本文選題：周期性張應(yīng)力　切入點(diǎn)：成肌細(xì)胞　出處：《青島大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：頜面部肌肉組織的適應(yīng)性改建在正畸功能矯形中都發(fā)揮了重要作用。成肌細(xì)胞是構(gòu)成頜面部肌肉組織的重要組成部分,是適應(yīng)性改建的主要體現(xiàn)者。目前多數(shù)學(xué)者的研究集中于成肌細(xì)胞的分化、增殖等方面,忽視了成肌細(xì)胞凋亡方面的研究。而成肌細(xì)胞的凋亡與成肌細(xì)胞的分化、增殖一樣,在頜面部肌肉組織的生長和改建中發(fā)揮著重要的作用。本研究通過構(gòu)建成肌細(xì)胞力學(xué)刺激模型研究周期性張應(yīng)力對大鼠L6成肌細(xì)胞凋亡的影響,明確CaN-NFAT信號通路在應(yīng)力介導(dǎo)的大鼠L6成肌細(xì)胞凋亡中的作用,從而闡明功能矯形時(shí)成肌細(xì)胞適應(yīng)性改建的分子機(jī)制,為正畸醫(yī)生治療錯(cuò)頜畸形提供理論支持。 方法：本實(shí)驗(yàn)應(yīng)用多通道細(xì)胞牽張應(yīng)力加載系統(tǒng)對大鼠L6成肌細(xì)胞構(gòu)建的力學(xué)刺激模型分別施加Oh、1h、2h、6h、12h、24h的周期性張應(yīng)力(0h組即為對照組),加載的力值為15%的細(xì)胞形變率；加載的頻率為10個(gè)循環(huán)/min,每個(gè)循環(huán)均包括3s拉伸和3s松弛；于倒置相差顯微鏡下對成肌細(xì)胞的形態(tài)學(xué)改變和生長狀況進(jìn)行觀察；經(jīng)過Hoechst33258熒光染料染色后,采用熒光顯微鏡觀察凋亡的成肌細(xì)胞；采用流式細(xì)胞術(shù)分別分析各組成肌細(xì)胞的凋亡率；運(yùn)用實(shí)時(shí)熒光定量PCI技術(shù)及Western blot技術(shù)分別檢測周期性張應(yīng)力作用下各組成肌細(xì)胞鈣調(diào)神經(jīng)磷酸酶(CaN)及NFAT的mRNA和蛋白含量；采用CaN的特異性抑制劑環(huán)孢素(CsA)明確CaN在周期性張應(yīng)力介導(dǎo)的L6成肌細(xì)胞凋亡中的作用,從而進(jìn)一步明確內(nèi)質(zhì)網(wǎng)應(yīng)激與應(yīng)力介導(dǎo)的大鼠L6成肌細(xì)胞凋亡之間的關(guān)系。采用統(tǒng)計(jì)學(xué)軟件SPSS17.0對所得的數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果： 1大鼠L6成肌細(xì)胞在受到大小為15%細(xì)胞形變率,頻率為10個(gè)循環(huán)/min的周期性張應(yīng)力后,貼壁生長情況良好,細(xì)胞無變性并且脫落率極低,說明成肌細(xì)胞體外培養(yǎng)-力學(xué)刺激模型構(gòu)建成功； 2當(dāng)大鼠L6成肌細(xì)胞受到長時(shí)間的周期性張應(yīng)力作用后,細(xì)胞排列方向由原先的雜亂無章變?yōu)轫槕?yīng)力場方向排列,并且隨著應(yīng)力作用時(shí)問變長,細(xì)胞排列方向變得更加明顯； 3Hoechst33258熒光染色及流式細(xì)胞術(shù)的檢測結(jié)果顯示15%的細(xì)胞形變率,10個(gè)循環(huán)/min的周期性張應(yīng)力誘導(dǎo)大鼠L6成肌細(xì)胞發(fā)生凋亡,并且在一定的作用時(shí)間范圍內(nèi),成肌細(xì)胞的凋亡率隨著應(yīng)力作用時(shí)間的延長而升高,在24h達(dá)到高峰； 4實(shí)時(shí)熒光定量PCR的檢測結(jié)果顯示,隨著應(yīng)力作用時(shí)間的延長,鈣調(diào)神經(jīng)磷酸酶(CaN)及NFAT的mRNA表達(dá)量均有所增加；Western blot的檢測結(jié)果顯示,隨著應(yīng)力作用時(shí)間的延長,NFAT的蛋白量也有所增加,這與PCR的檢測結(jié)果相符；可見,CaN-NFAT信號通路參與了周期性張應(yīng)力作用下的大鼠L6成肌細(xì)胞的力學(xué)信號傳導(dǎo)。 5CaN的特異性抑制劑環(huán)孢素(CsA)抑制CaN活性后,成肌細(xì)胞的凋亡率下降,鈣調(diào)神經(jīng)磷酸酶(CaN)和NFAT的mRNA表達(dá)量、NFAT的蛋白量下降,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論： 1.15%的細(xì)胞形變率,10個(gè)循環(huán)/min的周期性張應(yīng)力可以誘導(dǎo)大鼠L6成肌細(xì)胞發(fā)生凋亡； 2. CaN-NFAT信號通路可能參與周期性張應(yīng)力介導(dǎo)的大鼠L6成肌細(xì)胞凋亡。
[Abstract]:Objective: facial muscle tissue remodeling in orthodontic orthopedic have played an important role. Myoblasts constitute an important part of facial muscle tissue, is the main embodiment of adaptive remodeling. At present many scholars study focused on the differentiation of myoblast proliferation, etc., ignored the study muscle cell apoptosis and differentiation. The apoptosis of muscle cells, and the proliferation of skeletal muscle cells, plays an important role in the growth and reconstruction of maxillofacial muscle tissue. This study builds the myoblast mechanical stimulation effects of myoblasts on apoptosis of rat L6 model of cyclical stretch clearly, the CaN-NFAT signaling pathway of myoblast apoptosis in stress mediated L6 in rats, so as to elucidate the molecular mechanism of functional orthopedics into rebuilding muscle cell adaptability, for treatment of malocclusion orthodontic doctors Provide theoretical support.
Methods: the application of multi channel cell traction mechanical tensile stress loading system of myoblast construction on rat L6 stimulation model was applied to Oh, 1H, 2h, 6h, 12h, periodic Zhang Yingli 24h (0h group as the control group), the loading force value for cell deformation rate of 15%; loading the frequency of 10 cycles of /min, each cycle includes 3S stretch and 3S relaxation; in inverted on the morphology of myoblast changes and growth status were observed under microscope; after Hoechst33258 fluorescent dye staining, observation of myoblasts apoptosis by fluorescence microscope; the apoptosis rate of the muscle cells were analyzed by flow cytometry FCM; using real-time fluorescence quantitative PCI and Western blot technology were used to detect the cyclic tensile stress under the action of the muscle cells of calcineurin (CaN) and mRNA protein content and NFAT; the specificity of CaN suppression Preparation of cyclosporine A (CsA) clear CaN myoblasts apoptosis in periodic Zhang Yingli mediated by L6, so as to further clarify the endoplasmic reticulum stress and stress mediated the relationship between rat L6 muscle cells apoptosis. Statistical analysis was performed using SPSS17.0 statistical software for the data.
Result錛，

本文編號：1615211