磷脂酶C非依赖的蛋白激酶C信号通路在甲状旁腺素调节骨代谢中的作用

发布时间：2018-01-30 15:17

本文关键词： 甲状旁腺素信号转导通路成骨作用抗骨质疏松显微CT　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:甲状旁腺素(Parathyroid Hormone,PTH)是一种促进骨形成的激素类药物。前期研究显示,PTH通过nonPLC/PKC信号途径提升松质骨骨量,改善骨小梁的微结构,研究nonPLC/PKC的信号分子和途径以及骨代谢作用,有利于设计出更好的nonPLC/PKC模拟肽,提高药物的效能,扩大应用范围。前期研究发现该通路可以分成cAMP/PKA依赖途径和非依赖途径,但是该通路的生物学功能尚不清楚。目的:通过现存的PTH模拟肽验证cAMP/PKA依赖的nonPLC/PKC信号通路是否对甲状旁腺素在成骨细胞的增殖、凋亡和成骨分化中的起作用;通过前期合成的cAMP/PKA非依赖的nonPLC/PKC信号特异性模拟肽MY-1肽和MY-2肽,验证其信号通路是否为cAMP非依赖和PLC非依赖途径,并验证模拟肽的抗凋亡作用,体外细胞成骨分化和破骨分化效应;建立卵巢切除的小鼠骨质疏松模型,评价模拟肽MY-1和MY-2的抗骨质疏松作用。方法:取MC3T3E1细胞,低浓度血清处理12小时后,模拟肽间断刺激6小时后用MTT观察细胞的增殖情况,细胞饥饿处理12小时后,模拟肽处理4小时后流式细胞观察模拟肽的抗凋亡效果,成骨诱导分化14天观察碱性磷酸酶染色和酶活性检测,28天后观察钙结节的茜素红染色和钙定量;取小鼠原代颅骨细胞,行ELISA检测MY-1肽和MY-2肽的cAMP和PLC信号通路,并同时行细胞流式凋亡实验,取6周龄的C57BL小鼠的全骨髓细胞,行成骨分化培养和破骨分化培养;取6周龄的C57BL雌鼠,构建卵巢切除的骨质疏松模型,皮下注射PTH模拟肽,分别在4周和8周后处死小鼠,并行胫骨骨小梁结构显微CT分析。结果:对于cAMP/PKA依赖的nonPLC/PKC信号通路,可以促进成骨细胞增殖,抑制成骨细胞凋亡,促进成骨细胞的分化;对于cAMP/PKA非依赖的nonPLC/PKC信号通路,信号通路检测发现MY-1肽和MY-2肽都不能显著激活cAMP和PLC通路,MY-1和MY-2具有良好的抗凋亡效果,在间断刺激培养中,MY-1肽和MY-2肽可以显著促进细胞成骨分化,并表现出低破骨分化的特性,连续刺激同样表现出成骨分化作用和低破骨的特性;使用卵巢切除的小鼠模型,我们发现卵巢切除组的骨量较假手术组显著降低,注射MY-1肽和MY-2肽的单位骨量4周和8周组较卵巢切除组显著升高,表现出良好的抗骨质疏松效果。结论:cAMP/PKA依赖的nonPLC/PKC信号通路具有促进成骨细胞增殖、促进成骨细胞分化和抑制成骨细胞凋亡的作用;cAMP/PKA非依赖的nonPLC/PKC信号模拟肽MY-1和MY-2体外细胞间断和连续诱导都表现出促进成骨和低破骨分化的特性,并具有良好的抗凋亡作用;动物研究显示MY-1肽和MY-2肽具有良好的抗骨质疏松的作用。
[Abstract]:Background: parathyroid Hormoney (PTH) is a hormone that promotes bone formation. PTH enhances cancellous bone mass through nonPLC/PKC signaling pathway, improves the microstructure of trabecular bone, and studies signal molecules and pathways of nonPLC/PKC and bone metabolism. It is helpful to design a better nonPLC/PKC mimic peptide, improve the efficacy of the drug, and expand the scope of application. Previous studies found that the pathway can be divided into cAMP/PKA dependent pathway and non-dependent pathway. But the biological function of the pathway is not clear. The existing PTH mimic peptide was used to verify whether the cAMP/PKA dependent nonPLC/PKC signaling pathway could promote the proliferation of parathyroid hormone in osteoblasts. Apoptosis and osteogenic differentiation; The cAMP/PKA independent nonPLC/PKC signal specific mimic peptides MY-1 peptide and MY-2 peptide were synthesized. To verify whether the signaling pathway is cAMP independent and PLC independent, and to verify the antiapoptotic effect of mimic peptide, osteogenic differentiation and osteoclast differentiation in vitro. Ovariectomized mouse model of osteoporosis was established to evaluate the anti-osteoporosis effects of mimic peptides MY-1 and MY-2. Methods: MC3T3E1 cells were taken and treated with low concentration of serum for 12 hours. The cell proliferation was observed by MTT after intermittent stimulation of mimic peptide for 6 hours, and the anti-apoptotic effect of mimic peptide was observed by flow cytometry after 12 hours of starvation treatment and 4 hours of simulated peptide treatment. Alkaline phosphatase staining and enzyme activity were observed on the 14th day after osteogenic differentiation. After 28 days, alizarin red staining and calcium quantification of calcium nodules were observed. The primary skulls of mice were collected and the cAMP and PLC signaling pathways of MY-1 peptide and MY-2 peptide were detected by ELISA. The whole bone marrow cells of 6-week-old C57BL mice were cultured with osteogenic differentiation and osteoclast differentiation. Ovariectomized ovariectomized ovariectomized female C57BL mice were injected subcutaneously with PTH mimic peptide. The mice were killed after 4 and 8 weeks, respectively. Results: cAMP/PKA dependent nonPLC/PKC signaling pathway could promote the proliferation of osteoblasts and inhibit the apoptosis of osteoblasts. Promote the differentiation of osteoblasts; For cAMP/PKA independent nonPLC/PKC signaling pathway, both MY-1 peptide and MY-2 peptide could not activate cAMP and PLC pathway significantly. MY-1 and MY-2 have good anti-apoptotic effect. MY-1 peptide and MY-2 peptide can significantly promote osteogenic differentiation and show the characteristics of low osteoclastic differentiation in intermittent stimulation culture. Continuous stimulation also showed osteogenic differentiation and low osteoclast. Using the ovariectomized mouse model, we found that the bone mass of the ovariectomized group was significantly lower than that of the sham-operated group, and the bone mass per unit of MY-1 peptide and MY-2 peptide injected into the ovariectomized group was significantly higher than that of the ovariectomized group at 4 and 8 weeks. Conclusion the nonPLC/PKC signaling pathway dependent on CPP-PKA can promote the proliferation of osteoblasts. Promoting the differentiation of osteoblasts and inhibiting the apoptosis of osteoblasts; The discontinuous and continuous induction of cAMP/PKA independent nonPLC/PKC signal mimic peptide MY-1 and MY-2 showed the characteristics of promoting osteogenesis and low osteoclast differentiation. And has a good anti-apoptotic effect; Animal studies have shown that MY-1 peptide and MY-2 peptide have a good anti-osteoporosis effect.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R580

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