【摘要】：研究目的 在骨骼肌生長(zhǎng)、發(fā)育、損傷和修復(fù)過(guò)程中,骨骼肌衛(wèi)星細(xì)胞起著重要的作用。有在體研究表明,機(jī)械生長(zhǎng)因子(MGF)可能能激活骨骼肌衛(wèi)星細(xì)胞,并促進(jìn)其增殖。本研究在此基礎(chǔ)上通過(guò)離體實(shí)驗(yàn),觀察MGF對(duì)于骨骼肌衛(wèi)星細(xì)胞的激活作用及促進(jìn)其增殖的量效關(guān)系,探討MGF對(duì)骨骼肌衛(wèi)星細(xì)胞在細(xì)胞水平的調(diào)節(jié)機(jī)制,為骨骼肌損傷的修復(fù)提供了理論依據(jù)。 實(shí)驗(yàn)方法 原代骨骼肌衛(wèi)星細(xì)胞取自4周齡雄性SD大鼠(2只,120g)雙側(cè)的腓腸肌與比目魚(yú)肌。經(jīng)0.1%Ⅱ型膠原酶結(jié)合0.25%胰蛋白酶消化法,通過(guò)兩次差速貼壁純化骨骼肌衛(wèi)星細(xì)胞,使用橫紋肌肌動(dòng)蛋白鑒定骨骼肌衛(wèi)星細(xì)胞的純度。原代骨骼肌衛(wèi)星細(xì)胞經(jīng)兩次傳代后,使用不同濃度的MGF干預(yù)第三代細(xì)胞,濃度分別為10ng/ml,25ng/ml,50ng/ml, 100ng/ml,200ng/ml,在干預(yù)24h、48h、72h、96h后利用MTT法進(jìn)行增殖效果的檢測(cè)。使用25ng/ml的MGF對(duì)血清饑餓24h后的第三代骨骼肌衛(wèi)星細(xì)胞進(jìn)行干預(yù),對(duì)比DMEM干預(yù)的骨骼肌衛(wèi)星細(xì)胞,分別在8h、16h、24h、32h后使用流式細(xì)胞儀檢測(cè)其所處細(xì)胞周期,測(cè)定其激活情況。 實(shí)驗(yàn)結(jié)果 1.骨骼肌衛(wèi)星細(xì)胞活力達(dá)到98.8%。 2.骨骼肌衛(wèi)星細(xì)胞純度為97.4%。 3.MTT比色法的A值顯示：48h：25ng／ml組、50ng／ml組A值顯著高于對(duì)照組(P0.01), 10ng/ml組A值高于對(duì)照組(P0.05), 100ng/ml組、200ng/ml組與對(duì)照組之間差異沒(méi)有顯著性(P0.05), 100ng/ml與200ng/ml組的促增殖作用差異沒(méi)有顯著性(P0.05)；96h:10ng/ml與對(duì)照組之間差異沒(méi)有顯著性(P0.05),25ng/ml組、50ng/ml組、100ng/ml組、200ng/ml組A值顯著高于對(duì)照組和10ng/ml組(P0.01),25ng/ml組、50ng/ml組、100ng/ml組、200ng/ml組之間差異沒(méi)有顯著性(P0.05)。低劑量的MGF即可促進(jìn)骨骼肌衛(wèi)星細(xì)胞的增殖,隨濃度升高至25ng/ml至50ng/ml時(shí),MGF促骨骼肌衛(wèi)星細(xì)胞增殖作用相對(duì)更明顯,濃度繼續(xù)升高,會(huì)出現(xiàn)增殖速度減緩現(xiàn)象；各個(gè)時(shí)相檢測(cè)后發(fā)現(xiàn)MGF干預(yù)到96h增殖速度減緩。結(jié)合時(shí)間-劑量的結(jié)果顯示,在48h時(shí)25ng/ml組和50ng/ml組有交互作用。 4.使用不含血清的DMEM饑餓骨骼肌衛(wèi)星細(xì)胞24h后,處于GO期的細(xì)胞達(dá)到了86.76% 5.25ng/ml MGF干預(yù)GO期的衛(wèi)星細(xì)胞后,8h時(shí)MGF組和對(duì)照組之間差異沒(méi)有顯著性(P0.05),16h時(shí)MGF組G0／G 1期細(xì)胞含量低于對(duì)照組(P0.05),24h時(shí)MGF組低于對(duì)照組(P0.01),32h時(shí)MGF組低于對(duì)照組(P0.05)。 結(jié)論 1.MGF可以促進(jìn)骨骼肌衛(wèi)星細(xì)胞增殖。 2.MGF促進(jìn)骨骼肌衛(wèi)星細(xì)胞增殖呈現(xiàn)劑量和時(shí)間依賴性,較理想劑量范圍在25ng/ml至50ng/ml之間,干預(yù)24h后即開(kāi)始增殖。 3.不含血清的DMEM饑餓骨骼肌衛(wèi)星細(xì)胞24h可以使其退出細(xì)胞周期。 4.MGF可以激活四周齡SD大鼠的GO期骨骼肌衛(wèi)星細(xì)胞。
[Abstract]:Objective to study the role of skeletal muscle satellite cells in skeletal muscle growth, development, injury and repair. In vivo studies have shown that the mechanical growth factor (MGF) may activate skeletal muscle satellite cells and promote their proliferation. On the basis of in vitro experiments, the effects of MGF on the activation and proliferation of skeletal muscle satellite cells were observed, and the regulatory mechanism of MGF on skeletal muscle satellite cells at cell level was explored. It provides a theoretical basis for the repair of skeletal muscle injury. Methods the primary skeletal muscle satellite cells were obtained from the bilateral gastrocnemius and soleus muscles of 4 week old male SD rats (2 rats, 120g). Skeletal muscle satellite cells were purified by 0.1% collagenase and 0.25% trypsin digestion, and the purity of skeletal muscle satellite cells was identified by rhabdomyactin. After two passages of primary skeletal muscle satellite cells, different concentrations of MGF were used to interfere with the third generation cells. The concentrations were 10 ng / ml, 25 ng / ml, 100 ng / ml / ml, 200 ng / ml, respectively. After 96 hours, the proliferative effect was detected by MTT method. 25ng/ml MGF was used to intervene the third generation skeletal muscle satellite cells after 24 hours of serum starvation. Compared with those treated with DMEM, the cell cycle was measured by flow cytometry at 8 h, 16 h, 24 h and 32 h, respectively. Its activation was measured. Experimental results 1. The activity of skeletal muscle satellite cells was 98.8%. 2. The purity of skeletal muscle satellite cells was 97.4%. The A value of 3.MTT colorimetry showed that A value of 48h:25ng/ml group, 50ng/ml group was significantly higher than that of control group (P0.01), A value of 10ng/ml group was higher than that of control group (P0.05), 100ng/ml group, 100ng/ml group. There was no significant difference between 200ng/ml group and control group (P0.05), but there was no significant difference between 100ng/ml group and 200ng/ml group (P0.05). There was no significant difference between 96h:10ng/ml and control group (P0.05). The A value of 25ng/ml group, 50ng/ml group, 100ng/ml group and 200ng/ml group was significantly higher than that of control group and 10ng/ml group (P0.01), 25ng/ml group, 50ng/ml group, and so on. There was no significant difference between 100ng/ml group and 200ng/ml group (P0.05). Low dose of MGF could promote the proliferation of skeletal muscle satellite cells. When the concentration increased to 25ng/ml to 50ng/ml, the effect of MGF on the proliferation of skeletal muscle satellite cells was more obvious, and the increase of MGF concentration would slow down the proliferation of skeletal muscle satellite cells. It was found that the proliferation rate of MGF decreased at 96 h after each phase detection. The results of time-dose analysis showed that there was interaction between 25ng/ml group and 50ng/ml group at 48 h. 4. After using serum-free DMEM starved skeletal muscle satellite cells for 24 hours, the number of cells in GO phase reached 86.76% after intervention with GO phase satellite cells, and there was no significant difference between MGF group and control group at 8 h (P0.05). The cell content of G _ 0 / G _ 1 phase in MGF group was lower than that in control group at 16 h (P0.05), MGF group was lower than control group at 24 h (P0.01), and MGF group was lower than control group at 32 h (P0.05). Conclusion 1.MGF can promote the proliferation of skeletal muscle satellite cells. 2.MGF promoted the proliferation of skeletal muscle satellite cells in a dose-and time-dependent manner. The optimal dose range was between 25ng/ml and 50ng/ml, and the proliferation began after 24 hours of intervention. 3. DMEM starved skeletal muscle satellite cells without serum could withdraw from cell cycle for 24 h. 4.MGF can activate GO phase skeletal muscle satellite cells of four-week old SD rats.
【學(xué)位授予單位】：上海體育學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李宏偉;;干細(xì)胞技術(shù)在運(yùn)動(dòng)醫(yī)學(xué)研究中的應(yīng)用進(jìn)展[J];實(shí)用醫(yī)學(xué)雜志;2011年16期

2 ;[J];;年期

3 ;[J];;年期

4 ;[J];;年期

5 ;[J];;年期

6 ;[J];;年期

7 ;[J];;年期

8 ;[J];;年期

9 ;[J];;年期

10 ;[J];;年期

相關(guān)會(huì)議論文 前8條

1 吳]};陸耀飛;;機(jī)械生長(zhǎng)因子對(duì)骨骼肌衛(wèi)星細(xì)胞激活的影響研究[A];中國(guó)生理學(xué)會(huì)第23屆全國(guó)會(huì)員代表大會(huì)暨生理學(xué)學(xué)術(shù)大會(huì)論文摘要文集[C];2010年

2 覃立立;黎小葵;王，

本文編號(hào)：2351444