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The Research on Proliferation and Transdifferentiation of C2C12 and L6 Cells Infuced by the Fatty Acids

Author: LinLin
Tutor: ChenXiangGui
School: West China University
Course: Of Food Science
Keywords: aliphatic acid myoblast differentiation
CLC: R151
Type: Master's thesis
Year: 2011
Downloads: 182
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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.

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