本文選題：周期性張應力　切入點：成肌細胞　出處：《青島大學》2014年碩士論文　論文類型：學位論文

【摘要】：目的：頜面部肌肉組織的適應性改建在正畸功能矯形中都發(fā)揮了重要作用。成肌細胞是構(gòu)成頜面部肌肉組織的重要組成部分,是適應性改建的主要體現(xiàn)者。目前多數(shù)學者的研究集中于成肌細胞的分化、增殖等方面,忽視了成肌細胞凋亡方面的研究。而成肌細胞的凋亡與成肌細胞的分化、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)建成肌細胞力學刺激模型研究周期性張應力對大鼠L6成肌細胞凋亡的影響,明確CaN-NFAT信號通路在應力介導的大鼠L6成肌細胞凋亡中的作用,從而闡明功能矯形時成肌細胞適應性改建的分子機制,為正畸醫(yī)生治療錯頜畸形提供理論支持。 方法：本實驗應用多通道細胞牽張應力加載系統(tǒng)對大鼠L6成肌細胞構(gòu)建的力學刺激模型分別施加Oh、1h、2h、6h、12h、24h的周期性張應力(0h組即為對照組),加載的力值為15%的細胞形變率；加載的頻率為10個循環(huán)/min,每個循環(huán)均包括3s拉伸和3s松弛；于倒置相差顯微鏡下對成肌細胞的形態(tài)學改變和生長狀況進行觀察；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胞；采用流式細胞術分別分析各組成肌細胞的凋亡率；運用實時熒光定量PCI技術及Western blot技術分別檢測周期性張應力作用下各組成肌細胞鈣調(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在周期性張應力介導的L6成肌細胞凋亡中的作用,從而進一步明確內(nèi)質(zhì)網(wǎng)應激與應力介導的大鼠L6成肌細胞凋亡之間的關系。采用統(tǒng)計學軟件SPSS17.0對所得的數(shù)據(jù)進行統(tǒng)計學分析。 結(jié)果： 1大鼠L6成肌細胞在受到大小為15%細胞形變率,頻率為10個循環(huán)/min的周期性張應力后,貼壁生長情況良好,細胞無變性并且脫落率極低,說明成肌細胞體外培養(yǎng)-力學刺激模型構(gòu)建成功； 2當大鼠L6成肌細胞受到長時間的周期性張應力作用后,細胞排列方向由原先的雜亂無章變?yōu)轫槕龇较蚺帕?并且隨著應力作用時問變長,細胞排列方向變得更加明顯； 3Hoechst33258熒光染色及流式細胞術的檢測結(jié)果顯示15%的細胞形變率,10個循環(huán)/min的周期性張應力誘導大鼠L6成肌細胞發(fā)生凋亡,并且在一定的作用時間范圍內(nèi),成肌細胞的凋亡率隨著應力作用時間的延長而升高,在24h達到高峰； 4實時熒光定量PCR的檢測結(jié)果顯示,隨著應力作用時間的延長,鈣調(diào)神經(jīng)磷酸酶(CaN)及NFAT的mRNA表達量均有所增加；Western blot的檢測結(jié)果顯示,隨著應力作用時間的延長,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力作用下的大鼠L6成肌細胞的力學信號傳導。 5CaN的特異性抑制劑環(huán)孢素(CsA)抑制CaN活性后,成肌細胞的凋亡率下降,鈣調(diào)神經(jīng)磷酸酶(CaN)和NFAT的mRNA表達量、NFAT的蛋白量下降,差異具有統(tǒng)計學意義(P0.05)。 結(jié)論： 1.15%的細胞形變率,10個循環(huán)/min的周期性張應力可以誘導大鼠L6成肌細胞發(fā)生凋亡； 2. CaN-NFAT信號通路可能參與周期性張應力介導的大鼠L6成肌細胞凋亡。
[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