t10，c12-CLA对猪皮下和背最长肌脂肪沉积的差异性调控机理研究
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摘要
本论文采用皮下脂肪和背最长肌组织块培养及前脂肪细胞培养、细胞生物学及分子生物学等技术手段,系统研究了t10,c12-CLA对猪皮下和背最长肌组织脂肪沉积影响的生物学机制。研究共分三个部分：
1.本试验首先建立了30日龄猪皮下脂肪和背最长肌组织块培养及皮下和肌内前脂肪细胞体外培养体系,共设2个处理,处理1为阴性对照(添加0.1 mmol/LBSA),处理2添加100μmol/L的t10,c12-CLA,每个处理设6个平行,接种时即加入BSA与t10,c12-CLA进行处理至第10天。试验全期通过MTT法(采用上述前脂肪细胞培养体系)、GPDH活性检测及油红0染色技术(采用上述组织块培养体系)探讨了t10,c12-CLA对皮下和肌内脂肪前体细胞增殖与分化的影响。结果表明：(1)本试验中,100μmol/L t10,c12-CLA显著抑制了猪皮下脂肪前体细胞增殖,但对肌内脂肪前体细胞的增殖影响并不显著；(2)100μmol/L t10,c12-CLA显著抑制了猪皮下脂肪前体细胞分化,显著促进了肌内脂肪前体细胞的分化(P<0.05)；(3)本部分结果说明t10,c12-CLA对猪皮下和肌内脂肪前体细胞增殖与分化存在差异调控作用。
2.本试验猪皮下脂肪和背最长肌组织块体外培养及处理设计同第一部分,采用荧光定量PCR技术、酶活性测定及ELISA测定研究t10,c12-CLA对猪皮下和肌内前脂肪细胞分化关键转录因子、脂类代谢关键酶及调控激素、脂肪合成与分解速率的影响。结果表明：(1)本试验中,100μmol/L t10,c12-CLA显著提高了猪背最长肌的脂肪合成速率与猪皮下脂肪的脂肪分解速率(P<0.05),而背最长肌的脂肪分解速率与皮下脂肪的脂肪合成速率对照组与处理组差异并不显著(P>0.05)。同时100μmol/L t10,c12-CLA显著降低了猪皮下脂肪TG含量并提高了猪背最长肌的TG含量(P<0.05)；(2)100μmol/L t10,c12-CLA显著抑制了猪皮下脂肪PPARγ、FAS、INSR与背最长肌HSL和LPL的基因表达,促进了猪皮下脂肪HSL和背最长肌PPARγ、ADD1、INSR的基因表达(P<0.05)；(3)100μmol/L t10,c12-CLA显著降低了猪皮下脂肪HSL与背最长肌LPL活性及皮下INSR与肌内GHR蛋白表达水平,并提高了背最长肌ME活性和INSR蛋白表达水平(P<0.05)；(4)本部分研究结果从分化转录因子、代谢酶类与调控激素及脂肪合成与降解速率方面揭示了t10,c12-CLA对猪皮下和背最长肌组织脂肪沉积的差异性调控机制,进一步证实了t10,c12-CLA抑制猪皮下脂肪沉积同时提高肌内脂肪含量。
3.本试验猪皮下脂肪和背最长肌组织块体外培养及处理设计同第一部分,通过荧光定量PCR技术和ELISA方法研究t10,c12-CLA对肌肉—脂肪轴共调节细胞因子的影响。结果表明：(1)本试验中,100μmol/L t10,c12-CLA显著降低了猪背最长肌TNF-a、瘦素及IL-15的基因表达,并提高了猪皮下脂肪TNF-a、IL-6、瘦素与背最长肌脂联素、MSTN的基因表达水平(P<0.05)；(2)猪皮下脂肪TNF-a的t10,c12-CLA处理组与对照组的蛋白表达水平无显著差异,而皮下脂肪IGF-1的t10,c12-CLA处理组蛋白表达水平显著降低(P<0.05),其它因子变化规律与基因表达变化规律一致；(3)本部分研究结果揭示t10,c12-CLA很可能通过TNF-a、瘦素、脂联素、IL-15及IL-6这些细胞因子的介导从而发挥了对猪皮下脂肪和肌内脂肪的差异性调控作用,进一步证实TNF-a、瘦素、脂联素、IL-15及IL-6在脂肪细胞和肌细胞间的分子对话中发挥着重要作用。
全文总体结论如下：(1)本研究条件下,l00μmol/L t10,c12-CLA通过上调或下调猪皮下脂肪和背最长肌内部分关键转录因子(PPARν、ADD1)、脂肪代谢酶(LPL、HSL)与激素受体(INSR),脂肪合成与分解速率,细胞因子(leptin、TNF-a等)的基因表达和蛋白水平,进而抑制猪皮下前脂肪细胞增殖与分化并促进了猪背最长肌前脂肪细胞的分化(P<0.05),最终降低了猪皮下脂肪沉积并提高了背最长肌的肌内脂肪含量。(2)本研究结果初步表明TNF-a、瘦素、脂联素、IL-15及IL-6在脂肪组织和肌肉组织之间的分子对话中发挥着重要作用,进一步揭示t10, c12-CLA很可能通过TNF-a、瘦素、脂联素、IL-15及IL-6这些细胞因子的介导从而发挥了对猪皮下脂肪和背最长肌组织脂肪沉积的差异性调控作用。
In this thesis, subcutaneous(SC) adipose and longissimus muscle tissue culture and preadipocyte culture techniques, cell biology and molecular biology techniques were employed to investigate the effects of t10,cl2-CLA on regulation of lipid deposition between SC adipose and longissimus muscle tissues in pig. There are three parts in this study:
Part1:In vitro tissue culture system of SC adipose and longissimus muscle, and preadipocyte culture system taken from 30-days pig were constructed firstly,0.1 mmol/L bovine serum albumin(BSA, as control),100μmol/L t10,cl2-CLA were added to the cultures for 10 d, six replicates were performed for each treatment. At the end of a 10-d treatment period, cells were harvested and stored at-70℃. MTT assay(using preadipocyte culture system), GPDH activity assay and oil red O staining(using tissue culture system) were used to characterize the effects of t10,cl2-CLA on the proliferation and differentiation of pig SC and intramuscular(IM) preadipocytes in primary cultures. The results showed that:Proliferation and differentiation of SC preadipocytes were inhibited and differentiation of IM preadipocytes were promoted by adding 100μmol/L tl0.cl2-CLA(P<0.05), but no effect on proliferation of IM preadipocytes was observed(P>0.05). These results demonstrated that the effects of t10,cl2-CLA on proliferation and differentiation of SC preadipocytes differed from that on IM preadipocytes at cell morphology point of view.
Part2:In this trial, in vitro tissue culture system and experiment design were same to partl. Real-time RT-PCR, enzyme activities assays and ELISA assays were used to study the effects of t10,cl2-CLA on the preadipocyte differentiation transcription factors, lipogenic and lipolytic enzymes, regulation hormones, lipogenic and lipolytic rates in SC and longissimus muscle tissue. The results showed that:the mRNA abundance of PPARγ, FAS and INSR, HSL activities, INSR protein level, lipolytic rates,TG content in SC adipose tissue and the mRNA abundance of HSL and LPL, LPL activities,GHR protein level in longissimus muscle tissue were decreased by adding t10,c12-CLA(P<0.05); by contrast, HSL mRNA abundance in SC adipose tissue and the mRNA abundance of PPARγ, ADD1 and INSR, ME activities, INSR protein level, lipogenic rates, TG content in longissimus muscle tissue were increased by adding t10,c12-CLA(P<0.05). These results illustrated that the effects of t10,cl2-CLA on lipid deposition of SC adipose tissue differed from that on longissimus muscle tissue at the lipid metabolism point of view and confirmed t10,cl2-CLA inhibited the lipid deposition of SC adipose and promoted the lipid deposition of longissimus muscle in pig further.
Part3:In this trial, in vitro tissue culture system and experiment design were also same to partl. Real-time RT-PCR and ELISA assays were used to study the effects of t10,c12-CLA on the cytokines secreted by adipose and muscle tissue which exert the regulation action on both two tissues. The results showed that:the mRNA abundance and protein level of TNF-a, leptin and IL-15 in longissimus muscle tissue and IGF-1 protein level in SC adipose tissue were decreased by adding t10,c12-CLA(P<0.05); by contrast, the mRNA abundance and protein level of adiponectin, MSTN in longissimus muscle tissuee and TNF-a, IL- 6, leptin in SC adipose tissue were increased by adding t10,c12-CLA(P<0.05); But no effect on protein level of TNF-a in SC adipose tissue was observed (P>0.05). These results demonstrated that TNF-a, leptin, adiponectin, IL-15 and IL-6 play the important roles in molecular dialogue between adipocyte and myocyte, and these cytokines probably served as the mediator of tlO,cl2-CLA to exert different regulation effect on lipid deposition in SC and longissimus muscle tissue of pig.
The general conclusion:
In this study, the different mechanisms of effects of 100μmol/L t10,c12-CLA on regulation of lipid metabolism and deposition between SC adipose and longissimus muscle tissues in pig by affecting proliferation and differentiation of preadipocytes, differentiation transcription factors, lipogenic and lipolytic enzymes, regulation hormones, and cytokines secreted by two tissues were revealed, the inhibition effects of t10,cl2-CLA on lipid deposition in SC adipose and opposite effect on lipid deposition in longissimus muscle tissues were also confirmed further.
1.本试验首先建立了30日龄猪皮下脂肪和背最长肌组织块培养及皮下和肌内前脂肪细胞体外培养体系,共设2个处理,处理1为阴性对照(添加0.1 mmol/LBSA),处理2添加100μmol/L的t10,c12-CLA,每个处理设6个平行,接种时即加入BSA与t10,c12-CLA进行处理至第10天。试验全期通过MTT法(采用上述前脂肪细胞培养体系)、GPDH活性检测及油红0染色技术(采用上述组织块培养体系)探讨了t10,c12-CLA对皮下和肌内脂肪前体细胞增殖与分化的影响。结果表明：(1)本试验中,100μmol/L t10,c12-CLA显著抑制了猪皮下脂肪前体细胞增殖,但对肌内脂肪前体细胞的增殖影响并不显著；(2)100μmol/L t10,c12-CLA显著抑制了猪皮下脂肪前体细胞分化,显著促进了肌内脂肪前体细胞的分化(P<0.05)；(3)本部分结果说明t10,c12-CLA对猪皮下和肌内脂肪前体细胞增殖与分化存在差异调控作用。
2.本试验猪皮下脂肪和背最长肌组织块体外培养及处理设计同第一部分,采用荧光定量PCR技术、酶活性测定及ELISA测定研究t10,c12-CLA对猪皮下和肌内前脂肪细胞分化关键转录因子、脂类代谢关键酶及调控激素、脂肪合成与分解速率的影响。结果表明：(1)本试验中,100μmol/L t10,c12-CLA显著提高了猪背最长肌的脂肪合成速率与猪皮下脂肪的脂肪分解速率(P<0.05),而背最长肌的脂肪分解速率与皮下脂肪的脂肪合成速率对照组与处理组差异并不显著(P>0.05)。同时100μmol/L t10,c12-CLA显著降低了猪皮下脂肪TG含量并提高了猪背最长肌的TG含量(P<0.05)；(2)100μmol/L t10,c12-CLA显著抑制了猪皮下脂肪PPARγ、FAS、INSR与背最长肌HSL和LPL的基因表达,促进了猪皮下脂肪HSL和背最长肌PPARγ、ADD1、INSR的基因表达(P<0.05)；(3)100μmol/L t10,c12-CLA显著降低了猪皮下脂肪HSL与背最长肌LPL活性及皮下INSR与肌内GHR蛋白表达水平,并提高了背最长肌ME活性和INSR蛋白表达水平(P<0.05)；(4)本部分研究结果从分化转录因子、代谢酶类与调控激素及脂肪合成与降解速率方面揭示了t10,c12-CLA对猪皮下和背最长肌组织脂肪沉积的差异性调控机制,进一步证实了t10,c12-CLA抑制猪皮下脂肪沉积同时提高肌内脂肪含量。
3.本试验猪皮下脂肪和背最长肌组织块体外培养及处理设计同第一部分,通过荧光定量PCR技术和ELISA方法研究t10,c12-CLA对肌肉—脂肪轴共调节细胞因子的影响。结果表明：(1)本试验中,100μmol/L t10,c12-CLA显著降低了猪背最长肌TNF-a、瘦素及IL-15的基因表达,并提高了猪皮下脂肪TNF-a、IL-6、瘦素与背最长肌脂联素、MSTN的基因表达水平(P<0.05)；(2)猪皮下脂肪TNF-a的t10,c12-CLA处理组与对照组的蛋白表达水平无显著差异,而皮下脂肪IGF-1的t10,c12-CLA处理组蛋白表达水平显著降低(P<0.05),其它因子变化规律与基因表达变化规律一致；(3)本部分研究结果揭示t10,c12-CLA很可能通过TNF-a、瘦素、脂联素、IL-15及IL-6这些细胞因子的介导从而发挥了对猪皮下脂肪和肌内脂肪的差异性调控作用,进一步证实TNF-a、瘦素、脂联素、IL-15及IL-6在脂肪细胞和肌细胞间的分子对话中发挥着重要作用。
全文总体结论如下：(1)本研究条件下,l00μmol/L t10,c12-CLA通过上调或下调猪皮下脂肪和背最长肌内部分关键转录因子(PPARν、ADD1)、脂肪代谢酶(LPL、HSL)与激素受体(INSR),脂肪合成与分解速率,细胞因子(leptin、TNF-a等)的基因表达和蛋白水平,进而抑制猪皮下前脂肪细胞增殖与分化并促进了猪背最长肌前脂肪细胞的分化(P<0.05),最终降低了猪皮下脂肪沉积并提高了背最长肌的肌内脂肪含量。(2)本研究结果初步表明TNF-a、瘦素、脂联素、IL-15及IL-6在脂肪组织和肌肉组织之间的分子对话中发挥着重要作用,进一步揭示t10, c12-CLA很可能通过TNF-a、瘦素、脂联素、IL-15及IL-6这些细胞因子的介导从而发挥了对猪皮下脂肪和背最长肌组织脂肪沉积的差异性调控作用。
In this thesis, subcutaneous(SC) adipose and longissimus muscle tissue culture and preadipocyte culture techniques, cell biology and molecular biology techniques were employed to investigate the effects of t10,cl2-CLA on regulation of lipid deposition between SC adipose and longissimus muscle tissues in pig. There are three parts in this study:
Part1:In vitro tissue culture system of SC adipose and longissimus muscle, and preadipocyte culture system taken from 30-days pig were constructed firstly,0.1 mmol/L bovine serum albumin(BSA, as control),100μmol/L t10,cl2-CLA were added to the cultures for 10 d, six replicates were performed for each treatment. At the end of a 10-d treatment period, cells were harvested and stored at-70℃. MTT assay(using preadipocyte culture system), GPDH activity assay and oil red O staining(using tissue culture system) were used to characterize the effects of t10,cl2-CLA on the proliferation and differentiation of pig SC and intramuscular(IM) preadipocytes in primary cultures. The results showed that:Proliferation and differentiation of SC preadipocytes were inhibited and differentiation of IM preadipocytes were promoted by adding 100μmol/L tl0.cl2-CLA(P<0.05), but no effect on proliferation of IM preadipocytes was observed(P>0.05). These results demonstrated that the effects of t10,cl2-CLA on proliferation and differentiation of SC preadipocytes differed from that on IM preadipocytes at cell morphology point of view.
Part2:In this trial, in vitro tissue culture system and experiment design were same to partl. Real-time RT-PCR, enzyme activities assays and ELISA assays were used to study the effects of t10,cl2-CLA on the preadipocyte differentiation transcription factors, lipogenic and lipolytic enzymes, regulation hormones, lipogenic and lipolytic rates in SC and longissimus muscle tissue. The results showed that:the mRNA abundance of PPARγ, FAS and INSR, HSL activities, INSR protein level, lipolytic rates,TG content in SC adipose tissue and the mRNA abundance of HSL and LPL, LPL activities,GHR protein level in longissimus muscle tissue were decreased by adding t10,c12-CLA(P<0.05); by contrast, HSL mRNA abundance in SC adipose tissue and the mRNA abundance of PPARγ, ADD1 and INSR, ME activities, INSR protein level, lipogenic rates, TG content in longissimus muscle tissue were increased by adding t10,c12-CLA(P<0.05). These results illustrated that the effects of t10,cl2-CLA on lipid deposition of SC adipose tissue differed from that on longissimus muscle tissue at the lipid metabolism point of view and confirmed t10,cl2-CLA inhibited the lipid deposition of SC adipose and promoted the lipid deposition of longissimus muscle in pig further.
Part3:In this trial, in vitro tissue culture system and experiment design were also same to partl. Real-time RT-PCR and ELISA assays were used to study the effects of t10,c12-CLA on the cytokines secreted by adipose and muscle tissue which exert the regulation action on both two tissues. The results showed that:the mRNA abundance and protein level of TNF-a, leptin and IL-15 in longissimus muscle tissue and IGF-1 protein level in SC adipose tissue were decreased by adding t10,c12-CLA(P<0.05); by contrast, the mRNA abundance and protein level of adiponectin, MSTN in longissimus muscle tissuee and TNF-a, IL- 6, leptin in SC adipose tissue were increased by adding t10,c12-CLA(P<0.05); But no effect on protein level of TNF-a in SC adipose tissue was observed (P>0.05). These results demonstrated that TNF-a, leptin, adiponectin, IL-15 and IL-6 play the important roles in molecular dialogue between adipocyte and myocyte, and these cytokines probably served as the mediator of tlO,cl2-CLA to exert different regulation effect on lipid deposition in SC and longissimus muscle tissue of pig.
The general conclusion:
In this study, the different mechanisms of effects of 100μmol/L t10,c12-CLA on regulation of lipid metabolism and deposition between SC adipose and longissimus muscle tissues in pig by affecting proliferation and differentiation of preadipocytes, differentiation transcription factors, lipogenic and lipolytic enzymes, regulation hormones, and cytokines secreted by two tissues were revealed, the inhibition effects of t10,cl2-CLA on lipid deposition in SC adipose and opposite effect on lipid deposition in longissimus muscle tissues were also confirmed further.
引文
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