALP和BGP的分泌情况，并观察经典诱导液与柚皮苷的交互作用； 5．运用RT-PCR的方法检测各诱导体系诱导分化过程中与OB分化相关的信号蛋白BMP-2和Cbfa1mRNA的影响，分组同上。 结果： 1．BMSCs分离后24h基本贴壁，12-15d达到融合，经过成骨、成脂和成软骨诱导培养，BMSCs分别表现出成骨细胞、脂肪细胞和成软骨细胞表型； 2．不同浓度的柚皮苷作用后，MTT显示：低浓度柚皮苷组的OD值高于空白组，并存在一定的量效关系，与空白组相比有显著差异(P＜0.05)，且以1.0μg/ml和1.5μg/ml效果最好，中浓度柚皮苷组与空白组相比无明显差异(P＞0.05)，高浓度柚皮苷组的OD值远低于空白组(P＜0.05)，CFU-F显示的集落数结果和MTT类似； 3．ALP阳性细胞计数示，柚皮苷具有确定的诱导BMSCs向OB分化，其低中浓度对诱导分化的影响无显著差异(P＞0.05)，考虑成本选用2μg/ml为最佳浓
Objective: To explore the mechanism of Drynaria Rhizome on invigorating the kidney and building up bones on different levels. To identify the optimum conditions of the SD rat BMSCs cultured with the active component Naringin of Drynaria Rhizome in vitro. To observe the effects on proliferation, differentiation to osteoblast of BMSCs cultured by various concentrations of Naringinn and confirm the best concentrations and duration of action.To investigate the expression of BMP-2 and Cbfal mRNA in induced system with Naringin on molecular biology level. Methods:1. BMSCs from SD rats were isolated and purified by using different attachment method, then identified according to morphology and induced-differentiation potentiality. So we can establish a stable culture system in viro;2. Different concentrations of Naringin were added to BMSCs with high, medium and low concentrations. Proliferation between Naringin groups (different concentrations) and blank control group (standard medium) were detected by means of CFU-F and MTT( CFU-F: 2μg/ml～500μg/ml; MTT: 0.06μg/ml～500μg/ml);3. ALP-positive cell counts by the improvement Gomori"s calcium-cobalt staining was explored to observe the osteoblastic differentiation potentiality of BMSCs induced by different-concentration Naringin (2μg/ml～500μg/ml). Blank control is standard medium without Naringin and positive control is the same one with dexamethasone, sodium β-glycerophosphate and vitamine C. Through these we could confirm the most effective concentrations and duration of action with Naringin(2μg/ml, 15 days);4. 4 groups were divided: Naringin group ( induced by the most effective concentrations), blank control, classic group (classic induce bone formation group, positive control), Naringin +classic group; The ALP activity and the amount of osteocalcin in cells were measured to indicate the osteoblast function of different groups;
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