共軛亞油酸對(duì)C2C12肌細(xì)胞生脂和生肌分化的影響

齊仁立　王　琪　王　敬　楊飛云　劉作華　黃金秀

(重慶市畜牧科學(xué)院，農(nóng)業(yè)部養(yǎng)豬科學(xué)重點(diǎn)實(shí)驗(yàn)室，養(yǎng)豬科學(xué)重慶市市級(jí)重點(diǎn)試驗(yàn)室，榮昌402460)

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共軛亞油酸對(duì)C2C12肌細(xì)胞生脂和生肌分化的影響

齊仁立王琪*王敬楊飛云劉作華黃金秀**

(重慶市畜牧科學(xué)院，農(nóng)業(yè)部養(yǎng)豬科學(xué)重點(diǎn)實(shí)驗(yàn)室，養(yǎng)豬科學(xué)重慶市市級(jí)重點(diǎn)試驗(yàn)室，榮昌402460)

本研究分析了共軛亞油酸(CLA)對(duì)C2C12肌細(xì)胞生脂轉(zhuǎn)分化和生肌分化的影響。分別培養(yǎng)并誘導(dǎo)C2C12鼠源肌細(xì)胞生脂轉(zhuǎn)分化和正常的生肌分化，同時(shí)分別使用終濃度為50 μmol/L的c9,t11-CLA和t10,c12-CLA處理細(xì)胞，并設(shè)對(duì)照組，取生脂轉(zhuǎn)分化第10天和生肌分化第8天的細(xì)胞用于實(shí)時(shí)定量PCR檢測(cè)，觀察c9,t11-CLA和t10,c12-CLA對(duì)C2C12肌細(xì)胞不同分化的影響。結(jié)果表明：1)與對(duì)照組相比，c9,t11-CLA促進(jìn)了C2C12肌細(xì)胞的生脂轉(zhuǎn)分化，顯著增加了細(xì)胞內(nèi)甘油三酯(TG)含量(P<0.05)，顯著上調(diào)了細(xì)胞內(nèi)脂肪酸合成酶(FAS)、CCAAT增強(qiáng)子結(jié)合蛋白α(C/EBPα)、過(guò)氧化物酶體增殖劑激活受體γ(PPARγ)和脂肪酸結(jié)合蛋白4(FABP4)基因的表達(dá)水平(P<0.05)；與對(duì)照組相比，t10,c12-CLA則抑制了C2C12肌細(xì)胞的生脂轉(zhuǎn)分化，顯著減少了細(xì)胞內(nèi)TG含量(P<0.05)，顯著下調(diào)了細(xì)胞內(nèi)C/EBPα、PPARγ和FABP4基因的表達(dá)水平(P<0.05)。免疫印跡雜交結(jié)果顯示FAS和FABP4的蛋白質(zhì)表達(dá)水平也發(fā)生了與基因表達(dá)相一致的變化。2)與對(duì)照組相比，t10,c12-CLA抑制了C2C12肌細(xì)胞的生肌分化，顯著減少了細(xì)胞內(nèi)肌管數(shù)/細(xì)胞數(shù)(P<0.05)，顯著下調(diào)了細(xì)胞內(nèi)肌細(xì)胞生成素(MYOG)和成肌分化抗原(MYOD)基因的表達(dá)水平(P<0.05)；與對(duì)照組相比，c9,t11-CLA則顯著上調(diào)了細(xì)胞內(nèi)MYOG基因的表達(dá)水平(P<0.05)，對(duì)C2C12肌細(xì)胞的生肌分化有一定程度的促進(jìn)作用。免疫印跡雜交結(jié)果顯示MYOG和MYOD的蛋白質(zhì)表達(dá)水平也發(fā)生了與基因表達(dá)相一致的變化。以上結(jié)果表明，CLA對(duì)動(dòng)物骨骼肌細(xì)胞的正常生肌分化和生脂轉(zhuǎn)分化都具有重要的調(diào)節(jié)作用。

共軛亞油酸；C2C12肌細(xì)胞；生脂；生??；分化

由于哺乳動(dòng)物的肌肉細(xì)胞和脂肪細(xì)胞源于共同的多能間充質(zhì)干細(xì)胞，生肌細(xì)胞在特定條件下可以轉(zhuǎn)分化為脂肪細(xì)胞或脂肪細(xì)胞樣細(xì)胞，而脂肪細(xì)胞也可以轉(zhuǎn)分化為肌肉細(xì)胞[1-3]。生脂轉(zhuǎn)分化的肌細(xì)胞會(huì)失去生肌能力并獲得脂肪細(xì)胞的特性，細(xì)胞內(nèi)大量產(chǎn)生和沉積脂肪，表達(dá)脂肪細(xì)胞特異性基因及其編碼蛋白。

共軛亞油酸(conjugated linoleic acid，CLA)是一類含有共軛雙鍵的亞油酸異構(gòu)體，具有復(fù)雜的生理作用，包括促生長(zhǎng)、抗腫瘤、抗氧化和抗肥胖等[4-6]。CLA的抗肥胖作用包括抑制脂肪細(xì)胞分化、減少細(xì)胞內(nèi)脂肪生成和沉積、誘導(dǎo)脂肪細(xì)胞凋亡等[7]。c9,t11-CLA和t10,c12-CLA是2種主要的CLA異構(gòu)體，通常情況下二者的生理作用有很大差異。t10,c12-CLA對(duì)脂肪細(xì)胞的抗脂肪生成作用較為強(qiáng)烈，而c9,t11-CLA的作用則較弱[8-9]。有報(bào)道認(rèn)為c9,t11-CLA能夠改善脂肪細(xì)胞對(duì)胰島素的敏感性，增加動(dòng)物肌內(nèi)脂肪的生成和沉積[10-11]。此外，CLA對(duì)于動(dòng)物肌肉的生長(zhǎng)和發(fā)育也有一定影響[12]。肌細(xì)胞的生脂轉(zhuǎn)分化是與正常的脂肪細(xì)胞生脂分化或肌細(xì)胞生肌分化不同的分子事件，但目前關(guān)于CLA對(duì)肌細(xì)胞生脂轉(zhuǎn)分化的影響還不清楚。

本研究培養(yǎng)了C2C12肌細(xì)胞系并分別誘導(dǎo)其生脂和生肌分化，在此基礎(chǔ)上分別使用c9,t11-CLA和t10,c12-CLA處理細(xì)胞，研究2種CLA異構(gòu)體對(duì)C2C12肌細(xì)胞生脂轉(zhuǎn)分化以及正常生肌分化的影響。相關(guān)研究結(jié)果將會(huì)有助于我們更好地理解動(dòng)物肌肉與脂肪之間轉(zhuǎn)化的機(jī)理。

1　材料與方法

1.1試驗(yàn)材料

C2C12鼠源肌細(xì)胞系購(gòu)自于中科院上海細(xì)胞庫(kù)；DMEM培養(yǎng)基、DMEM-F12培養(yǎng)基、胎牛血清和馬血清均購(gòu)自于Life公司；c9,t11-CLA、t10,c12-CLA、油紅O染料和吉姆薩染料均購(gòu)自于Sigma-Aldrich公司；RNAiso Plus RNA試劑盒、cDNA合成試劑盒和SYBR Premix Ex TaqTMⅡ熒光定量PCR試劑盒均購(gòu)自于TaKaRa公司；免疫熒光染色和免疫印跡雜交(Western blotting)試驗(yàn)用脂肪酸結(jié)合蛋白4(fatty acid binding protein 4，F(xiàn)ABP4)、脂肪酸合成酶(fatty acid synthetase，F(xiàn)AS)和甘油醛-3-磷酸脫氫酶(glyceraldehyde-3-phosphate dehydrogenase，GAPDH)的一抗和二抗均購(gòu)自于CST公司，肌細(xì)胞生成素(myogenin，MYOG)和成肌分化抗原(myogenic differentiation antigen，MYOD)的抗體購(gòu)自于Santa Cruz公司。

1.2試驗(yàn)方法

1.2.1細(xì)胞培養(yǎng)與誘導(dǎo)分化

復(fù)蘇凍存的C2C12肌細(xì)胞，接種于DMEM-F12培養(yǎng)基(含10%胎牛血清)中培養(yǎng)。待細(xì)胞密度達(dá)到90%以上時(shí)傳代，傳代后的細(xì)胞接種于6孔板用于不同目的的誘導(dǎo)分化。正常生肌分化的細(xì)胞使用DMEM培養(yǎng)基(含2%馬血清)培養(yǎng)8～10 d。生脂轉(zhuǎn)分化的細(xì)胞使用DMEM-F12培養(yǎng)基(含10%胎牛血清)培養(yǎng)，待細(xì)胞密度達(dá)到70%時(shí)在培養(yǎng)基中加入生脂誘導(dǎo)物[1 μmol/L地塞米松、5 μg/mL胰島素和0.5 mmol/L 3-異丁基-1-甲基黃嘌呤(3-isobutyl-1-methylxanthine，IBMX)]培養(yǎng)2 d，更換為只含有胰島素的培養(yǎng)基再培養(yǎng)2 d，更換為正常培養(yǎng)基繼續(xù)培養(yǎng)4～6 d[2,11]。生脂轉(zhuǎn)分化的細(xì)胞和正常生肌分化的細(xì)胞在開始誘導(dǎo)分化的同時(shí)分別加入c9,t11-CLA和t10,c12-CLA(終濃度為50 μmol/L)，每個(gè)處理3個(gè)重復(fù)，并設(shè)對(duì)照組。

1.2.2油紅O染色和吉姆薩染色

油紅O染色用于觀察C2C12肌細(xì)胞內(nèi)脂肪生成情況。取生脂轉(zhuǎn)分化一定時(shí)間的細(xì)胞，棄掉培養(yǎng)基并用磷酸緩沖鹽(phosphate buffer saline，PBS)漂洗3次，然后用4%多聚甲醛固定15 min。細(xì)胞固定后，用PBS漂洗3次，然后用油紅O染色液在37 ℃條件下染色30 min。染色后的細(xì)胞用60%的異丙醇溶液迅速漂洗1次，再用PBS漂洗3次，倒置顯微鏡下拍照觀察。拍照后用100%異丙醇提取細(xì)胞內(nèi)的油紅O，用紫外分光光度計(jì)在540 nm下讀取吸光度，用于計(jì)算細(xì)胞內(nèi)甘油三脂(TG)的含量。

吉姆薩染色用于觀察C2C12肌細(xì)胞的生肌分化狀態(tài)。取生肌分化一定時(shí)間的細(xì)胞，棄掉培養(yǎng)基并用PBS漂洗3次，然后用4%多聚甲醛固定15 min。細(xì)胞固定后，用PBS漂洗3次，然后用吉姆薩染色液在室溫條件下染色2 min。染色后的細(xì)胞用去離子水漂洗3次，倒置顯微鏡下觀察拍照，并統(tǒng)計(jì)典型的多核細(xì)胞(肌管)數(shù)量，計(jì)算肌管數(shù)/細(xì)胞數(shù)。

1.2.3RNA提取和實(shí)時(shí)定量PCR

取生脂轉(zhuǎn)分化第10天和生肌分化第8天的細(xì)胞用于RNA提取和實(shí)時(shí)定量PCR檢測(cè)。使用RNAiso Plus RNA試劑盒提取細(xì)胞中的總RNA，核酸定量?jī)x檢測(cè)RNA的濃度，1%瓊脂糖凝膠電泳檢測(cè)RNA完整性。使用反轉(zhuǎn)錄試劑盒將總RNA反轉(zhuǎn)錄為cDNA。使用Step One實(shí)時(shí)定量PCR系統(tǒng)(ABI公司)檢測(cè)相關(guān)基因的表達(dá)水平，GAPDH為內(nèi)參基因，2-△△CT法計(jì)算基因的相對(duì)表達(dá)量。實(shí)時(shí)定量PCR的反應(yīng)條件：95 ℃預(yù)變性30 s，95 ℃變性5 s，60 ℃退火35 s，循環(huán)次數(shù)40次。PCR引物由生工生物工程(上海)有限公司設(shè)計(jì)合成，序列見(jiàn)表1。

1.2.4免疫熒光染色

使用免疫熒光染色法檢測(cè)生脂轉(zhuǎn)分化細(xì)胞中FABP4基因的表達(dá)情況和生肌分化細(xì)胞中MYOG基因的表達(dá)情況。取相應(yīng)的細(xì)胞爬片放置于35 mm細(xì)胞培養(yǎng)皿中，PBS漂洗3次；4%的多聚甲醛溶液固定細(xì)胞30 min，PBS漂洗3次；再用0.2% Triton X-100通透細(xì)胞10 min，PBS漂洗3次；用5%牛血清白蛋白(bovine serum albumin，BSA)溶液封閉細(xì)胞30 min并用PBS漂洗5次；用相應(yīng)的一抗(鼠源Anti-FABP4或者Anti-MYOG一抗)在4 ℃條件下孵育細(xì)胞過(guò)夜，然后用PBS漂洗3次；再用對(duì)應(yīng)的二抗(辣根過(guò)氧化物酶標(biāo)記羊抗鼠二抗)室溫孵育2 h(避光)；然后用4,6-二脒基-2-苯基吲哚(4,6-diamidino-2-phenylindole dihydrochloride，DAPI)溶液染細(xì)胞核5 min；最后用倒置熒光顯微鏡觀察并照相。

表1　實(shí)時(shí)定量PCR引物

1.2.5免疫印跡雜交

使用RIPA蛋白裂解液(碧云天公司)提取不同處理的細(xì)胞內(nèi)總蛋白。使用BCA蛋白濃度檢測(cè)試劑盒(天根公司)檢測(cè)樣品總蛋白濃度。10%的十二烷基硫酸鈉-聚丙烯酰胺凝膠(SDS-PAGE)進(jìn)行蛋白質(zhì)分離(60 V，4 h)，然后轉(zhuǎn)印到0.22 μm聚偏氟乙烯(poly vinylidene fluoride，PVDF)膜(Millipore公司)上；轉(zhuǎn)印完的PVDF膜用5%的脫脂牛奶溶液室溫封閉2 h，然后使用相應(yīng)的一抗(鼠源Anti-FAS、Anti-FABP4、Anti-MYOG和Anti-MYOD一抗)溶液4 ℃孵育過(guò)夜；Tris緩沖生理鹽水和吐溫20(tris-buffered saline and Tween 20，TBST)溶液漂洗干凈后使用二抗(辣根過(guò)氧化物酶標(biāo)記羊抗鼠二抗)溶液室溫孵育1 h；最后用增強(qiáng)化學(xué)發(fā)光(enhanced chemiluminescence，ECL)試劑(Millipore公司)和自動(dòng)發(fā)光曝光儀進(jìn)行拍照分析。GAPDH作為內(nèi)參抗體。

1.3數(shù)據(jù)統(tǒng)計(jì)分析

使用SPSS 19.0統(tǒng)計(jì)軟件(IBM公司)進(jìn)行數(shù)據(jù)統(tǒng)計(jì)和分析，組間差異采用單因素方差分析(one-way ANOVA)。P<0.05為差異顯著。

2　結(jié)果與分析

2.1C2C12肌細(xì)胞的生脂和生肌分化

試驗(yàn)觀察到生脂轉(zhuǎn)分化的C2C12肌細(xì)胞逐漸變?yōu)閳A形，細(xì)胞內(nèi)出現(xiàn)大量脂滴并且逐漸融合為大脂滴；免疫熒光染色顯示生脂轉(zhuǎn)分化的細(xì)胞大量表達(dá)脂肪特異蛋白FABP4(圖1)。上述結(jié)果說(shuō)明C2C12肌細(xì)胞已經(jīng)轉(zhuǎn)分化為具有生脂能力的脂肪細(xì)胞(或脂肪細(xì)胞樣細(xì)胞)。生肌分化的C2C12肌細(xì)胞有序排列并逐漸融合，形成多核細(xì)胞，并最終形成大量的肌管；免疫熒光染色顯示生肌分化的細(xì)胞大量表達(dá)肌肉特異蛋白MYOG(圖2)。

圖1　生脂轉(zhuǎn)分化的C2C12肌細(xì)胞(分化第10天)

圖2　生肌分化的C2C12肌細(xì)胞(分化第8天)

2.2CLA對(duì)C2C12肌細(xì)胞生脂轉(zhuǎn)分化的影響

兩種CLA異構(gòu)體對(duì)C2C12肌細(xì)胞生脂轉(zhuǎn)分化的影響見(jiàn)圖3。與對(duì)照組相比，c9,t11-CLA促進(jìn)了C2C12肌細(xì)胞的生脂轉(zhuǎn)分化，增加了細(xì)胞內(nèi)的脂肪沉積，c9,t11-CLA組細(xì)胞內(nèi)TG含量增加了83%(P<0.05)；c9,t11-CLA組細(xì)胞內(nèi)的FAS、CCAAT增強(qiáng)子結(jié)合蛋白α(CCAAT/enhancer binding protein α，C/EBPα)、過(guò)氧化物酶體增殖劑激活受體γ(peroxisome proliferators-activated receptors γ，PPARγ)、FABP4基因的表達(dá)水平均顯著上調(diào)(P<0.05)，而脂肪酸轉(zhuǎn)運(yùn)蛋白1(fatty acid transport protein 1，F(xiàn)ATP1)基因的表達(dá)水平則無(wú)顯著變化(P>0.05)。與對(duì)照組相比，t10,c12-CLA則抑制了C2C12肌細(xì)胞的生脂轉(zhuǎn)分化，減少了細(xì)胞內(nèi)的TG含量(P<0.05)，顯著下調(diào)了C/EBPα、PPARγ和FABP4基因的表達(dá)水平(P<0.05)。免疫印跡雜交結(jié)果顯示FAS和FABP4的蛋白質(zhì)表達(dá)水平也發(fā)生了與基因表達(dá)相一致的變化。

2.3CLA對(duì)C2C12肌細(xì)胞生肌分化的影響

2種CLA異構(gòu)體對(duì)C2C12肌細(xì)胞生肌分化的影響見(jiàn)圖4。與對(duì)照組相比，t10,c12-CLA抑制了C2C12肌細(xì)胞的生肌分化，顯著減少了肌管數(shù)/細(xì)胞數(shù)(P<0.05)，顯著下調(diào)了MYOG和MYOD基因的表達(dá)水平(P<0.05)。與對(duì)照組相比，c9,t11-CLA則顯著提高了細(xì)胞內(nèi)MYOG基因的表達(dá)水平(P<0.05)，對(duì)C2C12肌細(xì)胞的生肌分化有一定程度的促進(jìn)作用。免疫印跡雜交結(jié)果顯示MYOG和MYOD的蛋白質(zhì)表達(dá)水平也發(fā)生了與基因表達(dá)相一致的變化。

3　討　論

研究發(fā)現(xiàn)，生肌細(xì)胞在特定生理?xiàng)l件下或者藥物刺激下可以轉(zhuǎn)分化為脂肪細(xì)胞或者骨細(xì)胞[1-3]。反之，脂肪細(xì)胞和骨細(xì)胞也可以轉(zhuǎn)分化為肌細(xì)胞。與正常的分化相比，肌細(xì)胞和脂肪細(xì)胞之間轉(zhuǎn)分化的物質(zhì)代謝基礎(chǔ)和分子調(diào)控機(jī)制有很大差異，目前還未完全清楚。一方面，肌細(xì)胞的生脂轉(zhuǎn)分化影響了機(jī)體的能量蓄積和利用，過(guò)度的脂肪蓄積還會(huì)導(dǎo)致炎癥損傷、胰島素抵抗和糖尿病等代謝癥；肌細(xì)胞和脂肪細(xì)胞之間轉(zhuǎn)化的失衡對(duì)于肌肉的正常生理功能也會(huì)造成影響。另一方面，肌細(xì)胞的適度生脂轉(zhuǎn)分化對(duì)于提高動(dòng)物肌內(nèi)脂肪含量和提升動(dòng)物的肉品質(zhì)具有一定的幫助作用。

A.細(xì)胞油紅O染色(100倍放大)；B.細(xì)胞內(nèi)TG含量；C.生脂基因的相對(duì)表達(dá)水平(1：脂肪酸合成酶；2：CCAAT增強(qiáng)子結(jié)合蛋白α；3：過(guò)氧化物酶體增殖劑激活受體γ；4：脂肪酸結(jié)合蛋白4；5：脂肪酸轉(zhuǎn)運(yùn)蛋白1)；D.生脂因子蛋白質(zhì)表達(dá)水平。數(shù)據(jù)柱形標(biāo)注不同字母代表差異顯著(P<0.05)。

A. Oil red O staining cells (100 times magnification); B. intracellular TG content; C. relative expression levels of adipogenic genes (1:FAS, 2:C/EBPα, 3:PPARγ, 4:FABP4, 5:FATP1); D. protein expression levels of adipogenic factors. Data columns with different letters mean significant difference (P<0.05).

圖3CLA對(duì)C2C12肌細(xì)胞生脂轉(zhuǎn)分化的影響

Fig.3Effects of conjugated linoleic acid on the adipogenic trans-differentiation of C2C12 myoblasts

體內(nèi)和體外試驗(yàn)中CLA都表現(xiàn)出較強(qiáng)的抗脂肪生成能力，但不同CLA異構(gòu)體的作用差異較大。研究認(rèn)為t10,c12-CLA能夠顯著抑制人和動(dòng)物的體脂沉積，抑制前體脂肪細(xì)胞分化為成熟脂肪細(xì)胞，誘導(dǎo)脂肪細(xì)胞凋亡，阻礙細(xì)胞內(nèi)脂肪酸的酯化。與之相比，c9,t11-CLA的作用較為溫和，對(duì)于脂肪細(xì)胞的影響較弱[7-8]。此外，研究報(bào)道認(rèn)為CLA能夠調(diào)控動(dòng)物體內(nèi)脂肪的異位沉積，一方面減少脂肪組織沉積，另一方面增加肌內(nèi)脂肪含量[13]。與脂肪細(xì)胞上的研究結(jié)果相似，本試驗(yàn)結(jié)果表明t10,c12-CLA對(duì)于C2C12肌細(xì)胞的生脂轉(zhuǎn)分化和脂肪沉積同樣具有極強(qiáng)的抑制作用，而c9,t11-CLA則促進(jìn)了C2C12肌細(xì)胞的生脂轉(zhuǎn)分化和脂肪沉積。

關(guān)于CLA對(duì)動(dòng)物肌肉生長(zhǎng)和發(fā)育的研究也有不同的報(bào)道。有研究認(rèn)為CLA有益于肌肉的生長(zhǎng)和發(fā)育，可增加骨骼肌中線粒體的含量[14]，減少肌細(xì)胞的炎癥反應(yīng)并可改善由于高脂飼糧攝入引起的小鼠肌肉損傷[15-17]。課題組前期在豬上的試驗(yàn)研究發(fā)現(xiàn)飼糧補(bǔ)充CLA混合物能夠顯著增加豬背最長(zhǎng)肌肉塊的重量[12]。但是，關(guān)于CLA對(duì)骨骼肌細(xì)胞的增殖和分化影響的報(bào)道甚少。有試驗(yàn)發(fā)現(xiàn)c9,t11-CLA能夠促進(jìn)C2C12肌細(xì)胞的增殖和生肌分化，而t10,c12-CLA則起到相反的作用[18]。Hommelberg等[19]也證實(shí)了t10,c12-CLA能抑制骨骼肌細(xì)胞的分化，并降低葡萄糖轉(zhuǎn)運(yùn)蛋白4(glucose transporter-4，GLUT4)基因的表達(dá)水平。本研究結(jié)果同樣發(fā)現(xiàn)t10,c12-CLA抑制了C2C12肌細(xì)胞的生肌分化，但c9,t11-CLA則對(duì)生肌分化有輕微的促進(jìn)作用。這些試驗(yàn)結(jié)果表明CLA對(duì)于肌細(xì)胞的影響是多方面的，既能保護(hù)肌細(xì)胞由于氧化應(yīng)激和炎癥反應(yīng)帶來(lái)的損傷，又對(duì)肌細(xì)胞的增殖分化具有調(diào)節(jié)作用。

A.細(xì)胞吉姆薩染色(200倍放大)；B.細(xì)胞內(nèi)肌管數(shù)分析；C.生肌基因的相對(duì)表達(dá)水平(1：肌細(xì)胞生成素；2：成肌分化抗原；3：成肌調(diào)節(jié)因子5)；D.生肌因子蛋白質(zhì)表達(dá)水平。數(shù)據(jù)柱形標(biāo)注不同字母代表差異顯著(P<0.05)。

A. Giemsa staining cells (200 times magnification); B. intracellular myotube number analysis; C. relative expression levels of myogenic genes (1:MYOG, 2:MYOD, 3:MYF5); D. protein expression levels of myogenic factors. Data columns with different letters mean significant difference (P<0.05).

圖4CLA對(duì)C2C12肌細(xì)胞生肌分化的影響

Fig.4Effects of conjugated linoleic acid on the myogenic differentiation of C2C12 myoblasts

研究發(fā)現(xiàn)CLA的不同異構(gòu)體表現(xiàn)出的生理作用差異較大，但是其具體原因還不完全清楚。推測(cè)可能是由于以下原因：1)不同CLA異構(gòu)體的代謝速率和代謝產(chǎn)物不同。比較而言，t10,c12-CLA更易于參與β氧化，而c9,t11-CLA更易于在體內(nèi)蓄積[20]。c9,t11-CLA主要代謝形成C20∶3共軛脂肪酸及其衍生物，而t10,c12-CLA可以代謝形成C16∶2和C18∶3共軛脂肪酸及其衍生物[21]?；ㄉ南┧帷⑶傲邢偎睾湍獝和榈榷嗖伙柡椭舅岬拇x產(chǎn)物又能發(fā)揮不同的生物學(xué)作用。2)不同CLA異構(gòu)體及其代謝產(chǎn)物直接作為細(xì)胞信號(hào)分子激活或抑制的轉(zhuǎn)錄調(diào)控因子及信號(hào)傳導(dǎo)通路不同。如t10,c12-CLA可以激活核轉(zhuǎn)錄因子κB，而c9,t11-CLA則不能激活該轉(zhuǎn)錄因子，進(jìn)而對(duì)下游諸多功能基因的影響也會(huì)存在巨大的差異[19]。

總結(jié)本試驗(yàn)的結(jié)果，可以認(rèn)為CLA對(duì)肌細(xì)胞和脂肪細(xì)胞之間的轉(zhuǎn)化具有調(diào)控作用，但是不同CLA異構(gòu)體的作用具有差異。這些結(jié)果對(duì)相關(guān)疾病的治療以及提高動(dòng)物產(chǎn)品品質(zhì)等有一定的幫助。

4　結(jié)　論

c9,t11-CLA能夠促進(jìn)C2C12肌細(xì)胞的生脂轉(zhuǎn)分化，而t10,c12-CLA則抑制了C2C12肌細(xì)胞的生脂轉(zhuǎn)分化。此外，t10,c12-CLA抑制了C2C12肌細(xì)胞的生肌分化，而c9,t11-CLA則發(fā)揮了一定的促進(jìn)作用。

[1]TEBOUL L,GAILLARD D,STACCINI L,et al.Thiazolidinediones and fatty acids convert myogenic cells into adipose-like cells[J].The Journal of Biological Chemistry,1995,270(47):28183-28187.

[2]ASAKURA A,RUDNICKI M A,KOMAKI M.Muscle satellite cells are multipotential stem cells that exhibit myogenic,osteogenic,and adipogenic differentiation[J].Differentiation,2001,68(4/5):245-253.

[3]SINGHN K,CHAE H S,HWANG I H,et al.Transdifferentiation of porcine satellite cells to adipoblasts with ciglitizone[J].Journal of Animal Science,2007,85(5):1126-1135.

[4]LARSEN T M,TOUBRO S,ASTRUP A.Efficacy and safety of dietary supplements containing CLA for the treatment of obesity evidence from animal and human studies[J].Journal of Lipid Research,2003,44(12):2234-2241.

[5]MACDONALD H B.Conjugated linoleic acid and disease prevention:a review of current knowledge[J].Journal of the American College of Nutrition,2000,19(Suppl.2):111S-118S.

[6]HUANG J X,QI R L,CHEN X L, et al.Improvement in the carcass traits and meat quality of growing-finishing Rongchang pigs by conjugated linoleic acid through alteredgene expression of muscle fiber types[J].Genetics and Molecular Research,2014,13(3): 7061-7069.

[7]CHUNG S,BROWN J M,SANDBERG M B,et al.Trans-10,cis-12 CLA increases adipocyte lipolysis and alters lipid droplet-associated proteins:role of mTOR and ERK signaling[J].Journal of Lipid Research,2005,46(5):885-895.

[8]DECLERCQ V,ZAHRADKA P,TAYLOR C G.Dietaryt10,c12-CLA but notc9,t11 CLA reduces adipocyte size in the absence of changes in the adipose renin-angiotensin system infa/faZucker rats[J].Lipids,2010,45(11):1025-1033.

[9]LIN Y G,KREEFT A,SCHUURBIERS J A E,et al.Different effects of conjugated linoleic acid isomers on lipoprotein lipase activity in 3T3-L1 adipocytes[J].The Journal of Nutritional Biochemistry,2001,12(3):183-189.

[10]PARK Y,STORKSON J M,ALBRIGHT K J,et al.Evidence that thetrans-10,cis-12 isomer of conjugated linoleic acid induces body composition changes in mice[J].Lipids,1999,34(3):235-241.

[11]ZHOU X,LI D F,YIN J D,et al.CLA differently regulates adipogenesis in stromal vascular cells from porcine subcutaneous adipose and skeletal muscle[J].Journal of Lipid Research,2007,48(8):1701-1709.

[12]QI R L,CHEN Y,PENG H,et al.Conjugated linoleic acid supplementation during late gestation and lactation of sows affects myofiber type in their litters[J].Livestock Science,2015,178:322-329.

[13]JIANG Z Y,ZHONG W J,ZHENG C T,et al.Conjugated linoleic acid differentially regulates fat deposition in backfat and longissimus muscle of finishing pigs[J].Journal of Animal Science,2010,88(5):1694-1705.

[14]VAUGHAN R A,GARCIA-SMITH R,BISOFFI M,et al.Conjugated linoleic acid or omega 3 fatty acids increase mitochondrial biosynthesis and metabolism in skeletal muscle cells[J].Lipids In Health And Disease,2012,11:142.

[15]LARSEN A E,CAMERON-SMITH D,CROWE T C.Conjugated linoleic acid suppresses myogenic gene expression in a model of human muscle cell inflammation[J].The Journal of Nutrition,2008,138(1):12-16.

[16]LARSEN A E,CROWE T C.Effects of conjugated linoleic acid on myogenic and inflammatory responses in a human primary muscle and tumor coculture model[J].Nutrition and Cancer,2009,61(5):687-695.

[17]LEE S R,JO E,KHAMOUI A V,et al.Resistance training and CLA/n-3 administration improve myofiber size and myogenic capacity in high fat diet-fed mice[J].The FASEB Journal,2013,27(7):1093.

[18]LEE J H,TACHIBANA H,MORINAGA Y,et al.Modulation of proliferation and differentiation of C2C12 skeletal muscle cells by fatty acids[J].Life Sciences,2009,84(13/14):415-420.

[19]HOMMELBERG P H H,PLAT J,REMELS A H V,et al.Trans-10,cis-12 conjugated linoleic acid inhibits skeletal muscle differentiation and GLUT4 expression independently from NF-κB activation[J].Molecular Nutrition & Food Research,2010,54(12):1763-1772.

[20]MARTIN J C,GRéGOIRE S,SIESS M H,et al.Effects of conjugated linoleic acid isomers on lipid-metabolizing enzymes in male rats[J].Lipids,2000,35(1):91-98.

[21]SéBéDIO J L,ANGIONI E,CHARDIGNY J M,et al.The effect of conjugated linoleic acid isomers on fatty acid profiles of liver and adipose tissues and their conversion to isomers of 16∶2 and 18∶3 conjugated fatty acids in rats[J].Lipids,2001,36(6):575-582.

*Contributed equally

**Corresponding author, professor, E-mail: short00@163.com

(責(zé)任編輯李慧英)

Effects of Conjugated Linoleic Acid on Adipogenic and Myogenic Differentiation of C2C12 Myoblasts

QI RenliWANG Qi*WANG JingYANG FeiyunLIU ZuohuaHUANG Jinxiu**

( Chongqing Academy of Animal Sciences, Key Laboratory of Pig Industry Sciences, Ministry of Agriculture,Chongqing Key Laboratory of Pig Industry Sciences, Rongchang 402460, China)

This study was conducted to evaluated the effects of conjugated linoleic acid (CLA) on adipogenic trans-differentiation and myogenic differentiation of C2C12 myoblasts. Eitherc9,t11-CLA ort10,c12-CLA (the final concentration was 50 μmol/L) was used to treat the C2C12 mouse origin myoblasts which were cultured and induced adipogenic trans-differentiation and normal myogenic differentiation, respectively. And set the control group. The C2C12 myoblasts adipogenic trans-differentiation on the tenth day and myogenic differentiation on the eighth day were used for real-time quantitative PCR detection, and to observed the effects ofc9,t11-CLA andt10,c12-CLA on different differentiation of C2C12 myoblasts. The results showed as follows: 1) compared with the control group,c9,t11-CLA promoted adipogenic trans-differentiation of C2C12 myoblasts, significantly increased the intracellular triglycerides (TG) content and up-regulated intracellular genes expression levels of the fatty acid synthetase (FAS), CCAAT/enhancer binding protein α (C/EBPa), peroxisome proliferators-activated receptors γ (PPARγ) and fatty acid binding protein 4 (FABP4) (P<0.05). Compared with the control group,t10,c12-CLA inhibited the adipogenic trans-differentiation of C2C12 myoblasts, significantly decreased the intracellular TG content and down-regulated intracellular genes expression levels ofC/EBPa,PPARγandFABP4 (P<0.05). The results of Western blotting showed that protein expression levels of FAS and FABP4 also changed as same as the gene expression levels. 2) Compared with the control group,t10,c12-CLA inhibited the myogenic differentiation of C2C12 myoblasts, significantly decreased the intracellular myotube numbers/cell numbers and down-regulated intracellular genes expression levels of myogenin (MYOG) and myogenic differentiation antigen (MYOD) (P<0.05). Compared with the control group,c9,t11-CLA significantly down-regulated gene expression level ofMYOG(P<0.05) and promoted the myogenic differentiation of C2C12 myoblasts to some degree. The results of Western blotting showed that protein expression levels of MYOG and MYOD also changed as same as the gene expression levels. All these results suggest that CLA has important regulation on normal myogenic differentiation and adipogenesis trans-differentiation of skeletal muscle cells in animals.[ChineseJournalofAnimalNutrition, 2016, 28(9)：2778-2785]

conjugated linoleic acid; C2C12 myoblasts; adipogenic; myogenic; differentiation

10.3969/j.issn.1006-267x.2016.09.016

2016-03-03

國(guó)家“973”計(jì)劃項(xiàng)目(2012CB124702)；國(guó)家自然科學(xué)基金(31302055)；重慶市畜牧科學(xué)院基本科研業(yè)務(wù)(14441)

齊仁立(1982—)，男，河南南陽(yáng)人，助理研究員，博士，從事動(dòng)物脂肪代謝相關(guān)研究。E-mail: qirenli1982@163.com

S811.2

A

1006-267X(2016)09-2778-08

*同等貢獻(xiàn)作者

**通信作者：黃金秀，研究員，E-mail: short00@163.com