蛇床子素对大鼠骨髓间充质干细胞增殖和骨向分化的影响
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摘要
目的:
建立大鼠骨髓间充质干细胞(MSCs)的体外培养体系,观察大鼠MSCs生物学特性,同时研究蛇床子素对体外培养的MSCs增殖和骨向分化的影响,以及在诱导过程中向成骨细胞(OB)分化相关的蛋白BMP-2mRNA的影响,从干细胞角度探讨蛇床子素防治OP的可能作用机制。
方法:
1.运用全骨髓贴壁法分离培养鉴定SD大鼠的MSCs。
2.用MTT法检测不同浓度蛇床子素对MSCs增殖的影响,了解量效关系。
3.用改良钙钴法对加入蛇床子素诱导后的细胞进行检测碱性磷酸酶(ALP)染色,确定蛇床子素是否具有成骨诱导作用,并通过ALP阳性细胞计数,确定最佳蛇床子素添加浓度。
4.用最佳促骨向分化浓度的蛇床子素诱导MSCs向OB分化,分蛇床子素组、空白组、经典诱导组、蛇床子素和经典诱导液协同作用组四组,检测ALP活性和矿化结节数目。
5.运用RT-PCR的方法检测成骨诱导分化过程中各组与OB分化相关信号蛋白BMP-2mRNA的影响,分组同上。
结果:
1.MSCs分离后24 h基本贴壁,10-12d达到融合,经过成骨、成脂诱导培养,MSCs分别表现出成骨细胞、脂肪细胞:流式细胞仪检测分离到的SD大鼠MSCs表面标志抗原CD44、CD29表达阳性;CD45及CD34表达为阴性。
2.经MTT法检测蛇床子素对MSCs增殖的作用,蛇床子素浓度在1×10~(-5)mol/L,1×10~(-6)mol/L,1×10~(-7)mol/L有明显促进MSCs增殖的作用,与空白组相比较有显著差异(P＜0.05),其中以10~(-6)mol/L效果最好。
3.ALP染色阳性表达率结果显示:与空白组比较蛇床子素在1×10~(-6)mol/L具有统计学意义(P＜0.05),1×10~(-4)mol/L,1×10~(-5)mol/L,1×10~(-6)mol/L,1×10~(-7)mol/L无统计学意义。(P＞0.05)
4.细胞培养12d后,蛇床子素干预组培养液中的ALP活性与空白组之间差异有显著性(P＜0.05),而经典诱导组与协同组组间无显著性差异(P＞0.05)。
5.细胞培养21d后,矿化结节计数结果显示:蛇床子素干预组、空白组之间差异有显著性(P＜0.05),而经典诱导组与协同组、蛇床子素组之间均无显著性差异(P＞0.05)。
6.蛇床子素对干预体系中BMP-2mRNA的表达有上调作用,BMP-2 mRNA的表达,蛇床子素干预组与空白组之间有显著性差异(P＜0.05),经典诱导组与协同组之间无显著性差异(P＞0.05)。
结论:
1.采用全骨髓贴壁法可稳定获得均质性良好的大鼠MSCs。
2.1×10~(-5)mol/L、1×10~(-6)mol/L、1×10~(-7)mol/L的蛇床子素具有促进MSCs增殖的作用。
3.1×10~(-6)mol/L蛇床子素具有诱导MSCs向OB分化,并能提高反映OB功能的ALP活性和矿化节结数目。
4.1×10~(-6)mol/L蛇床子素可以上调BMP-2mRNA的表达,提示蛇床子素诱导MSCs骨向分化的可能作用机制。
Objective:
To establish cultivation system in vitro of bone marrow stromal cells from SD rats,observe their growth and characteristics and investigate the effects of osthole on their proliferation and osteoblast-differentiation ability.And to investigate the expression of BMP-2mRNA in induced procedure with osthole.The study may provide a probable action mechanism of osthole prevent and cure OP in stem cell aspect.
Methods:
1.MSCs from SD rats were isolated and purified by using differential attachment method,then identified according to morphology,induced-differentiation potentiality and the cell surface antigens.So we can establish a stable culturing system in viro.
2.MTT method was used to investigate the proliferation effects different concentrations Osthole on MSCs in SD rats,and blank control group.
3.ALP-positive cell counts by the improvement Gomori's calcium-cobalt staining was explored to observe.Osteoblastic differentiation potentiality of MSCs induced by Different-concentration osthole.Blank control is standard medium without Osthole Through these we confirm the most effective Concentrations.
4.Groups were divided:osthole group(induced by the most effective Concentrations),Blank control,classics group(classics induce bone formation group,positive control),Osthole +classics induce bone formation group;The ALP activity and the number of mineralized nodesls were measured to indicate the osteoblast function of different groups.
5.Semi-quantitative analysis was used to detect the expression of BMP-2 during osteodifferentiation through RT-PCR method,Grouping was same as above.
Results:
1.Primary cultured MSCs adhered to plastic surface within 24 hours and reached confluence within 10-12days.To be conditioned induced,MSCs were successfully differentiated into osteoblast and adipocyte.And confirmed that there was expression of CD29 and CD44, and no expression of CD34 and CD45 on the surface of MSCs by flow cytometry(FCM).
2.OD values of MTT shows Osthole groups of 1×10~(-5),1×10~(-6),1×10~(-7) mol/L were higher than blank groups,significantly(P＜0.05),especiallyl×10~(-6) mol/L.
3.Results of ALP staining for ALP-positive cell counts showed Osthole of 1×10~(-6)mol/L addition had a definit induction to osteoblast differentiation compared with the blank with significant difference(P＜0.05).1×10~(-4),1×10~(-5),1×10~(-7),1×10~(-8) mol/L with nosignificant difference(P＞0.05).
4.ALP activity values in MSCs 12 days of osthole groups were higher than that in blank groups. But there were no significant Differences between classics group and Osthole +classics induce bone formation group(P＞0.05).
5.The average number of mineralized nodes in MSCs 21days of osthole groups were higher than that in blank group.But there were no significant differences between classics group and Osthole +classics induce bone formation group(P＞0.05).
6.The expression of the BMP-2 mRNA was stronger than bank group.There were significant differences between Osthole group and classics group(P＜0.05).But there were no significant differences between classics group and Osthole +classics induce bone formation group P＞0.05).
Conclusion:
1.MSCs can be isolated from rat bone marrow by the method of Differential attachment.
2.Osthole in all these concentrations of 1×10~(-5) mol/L、1×10~(-6) mol/L、1×10~(-7)mol/L all can promote the proliferation of primary mouse bone MSCs
3.Osthole in the concentration of 1×10~(-6) mol/L can induce differentiation to osteoblast of MSCs;can improve the activity of ALP and the number of mineralized nodes.
4.Osthole in the concentration of 1×10~(-6) mol/L can enhance the expression of BMP-2 and mRNA,which was may be the mechanism of inducing differentiation to osteoblast of MSCs.
建立大鼠骨髓间充质干细胞(MSCs)的体外培养体系,观察大鼠MSCs生物学特性,同时研究蛇床子素对体外培养的MSCs增殖和骨向分化的影响,以及在诱导过程中向成骨细胞(OB)分化相关的蛋白BMP-2mRNA的影响,从干细胞角度探讨蛇床子素防治OP的可能作用机制。
方法:
1.运用全骨髓贴壁法分离培养鉴定SD大鼠的MSCs。
2.用MTT法检测不同浓度蛇床子素对MSCs增殖的影响,了解量效关系。
3.用改良钙钴法对加入蛇床子素诱导后的细胞进行检测碱性磷酸酶(ALP)染色,确定蛇床子素是否具有成骨诱导作用,并通过ALP阳性细胞计数,确定最佳蛇床子素添加浓度。
4.用最佳促骨向分化浓度的蛇床子素诱导MSCs向OB分化,分蛇床子素组、空白组、经典诱导组、蛇床子素和经典诱导液协同作用组四组,检测ALP活性和矿化结节数目。
5.运用RT-PCR的方法检测成骨诱导分化过程中各组与OB分化相关信号蛋白BMP-2mRNA的影响,分组同上。
结果:
1.MSCs分离后24 h基本贴壁,10-12d达到融合,经过成骨、成脂诱导培养,MSCs分别表现出成骨细胞、脂肪细胞:流式细胞仪检测分离到的SD大鼠MSCs表面标志抗原CD44、CD29表达阳性;CD45及CD34表达为阴性。
2.经MTT法检测蛇床子素对MSCs增殖的作用,蛇床子素浓度在1×10~(-5)mol/L,1×10~(-6)mol/L,1×10~(-7)mol/L有明显促进MSCs增殖的作用,与空白组相比较有显著差异(P＜0.05),其中以10~(-6)mol/L效果最好。
3.ALP染色阳性表达率结果显示:与空白组比较蛇床子素在1×10~(-6)mol/L具有统计学意义(P＜0.05),1×10~(-4)mol/L,1×10~(-5)mol/L,1×10~(-6)mol/L,1×10~(-7)mol/L无统计学意义。(P＞0.05)
4.细胞培养12d后,蛇床子素干预组培养液中的ALP活性与空白组之间差异有显著性(P＜0.05),而经典诱导组与协同组组间无显著性差异(P＞0.05)。
5.细胞培养21d后,矿化结节计数结果显示:蛇床子素干预组、空白组之间差异有显著性(P＜0.05),而经典诱导组与协同组、蛇床子素组之间均无显著性差异(P＞0.05)。
6.蛇床子素对干预体系中BMP-2mRNA的表达有上调作用,BMP-2 mRNA的表达,蛇床子素干预组与空白组之间有显著性差异(P＜0.05),经典诱导组与协同组之间无显著性差异(P＞0.05)。
结论:
1.采用全骨髓贴壁法可稳定获得均质性良好的大鼠MSCs。
2.1×10~(-5)mol/L、1×10~(-6)mol/L、1×10~(-7)mol/L的蛇床子素具有促进MSCs增殖的作用。
3.1×10~(-6)mol/L蛇床子素具有诱导MSCs向OB分化,并能提高反映OB功能的ALP活性和矿化节结数目。
4.1×10~(-6)mol/L蛇床子素可以上调BMP-2mRNA的表达,提示蛇床子素诱导MSCs骨向分化的可能作用机制。
Objective:
To establish cultivation system in vitro of bone marrow stromal cells from SD rats,observe their growth and characteristics and investigate the effects of osthole on their proliferation and osteoblast-differentiation ability.And to investigate the expression of BMP-2mRNA in induced procedure with osthole.The study may provide a probable action mechanism of osthole prevent and cure OP in stem cell aspect.
Methods:
1.MSCs from SD rats were isolated and purified by using differential attachment method,then identified according to morphology,induced-differentiation potentiality and the cell surface antigens.So we can establish a stable culturing system in viro.
2.MTT method was used to investigate the proliferation effects different concentrations Osthole on MSCs in SD rats,and blank control group.
3.ALP-positive cell counts by the improvement Gomori's calcium-cobalt staining was explored to observe.Osteoblastic differentiation potentiality of MSCs induced by Different-concentration osthole.Blank control is standard medium without Osthole Through these we confirm the most effective Concentrations.
4.Groups were divided:osthole group(induced by the most effective Concentrations),Blank control,classics group(classics induce bone formation group,positive control),Osthole +classics induce bone formation group;The ALP activity and the number of mineralized nodesls were measured to indicate the osteoblast function of different groups.
5.Semi-quantitative analysis was used to detect the expression of BMP-2 during osteodifferentiation through RT-PCR method,Grouping was same as above.
Results:
1.Primary cultured MSCs adhered to plastic surface within 24 hours and reached confluence within 10-12days.To be conditioned induced,MSCs were successfully differentiated into osteoblast and adipocyte.And confirmed that there was expression of CD29 and CD44, and no expression of CD34 and CD45 on the surface of MSCs by flow cytometry(FCM).
2.OD values of MTT shows Osthole groups of 1×10~(-5),1×10~(-6),1×10~(-7) mol/L were higher than blank groups,significantly(P＜0.05),especiallyl×10~(-6) mol/L.
3.Results of ALP staining for ALP-positive cell counts showed Osthole of 1×10~(-6)mol/L addition had a definit induction to osteoblast differentiation compared with the blank with significant difference(P＜0.05).1×10~(-4),1×10~(-5),1×10~(-7),1×10~(-8) mol/L with nosignificant difference(P＞0.05).
4.ALP activity values in MSCs 12 days of osthole groups were higher than that in blank groups. But there were no significant Differences between classics group and Osthole +classics induce bone formation group(P＞0.05).
5.The average number of mineralized nodes in MSCs 21days of osthole groups were higher than that in blank group.But there were no significant differences between classics group and Osthole +classics induce bone formation group(P＞0.05).
6.The expression of the BMP-2 mRNA was stronger than bank group.There were significant differences between Osthole group and classics group(P＜0.05).But there were no significant differences between classics group and Osthole +classics induce bone formation group P＞0.05).
Conclusion:
1.MSCs can be isolated from rat bone marrow by the method of Differential attachment.
2.Osthole in all these concentrations of 1×10~(-5) mol/L、1×10~(-6) mol/L、1×10~(-7)mol/L all can promote the proliferation of primary mouse bone MSCs
3.Osthole in the concentration of 1×10~(-6) mol/L can induce differentiation to osteoblast of MSCs;can improve the activity of ALP and the number of mineralized nodes.
4.Osthole in the concentration of 1×10~(-6) mol/L can enhance the expression of BMP-2 and mRNA,which was may be the mechanism of inducing differentiation to osteoblast of MSCs.
引文
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