云南白药对PRP促兔骨髓基质干细胞增殖分化影响的研究
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摘要
【目的】1.研究云南白药对富血小板血浆促兔骨髓基质干细胞增殖和分化的影响;2.研究经口服云南白药干预后的富血小板血浆对兔骨髓基质干细胞增殖和分化的影响。
【方法】取4月龄新西兰大耳白兔骨髓,采用贴壁筛选法并结合传代对其进行纯化,并采用流式细胞术对其进行鉴定;观察第1代、第2代、第3代、第4代、第5代、第6代、第7代、第8代细胞的增殖情况,绘制生长曲线;以100mg/kg,1次/d,给兔灌药,连续7d,分别于给药第0、3、5、7d采静脉血;采用Landesberg法分别制备未经云南白药干预的PRP和经全身用药后的PRP;分别用不同浓度懂得云南白药配合未干预的PRP及用口服云南白药干预后的PRP培养rBMSCs;用四唑盐比色法(MTT)和酶动力学方法检测云南白药对PRP促rBMSCs增殖和分化的影响。
【结果】云南白药体外直接干预组与对照组相比,60μg/ml(0.53±0.03)、65μg/ml(0.56±0.05)组对rBMSCs的增殖作用有显著性差异(P＜0.05);65μg/ml(1.21±0.08)、70μg/ml(1.28±0.05)、75μg/ml(1.24±0.06)组云南白药可显著增强rBMSCs的ALP活性(P＜0.05)。口服云南白药干预组与对照组相比,服药5d(0.30±0.02)和7d(0.34±0.08)组对rBMSCs增殖作用有显著性差异(P＜0.05);服药7d(0.88±0.09)组可显著增强rBMSCs的ALP活性(P＜0.05)。
【结论】云南白药能够促进PRP对rBMSCs的增殖和分化作用,其中,60μg/m、65μg/m的云南白药能够促进PRP对rBMSCs的增殖作用;65μg/ml～75μg/m的云南白药能够促进PRP对rBMSCs的分化:服用云南白药5d和服用云南白药7d能够促进PRP对rBMSCs的增殖作用;服用云南白药7d能够促进PRP对rBMSCs的分化。
[Objective]To study the direct impact of YunnanBaiyao on platelet-rich plasma (PRP) for proliferation and differentiation of the bone marrow stromal cells of the rabbits;to study the impact of YunnanBaiyao which were taken though oral on PRP for proliferation and differentiation of the bone marrow stromal cells of the rabbits.
[Methods]The bone marrow stromal cells were isolated from the 4-month-old New Zealand rabbits as samples by using the adherent method.The Microscopy was used to study the cells's shape,and the flow cytometer was used to identify the cells.
Observed the proliferation status of the 1~(st),2~(nd),3~(rd),4~(th),5~(th),6~(th),7~(th),and 8~(th) generation cells,and drew the growth curve of the cells.Fed the rabbit with 100mg/Kg YunnanBaiyao once per day for 7 days.Obtain the blood samples of the rabbit on the 3~(rd),5~(th) and 7~(th) dav respectively.Obtained one pure PRP sample from the rabbit that has not been fed with YunnanBaiyao and one PRP sample from the rabbit that has been fed with YunnanBaiyao by using the Landesberg method.Then,check the jinfluence of YunnanBaiyao on the PRP for the proliferation and differentiation of rBMSCs by the MTT and enzyme kinetics methods.
[Results]Comparison between the YunnanBaiyao in vitro direct intervention groups and the control group:The difference between 60μg/m(0.53±0.03) and 65ug/m(0.56±0.051 sample goups on proliferation of rBMSCs(P＜0.05) is distinct.
Sample goup 65μg/ml(1.21±0.08).70μg/ml(1.28±0.05),and 75μg / ml(1.24±0.06) proves that YunnanBaiyao can enchance the ALP activity(P＜0.05) of the rBMSCs.
Comparison between the YunnanBaiyao oral intervention group and the control group: For the sample groups that have taken YunnanBaiyao for 5 days(0.30±0.02) and 7days(0.34±0.08),the one that has taken YunnanBaiyao for 7 days(0.88±0.06)proves that YunnanBaiyao can better enhance the ALP activity(P＜0.05) of the rBMSCs.
[Conclusion]PRP,activated by YunnanBaiyao,can promote the proliferation and differentiation of rBMSCs.The 60μg / ml and 65μg / ml sample group and the sample group that has taken YunnanBaiyao for 5 davs and 7 days show the best effect on proliferation of rBMSCs;and the sample group 65μg/ml,70μg / ml,and 75μg / ml that have taken YunnanBaiyao for 7days show the best effect on differentiation of rBMSCs.
【方法】取4月龄新西兰大耳白兔骨髓,采用贴壁筛选法并结合传代对其进行纯化,并采用流式细胞术对其进行鉴定;观察第1代、第2代、第3代、第4代、第5代、第6代、第7代、第8代细胞的增殖情况,绘制生长曲线;以100mg/kg,1次/d,给兔灌药,连续7d,分别于给药第0、3、5、7d采静脉血;采用Landesberg法分别制备未经云南白药干预的PRP和经全身用药后的PRP;分别用不同浓度懂得云南白药配合未干预的PRP及用口服云南白药干预后的PRP培养rBMSCs;用四唑盐比色法(MTT)和酶动力学方法检测云南白药对PRP促rBMSCs增殖和分化的影响。
【结果】云南白药体外直接干预组与对照组相比,60μg/ml(0.53±0.03)、65μg/ml(0.56±0.05)组对rBMSCs的增殖作用有显著性差异(P＜0.05);65μg/ml(1.21±0.08)、70μg/ml(1.28±0.05)、75μg/ml(1.24±0.06)组云南白药可显著增强rBMSCs的ALP活性(P＜0.05)。口服云南白药干预组与对照组相比,服药5d(0.30±0.02)和7d(0.34±0.08)组对rBMSCs增殖作用有显著性差异(P＜0.05);服药7d(0.88±0.09)组可显著增强rBMSCs的ALP活性(P＜0.05)。
【结论】云南白药能够促进PRP对rBMSCs的增殖和分化作用,其中,60μg/m、65μg/m的云南白药能够促进PRP对rBMSCs的增殖作用;65μg/ml～75μg/m的云南白药能够促进PRP对rBMSCs的分化:服用云南白药5d和服用云南白药7d能够促进PRP对rBMSCs的增殖作用;服用云南白药7d能够促进PRP对rBMSCs的分化。
[Objective]To study the direct impact of YunnanBaiyao on platelet-rich plasma (PRP) for proliferation and differentiation of the bone marrow stromal cells of the rabbits;to study the impact of YunnanBaiyao which were taken though oral on PRP for proliferation and differentiation of the bone marrow stromal cells of the rabbits.
[Methods]The bone marrow stromal cells were isolated from the 4-month-old New Zealand rabbits as samples by using the adherent method.The Microscopy was used to study the cells's shape,and the flow cytometer was used to identify the cells.
Observed the proliferation status of the 1~(st),2~(nd),3~(rd),4~(th),5~(th),6~(th),7~(th),and 8~(th) generation cells,and drew the growth curve of the cells.Fed the rabbit with 100mg/Kg YunnanBaiyao once per day for 7 days.Obtain the blood samples of the rabbit on the 3~(rd),5~(th) and 7~(th) dav respectively.Obtained one pure PRP sample from the rabbit that has not been fed with YunnanBaiyao and one PRP sample from the rabbit that has been fed with YunnanBaiyao by using the Landesberg method.Then,check the jinfluence of YunnanBaiyao on the PRP for the proliferation and differentiation of rBMSCs by the MTT and enzyme kinetics methods.
[Results]Comparison between the YunnanBaiyao in vitro direct intervention groups and the control group:The difference between 60μg/m(0.53±0.03) and 65ug/m(0.56±0.051 sample goups on proliferation of rBMSCs(P＜0.05) is distinct.
Sample goup 65μg/ml(1.21±0.08).70μg/ml(1.28±0.05),and 75μg / ml(1.24±0.06) proves that YunnanBaiyao can enchance the ALP activity(P＜0.05) of the rBMSCs.
Comparison between the YunnanBaiyao oral intervention group and the control group: For the sample groups that have taken YunnanBaiyao for 5 days(0.30±0.02) and 7days(0.34±0.08),the one that has taken YunnanBaiyao for 7 days(0.88±0.06)proves that YunnanBaiyao can better enhance the ALP activity(P＜0.05) of the rBMSCs.
[Conclusion]PRP,activated by YunnanBaiyao,can promote the proliferation and differentiation of rBMSCs.The 60μg / ml and 65μg / ml sample group and the sample group that has taken YunnanBaiyao for 5 davs and 7 days show the best effect on proliferation of rBMSCs;and the sample group 65μg/ml,70μg / ml,and 75μg / ml that have taken YunnanBaiyao for 7days show the best effect on differentiation of rBMSCs.
引文
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[3]吴在德.外科学.第5版,北京:人民卫生出版社,2000:804-806
[4]刘兴文.PRP在骨组织修复再生中的应用.国外医学生物医学工程分册.2003,26(6):251-254
[5]Huang LH,Neiva RE,Soehren SE,et al.The effect of platelet-rich plasma on the coronally advanced flap root coverage procedure:a pilot human trial.J Periodontol 2005,76(10):1768-1777
[6]Belli E,Longo B,Balestra FM.Autogenous platelet-rich plasma in combination,with bovine-derived hydroxyapatite xenograft for treatment of a cystic lesion of the jaw.J Craniofac Surg 2005,16(6):978-980
[7]张晔,曾炳芳,张长青等.富血小板血浆对体外培养骨髓间充质干细胞增殖及成骨活性的作用.中国修复重建外科杂志,2005,19(2):109-113
[8]刘兴文,刘宏伟,张秀白等.富血小板血浆对骨髓基质细胞BMSCs碱性磷酸酶活性和总蛋白含良的影响.临床口腔医学杂志,2004,20(5):259-261
[9]张卫兵,洪光祥,康皓.富血小板血浆对兔骨髓间充质干细胞增殖的作用.组织工程研究与临床康复.2008,12(34):6630-6642
[10]李威,阎福华,卢友光等.富血小板血浆对牙髓成纤维细胞附着、增殖的影响.口腔医学研究,2004,20(2):126-128
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