EGF对牛前脂肪细胞增殖和分化的影响
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摘要
表皮生长因子(Epidemal growth factor,EGF)是一类重要的生长因子,通过与细胞膜上的受体结合而发挥作用。它主要通过与其受体结合而使结合区域结合发生构型变化,从而改变细胞的骨架结构,使细胞分化、分裂和增殖。多项研究均表明EGF可以促进前体脂肪细胞的增殖。但是EGF对前体脂肪细胞分化的影响,各家众说纷纭。并且还未见EGF对于牛前体脂肪细胞增殖和分化的影响。我国对于脂肪细胞的研究刚刚起步,大部分是关于不同种属的脂肪细胞形态学的研究。对于牛前体脂肪细胞体外培养模式及其培养过程中影响因素都少见报道。本文对于表皮生长因子的结构、生理功能、脂肪细胞来源、脂肪细胞分化的体外系统、脂肪细胞分化增殖的过程及影响因素作一综述。本实验通过建立牛前体脂肪细胞原代培养,采用细胞生物学基本方法,观察了牛前脂肪细胞增殖分化过程中的形态变化,以及采用RT-PCR方法检测了不同培养基培养过程中脂肪细胞分化标志基因的表达量的变化。通过添加不同浓度(10 ng/mL、50 ng/mL、100 ng/mL、200 ng/mL)的EGF到前脂肪细胞培养基中,用MTT比色法、油红O提取比色法和RT-PCR法检测了不同浓度的EGF对脂肪细胞增殖及分化的影响。获得的研究主要结果如下:
1.建立了本实验室成熟的牛前脂肪细胞培养体系。用I型胶原酶,将剪碎的脂肪组织块在37℃水浴恒温振荡器中消化50 min( 120次/ min),即可得到脂肪细胞。组织块法与胶原酶消化法相比也可得到脂肪细胞,且剪的越碎,培养的成功率越高,操作简便;但细胞生长缓慢、细胞数量少。胶原酶法可得到大量的前体脂肪细胞。并且在培养过程中,用分化培养基培养与完全培养基培养通过油红O提取比色法及分化标志基因表达量的变化的比较,证明分化培养基可促进脂肪细胞分化。
2、通过MTT比色法检测发现,10 ng/mL～200 ng/mL浓度的EGF均可促进牛前脂肪细胞的增殖,且10 ng/mL的EGF与各组相比均能显著促进牛脂肪细胞的增殖。
3、牛前脂肪细胞在分化诱导培养两天后,无血清培养基中添加10 ng/mL～200 ng/mL不同浓度的EGF处理均可促进脂肪细胞分化,测定脂肪细胞分化标志基因LPL、PPARγ和A-FABP mRNA的相对表达量发现各浓度均能能促进脂肪细胞分化。并且200 ng/mL与各处理组相比均可在第8 d显著促进脂肪细胞分化。
Epidemal growth factor is one kind of important growth factors, which play its role through binding the repector of the membrane.After binded the repector, the structure of binding domain was changed, it made the structure of cytoskeleton changing,that improved the cell differentiation, division and proliferation. More study show that EGF could improved proliferation of the preadipocytes. but effect of EGF on preadipocyte differentiation has not been studied. Our country just started to the fatty cell research,majority of was about the fatty cell morphology research .but has litter reporeted about bovine preadipocyte culture and effections factors during culturing process. this expriment about the structure, physiological function,origin of adipocyte,the system of adipocyte differentiation in vivo and influence factors of the adipocyte proliferation and differentiation process .This study was to establish the method of bovine preadipocyte cultruing,on the basis of this, using biology essential method,observed the shape change in preadipocyte differentiation,and using RT-PCR determind the expression of differentiation marker genes cultured with different medium. And invesstigated the effect of different doses EGF on preadipocyte proliferation and differentiation by MTT,oil red O incorporction and RT-PCR . Experimental results as follows:
1. Established the mature model of bovine preadipocyte of our laboratory. Adipocyte were cuted into pieces then digested in Type I collagenase for 50 min at 37℃with shaking at 120 cycles/min.then we could collected preadipocytes. Tissue culture method comparaed with by Type I collagenase digested method, which could collect adipocyte and when cuted into more pieces, the higher success rate,and it easily operated.But using this method, the cell grew slowlier and shew fewer comparaed with the Type I collagenase digested method .through Oil red O incorporction and changing of differentiation maker genes expression of the cell cultured by complete medium and differentiation medium ,we found that differentiation medium could promote the preadipocyte differentiation.
2. Preadipocyte proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), which investgated that 10ng/mL～200ng/mL doses EGF could promote the bovine preadipocyte proliferation.especially the dose of 10 ng/mL EGF could significantly promoted bovine preadipocytes proliferation comparaed with other treated groups.
3. After the preadipocytes confluence, the preadipocyte were induced by differentiation medium two days, different doses EGF (10 ng/mL、50 ng/mL、100 ng/mL、200 ng/mL) added into complete medium without FBS. And determined the expression of differentiation marker genes LPL、PPARγand A-FABP mRNA at 8 day, found that each treated groups could promoted the adipocyte differentiation comparaed with control group. especially the dose of 200 ng/mL could significantly promoted adipocyte differentiation.
1.建立了本实验室成熟的牛前脂肪细胞培养体系。用I型胶原酶,将剪碎的脂肪组织块在37℃水浴恒温振荡器中消化50 min( 120次/ min),即可得到脂肪细胞。组织块法与胶原酶消化法相比也可得到脂肪细胞,且剪的越碎,培养的成功率越高,操作简便;但细胞生长缓慢、细胞数量少。胶原酶法可得到大量的前体脂肪细胞。并且在培养过程中,用分化培养基培养与完全培养基培养通过油红O提取比色法及分化标志基因表达量的变化的比较,证明分化培养基可促进脂肪细胞分化。
2、通过MTT比色法检测发现,10 ng/mL～200 ng/mL浓度的EGF均可促进牛前脂肪细胞的增殖,且10 ng/mL的EGF与各组相比均能显著促进牛脂肪细胞的增殖。
3、牛前脂肪细胞在分化诱导培养两天后,无血清培养基中添加10 ng/mL～200 ng/mL不同浓度的EGF处理均可促进脂肪细胞分化,测定脂肪细胞分化标志基因LPL、PPARγ和A-FABP mRNA的相对表达量发现各浓度均能能促进脂肪细胞分化。并且200 ng/mL与各处理组相比均可在第8 d显著促进脂肪细胞分化。
Epidemal growth factor is one kind of important growth factors, which play its role through binding the repector of the membrane.After binded the repector, the structure of binding domain was changed, it made the structure of cytoskeleton changing,that improved the cell differentiation, division and proliferation. More study show that EGF could improved proliferation of the preadipocytes. but effect of EGF on preadipocyte differentiation has not been studied. Our country just started to the fatty cell research,majority of was about the fatty cell morphology research .but has litter reporeted about bovine preadipocyte culture and effections factors during culturing process. this expriment about the structure, physiological function,origin of adipocyte,the system of adipocyte differentiation in vivo and influence factors of the adipocyte proliferation and differentiation process .This study was to establish the method of bovine preadipocyte cultruing,on the basis of this, using biology essential method,observed the shape change in preadipocyte differentiation,and using RT-PCR determind the expression of differentiation marker genes cultured with different medium. And invesstigated the effect of different doses EGF on preadipocyte proliferation and differentiation by MTT,oil red O incorporction and RT-PCR . Experimental results as follows:
1. Established the mature model of bovine preadipocyte of our laboratory. Adipocyte were cuted into pieces then digested in Type I collagenase for 50 min at 37℃with shaking at 120 cycles/min.then we could collected preadipocytes. Tissue culture method comparaed with by Type I collagenase digested method, which could collect adipocyte and when cuted into more pieces, the higher success rate,and it easily operated.But using this method, the cell grew slowlier and shew fewer comparaed with the Type I collagenase digested method .through Oil red O incorporction and changing of differentiation maker genes expression of the cell cultured by complete medium and differentiation medium ,we found that differentiation medium could promote the preadipocyte differentiation.
2. Preadipocyte proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), which investgated that 10ng/mL～200ng/mL doses EGF could promote the bovine preadipocyte proliferation.especially the dose of 10 ng/mL EGF could significantly promoted bovine preadipocytes proliferation comparaed with other treated groups.
3. After the preadipocytes confluence, the preadipocyte were induced by differentiation medium two days, different doses EGF (10 ng/mL、50 ng/mL、100 ng/mL、200 ng/mL) added into complete medium without FBS. And determined the expression of differentiation marker genes LPL、PPARγand A-FABP mRNA at 8 day, found that each treated groups could promoted the adipocyte differentiation comparaed with control group. especially the dose of 200 ng/mL could significantly promoted adipocyte differentiation.
引文
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