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  本文關鍵詞：骨骼肌兩型肌纖維體外培養(yǎng)條件的初探　出處：《遵義醫(yī)學院》2014年碩士論文　論文類型：學位論文

  更多相關文章： 骨骼肌細胞 肌球蛋白重鏈(MHC) 體外培養(yǎng) 逆轉(zhuǎn)錄多聚酶鏈式反應

【摘要】：目的：通過分別對體外培養(yǎng)條件下的骨骼肌兩型肌纖維細胞的標志蛋白MHC基因同功型的檢測，以探索適合骨骼肌兩型肌纖維細胞的培養(yǎng)條件，為進一步研究兩型肌纖維細胞的區(qū)別及轉(zhuǎn)化機制奠定基礎。 方法：實驗以18只家兔作為研究對象，不拘雌雄,4-6周，體重500-1000g，每項指標檢測各用3只。動物分四組：Ⅰ型和Ⅱ型骨骼肌組織組，Ⅰ型和Ⅱ型骨骼肌消化細胞組，Ⅰ型和Ⅱ型骨骼肌細胞常氧（1、3、7天）型和Ⅰ型和Ⅱ型骨骼肌細胞低氧（3%O2）培養(yǎng)組。取家兔后肢右側半膜肌的固有半膜肌亞部和副半膜肌背側亞部，采用胰蛋白酶一步消化法，用含15%胎牛血清的高糖DMEM作為細胞培養(yǎng)液進行常氧和低氧培養(yǎng)，兩型肌纖維培養(yǎng)細胞分別在培養(yǎng)1天、3天、7天取材進行觀察。最后用常氧及低氧培養(yǎng)3天的兩型骨骼肌細胞做反轉(zhuǎn)錄聚合酶鏈式反應（Rt-PCR）鑒定和灰度值測定作對照，以分析骨骼肌兩型肌纖維細胞mRNA標志蛋白MHC的表達變化。 結果：1.骨骼肌組織檢測結果：固有半膜肌表達MHCⅠ及少量的MHCⅡ型，副半膜肌表達MHCⅡa, MHCⅡb型。 2.消化后未經(jīng)培養(yǎng)的細胞檢測結果：固有半膜肌細胞表達MHCⅠ型、MHCⅡa和MHCⅡb型，副半膜肌表達MHCⅡa、 MHCⅡb型。 3.培養(yǎng)細胞檢測結果：在常氧培養(yǎng)組固有半膜肌表達MHCⅡa、 MHCⅡb，，不表達MHCⅠ型；副半膜肌表達MHCⅡa, MHCⅡb型，不表達MHCⅠ型。在低氧培養(yǎng)組兩型骨骼肌細胞均表達為MHCⅡa, MHCⅡb，不表達MHCⅠ型。 4.我們選取骨骼肌組織組、培養(yǎng)后第3天的常氧培養(yǎng)組和低氧培養(yǎng)組進行單因素方差分析統(tǒng)計，結果顯示：Ⅰ型肌纖維細胞在常氧及低氧條件的MHCⅠ型基因表達與組織相比較存在明顯差異（P＜0.05），而MHCⅡa、MHCⅡb和MHCⅡx型基因表達無明顯差異(P＞0.05)。低氧組與常氧組相比，MHCⅠ、MHCⅡa較高，而MHCⅡb較低。Ⅱ型肌纖維細胞在常氧及低氧條件的基因MHCⅠ、MHCⅡa、MHCⅡb和MHCⅡx型表達與組織相比較無明顯差異(P＞0.05)。低氧組與常氧組比較MHCⅠ、MHCⅡb較高，MHCⅡa較低。 結論：1.含15%胎牛血清高糖DMEM適合骨骼肌細胞生長。 2.骨骼肌兩型肌纖維在上述培養(yǎng)條件下均有由Ⅰ型纖維向Ⅱ型纖維轉(zhuǎn)化的趨勢。 3．低氧培養(yǎng)條件有促進Ⅰ型纖維向Ⅱ型纖維轉(zhuǎn)化的作用。
[Abstract]:Objective: through detecting signs under the conditions of the skeletal muscle of type two muscle fibers in vitro protein MHC gene isoform, culture conditions to explore for skeletal muscle of type two muscle fiber cells, and lay the foundation for the further study of the mechanism of transformation and difference between two types of muscle fiber cells.
Methods: the experiment with 18 rabbits as the research object, both male and female, 4-6 weeks, weight 500-1000g, each index to detect the 3. The animal were divided into four groups: type I and type II skeletal muscle group, type I and type II skeletal muscle of the digestive cells group, type I and type II skeletal muscle cells often oxygen (1,3,7 days) and type I and type II skeletal muscle cell hypoxia (3%O2) group. The rabbit right hindlimb culture Semimembranous muscle dorsal inherent semimembranosus muscle compartment and vice semimembranosus sub department, step by trypsin digestion method, containing 15% fetal bovine serum glucose as DMEM cell culture fluid in normoxia and hypoxia, type two muscle fibers in cultured cells were cultured for 1 days, 3 days, 7 days were observed. In normoxia and hypoxia training 3 days of type two skeletal muscle cells do reverse transcription polymerase chain reaction (Rt-PCR) identification and gray value was measured as control. In order to analyze the skeletal muscle of type two muscle fibers Changes in the expression of mRNA marker protein MHC.
Results: the testing results of 1. skeletal muscle tissue: MHC I and a small amount of MHC type II expression inherent semimembranosus, expression of MHC II a vice semimembranosus, MHC type.
2. after digestion without cell culture test results: the expression of the inherent semimembranosus cell type MHC, a and MHC II MHC II B, MHC II a expression of vice semimembranosus, MHC type.
3. test results: cultured cells cultured in normoxic group inherent semimembranosus muscle expression of MHC II A, MHC II B, the expression of MHC type 1; MHC II a expression of vice semimembranosus, MHC type, MHC type. The expression of cultured type two skeletal muscle cells were expressed as MHC II A in hypoxia MHC, of B, the expression of MHC type.
We selected 4. groups of skeletal muscle tissue, cultured after third days of normoxia group and hypoxia group were analyzed by one-way ANOVA statistics, the results show: compared with the organization difference expression of MHC type I type I muscle cells under normoxia and hypoxia condition gene (P < 0.05), and MHC II A, B and MHC II MHC II type X gene expression had no significant difference (P > 0.05). Compared with normoxia group and hypoxia group, MHC I, MHC II and MHC B II a high, low. Gene MHC I type II muscle cells under normoxia and hypoxia conditions, MHC II a. MHC II B and MHC II X expression compared with tissue had no significant difference (P > 0.05). Hypoxia group and normoxia group MHC 1, MHC 2 B high MHC II A is low.
Conclusion: 1. high glucose DMEM containing 15% fetal bovine serum is suitable for the growth of skeletal muscle cells.
2. the two muscle fibers of skeletal muscle were transformed from type I fiber to type II fiber under the above culture conditions.
3. the condition of low oxygen culture can promote the conversion of type I fiber to type II fiber.

【學位授予單位】：遵義醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R336

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