Chinese Journal of Tissue Engineering Research ›› 2013, Vol. 17 ›› Issue (6): 1101-1106.doi: 10.3969/j.issn.2095-4344.2013.06.025
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Lei Shuan-hu1,2, Yue Hai-yuan1, Wang Jing2, Wang Yu-liang1
Received:
2012-04-19
Revised:
2012-05-18
Online:
2013-02-05
Published:
2013-02-05
Contact:
Wang Yu-liang, Master’s supervisor, Professor, Department of Orthopedics, Second Affiliated Hospital of Lanzhou University, Lanzhou 730000, Gansu Province, China
About author:
Lei Shuan-hu★, Studying for master’s degree, Department of Orthopedics, Second Affiliated Hospital of Lanzhou University, Lanzhou 730000, Gansu Province, China 2006leihc@163.com
CLC Number:
Lei Shuan-hu, Yue Hai-yuan, Wang Jing, Wang Yu-liang. Research progress in differentiation of bone marrow mesenchymal stem cells into osteoblasts[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(6): 1101-1106.
2.1 电磁场 正弦电磁场15 Hz,1 mT可以刺激骨髓间充质干细胞向成骨细胞分化,同时抑制了脂肪细胞的形成[11]。具体方法:将8周龄SD鼠(雌雄均可,80-120 g)脱颈处死,无菌条件下取其股骨、胫骨,分离培养骨髓间充质干细胞后,将骨髓间充质干细胞暴露于连续正弦电磁场(Bm= 1 mT,f=15 Hz)。对照组除未使用电磁场外,其余处理措施均同实验组。结论:电磁场增强了核心结合蛋白因子2、骨涎蛋白、远端缺失同源盒5和骨形态发生蛋白2基因的表达,这些基因被认为是成骨细胞分化的标志物[12-14]。同时,电磁场降低了过氧化物酶体增殖物激活受体γ,脂肪酶,AP-2基因的表达,这3个转录因子和脂肪细胞的分化相关[15-16]。 2.2 前列腺素E2受体选择性激动剂(prostaglandin E2 receptor EP4-selective agonist)(ONO-4819) ONO-4819通过诱导骨髓间充质干细胞向成骨细胞分化的作用而增加了骨的生成[17]。具体方法:选用7周龄SD雄鼠,每只雄鼠给予ONO-4819(10 μg/kg,皮下注射),2次/d,一共5周[18],对照组使用同等剂量的盐水代替ONO-4819。分别于实验开始后第1,3,5,7,14,21,28和35天处死SD鼠(5只/次),观察实验结果。实验结果:骨形态测量学分析显示实验组小鼠其成骨细胞数量、骨体积、骨生成率均显著增加;免疫组织化学检测证实ONO-4819增加了核心结合蛋白因子2和成骨细胞特异性转录因子阳性率的表达;体外研究还表明,ONO-4819诱导碱性磷酸酶积极表达,上调了碱性磷酸酶和成骨细胞特异性转录因子相关mRNA的表达。与此相反,ONO-4819抑制了骨髓间充质干细胞向脂肪细胞的分化,降低了过氧化物酶体增殖物激活受体γ相关mRNA的表达。 2.3 骆驼蓬碱 骆驼蓬碱增强了骨髓间充质干细胞向成骨细胞分化的能力,其作用可能是通过诱导、活化骨形成蛋白和核心结合蛋白因子2的表达这一途径实现的[19]。4-6周龄ddy鼠中分离骨髓间充质干细胞,将骨髓间充质干细胞放入加有维生素C (50 mg /L)、b-甘油磷酸钠(10 mmol/L)和骆驼蓬碱的培养基培养14 d,结果显示:骆驼蓬碱显著诱导提高了成骨细胞标志酶碱性磷酸酶的活性和骨钙素分泌量。 2.4 纤维增强复合水凝胶纳米复合材料(fiber-reinforced laminated hydrogel nanocomposites) 选用年轻、成熟Wistar雄鼠,取其股骨和胫骨并从中分离出骨髓间充质干细胞[20-22]。用含有100 nmol/L 地塞米松,50 mg /L维生素C,10 mmol/L β-甘油磷酸钠的培养基诱导骨髓间充质干细胞,并将其置于纤维增强复合水凝胶纳米复合材料之中。结果:纤维增强复合水凝胶纳米复合材料通过模拟骨单位结构,为再生区域提供了机械强度,同时促进了骨髓间充质干细胞向成骨细胞分化的能力,显著提升了骨桥蛋白和骨钙素的表达[23]。 2.5 枸橘苷 采用C3H10T1/2细胞系,来源于C3H鼠胚胎的骨髓间充质干细胞。用含有枸橘苷1 μmol/L、体积分数10% FBS,50 mg /L维生素C,和10 mmol/L β-甘油磷酸钠的α-MEM培养液诱导分化[24]。培养24 h后检测核心结合蛋白因子2 mRNA的表达,2 d后检测核心结合蛋白因子2蛋白量,6 d后检测碱性磷酸酶的活性、7 d后检测、骨钙素、Ⅰ型胶原及骨桥蛋白mRNA的表达,30 d后观察矿化结节。实验结果:核心结合蛋白因子2 mRNA及其产物蛋白质表达量显著增加,碱性磷酸酶活性亦明显增加,骨钙素、Ⅰ型胶原和骨桥蛋白mRNA表达水平也显著提升。 2.6 骨形成态发生蛋白2活性多肽 取4周龄Wistar大鼠培养骨髓间充质干细胞,传至第3代改用条件培养基,培养基中加入200 mg/L的骨形成蛋白2活性多肽,细胞培养至5,10,15及20 d,检测碱性磷酸酶活性,测定钙含量,检测Ⅰ型胶原、骨桥蛋白、骨钙素mRNA的表达[25]。结果:骨形成蛋白2活性多肽促使碱性磷酸酶活性和钙含量明显增加;Ⅰ型胶原、骨桥蛋白和骨钙素mRNA的表达增强,有效地促进了骨髓间充质干细胞向成骨方向分化。 2.7 肉苁蓉含药血清 选用SD大鼠(体质量150- 200 g,雌雄不限),取其股骨和胫骨并从中分离出骨髓间充质干细胞并用含10%内苁蓉舍药血清的L-DMEM培养诱导骨髓间充质干细胞。第10天做碱性磷酸酶染色,第20天做茜素红染色[26]。结果:第10天碱性磷酸酶染色阳性,第12天可见白色钙结节,第20天茜素红染色阳性。实验结果表明肉苁蓉具有促进骨髓间充质干细胞增殖和诱导骨髓间充质干细胞向成骨分化的作用。 2.8 其他 除上述方诱导方法外,近年来也有一些特殊方法诱导骨髓间充质干细胞向骨细胞分化。如利用低能冲击波作用诱导骨髓基质细胞向成骨细胞的分化[27-28],还有利用转基因技术把具有成骨作用的基因转入靶细胞促进成骨分化[29-30]。"
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