CNR1基因的表达对肌纤维类型的影响及其机制研究
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摘要
肌纤维的类型和组成是肌肉生长和肉品品质的重要生化和分子生物学基础。不同的肌纤维类型具有不同的能量代谢特点和生理生化特性。在哺乳动物骨骼肌中表达的肌纤维类型主要有MyHCⅠ、MyHCⅡa、MyHCⅡx和MyHCⅡb四型,不同的肌纤维类型之间可以相互转化。本研究在课题组前期基因芯片筛选的基础上,以在肉质优良的金华猪和肉质欠佳的长白猪中差异表达的基因--内源性大麻素受体1(CNR1)为研究对象。利用荧光定量PCR,免疫组化,VWestern Blot等技术,建立体内、体外超表达和基因沉默的细胞或动物模型,较系统的研究了CNR1在骨骼肌纤维类型转化中的调控作用及可能的机制。主要研究结果如下：
1. CNR1在猪肌肉组织和肌卫星细胞中的表达规律研究
本实验以金华猪、长白猪和分离的猪肌卫星细胞为研究对象,比较研究了CNR1基因在猪不同肌肉组织如背最长肌(快肌纤维为主)、腓肠肌(混合型)和比目鱼肌中(慢肌纤维)表达的组织差异,发育性变化规律和品种差异,及其在肌卫星细胞诱导分化中的表达规律。结果显示：在不同的肌肉组织中,CNR1基因在背最长肌中的表达量较高(p<0.01)；发育性变化规律研究表明,CNR1基因表达存在着随日龄的增长而下降的趋势；品种差异显示,CNR1基因在长白猪背最长肌和比目鱼肌中的表达量均高于金华猪(p<0.01),而在腓肠肌中未发现显著的品种差异。在分离的猪肌卫星细胞中,CNR1在诱导前表达量较高,随着诱导呈现显著下降的趋势(p<0.05)。从以上结果可以看出CNR]基因在背最长肌中相对高表达；并有随日龄的增长而呈现显著下降的趋势；同时CNR1在长白猪中的表达量高于金华猪。在分离的肌卫星细胞中有随着诱导分化呈现显著下降的趋势。提示CNR1基因的表达可能与肌纤维的发育和类型相关。
2. CNR1特异性激动剂和抑制剂对肌纤维类型的影响及其分子机制
以大鼠成肌细胞L6为模型,首先研究了随着L6细胞的诱导分化,CNR1基因和肌纤维分型相关基因MyHCⅠ、MyHCⅡa、MyHCⅡx和MyHCⅡb的表达规律；进一步用CNR1的激动剂MAEA和抑制剂AM251处理L6细胞,荧光定量PCR检测CNR1基因、肌纤维类型相关基因(MyHCⅠ、MyHCⅡa、MyHCⅡx和MyHCⅡb)、能量代谢类型的关键酶基因(LDH、SDH和MDH)和肌纤维类型转化调控的关键基因(AMPKa1、PGC-1、ERK1和ERK2)的表达。研究结果显示：诱导0、1、3、5、7、9天的L6细胞中,CNR1基因的表达在诱导的第3天和第7天较高(p<0.05)。MyHCI和MyHCIIa型的表达量在第3天达到峰值,随后显著下降。MyHCIIx在诱导的前7天有随着诱导升高的趋势,第3天和第7天的表达量较高(P<0.05)。MyHCIIb基因在诱导的第2天即显著升高(P<0.05),第3天达到峰值后极显著下降(P<0.05)。不同浓度(0、5、10、15μM/L)的CNR1激动剂MAEA处理L6细胞,CNR1基因的表达在15μM的浓度下显著提高。进一步在15μM的浓度下处理不同时间,CNR1基因的表达在第1天和第3天提高。无论是MAEA不同浓度处理还是在相同浓度下MAEA处理不同时间,CNR1的激动剂MAEA均能显著降低MyHCIIb基因的表达,MyHCIIa基因的表达量有上升的趋势；对MyHCI和MyHCIIx的表达无显著影响。能量代谢关键酶基因发面,MAEA处理后,糖酵解关键酶LDH基因的表达下降(p<0.05)。有氧氧化关键酶SDH和MDH无显著变化。肌纤维类型转化调控基因ERK1的表达量显著升高。而不同浓度(0、0.1、1、10μM/L)的抑制剂AM251处理L6细胞后,10μM/L的AM251显著降低了CNR1基因的表达；进一步用10μM/L的浓度下处理不同时间(1、3、5天),CNR1基因的表达均呈现下降趋势。无论是AM251不同浓度处理还是在相同浓度下处理不同时间,肌纤维类型相关基因MyHCIIa和MyHCIIx的表达量下降(P<0.05),MyHCIIb基因的表达增加(P<0.01)。能量代谢关键酶基因LDH的表达量升高,MDH和SDH的基因表达则显著下降。肌纤维类型转化调控的关键基因(?)MPKal和ERK1的表达量降低(P<0.01)。上述述结果初步揭示CNR1激动剂可以显著降低酵解型肌纤维MyHCIIb基因的表达,增加快氧化型肌纤维基因的表达；显著降低酵解关键酶基因LDH的表达和升高氧化型酶SDH和MDH的表达,同时ERK1基因的表达也增强。而添加抑制剂后得到了与之相反的实验结果。
3.干扰CNR1基因对肌纤维类型的影响及其分子机制
本实验中首先设计合成三对CNR1基因的特异性发夹siRNA干扰片段,将其克隆入干扰载体pYr-1.1,构建可沉默(CNR1基因的siRNA表达载体CNR1-1、CNR1-2和CNR1-3。并采用LipofectamineTM (Lip)2000介导质粒转染L6细胞,绿色荧光蛋白的表达和流式细胞仪监测转染效率,通过实时荧光定量分析siRNA表达载体的干扰效果。并进一步用G418进行了稳定转染siRNA的L6细胞筛选。结果显示,CNR1基因的siRNA表达载体构建正确,瞬时转染L6细胞的转染效率分别为10.45%(P<0.01)、8.57%(P<0.01)和8.71%(P<0.01)；干扰效率为39%(P<0.05)、64%(P<0.01)及68%(p<0.01)。稳定筛选的最佳G418浓度为800ug/ml,稳定筛选后干扰效率分别为43%(P<0.05)、78%(P<0.01)及91%(P<0.01)。干扰效率较高的CNR1-3表达载体和稳定转染CNR1-3的细胞为构建筛选成功的siRNA表达载体和阳性细胞。
有效沉默CNR1基因后,MyHCIIa基因的表达显著下降；MyHCIIb基因的表达则显著上升。进一步对稳定干扰细胞进行诱导,在诱导后1、3、5天MyHCI和MyHCIIx基因的表达均无显著变化；MyHCIIb基因的表达在诱导1、3、5天分别显著提高(P<0.05)；MyHCIIa基因的表达均呈现下降,在诱导第3天显著下降(P<0.05)。在稳定干扰的L6细胞系中,糖酵解关键酶基因LDH的表达显著升高(P<0.05)；有氧氧化关键酶基因MDH和SDH基因的表达下降,但无显著差异。ERK2基因的表达量极显著的升高(P<0.05)。而AMPKa1、PGC-1和ERK1的基因表达无显著变化。这些变化趋势和CNR1基因的抑制剂作用效果基本一致。这些实验结果揭示,在L6细胞中抑制和沉默CNR1基因后可以显著升高酵解型肌纤维MyHCIIb基因的表达,显著降低快氧化型肌纤维MyHCIIa基因的表达：显著升高能量代谢关键酶基因LDH基因的表达；ERK1基因的表达显著降低而ERK2基因的表达显著升高。
4.超表达CNR1对肌纤维类型的影响及其分子机制
4.1体外超表达CNR1对肌纤维类型的影响及其分子机制
质粒载体pCDNA3.0具有G418抗性,用800ug/ml的G418对转染超表达质粒的L6细胞进行稳转筛选。筛选出的稳定转染细胞系中CNR1基因和蛋白的相表达量均显著增加,稳定超表达CNR1细胞模型建立成功。稳定超表达CNR1后,MyHCIIa基因的表达显著升高(P<0.01),MyHCIIb基因的表达显著下降(P<0.01), MyHCI和MyHCIIx的表达无显著变化。进一步对稳定超表达CNR1的细胞进行诱导后,MyHCI、MyHCIIb基因的表达在诱导的1、3、5天均无显著变化。MyHCIIa基因的表达在诱导后均显著提高(P<0.05)。糖酵解关键酶基因LDH的表达显著下降(P<0.01),有氧氧化代谢的关键酶MDH (P<0.01)和SDH (P<0.05)显著升高。肌纤维转型关键基因ERK1基因的表达显著升高(P<0.05), AMPKal、PGC-1和ERK2的表达无显著差异。这些变化趋势与CNR1激动剂MAEA的作用效果基本一致。综合上述结果,CNR1激动剂和体外超表达CNR1可显著降低MyHCIIb基因的表达,显著增加快氧化型MyHCIIa的表达。同时显著升高了有氧氧化的关键酶SDH和MDH的表达而酵解关键酶LDH表达显著降低,肌纤维类型转变的关键调控因子ERK1的表达显著上升。
4.2大鼠体内超表达CNR1对肌纤维类型的影响及其调控机制
利用课题组前期建立的大鼠活体肌肉电击转化实验平台,将CNR1超表达质粒导入大鼠腓肠肌和比目鱼肌中,CNR1的基因和蛋白表达量均显著提高,CNR1超表达体内模型建立成功。肌肉中超表达CNR1后肌纤维类型的变化,腓肠肌和比目鱼肌中MyHCIIa的基因表达均显著上调,MyHCIIa的蛋白表达量升高,但在比目鱼肌中差异不显著。肌肉中超表达CNR1后能量代谢关键酶基因和酶活性的变化,腓肠肌中和比目鱼肌中糖酵解关键酶LDH的活性降低(p<0.05),有氧代谢的关键酶MDH的基因表达和酶活性显著升高(p<0.05),SDH的基因表达和酶活升高,仅在比目鱼肌中达到显著水平。肌肉中超表达CNR1后肌纤维转化关键基因表达的变化,腓肠肌中和比目鱼肌中ERK1和PGC-1基因的表达量均显著升高。从以上结果可以看出,体内超表达CNR1后升高了MyHCIIa的基因和蛋白表达；显著降低了酵解关键酶LDH酶活,显著增加了有氧氧化关键酶MDH和SDH的基因表达和酶活。同时调控肌纤维类型转化的关键基因PGC和ERK1的表达均显著升高。
综合体外和体内的实验结果,CNR1可以增加肌肉细胞和肌肉组织中MyHCII a的表达,促使肌纤维向快氧化型转化。上调有氧氧化关键酶MDH和SDH,下调酵解代谢的关键酶LDH,影响肌肉细胞和肌肉组织中的能量代谢,在这个过程中ERK1/2可能发挥着重要作用。另外,(CNR1对肌纤维类型的调控及机制存在体内和体外差异。
One of the biochemical and molecular basis factors determing muscle growth and meat quality is the muscle fiber types.Different muscle fiber types have showed different characteristics of energy metabolism and biochemical characteristics. There are four main muscle fiber types (MyHCI, MyHCIIa, MyHCIIx and MyHCIIb) in mammalian skeletal muscle.Furthermore, different muscle fiber types can be transformed into each other.Taking advantages of the global microarray technology, cannabinoid receptor1(CNR1) have identified as fifferently expression between Jinhua with superior meat quality and Landrace pigs with lower meat quality.In order to inverstgate the effects and mechanism of CNR1on muscle fiber types, Quantitative PCR, immunohistochemistry, Western Blot and other technologies were used to establish the in vitro and in vivo overexpression and gene silencing in cells or animal models. The findings are as follows:
1. Expression patterns of the cannabinoid receptor1gene in pig skeletal muscle and skeletal satellite muscle cells
Up to now, detailed analysis of CNR1gene expression in pig muscles and muscle cells has not been reported. Here, quantitative PCR were used to characterize the CNR1in different muscles of pig and isolation of porcine myogenic satellite cells. Results showed that the expression of CNR1mRNA was greatest in longissimus dorsi muscle (p<0.01)among that in the gastrocnemius (mix), soleus muscle (slow-type) and longissimus doris muscle (fast-type). As to age-related changes of CNR1gene in different pig muscles, the higher expression was found at the30th day, then declined (p<0.05) in all three kinds of muscles to the90th day. At the150th day, the expression of CNR1steadily decreased (p<0.05) in the gastrocnemius compared to the90th day, but not in longissimus doris muscle and soleus. Meanwhile the breed differences in the expression of CNR1mRNA between Jinhua pig and Landrace were also identified. The mRNA abundance of CNR1in Landrace was higher compared with that of Jinhua pigs and strong differences were observed in longissimus doris muscle (p<0.05) and soleus (p<0.01). In vitro, with the differentiation of the satellite cells, the mRNA expression of the CNR1was down-regulated (p<0.05). The lowest mRNA expression was observed at the9th day. These data provide a framework of CNR1gene expression patterns and implicate a novel gene for regulating muscle fiber types.
2. Effects and molecular mechanism of CNR1agonist and antagonist on muscle fiber types
we analyzed the expression patterns of CNR1and MyHC isforms types includingⅠ,Ⅱa,Ⅱx Ⅱb with the cell differentiate in L6cells.After that,L6cell were coped with CNR1selective agonist MAEA and antagonist AM251.The changes of CNR1expression, MyHC isforms(MyHCⅠ, MyHCⅡa, MyHCⅡx and MyHCⅡb) and the key enzyme gene (LDH,SDH and MDH)as well as the key gene of muscle fiber types transition(AMPKal, PGC-1, ERK1and ERK2) were detected using quantitative PCR.The results show that:CNR1gene expression was higher at the3th and7th day. The levels of MyHCI, and MyHCⅡa expression peaked at the3thday, then dramatically decreased. MyHCⅡx gene expression showed the increased trend before the7th day (P<0.05). MyHClIb gene was significantly increased at the first two2days, at the3th day,reached the higest level and then significant decreased.
Treated L6cells with different concentrations of MAEA(0,5,10,15μM/L), we found that CNR1expression was significantly increased at15μM/L. Further treated with different time at15μM/L.Under the both conditions, the MyHCⅡb gene expression was reduced (p<0.01). but MyHCIIa gene expression was increased The gege expression of MyHCl and MyHCⅡx had no significant difference. Meanwhile gene expression of glycolytic key enzyme LDH were decreased (p<0.05). Key enzyme genes of oxidation SDH and MDH had no significant change.The key gene of muscle fiber types transition ERK1expression was increased (P<0.05).
On the contrary, treated L6cells with different concentrations of AM251(0,0.1,1,10μM/L), the results showed that CNR1expression was significantly down regulated at10μM/L. Further treated with different time at10μM/L.Under the both conditions, the gene expression MyHCⅡa, and MyHCⅡx was significantly decreased (P<0.05), MyHCⅡb gene expression significantly increased (P<0.01). The expression level of LDH was elevated (P>0.05), MDH and SDH gene expression were significantly decreased (P<0.05). AMPKal and ERK1expression was significantly down regulated (P<0.01).
3. Effects and molecular mechanism of interference CNR1on muscle fiber types in vitro
This study was conducted to construct and identify CNRl gene siRNA expression vectors and screen the stable CNR1-interference positive L6cell clones. Three pairs of CNR1-specific double-strand siRNAs were designed, synthesized, annealed and inserted into the pYr-1.1vector.The CNR1gene siRNA expression vectors were identified by restriction enzyme digestion and sequencing. After that siRNAs were transfected with L6cells by LipofectamineTM (Lip)2000. Then, the transfection efficiency was detected by EGFP and FCM.CNRI gene expression was determined by real-time PCR and the stable transgenic L6cell clones were screened by G418. The results revealed that the CNR1gene siRNA expression vectors have been constructed successfully. The transient transfection efficiency to L6cells were10.45%(P<0.01)、8.57%(P<0.01) and8.71%(P<0.01),and the silencing efficacy of the transient transfected L6cells was39%(P<0.05)、64%(P<0.01) and68%(p<0.01),respectively. The optimal selection concentration of G418for stable transfected L6cell clones was800ug/ml. The silencing efficacy of CNR-1-positive transgenic cell clones were43%(P<0.05).78%(P<0.01) and91%(P<0.01). The results showed that CNR1-3expression vector is the optimal silencing vector and CNR1-3stable transgenic cell clones is best silencing cell line. This study provides a successful CNR1gene silencing method by siRNA and the screening of CNR1-interference positive L6cell clones renders basic tools for further studying the functions of CNR1gene.
After effectively silence CNR1gene by RNA interference. The gene expression of MyHCⅡa was decreased (P<0.05); the levels of MyHCⅡb was increased significantly (P<0.05), stable interference cells inducted by1,3,5days; gene expression of MyHCⅡb was significantly increased (P<0.05); Meanwhile,MyHCⅡa gene expression showed a decline trend. At the3th day, MyHCIIa gene expression decreased significantly (P<0.05). Expression of the glycolytic key enzyme gene LDH was significantly increased (P<0.05); the key enzyme genes of oxidation MDH and SDH expression was decreased, but no significant difference. ERK2gene expression was significantly increased (P<0.05). These results were consistent with treated with AM251.
4. Effects and molecular mechanism of overexpression CNR1on muscle fiber types in vitro
4.1. Effects and molecular mechanism of overexpression CNR1on muscle fiber types in vitro
Stable CNR1-overexpression positive L6cell clones were screened by800ug/ml G418.The level of CNR1gene and protein both was increased(P<0.05),it means that stable CNR1overexpression cell model was eatablished successfully. In stable overexpression CNR1cells, gene expression of MyHCⅡa was increased (P<0.01), MyHCIIb gene expression was decreased (P<0.01), The expression of MyHCI and MyHCIIX had no significant changes.While differentiated stable overexpression cells differentiated, gene expression of MyHCIIa was increased (P<0.05). In addition, the expression of glycolytic key enzyme gene LDH was decreased (P<0.01), key enzyme gene of oxidative metabolism including MDH (P<0.01) and SDH (P<0.05) were significantly increased. Key genes involved in Muscle fiber transformation, gene expression of ERK1significantly increased (P<0.05), AMPKa1, PGC-1and ERK2expression had no significant difference. These results were consistent with treated with MAEA in L6cells.
4.2. Effects and molecular mechanism of overexpression CNR1on muscle fiber types in vivo
In vivo, gene overexpression system of rat gastrocnemius and soleus electroporation was established successfully, the expression of both gene and protein of CNR1were increased(P<0.05).Using the CNR1gene force expression technique to explore the effect and the molecular mechanism of CNR1on the muscle fiber types and energy metabolism.The result showed that the expression of gene and protein of MyHCⅡa significantly higher (P<0.01) than that of control group (empty vector group and saline injected group) in gastrocnemius.In selous.the gene expression of MyHCⅡa and MyHCⅡx were also significantly increased (P<0.05), the protein expression of MyHCⅡa was increased,but had no significantly differences. The Glycolysis key enzyme LDH activity was significantly decresed both in gastrocnemius and soleus (p<0.05);The gene and enzyme activity of MDH in gastrocnemius and soleus were significantly increased (P<0.05); SDH activity increased but had no significandifference in gastrocnemius; But in soleus,the RNA level and activity of SDH were significantly increased (P<0.01). The key gene which involved inmuscle fiber type transition, gene expression of ERK1(p<0.01) and PGC-1(p-<0.05) gene expression were significantly increased in both gastrocnemius and soleus.
Based on the data of vitro and vivo experimental results, we can conclude that CNR1has the function of increasing the expression of MyHCⅡa, promoting the conversion of muscle fibers to fast oxidative type. It also can effect energy metabolism with increasing the oxidation enzyme MDH and SDH and decreasing glycolysis key enzyme LDH. ERK1/2may play an important role in this process. In addition, the regulation and mechanism of CNR1in muscle fiber types exist differences between vivo and vitro.
1. CNR1在猪肌肉组织和肌卫星细胞中的表达规律研究
本实验以金华猪、长白猪和分离的猪肌卫星细胞为研究对象,比较研究了CNR1基因在猪不同肌肉组织如背最长肌(快肌纤维为主)、腓肠肌(混合型)和比目鱼肌中(慢肌纤维)表达的组织差异,发育性变化规律和品种差异,及其在肌卫星细胞诱导分化中的表达规律。结果显示：在不同的肌肉组织中,CNR1基因在背最长肌中的表达量较高(p<0.01)；发育性变化规律研究表明,CNR1基因表达存在着随日龄的增长而下降的趋势；品种差异显示,CNR1基因在长白猪背最长肌和比目鱼肌中的表达量均高于金华猪(p<0.01),而在腓肠肌中未发现显著的品种差异。在分离的猪肌卫星细胞中,CNR1在诱导前表达量较高,随着诱导呈现显著下降的趋势(p<0.05)。从以上结果可以看出CNR]基因在背最长肌中相对高表达；并有随日龄的增长而呈现显著下降的趋势；同时CNR1在长白猪中的表达量高于金华猪。在分离的肌卫星细胞中有随着诱导分化呈现显著下降的趋势。提示CNR1基因的表达可能与肌纤维的发育和类型相关。
2. CNR1特异性激动剂和抑制剂对肌纤维类型的影响及其分子机制
以大鼠成肌细胞L6为模型,首先研究了随着L6细胞的诱导分化,CNR1基因和肌纤维分型相关基因MyHCⅠ、MyHCⅡa、MyHCⅡx和MyHCⅡb的表达规律；进一步用CNR1的激动剂MAEA和抑制剂AM251处理L6细胞,荧光定量PCR检测CNR1基因、肌纤维类型相关基因(MyHCⅠ、MyHCⅡa、MyHCⅡx和MyHCⅡb)、能量代谢类型的关键酶基因(LDH、SDH和MDH)和肌纤维类型转化调控的关键基因(AMPKa1、PGC-1、ERK1和ERK2)的表达。研究结果显示：诱导0、1、3、5、7、9天的L6细胞中,CNR1基因的表达在诱导的第3天和第7天较高(p<0.05)。MyHCI和MyHCIIa型的表达量在第3天达到峰值,随后显著下降。MyHCIIx在诱导的前7天有随着诱导升高的趋势,第3天和第7天的表达量较高(P<0.05)。MyHCIIb基因在诱导的第2天即显著升高(P<0.05),第3天达到峰值后极显著下降(P<0.05)。不同浓度(0、5、10、15μM/L)的CNR1激动剂MAEA处理L6细胞,CNR1基因的表达在15μM的浓度下显著提高。进一步在15μM的浓度下处理不同时间,CNR1基因的表达在第1天和第3天提高。无论是MAEA不同浓度处理还是在相同浓度下MAEA处理不同时间,CNR1的激动剂MAEA均能显著降低MyHCIIb基因的表达,MyHCIIa基因的表达量有上升的趋势；对MyHCI和MyHCIIx的表达无显著影响。能量代谢关键酶基因发面,MAEA处理后,糖酵解关键酶LDH基因的表达下降(p<0.05)。有氧氧化关键酶SDH和MDH无显著变化。肌纤维类型转化调控基因ERK1的表达量显著升高。而不同浓度(0、0.1、1、10μM/L)的抑制剂AM251处理L6细胞后,10μM/L的AM251显著降低了CNR1基因的表达；进一步用10μM/L的浓度下处理不同时间(1、3、5天),CNR1基因的表达均呈现下降趋势。无论是AM251不同浓度处理还是在相同浓度下处理不同时间,肌纤维类型相关基因MyHCIIa和MyHCIIx的表达量下降(P<0.05),MyHCIIb基因的表达增加(P<0.01)。能量代谢关键酶基因LDH的表达量升高,MDH和SDH的基因表达则显著下降。肌纤维类型转化调控的关键基因(?)MPKal和ERK1的表达量降低(P<0.01)。上述述结果初步揭示CNR1激动剂可以显著降低酵解型肌纤维MyHCIIb基因的表达,增加快氧化型肌纤维基因的表达；显著降低酵解关键酶基因LDH的表达和升高氧化型酶SDH和MDH的表达,同时ERK1基因的表达也增强。而添加抑制剂后得到了与之相反的实验结果。
3.干扰CNR1基因对肌纤维类型的影响及其分子机制
本实验中首先设计合成三对CNR1基因的特异性发夹siRNA干扰片段,将其克隆入干扰载体pYr-1.1,构建可沉默(CNR1基因的siRNA表达载体CNR1-1、CNR1-2和CNR1-3。并采用LipofectamineTM (Lip)2000介导质粒转染L6细胞,绿色荧光蛋白的表达和流式细胞仪监测转染效率,通过实时荧光定量分析siRNA表达载体的干扰效果。并进一步用G418进行了稳定转染siRNA的L6细胞筛选。结果显示,CNR1基因的siRNA表达载体构建正确,瞬时转染L6细胞的转染效率分别为10.45%(P<0.01)、8.57%(P<0.01)和8.71%(P<0.01)；干扰效率为39%(P<0.05)、64%(P<0.01)及68%(p<0.01)。稳定筛选的最佳G418浓度为800ug/ml,稳定筛选后干扰效率分别为43%(P<0.05)、78%(P<0.01)及91%(P<0.01)。干扰效率较高的CNR1-3表达载体和稳定转染CNR1-3的细胞为构建筛选成功的siRNA表达载体和阳性细胞。
有效沉默CNR1基因后,MyHCIIa基因的表达显著下降；MyHCIIb基因的表达则显著上升。进一步对稳定干扰细胞进行诱导,在诱导后1、3、5天MyHCI和MyHCIIx基因的表达均无显著变化；MyHCIIb基因的表达在诱导1、3、5天分别显著提高(P<0.05)；MyHCIIa基因的表达均呈现下降,在诱导第3天显著下降(P<0.05)。在稳定干扰的L6细胞系中,糖酵解关键酶基因LDH的表达显著升高(P<0.05)；有氧氧化关键酶基因MDH和SDH基因的表达下降,但无显著差异。ERK2基因的表达量极显著的升高(P<0.05)。而AMPKa1、PGC-1和ERK1的基因表达无显著变化。这些变化趋势和CNR1基因的抑制剂作用效果基本一致。这些实验结果揭示,在L6细胞中抑制和沉默CNR1基因后可以显著升高酵解型肌纤维MyHCIIb基因的表达,显著降低快氧化型肌纤维MyHCIIa基因的表达：显著升高能量代谢关键酶基因LDH基因的表达；ERK1基因的表达显著降低而ERK2基因的表达显著升高。
4.超表达CNR1对肌纤维类型的影响及其分子机制
4.1体外超表达CNR1对肌纤维类型的影响及其分子机制
质粒载体pCDNA3.0具有G418抗性,用800ug/ml的G418对转染超表达质粒的L6细胞进行稳转筛选。筛选出的稳定转染细胞系中CNR1基因和蛋白的相表达量均显著增加,稳定超表达CNR1细胞模型建立成功。稳定超表达CNR1后,MyHCIIa基因的表达显著升高(P<0.01),MyHCIIb基因的表达显著下降(P<0.01), MyHCI和MyHCIIx的表达无显著变化。进一步对稳定超表达CNR1的细胞进行诱导后,MyHCI、MyHCIIb基因的表达在诱导的1、3、5天均无显著变化。MyHCIIa基因的表达在诱导后均显著提高(P<0.05)。糖酵解关键酶基因LDH的表达显著下降(P<0.01),有氧氧化代谢的关键酶MDH (P<0.01)和SDH (P<0.05)显著升高。肌纤维转型关键基因ERK1基因的表达显著升高(P<0.05), AMPKal、PGC-1和ERK2的表达无显著差异。这些变化趋势与CNR1激动剂MAEA的作用效果基本一致。综合上述结果,CNR1激动剂和体外超表达CNR1可显著降低MyHCIIb基因的表达,显著增加快氧化型MyHCIIa的表达。同时显著升高了有氧氧化的关键酶SDH和MDH的表达而酵解关键酶LDH表达显著降低,肌纤维类型转变的关键调控因子ERK1的表达显著上升。
4.2大鼠体内超表达CNR1对肌纤维类型的影响及其调控机制
利用课题组前期建立的大鼠活体肌肉电击转化实验平台,将CNR1超表达质粒导入大鼠腓肠肌和比目鱼肌中,CNR1的基因和蛋白表达量均显著提高,CNR1超表达体内模型建立成功。肌肉中超表达CNR1后肌纤维类型的变化,腓肠肌和比目鱼肌中MyHCIIa的基因表达均显著上调,MyHCIIa的蛋白表达量升高,但在比目鱼肌中差异不显著。肌肉中超表达CNR1后能量代谢关键酶基因和酶活性的变化,腓肠肌中和比目鱼肌中糖酵解关键酶LDH的活性降低(p<0.05),有氧代谢的关键酶MDH的基因表达和酶活性显著升高(p<0.05),SDH的基因表达和酶活升高,仅在比目鱼肌中达到显著水平。肌肉中超表达CNR1后肌纤维转化关键基因表达的变化,腓肠肌中和比目鱼肌中ERK1和PGC-1基因的表达量均显著升高。从以上结果可以看出,体内超表达CNR1后升高了MyHCIIa的基因和蛋白表达；显著降低了酵解关键酶LDH酶活,显著增加了有氧氧化关键酶MDH和SDH的基因表达和酶活。同时调控肌纤维类型转化的关键基因PGC和ERK1的表达均显著升高。
综合体外和体内的实验结果,CNR1可以增加肌肉细胞和肌肉组织中MyHCII a的表达,促使肌纤维向快氧化型转化。上调有氧氧化关键酶MDH和SDH,下调酵解代谢的关键酶LDH,影响肌肉细胞和肌肉组织中的能量代谢,在这个过程中ERK1/2可能发挥着重要作用。另外,(CNR1对肌纤维类型的调控及机制存在体内和体外差异。
One of the biochemical and molecular basis factors determing muscle growth and meat quality is the muscle fiber types.Different muscle fiber types have showed different characteristics of energy metabolism and biochemical characteristics. There are four main muscle fiber types (MyHCI, MyHCIIa, MyHCIIx and MyHCIIb) in mammalian skeletal muscle.Furthermore, different muscle fiber types can be transformed into each other.Taking advantages of the global microarray technology, cannabinoid receptor1(CNR1) have identified as fifferently expression between Jinhua with superior meat quality and Landrace pigs with lower meat quality.In order to inverstgate the effects and mechanism of CNR1on muscle fiber types, Quantitative PCR, immunohistochemistry, Western Blot and other technologies were used to establish the in vitro and in vivo overexpression and gene silencing in cells or animal models. The findings are as follows:
1. Expression patterns of the cannabinoid receptor1gene in pig skeletal muscle and skeletal satellite muscle cells
Up to now, detailed analysis of CNR1gene expression in pig muscles and muscle cells has not been reported. Here, quantitative PCR were used to characterize the CNR1in different muscles of pig and isolation of porcine myogenic satellite cells. Results showed that the expression of CNR1mRNA was greatest in longissimus dorsi muscle (p<0.01)among that in the gastrocnemius (mix), soleus muscle (slow-type) and longissimus doris muscle (fast-type). As to age-related changes of CNR1gene in different pig muscles, the higher expression was found at the30th day, then declined (p<0.05) in all three kinds of muscles to the90th day. At the150th day, the expression of CNR1steadily decreased (p<0.05) in the gastrocnemius compared to the90th day, but not in longissimus doris muscle and soleus. Meanwhile the breed differences in the expression of CNR1mRNA between Jinhua pig and Landrace were also identified. The mRNA abundance of CNR1in Landrace was higher compared with that of Jinhua pigs and strong differences were observed in longissimus doris muscle (p<0.05) and soleus (p<0.01). In vitro, with the differentiation of the satellite cells, the mRNA expression of the CNR1was down-regulated (p<0.05). The lowest mRNA expression was observed at the9th day. These data provide a framework of CNR1gene expression patterns and implicate a novel gene for regulating muscle fiber types.
2. Effects and molecular mechanism of CNR1agonist and antagonist on muscle fiber types
we analyzed the expression patterns of CNR1and MyHC isforms types includingⅠ,Ⅱa,Ⅱx Ⅱb with the cell differentiate in L6cells.After that,L6cell were coped with CNR1selective agonist MAEA and antagonist AM251.The changes of CNR1expression, MyHC isforms(MyHCⅠ, MyHCⅡa, MyHCⅡx and MyHCⅡb) and the key enzyme gene (LDH,SDH and MDH)as well as the key gene of muscle fiber types transition(AMPKal, PGC-1, ERK1and ERK2) were detected using quantitative PCR.The results show that:CNR1gene expression was higher at the3th and7th day. The levels of MyHCI, and MyHCⅡa expression peaked at the3thday, then dramatically decreased. MyHCⅡx gene expression showed the increased trend before the7th day (P<0.05). MyHClIb gene was significantly increased at the first two2days, at the3th day,reached the higest level and then significant decreased.
Treated L6cells with different concentrations of MAEA(0,5,10,15μM/L), we found that CNR1expression was significantly increased at15μM/L. Further treated with different time at15μM/L.Under the both conditions, the MyHCⅡb gene expression was reduced (p<0.01). but MyHCIIa gene expression was increased The gege expression of MyHCl and MyHCⅡx had no significant difference. Meanwhile gene expression of glycolytic key enzyme LDH were decreased (p<0.05). Key enzyme genes of oxidation SDH and MDH had no significant change.The key gene of muscle fiber types transition ERK1expression was increased (P<0.05).
On the contrary, treated L6cells with different concentrations of AM251(0,0.1,1,10μM/L), the results showed that CNR1expression was significantly down regulated at10μM/L. Further treated with different time at10μM/L.Under the both conditions, the gene expression MyHCⅡa, and MyHCⅡx was significantly decreased (P<0.05), MyHCⅡb gene expression significantly increased (P<0.01). The expression level of LDH was elevated (P>0.05), MDH and SDH gene expression were significantly decreased (P<0.05). AMPKal and ERK1expression was significantly down regulated (P<0.01).
3. Effects and molecular mechanism of interference CNR1on muscle fiber types in vitro
This study was conducted to construct and identify CNRl gene siRNA expression vectors and screen the stable CNR1-interference positive L6cell clones. Three pairs of CNR1-specific double-strand siRNAs were designed, synthesized, annealed and inserted into the pYr-1.1vector.The CNR1gene siRNA expression vectors were identified by restriction enzyme digestion and sequencing. After that siRNAs were transfected with L6cells by LipofectamineTM (Lip)2000. Then, the transfection efficiency was detected by EGFP and FCM.CNRI gene expression was determined by real-time PCR and the stable transgenic L6cell clones were screened by G418. The results revealed that the CNR1gene siRNA expression vectors have been constructed successfully. The transient transfection efficiency to L6cells were10.45%(P<0.01)、8.57%(P<0.01) and8.71%(P<0.01),and the silencing efficacy of the transient transfected L6cells was39%(P<0.05)、64%(P<0.01) and68%(p<0.01),respectively. The optimal selection concentration of G418for stable transfected L6cell clones was800ug/ml. The silencing efficacy of CNR-1-positive transgenic cell clones were43%(P<0.05).78%(P<0.01) and91%(P<0.01). The results showed that CNR1-3expression vector is the optimal silencing vector and CNR1-3stable transgenic cell clones is best silencing cell line. This study provides a successful CNR1gene silencing method by siRNA and the screening of CNR1-interference positive L6cell clones renders basic tools for further studying the functions of CNR1gene.
After effectively silence CNR1gene by RNA interference. The gene expression of MyHCⅡa was decreased (P<0.05); the levels of MyHCⅡb was increased significantly (P<0.05), stable interference cells inducted by1,3,5days; gene expression of MyHCⅡb was significantly increased (P<0.05); Meanwhile,MyHCⅡa gene expression showed a decline trend. At the3th day, MyHCIIa gene expression decreased significantly (P<0.05). Expression of the glycolytic key enzyme gene LDH was significantly increased (P<0.05); the key enzyme genes of oxidation MDH and SDH expression was decreased, but no significant difference. ERK2gene expression was significantly increased (P<0.05). These results were consistent with treated with AM251.
4. Effects and molecular mechanism of overexpression CNR1on muscle fiber types in vitro
4.1. Effects and molecular mechanism of overexpression CNR1on muscle fiber types in vitro
Stable CNR1-overexpression positive L6cell clones were screened by800ug/ml G418.The level of CNR1gene and protein both was increased(P<0.05),it means that stable CNR1overexpression cell model was eatablished successfully. In stable overexpression CNR1cells, gene expression of MyHCⅡa was increased (P<0.01), MyHCIIb gene expression was decreased (P<0.01), The expression of MyHCI and MyHCIIX had no significant changes.While differentiated stable overexpression cells differentiated, gene expression of MyHCIIa was increased (P<0.05). In addition, the expression of glycolytic key enzyme gene LDH was decreased (P<0.01), key enzyme gene of oxidative metabolism including MDH (P<0.01) and SDH (P<0.05) were significantly increased. Key genes involved in Muscle fiber transformation, gene expression of ERK1significantly increased (P<0.05), AMPKa1, PGC-1and ERK2expression had no significant difference. These results were consistent with treated with MAEA in L6cells.
4.2. Effects and molecular mechanism of overexpression CNR1on muscle fiber types in vivo
In vivo, gene overexpression system of rat gastrocnemius and soleus electroporation was established successfully, the expression of both gene and protein of CNR1were increased(P<0.05).Using the CNR1gene force expression technique to explore the effect and the molecular mechanism of CNR1on the muscle fiber types and energy metabolism.The result showed that the expression of gene and protein of MyHCⅡa significantly higher (P<0.01) than that of control group (empty vector group and saline injected group) in gastrocnemius.In selous.the gene expression of MyHCⅡa and MyHCⅡx were also significantly increased (P<0.05), the protein expression of MyHCⅡa was increased,but had no significantly differences. The Glycolysis key enzyme LDH activity was significantly decresed both in gastrocnemius and soleus (p<0.05);The gene and enzyme activity of MDH in gastrocnemius and soleus were significantly increased (P<0.05); SDH activity increased but had no significandifference in gastrocnemius; But in soleus,the RNA level and activity of SDH were significantly increased (P<0.01). The key gene which involved inmuscle fiber type transition, gene expression of ERK1(p<0.01) and PGC-1(p-<0.05) gene expression were significantly increased in both gastrocnemius and soleus.
Based on the data of vitro and vivo experimental results, we can conclude that CNR1has the function of increasing the expression of MyHCⅡa, promoting the conversion of muscle fibers to fast oxidative type. It also can effect energy metabolism with increasing the oxidation enzyme MDH and SDH and decreasing glycolysis key enzyme LDH. ERK1/2may play an important role in this process. In addition, the regulation and mechanism of CNR1in muscle fiber types exist differences between vivo and vitro.
引文
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