【摘要】：目的:探討瘦素在大鼠跟腱干細(xì)胞(TDSCs)成骨分化及跟腱異位骨化形成過程中的作用及機(jī)制,為異位骨化的預(yù)防和治療提供理論支持。方法:體外實驗,提取大鼠TDSCs進(jìn)行成骨誘導(dǎo)培養(yǎng),用不同濃度的瘦素(1、10、100 ng/ml)或100ng/ml瘦素+10nM雷帕霉素處理,3天后通過CCK-8實驗檢測細(xì)胞增殖情況,14天后采用堿性磷酸酶(ALP)染色/活性實驗檢測ALP表達(dá),茜紅素染色檢測礦化結(jié)節(jié)形成,熒光定量PCR檢測ALP、Runt相關(guān)轉(zhuǎn)錄因子2(Runx2)、成骨相關(guān)轉(zhuǎn)錄因子(OSX)及骨鈣素(OCN),蛋白免疫印跡檢測Runx2、OSX以及哺乳動物雷帕霉素靶蛋白復(fù)合物1(mTORC1)信號通路下游的磷酸化核糖體S6蛋白激酶(PS6K1)和磷酸化核糖體S6蛋白(PS6)的表達(dá)量。體內(nèi)實驗,選取6周齡雄性Sprague-Dawley大鼠建立跟腱中段切斷術(shù)模型,隨機(jī)分為對照組(control)、異位骨化組(HO)、異位骨化+雷帕霉素組(HO+RA)、異位骨化+瘦素組(HO+LEP)、異位骨化+瘦素+雷帕霉素組(HO+LEP+RA),采用瘦素及雷帕霉素處理10周,跟腱取材行伊紅-蘇木素染色檢測鈣化面積,micro-CT檢測異位骨化體積,免疫組織化學(xué)檢測瘦素、Runx2、OSX及PS6的表達(dá)量。結(jié)果:體外實驗:瘦素在1-1OOng/ml濃度范圍內(nèi)對TDSCs的增殖沒有顯著的毒性作用,差異無統(tǒng)計學(xué)意義(p0.05);不同濃度的瘦素(1-1OOng/ml)促進(jìn)TDSCs成骨分化過程中ALP表達(dá)及礦化結(jié)節(jié)形成,并呈濃度依賴性增加,在1OOng/ml瘦素時達(dá)到最大作用,差異具有統(tǒng)計學(xué)意義(p0.05);不同濃度的瘦素(1-100ng/ml)促進(jìn)ALP、Runx2、OSX及OCN的mRNA表達(dá),并隨著瘦素濃度的增加而增加,在100ng/ml瘦素時表達(dá)最強(qiáng),差異具有統(tǒng)計學(xué)意義(p0.05);不同濃度的瘦素(1-100ng/ml)促進(jìn) Runx2、OSX、PS6K1 及 PS6蛋白的表達(dá),并隨著瘦素濃度的增加而增加,在1OOng/ml瘦素時表達(dá)最強(qiáng),差異具有統(tǒng)計學(xué)意義(p0.05);瘦素+雷帕霉素組較瘦素組的Runx2、OSX、PS6K1及PS6蛋白表達(dá)量均顯著降低,差異具有統(tǒng)計學(xué)意義(p0.05)。體內(nèi)試驗:HO組較contro1組的瘦素表達(dá)明顯增高,差異具有統(tǒng)計學(xué)意義(p0.05);HO+LEP組較HO組和HO+LEP+RA組的鈣化面積、異位骨化體積顯著增多,差異具有統(tǒng)計學(xué)意義(p0.05);HO+RA組較HO組的鈣化面積和異位骨化體積顯著降低,差異具有統(tǒng)計學(xué)意義(p0.05);HO+LEP組較HO組和HO+LEP+RA組的Runx2、OSX及PS6表達(dá)顯著增強(qiáng),差異具有統(tǒng)計學(xué)意義(p0.05);HO+RA組較HO組的Runx2、OSX及PS6表達(dá)顯著降低,差異具有統(tǒng)計學(xué)意義(p0.05)。結(jié)論:瘦素上調(diào)mTORC1信號通路加速TDSCs成骨分化和跟腱異位骨化的形成,mTORC1信號通路抑制劑雷帕霉素可有效抑制瘦素誘導(dǎo)的TDSCs成骨分化和跟腱異位骨化形成,為跟腱異位骨化的預(yù)防及治療提供了新思路。
[Abstract]:Aim: to investigate the role and mechanism of leptin in the process of (TDSCs) osteogenic differentiation and heterotopic ossification of Achilles tendon stem cells in rats, and to provide theoretical support for the prevention and treatment of ectopic ossification. Methods: rat TDSCs was extracted for osteoblast induction culture in vitro and treated with different concentrations of leptin (1 10 100 ng/ml) or 100ng/ml leptin 10nM rapamycin. After 3 days, cell proliferation was detected by CCK-8 assay. After 14 days, ALP expression was detected by alkaline phosphatase (ALP) staining / activity assay, mineralized nodule formation was detected by hematoxylin staining, and ALP,Runt related transcription factor 2 (Runx2) was detected by fluorescence quantitative PCR. Detection of Runx2, by Western blotting of osteoblast-associated transcription factor (OSX) and osteocalcin (OCN), Expression of phosphorylated ribosomal S6 protein kinase (PS6K1) and phosphorylated ribosomal S6 protein (PS6) in OSX and mammalian rapamycin target protein complex 1 (mTORC1) signaling pathway. In vivo experiment, 6-week-old male Sprague-Dawley rats were selected to establish the model of amputation of Achilles tendon. They were randomly divided into control group, (control), ectopic ossification group, (HO), ectopic ossification group, rapamycin group, (HO RA), ectopic ossification leptin group, (HO LEP), group. In the ectopic ossification leptin group, (HO LEP RA), was treated with leptin and rapamycin for 10 weeks. The calcareous area was detected by eosinolin-hematoxylin staining, the volume of ectopic ossification was detected by micro-CT and leptin was detected by immunohistochemistry. The expression of Runx2,OSX and PS6. Results: in vitro, leptin had no significant toxic effect on the proliferation of TDSCs in the range of 1-1OOng/ml concentration, and the difference was not statistically significant (p0.05). Different concentrations of leptin (1-1OOng/ml) promoted the expression of ALP and the formation of mineralized nodules in the process of osteogenic differentiation of TDSCs, and increased in a dose-dependent manner, reaching the maximum effect at the time of 1OOng/ml leptin. The difference was statistically significant (p0.05). Different concentrations of leptin (1-100ng/ml) promoted the expression of mRNA in ALP,Runx2,OSX and OCN, and increased with the increase of leptin concentration. The expression of leptin in 100ng/ml was the strongest (p0.05). Different concentrations of leptin (1-100ng/ml) promoted the expression of Runx2,OSX,PS6K1 and PS6 protein, and increased with the increase of leptin concentration. The expression of leptin in 1OOng/ml was the strongest (p0.05). The expression of Runx2,OSX,PS6K1 and PS6 in leptin group was significantly lower than that in leptin group (p0.05). In vivo test: the expression of leptin in HO group was significantly higher than that in contro1 group (p0.05). The calcification area and ectopic ossification volume in HO LEP group were significantly larger than those in HO group and HO LEP RA group (p0.05). The calcification area and ectopic ossification volume in HO RA group were significantly lower than those in HO group, and the difference was statistically significant (p0.05); HO LEP group was significantly higher than HO group and HO LEP RA group in Runx2,OSX and PS6 expression, the difference was statistically significant (p0.05). The expression of Runx2,OSX and PS6 in HO RA group was significantly lower than that in HO group (p0.05). Conclusion: leptin upregulated mTORC1 signaling pathway to accelerate the formation of TDSCs osteogenic differentiation and heterotopic calcaneal ossification. Rapamycin, an inhibitor of mTORC1 signaling pathway, can effectively inhibit leptin induced TDSCs osteogenic differentiation and Achilles tendon ectopic ossification. It provides a new idea for the prevention and treatment of heterotopic ossification of Achilles tendon.
【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】：R681

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