摘要
背景:传统诱导剂只具有单纯诱导成骨或成脂的分化作用,应用较为局限。杜仲叶提取物具有提高骨密度、抑制骨吸收、调节骨代谢功能的药效作用,可促进成骨细胞增殖和碱性磷酸酶分泌。目的:探讨杜仲叶提取物诱导羊骨髓间充质干细胞向成骨细胞分化的同时,是否具有抑制成脂肪分化的作用。设计、时间及地点:细胞学体外观察,于2008-02/10在南昌大学第二附属医院分子中心实验室完成。材料:健康12月龄羊6只,由南昌大学提供。杜仲叶提取物由江西师大生物技术公司制备。方法:采用全骨髓法体外分离培养羊骨髓间充质干细胞,取第3代细胞,设立6组:空白对照组,加入DMEM+胎牛血清;传统成骨诱导剂组,加入含地塞米松、β-甘油磷酸钠、抗坏血酸的DMEM高糖培养基;传统成骨诱导剂+杜仲叶提取物组,在传统诱导剂组基础上加入10-5g/mL杜仲叶提取物;10-4,10-5,10-6g/mL杜仲叶提取物组分别加入对应质量浓度的杜仲叶提取物。同时另取第3代细胞行成脂肪诱导,分组同上,仅将传统成骨诱导剂更换为传统成脂诱导剂。主要观察指标:成骨诱导后,钙钴法检测碱性磷酸酶活性,VonKossa法观察矿化结节的形成,RT-PCR法检测成骨细胞中Cbfa1mRNA基因的表达;成脂诱导后,油红O染色观察骨髓间充质干细胞成脂分化情况。结果:与空白对照组比较,各成骨诱导组细胞内碱性磷酸酶合成增多,形成矿化结节,且传统成骨诱导剂+杜仲叶提取物组、10-4,10-5g/mL杜仲叶提取物组诱导成骨情况优于传统成骨诱导剂组、10-6g/mL杜仲叶提取物组;RT-PCR显示各成骨诱导组均能高效扩增出171bp的Cbfa1基因转录片段,且成骨细胞中Cbfa1mRNA表达水平差异无显著性意义(P>0.05)。油红O染色结果显示,加入杜仲叶提取物的各成脂诱导组,其脂滴数量明显少于传统成脂诱导剂组。结论:杜仲叶提取物能诱导体外分离纯化培养的羊骨髓间充质干细胞向成骨细胞方向分化增殖,同时抑制其向脂肪细胞分化,实现双向调节作用。
BACKGROUND： Traditional inductor only has osteogenic or adipogenic effects. Extracting solution from Eucommia ulmoides Oily. can elevate bone density, inhibit bone resorption, adjust bone metabolism, and promote osteoblast proliferation and alkaline phosphatase secretion. OBJECTIVE： To explore whether extracting solution from Eucommia ulmoides Oliv. induced goat bone marrow mesenchymal stem cells （BMSCs） differentiation into osteoblasts and inhibited it into adipocytes. DESIGN, TIME AND SETTING： The cytology, in vitro, observation experiment was performed at the Molecule Central Laboratory, Second Affiliated Hospital, Nanchang University from February to October 2008. MATERIALS： A total of 6 healthy goats aged 12 months were supplied by Nanchang University. Extracting solution from Eucommia ulmoides Oliv. was prepared by Shida, Jiangxi, China. METHODS： Goat BMSCs were isolated and cultured by the whole bone marrow method in vitro. At the third passage, BMSCs were assigned into six groups. In the blank control group, BMSCs were incubated in DMEM containing fetal bovine serum. In the osteogenic inductor group, BMSCs were incubated in high-glucose DMEM, supplemented with dexamethasone,β -sodium glycerophosphate and ascorbic acid. In the osteogenic inductor ＋ extracting solution from Eucommia ulrnoides Oliv. group, 10^-5 g/mL extracting solution from Eucommia ulmoides Oliv. was added on the basis of traditional osteogenic inductor. In the 10^-4, 10^-5, 10^-6g/mL extracting solution from Eucommia ulmoides Oliv. groups, extracting solution from Eucommia ulmoides Oliv. at corresponding mass concentrations was added. BMSCs at passage 3 received adipogenic induction. BMSCs were assigned into identical groups as above mentioned, with the exception of traditional adipogenic inductor instead of traditional osteogenic inductor. MAIN OUTCOME MEASURES： Following osteogenic induction, alkaline phosphatase activities were examined by the calcium-cobalt method. Mineralized nodules were observed using the Von Kossa assay. Cbfal mRNA gene expression was quantified in osteoblasts by RT-PCR. Following adipogenic induction, the adipogenic differentiation of BMSCs was observed using the Oil red O staining. RESULTS： Compared with the blank control group, alkaline phosphatase activities increased and mineralized nodules formed in each osteogenic induction groups. Osteogenic induction was better in the osteogenic inductor ＋ extracting solution from Eucommia ulmoides Oliv. group, 10^-4, 10^-5 g/mL extracting solution from Eucommia ulmoides Oliv. groups compared with the osteogenic inductor group, 10^-6g/mL extracting solution from Eucommia ulmoides Oliv. group. RT-PCR showed that the osteoblasts could be induced in each osteogenic inductor group amplified fragment 171 bp Cbfal gene transcription. No significant differences in Cbfal mRNA expression in osteoblasts were detected （P 〉 0.05）. Oil red O staining demonstrated that lipid droplet number was significantly less in the each adipogenic inductor ＋ extracting solution from Eucommia ulmoides Oily. groups compared with the adipogenic inductor group. CONCLUSION： Extracting solution from Eucomrnia ulmoides Oily. can induce the differentiation of in vitro cultured goat BMSCs into osteoblasts, and inhibit their differentiation into adipocytes.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第10期1960-1964,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
江西省教育厅资助项目(GJJ09123)~~
