成骨生长肽（10-14）对大鼠骨髓间充质干细胞成脂、成骨分化的影响

发布时间：2018-05-09 14:23

本文选题：成骨生长肽(10-14) + 骨髓间充质干细胞　；参考：《兰州大学》2007年博士论文

【摘要】： 以往的实验证据表明，骨髓间充质干细胞(MSCs)具有多向分化潜能，，在一定条件下可定向分化为成骨细胞和脂肪细胞，此已分化的成骨细胞、脂肪细胞可以去分化再分化，并且二者之间存在“此消彼长”的关系。研究发现，在各种类型的骨质疏松病人中，其骨髓基质中脂肪细胞的增多并伴随松质骨骨量低下。成骨生长肽(OGP)可促进骨形成、刺激骨折愈合、增加小梁骨骨密度；过度表达OGP基因的转基因小鼠，尤其是雌性鼠，因其骨形成增加而有着较高的骨量峰值；在成骨性细胞及起源于人及其它哺乳动物的骨髓基质细胞培养中，OGP调节细胞的增殖、碱性磷酸酶活性及基质矿化。正常生理状态下，在啮齿类动物和人类血液中80～90％的OGP是以与成骨生长肽结合蛋白(OGPBP)相结合的形式存在的。OGP从OGP-OGPBP复合物中分离出来后，在蛋白水解酶作用下，产生OGP的C末端五肽——OGP(10-14)。OGP(10-14)是具有OGP全部生物活性的最小片段，它通过激活靶细胞内Gi蛋白-促有丝分裂素激动蛋白(MAP)激酶信号途径发挥其生物学作用。以上实验结果促使我们对OGP(10-14)预防和治疗骨质疏松的机理进行探讨。OGP(10-14)是否通过调节MSCs的成骨、成脂分化来影响骨的形成尚未可知。在本研究中，OGP(10-14)采用Boc策略和Fmoc策略的固相多肽合成法手工合成，经分析型HPLC鉴定纯度＞95％，质谱鉴定分子量与理论值相符。生物活性鉴定结果显示，OGP(10-14)对NIH3T3细胞具有促增殖作用，剂量反应曲线呈一“近似钟形”，发挥最大生物活性的浓度为10~(-10)M。用贴壁筛选法分离MSCs，传3代后再进行成脂、成骨诱导，通过相差显微镜下观察细胞生长形态学变化，并行油红O染色、碱性磷酸酶(ALP)染色、Von Kossa钙染色、细胞内甘油三酯(TG)和ALP活性的测定，半定量RT-PCR分析成脂相关性基因PPARγ2,、LPL,、aP_2、C/EBP及成骨相关性基因cbfal、ALP、OC的表达情况，进而观察OGP(10-14)在大鼠MSCs成骨、成脂分化中的作用。本实验结果显示，不管是对rMSCs用10~(-10)M OGP(10-14)预处理后再成脂诱导还是在成脂诱导培养体系中直接加入10~(-10)M OGP(10-14)，其成脂分化与对照组相比明显受抑，表现为所形成的脂肪细胞数量减少、细胞内甘油三酯水平降低。同样，对rMSCs用10~(-10)M OGP(10-14)预处理后再成骨诱导或在成骨诱导培养体系中直接加入10~(-10)MOGP(10-14)，均可促进其成骨分化，ALP阳性细胞及所形成的骨样小结明显增多，细胞内ALP活性也明显高，且OGP(10-14)促进rMSCs成骨分化可不依赖于地塞米松的存在，但地塞米松(10~(-8)M)与OGP(10-14)(10~(-10)M)联合作用时，其效应大于单一因素的作用，呈现出显著的协同作用。不管是OGP(10-14)处理组还是对照组，均可探及cbfal和PPARγ2基因的表达；至于ALP，OC，LPL，aP_2和C/EBP，其表达水平非常低或未探及表达；与对照组比较，对rMSCs用10~(-10)M OGP(10-14)预处理7天可显著提高cbfal基因的表达水平，同时抑制PPARγ2基因的表达。本实验证实成骨细胞与脂肪细胞均可由MSCs分化而来；OGP(10-14)可以通过抑制MSCs向脂肪细胞的分化并促进其分化为更多的成骨细胞，从而有效地促进骨形成；OGP(10-14)对rMSCs成脂、成骨分化的影响可能在细胞定向分化阶段而非细胞成熟阶段；OGP(10-14)具有预防和治疗骨质疏松的潜在临床应用价值。
[Abstract]:Previous experimental evidence suggests that bone marrow mesenchymal stem cells (MSCs) have multiple differentiation potential and can differentiate into osteoblasts and adipocytes under certain conditions. The differentiated osteoblasts, adipocytes can dedifferentiated and redifferentiated, and the relationship between the two is "this elimination" relationship. Studies have found that various types of bone are found. In the patients with osteoporosis, the increase of fat cells in the bone marrow stroma and the low bone mass of the cancellous bone. Osteogenic growth peptide (OGP) promotes bone formation, stimulates fracture healing and increases the bone mineral density of the trabecular bone; the transgenic mice that overexpress the OGP gene, especially the female mice, have a higher bone peak value because of the increase of bone formation; and in osteogenesis. In the culture of cells and bone marrow stromal cells derived from human and other mammals, OGP regulates cell proliferation, alkaline phosphatase activity and matrix mineralization. Under normal physiological conditions, 80 to 90% of OGP in rodents and human blood are in the form of.OGP from OGP-OGPBP in the form of a combination of osteogenic growth peptide binding protein (OGPBP). After the separation, the C terminal five peptide of OGP, OGP (10-14).OGP (10-14), is the smallest fragment of all OGP bioactivity under the action of protein hydrolase. It plays its biological role by activating the Gi protein in the target cells - the signal pathway of the mitogen activator kinkinin (MAP) kinase (MAP) kinase. The results of the above results urge us on O GP (10-14) the mechanism of prevention and treatment of osteoporosis is discussed..OGP (10-14) is not yet known whether the formation of bone can be affected by adipogenic differentiation by regulating MSCs osteogenesis.
In this study, OGP (10-14) was manually synthesized by the Boc strategy and the solid-phase polypeptide synthesis of Fmoc strategy. The purity of the analytical HPLC was more than 95%, and the molecular weight identified by the mass spectrometry was in accordance with the theoretical value. The results of bioactivity identification showed that OGP (10-14) had a proliferation promoting effect on NIH3T3 cells, and the dose response curve showed a "approximate bell shape" and played the most important role. The concentration of large biological activity was 10~ (-10) M. to separate MSCs with adherent screening method. After 3 generations, lipid formation and osteogenesis were induced. The morphological changes of cell growth were observed under phase contrast microscope, oil red O staining, alkaline phosphatase (ALP) staining, Von Kossa calcium staining, determination of intracellular triglyceride (TG) and ALP activity, semi quantitative RT-PCR analysis. Lipid related gene PPAR gamma 2, LPL, aP_2, C/EBP and the expression of cbfal, ALP, OC in bone related genes, and then observe the role of OGP (10-14) in rat MSCs osteogenesis and lipid differentiation.
The results showed that the lipid differentiation of rMSCs 10~ (-10) M OGP (10-14) was induced or 10~ (-10) M OGP (10-14) was directly added to the lipid induced culture system, and its lipid differentiation was obviously inhibited compared with the control group, which showed that the number of adipocytes formed and the level of triglyceride in the cells decreased. The same, rMSCs. 10~ (-10) M OGP (10-14) was pretreated with bone induction or direct addition of 10~ (-10) MOGP (10-14) into the osteogenic induction culture system, which could promote osteogenesis. ALP positive cells and bone like nodules were significantly increased, and the intracellular ALP activity was significantly higher. OGP (10-14) promoted rMSCs osteogenesis to be dependent on the survival of dexamethasone. At the same time, the effect of dexamethasone (10~ (-8) M) and OGP (10-14) (10~ (-10) M) was greater than that of a single factor, showing a significant synergistic effect. Both OGP (10-14) treatment group and control group could detect the expression of cbfal and PPAR gamma 2 genes. Compared with the control group, the pretreatment of rMSCs with 10~ (-10) M OGP (10-14) for 7 days could significantly increase the expression level of cbfal gene and inhibit the expression of PPAR gamma 2 gene.
It is verified that osteoblasts and adipocytes can be differentiated by MSCs; OGP (10-14) can effectively promote bone formation by inhibiting the differentiation of MSCs into adipocytes and promoting its differentiation into more osteoblasts, and the effect of OGP (10-14) on rMSCs formation and osteogenic differentiation may be in the phase of differentiation of cells instead of cell maturity. OGP (10-14) has potential clinical value in the prevention and treatment of osteoporosis.

【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R329

【引证文献】