發(fā)布時間：2018-01-30 15:17

  本文關(guān)鍵詞： 甲狀旁腺素 信號轉(zhuǎn)導(dǎo)通路 成骨作用 抗骨質(zhì)疏松 顯微CT　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景:甲狀旁腺素(Parathyroid Hormone,PTH)是一種促進(jìn)骨形成的激素類藥物。前期研究顯示,PTH通過nonPLC/PKC信號途徑提升松質(zhì)骨骨量,改善骨小梁的微結(jié)構(gòu),研究nonPLC/PKC的信號分子和途徑以及骨代謝作用,有利于設(shè)計出更好的nonPLC/PKC模擬肽,提高藥物的效能,擴(kuò)大應(yīng)用范圍。前期研究發(fā)現(xiàn)該通路可以分成cAMP/PKA依賴途徑和非依賴途徑,但是該通路的生物學(xué)功能尚不清楚。目的:通過現(xiàn)存的PTH模擬肽驗證cAMP/PKA依賴的nonPLC/PKC信號通路是否對甲狀旁腺素在成骨細(xì)胞的增殖、凋亡和成骨分化中的起作用;通過前期合成的cAMP/PKA非依賴的nonPLC/PKC信號特異性模擬肽MY-1肽和MY-2肽,驗證其信號通路是否為cAMP非依賴和PLC非依賴途徑,并驗證模擬肽的抗凋亡作用,體外細(xì)胞成骨分化和破骨分化效應(yīng);建立卵巢切除的小鼠骨質(zhì)疏松模型,評價模擬肽MY-1和MY-2的抗骨質(zhì)疏松作用。方法:取MC3T3E1細(xì)胞,低濃度血清處理12小時后,模擬肽間斷刺激6小時后用MTT觀察細(xì)胞的增殖情況,細(xì)胞饑餓處理12小時后,模擬肽處理4小時后流式細(xì)胞觀察模擬肽的抗凋亡效果,成骨誘導(dǎo)分化14天觀察堿性磷酸酶染色和酶活性檢測,28天后觀察鈣結(jié)節(jié)的茜素紅染色和鈣定量;取小鼠原代顱骨細(xì)胞,行ELISA檢測MY-1肽和MY-2肽的cAMP和PLC信號通路,并同時行細(xì)胞流式凋亡實驗,取6周齡的C57BL小鼠的全骨髓細(xì)胞,行成骨分化培養(yǎng)和破骨分化培養(yǎng);取6周齡的C57BL雌鼠,構(gòu)建卵巢切除的骨質(zhì)疏松模型,皮下注射PTH模擬肽,分別在4周和8周后處死小鼠,并行脛骨骨小梁結(jié)構(gòu)顯微CT分析。結(jié)果:對于cAMP/PKA依賴的nonPLC/PKC信號通路,可以促進(jìn)成骨細(xì)胞增殖,抑制成骨細(xì)胞凋亡,促進(jìn)成骨細(xì)胞的分化;對于cAMP/PKA非依賴的nonPLC/PKC信號通路,信號通路檢測發(fā)現(xiàn)MY-1肽和MY-2肽都不能顯著激活cAMP和PLC通路,MY-1和MY-2具有良好的抗凋亡效果,在間斷刺激培養(yǎng)中,MY-1肽和MY-2肽可以顯著促進(jìn)細(xì)胞成骨分化,并表現(xiàn)出低破骨分化的特性,連續(xù)刺激同樣表現(xiàn)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,我們發(fā)現(xiàn)卵巢切除組的骨量較假手術(shù)組顯著降低,注射MY-1肽和MY-2肽的單位骨量4周和8周組較卵巢切除組顯著升高,表現(xiàn)出良好的抗骨質(zhì)疏松效果。結(jié)論:cAMP/PKA依賴的nonPLC/PKC信號通路具有促進(jìn)成骨細(xì)胞增殖、促進(jìn)成骨細(xì)胞分化和抑制成骨細(xì)胞凋亡的作用;cAMP/PKA非依賴的nonPLC/PKC信號模擬肽MY-1和MY-2體外細(xì)胞間斷和連續(xù)誘導(dǎo)都表現(xiàn)出促進(jìn)成骨和低破骨分化的特性,并具有良好的抗凋亡作用;動物研究顯示MY-1肽和MY-2肽具有良好的抗骨質(zhì)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R580

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 胡少宇;童國軍;孟越;郝松;李威;許伏龍;何友華;陳建庭;楊德鴻;;甲狀旁腺激素通過非依賴PLC的PKC途徑抑制成骨細(xì)胞的凋亡[J];南方醫(yī)科大學(xué)學(xué)報;2016年06期

2 楊德鴻;胡少宇;孟越;童國軍;陳建庭;;特立帕肽保守治療骨質(zhì)疏松性脊柱骨折:12例報告[J];南方醫(yī)科大學(xué)學(xué)報;2016年03期

3 郝松;孟越;李威;胡少宇;楊德鴻;;甲狀旁腺素非PLC依賴PKC通路激活增強(qiáng)成骨細(xì)胞CITED1表達(dá)[J];南方醫(yī)科大學(xué)學(xué)報;2015年04期

4 袁亮;林振;付兆宗;孟越;黃志平;吳秀華;楊德鴻;江建明;;信號選擇性甲狀旁腺素模擬肽促進(jìn)去勢雄性小鼠的骨折愈合[J];南方醫(yī)科大學(xué)學(xué)報;2013年02期

5 毛文晴;田甜;;原發(fā)性骨質(zhì)疏松的病因及發(fā)病機(jī)制[J];中國骨質(zhì)疏松雜志;2011年10期

6 于美娟;張雅妮;馮善偉;熊符;張成;;小鼠骨髓間充質(zhì)干細(xì)胞分離培養(yǎng)方法的建立[J];解剖學(xué)研究;2009年06期

7 邱明琪;姜欣;;內(nèi)分泌代謝疾病與骨質(zhì)疏松研究進(jìn)展[J];實用臨床醫(yī)學(xué);2006年09期

相關(guān)碩士學(xué)位論文 前1條

1 孟越;甲狀旁腺素通過PKA依賴及非依賴途徑激活PKC信號通路的實驗研究[D];南方醫(yī)科大學(xué);2014年

，

本文編號：1476544