【作者】 林琳；

【导师】 陈祥贵；

【作者基本信息】 西华大学 ， 食品科学， 2011， 硕士

【摘要】 目的:膳食脂肪对于人体具有很重要的生理功能,既是能量来源,同时又提供必需脂肪酸及脂溶性维生素等营养成分。但大量文献说明,过量摄入膳食脂肪是不可取的,膳食中的脂肪含量与种类与胰岛素抵抗的发生关系密切;骨骼肌是由成肌细胞发育而来,成肌细胞是一种干细胞,具有多向分化的潜力,除分化为骨骼肌细胞外,在适当的诱导条件下可分化为脂肪细胞和成骨细胞。因此,研究不同种类的脂肪酸对成肌细胞细胞的增殖和转分化的影响对于揭示食物中的脂肪成分与胰岛素抵抗发生的关系提供科学依据。通过脂肪酸暴露,对成肌细胞C2C12和L6在不同条件诱导下的培养和分化,比较成肌细胞增殖分化特性及其对不同种类脂肪酸的反应,从骨骼肌发育的角度探索不同种类的脂肪酸对成肌细胞的成肌分化或成脂分化可能具的有不同作用,为相关研究提供新的策略、方法和模型,为胰岛素抵抗相关疾病的预防和治疗提供重要线索。方法:1、脂肪酸对细胞增殖的剂量效应:选取对数生长期的C2C12、L6、3T3-L1细胞,胰酶消化制成单细胞悬液,调整细胞浓度为10×10⁵/mL,每孔接种200μL于96孔板,置于37℃、5%CO2的细胞培养箱中培养24h,按实验分组依次加入含3%血清,终浓度为200μmol/L、100μmol/L、50μmol/L、25μmol/L和1.25μmol/L的棕榈酸（palmitate,PA）、油酸（oleic acid,OA）、亚麻酸（linolenate,LA）的培养基以及含3%血清终浓度为0.1%DMSO的培养基,每天换液50%,4天后用MTT比色法计算五种剂量三种脂肪酸对细胞的生长抑制率（IR）,IR=[(对照组平均OD₄₉₀值-实验组平均OD₄₉₀值)/对照组平均OD₄₉₀]×100%,来观察不同脂肪酸对细胞增殖的剂量效应。2、脂肪酸对细胞增殖的时间效应:实验分组与剂量效应实验分组相同,但C2C12、L6、3T3-L1细胞要分别接种5张板,培养24 h后随机抽取其中一张板做MTT实验测定光吸收值,作为第0天的数据,其余4张班每孔按实验分组依次加人含3%FCS和0.1%DMSO的培养基,含3%FCS终浓度为分别为200μmol/L、100μmol/L、50μmol/L、25μmol/L、12.5μmol/L的PA、OA、LA的培养基,于2天,4天,6天和8天后各取一板做MTT实验测定光吸收值,记录结果,并按公式计算IR,来观察不同脂肪酸对细胞增殖的时间效应。3、脂肪酸对细胞分化的影响:选取对数生长期的C2C12、L6细胞,胰酶消后调整细胞浓度50×10⁵/mL,每孔接种400μL于48孔板,培养24 h,按实验分组于分化开始的前、分化初期和分化中期分别加25μmol/L的PA、OA、LA处理2天,其余时间用含2%马血清的分化培养基培养,共计培养10天。分别通过油红O、姬姆萨染色观察不同脂肪酸处理队细胞形态上的影响,通过计算肌酸激酶（CK）量的相对值,进一步确定脂肪酸对细胞分化的影响。结果:1、脂肪酸对细胞增殖剂量效应:三种脂肪酸在高浓度（200μmol/L）时都会显著抑制C2C12、L6细胞的增殖,抑制率在60%左右,而对3T3-L1细胞增殖的影响较小,抑制率<35%。低浓度（12.5μmol/L）的PA对成肌细胞的抑制大于对脂肪前体细胞的抑制;OA五个浓度对两种成肌细胞增殖的抑制基本相同,但对3T3-L1细胞增殖的抑制在40%左右,小于对成肌细胞的抑制;LA在低浓度（12.5μmol/L）时对三种细胞的抑制率显著对于PA、OA。2、脂肪酸对细胞增殖的时间效应:随着时间的延长,脂肪酸随着对细胞增殖的抑制率不断加大;脂肪酸对细胞抑制的强度3T3-L1<L6<C2C12;脂肪酸对C2C12、L6细胞增殖的抑制率趋势相同,脂肪酸对L6细胞增殖的抑制随着时间延长增加较C2C12细胞缓慢,抑制率增加的幅度不断减小;脂肪酸对C2C12、L6细胞增殖的抑制强度表现为200μmol/L、100μmol/L、50μmol/L时PA>OA=LA,25μmol/L、12.5μmol/L时PA>OA>LA;脂肪酸对3T3-L1细胞增殖的抑制强度表现为PA>OA=LA。3、脂肪酸对细胞分化的影响:从油红O、姬姆萨染色以及CK相对含量的测定的结果可见分化前加处理组与分化初期处理组、分化中期处理组表现出明显差异,后两者在细胞形态及CK含量方面与对照基本相同。经油红O、姬姆萨染色后对细胞形态进行观察,PA、OA处理组处理的细胞只是有向同一个方向生长的趋势,但细胞没有融合,或只能见到两个细胞融合,而没有出现多核肌管,在细胞两端胞浆中大面积出现大小不同的脂滴,LA处理的细胞,胞浆中脂滴数较少,脂滴分散,形成明显的多核肌管,肌管体积比单个肌细胞的体积大很多,沿细胞中心轴线排列形成中心核链有10～35个核不等;肌酸激酶相对量,PA处理的细胞是最少的,仅有阴性对照组的060%⁷0%,OA处理的细胞肌酸激酶的相对量是阴性对照组的80%多,LA处理的细胞酸激酶的相对量分别是阴性对照组的1.7¹.9倍之间。结论:1、不同浓度的脂肪酸对细胞增殖的影响不同,随着浓度的减小,脂肪酸对细胞增殖的抑制率有所减少。不同种类脂肪酸对同种细胞增殖的影响不同,不饱和脂肪酸对细胞增殖的抑制率小于饱和脂肪酸;同种脂肪酸对不同种细胞增殖的影响不同,脂肪酸对脂肪前体细胞增殖的抑制率小于成肌细胞。2、随着时间的延长,脂肪酸随着对细胞增殖的抑制强度不断加大。3、在分化的前期脂肪酸处理对成肌细胞分化有影响,而分化初期和中期脂肪酸处理不影响细胞分化;分化前期多不饱和脂肪酸处理促进成肌细胞分化成肌管,饱和脂肪酸和单不饱和脂肪酸处理促使成肌细胞胞浆中出现脂滴,抑制细胞分化为肌管。更多还原

【Abstract】 Purpose:Dietary fat for the human body has very important physiological functions, which is both energy source and provides essential fatty acids and fat-soluble vitamins nutrients. However, plenty of literature shows that intake of dietary fat excessively is not desirable, because the fat content and species of dietary fat have close relationship with insulin resistance. The insulin sensitivity of skeletal muscles plays a very important role in high-fat insulin resistance. The lip toxicity of skeletal muscle is higher than the key point of insulin resistance and diabetesⅡ. The skeletal muscles develop from muscle cell that is one kind of stem cells with multiple differentiations. Besides, in some conditions, muscle cell can be differentiated into adipocyte and osteoblast. Therefore, different kinds of aliphatic acids have influence on myoblast proliferation and differentiation, and it shows the evidence of the connection between fat in food and insulin resistance. As for the skeletal development, we may draw a conclusion that effects on the differentiation of myoblast or lipoblast vary from different kinds of aliphatic acids by the exposure of aliphatic acid, by the differentiation of C2C12 and L6 myoblast cells, and by the comparison of the proliferation differentiation and the results of different kinds of aliphatic acids, which is taken as a very important clue to the prophylaxis and the cure of some diseases by insulin and provides some strategies, methods and models of the relative studies.Methods:1、Dose Effect of Aliphatic Acid on Cell Proliferation.Select C2C12, L6, 3T3-L1 cells in logarithmic growth period, and add trypsinization as single cell suspension. Change the cell concentration as 10×10⁵/ml, and put 200μL suspension in each hole of 96 orifice plate in the incubator with 37℃and 5%CO2 for 24 hours. Then 3% serum is added, and the final concentrations of media are 200μmol/L, 100μmol/L, 50μmol/L, 25μmol/L, and 1.25μmol/L palmitate acid （PA）, oleic acid （OA）, and linolenate acid （LA）, and a medium with 3% serum and 0.1%DMSO, which need to be change 50% part. After four days, the cell IR of three aliphatic acids in five doses is calculated by MTT method. The formula is IR= [(average number of OD₄₉₀ in control group– average number of OD₄₉₀ in test group)/ average number of OD₄₉₀ in control group]×100%, through which observe dose effect on cell proliferation.2、Time Effect of Aliphatic Acid on Cell Proliferation.The test groups are the same as those of dose effect of aliphatic acid on cell proliferation, but C2C12、L6、3T3-L1 need to put on five papers that cultivate for 24 hours. And pick any one of them to conduct MTT test to get the light absorption as the data of the 0 day. as for other four papers, 3%FCS and 0.1%DMSO are add with the final concentration in 200μmol/L、100μmol/L、50μmol/L、25μmol/L、12.5μmol/L PA, OA, and LA. After two days, four days, six days and eight days separately, MTT test is conducted to get the result of light absorption, and IR is calculated according to the formula above. 3、Influence of Aliphatic Acid on Cell Differentiation.Select C2C12, L6, cells in logarithmic growth period, and add trypsinization in the 50×10⁵/mL concentration. Put 400μL of them into each hole on 48 orifice plate. On the two days before the differentiations of C2C12 and L6 cells, three stages are defined as incipient, medium and late stages, during which the 25μmol/L PA, OA, and LA are added to process those cells for two days, and 2% horse serous is used. After 10 days in all, we use oil red O and Giemsa Stain for observation. The final result can be obtain by calculating CK.Results:1、Dose Effect of Aliphatic Acid on Cell Proliferation.Three aliphatic acids will restrict C2C12, L6 cell proliferation in high concentration （200μmol/L）, and the inhibition rate is about 60%, which show little influence on 3T3-L1 with the inhibition rate <35%. PA in low concentration （12.5μmol/L） has more inhibition in myoblast than that in preadipocytes. Five different concentration of OA has the same effect on two myoblasts proliferation. But the number of 3T3-L1 cell inhibition is about 40%, which is less than that of myoblasts. LA in 12.5μmol/L has obvious effect of inhibition than those of PA and OA. 2、Time Effect of Aliphatic Acid on Cell Proliferation.With the increase of time, aliphatic acid shows more inhibition rate on cell proliferation, and the rank is 3T3-L1<L6<C2C12. The tendency of aliphatic acid IR to C2C12 is the same, which maximizes at the sixth day but reduces at the eighth day. IR of aliphatic acid to L6 cells increases slowly with the time and its range decreases. The data that aliphatic acid IR to C2C12 and to L6 cells are PA>OA=LA in 200μmol/L, 100μmol/L, and 50μmol/L, and PA>OA>LA in 25μmol/L, 12.5μmol/L. IR of aliphatic acid to 3T3-L1 is PA>OA=LA. 3、Influence of Aliphatic Acid on Cell Differentiation.From the results that contain oil red O, Giemsa Stain and the relative CK result, differences can be distinguished in incipient, medium and late stage. The two latter is the same as cell appearance and CK. After the process of red oil O and Giemsa Stain, we observe the cell appearance and find that cells processed by PA and OA has the tendency to grow in one direction without cell fusion or with only two cell fusion. Meanwhile, the polymorphonuclear myotube does not arise. In the cytoplasm cells appear on both ends of different size large LA processing, lipid drops in the cytoplasm and cell, small number of lipid drops scattered lipid drops, forming more noticeable nuclear muscle tube, muscle tube volume than that of a single muscle cells along the big volume many central spindle cells arranged forming centre nuclear chain has 10 35 nuclear differ; Creatine kinase relative amounts, PA processing cells are least, only negative 060% 70% compared with cells, OA creatine kinase control the relative amount is negative, LA more than 80 per cent of disposal of the relative amount cell acid kinase were negative control between 1.7 1.9 times. Conclusion:1、Different concentrations of fatty acid on cell proliferation are different. With he influence of the decrease of the different concentration of cell proliferation, fatty acid on the inhibition rate reduced. Different kinds of fatty acid on the same cell proliferation affect differently, and unsaturated fatty acids to cell proliferation inhibition rate is less than saturated fatty acid; The same fatty acid on different kinds of cell proliferation, fatty acid on fats affect different inhibition rate before the proliferation of cells than into muscle cells. 2、With the extension of time, the intensity of fatty acids with the inhibition of cell proliferation increase. 3、Differentiate into muscle of the fatty acid treatment has effects and cell differentiation and differentiation in initial period and fatty acid treatment in middle period do not affect cell differentiation; Differentiation polyunsaturated fatty acid treatment in the prophase period promotes into muscle cells to differentiate into muscle tube, and saturated fatty acid and monounsaturated fatty acid treatment prompte into muscle cells appeared in the cytoplasm of inhibiting cell differentiation, lipid drops for muscle tube.更多还原