【作者】 孙永刚；

【导师】 昝林森；

【作者基本信息】 西北农林科技大学 ， 细胞生物学， 2009， 硕士

【摘要】 本研究以荷斯坦新生公犊牛为实验材料,无菌采集腹股沟和附睾周围的脂肪组织,用Ⅰ型胶原酶消化法和组织块培养法分离培养牛前脂肪细胞,通过对所培养的脂肪细胞进行形态学观察、生长曲线绘制、胰岛素诱导分化、油红O染色及前脂肪细胞和成熟脂肪细胞标志基因检测,构建了牛前脂肪细胞体外增殖及分化模型。检测adiponectin、AdipoR1、AdipoR2、Pref-1和PPAR-γmRNA在牛前脂肪细胞分化过程中的表达规律。同时,以分化9 d的脂肪细胞为对象,研究了不同浓度和不同处理时间,胰岛素对adiponectin、AdipoR1和AdipoR2 mRNA表达的影响及可能影响的代谢通路。本研究初步揭示牛前脂肪细胞增殖和分化规律,探讨了脂联素及其受体基因在牛脂肪细胞增殖分化过程中的可能调控机制,为提高我国地方黄牛肌内脂肪含量奠定了基础。主要研究结果如下:1.初步构建了牛前体脂肪细胞体外增殖及分化模型用Ⅰ型胶原酶消化法可从新生牛脂肪组织中分离大量的前脂肪细胞,经胰岛素诱导分化、油红O染色和前脂肪细胞标志基因Pref-1和脂肪细胞标志基因PPAR-γ检测,体外分离培养的前脂肪细胞生长汇合后诱导9 d可分化成脂肪细胞。2.阐明了adiponectin、AdipoRs、Pref-1和PPAR-γ在牛脂肪细胞分化过程中的表达规律Adiponectin在牛前脂肪细胞中不表达,随前脂肪细胞的诱导分化adiponectin和AdipoR2 mRNA的表达显著增加(P<0.05),AdipoR1在细胞分化过程中持续表达且表达水平变化不显著(P>0.05)。Pref-1 mRNA在前脂肪细胞中高表达随脂肪细胞分化显著降低,在8 d分化的细胞中不表达,同时,在诱导分化的细胞中Pref-1的表达显著低于正常培养的细胞(P<0.05)。PPAR-γ随前脂肪细胞分化表达增加,且在诱导分化的细胞中显著高于正常培养细胞(P<0.05)。3.研究了不同胰岛素浓度及处理时间对脂联素及其受体基因的表达变化诱导分化9 d的牛脂肪细胞中不同处理浓度胰岛素均能抑制adiponectin mRNA的表达且呈剂量依赖性抑制,同时,100 nmol/L呈时间依赖性抑制adiponectin的表达。不同浓度胰岛素均能抑制AdipoR2的表达而且各剂量的抑制效果差异不显著,100 nmol/L胰岛素处理24 h能显著抑制AdipoR2的表达。不同浓度胰岛素和胰岛素处理时间对AdipoR1的表达没有影响。4.初步揭示了牛脂肪细胞中胰岛素能通过PI3K/Akt信号通路抑制adiponectin和AdipoR2的转录,而且抑制是可逆的。更多还原

【Abstract】 Preadipocytes were obtained from perirenal adipose tissue and epididymal adipose tissue of newborn Chinese Holstein calves by type I collagenase digestion and tissue explants methods. Culutred preadipocytes were identified by their morphological changes, growth curve, oil red O staining and preadipocytes marker gene, adipocytes marker gene mRNA expression. At the same time, the culture model for proliferation and differentiation of bovine preadipocytes was established in vitro. The expression pattern of adiponectin, AdipoR1, AdipoR2, Pref-1 and PPAR-γwas investigated during bovine preadipocytes differentiation using semi-quantitative RT-PCR method. Furthermore, To reveal the potential regulation of adiponectin and adiponectin receptors by insulin in bovine adipocytes, we used various concentrations of insulin to treat 9-day-differentiated bovine adipocytes for 24 h and 100 nmol/L insulin to treat for different time and examined the effect of insulin on adiponectin, AdipoR1 and AdipoR2 genes expression in bovine adipocytes in vitro. The obtained results provided the information and basis for regulation of bovine preadipocytes proliferation and differentiation and intramuscular fat deposition. The main results were as follows:1. Establishment of the culture model for proliferation and differentiation of bovine preadipocytes in vitro.A large number preadipocytes was isolated from adipose tissue of newborn Chinese Holstein calves by type I collagenase digestion. Preadipocytes can induced into mature adipocytes by insulin for 9 d differentiation and the cells were identified by their oil red O staining and preadipocytes marker gene (preadipocyte factor -1, Pref-1), adipocytes marker gene (peroxisome proliferator-activated receptor-γ, PPAR-γ) mRNA expression.2. Investigation on expression pattern of adiponectin, AdipoR1, AdipoR2, Pref-1 and PPAR-γduring bovine preadipocytes differentiation.Adiponectin mRNA was not expressed in preadipocytes but adiponectin and AdipoR2 mRNA increased as differentiation proceeded and reached its highest level on day 8 of induced differentiation(P<0.05). AdipoR1 mRNA was stably expressed throughout the differentiation period (P>0.05). Pref-1 mRNA was highly expressed in preadipocytes but not expressed in 8-day-differentiated adipocytes. Meanwhile, the expression abundance of Pref-1 mRNA in differentiated cells by insulin was significantly lower than normal cultured preadicytes. The expression of PPAR-γmRNA was increased significantly as preadipocytes differentiation proceeded, also significantly higher than the expression abundance of PPAR-γmRNA in differentiated cells was significantly higher than normal cultured preadicytes.3. Study of the effects of insulin concentrations and treatment time on adiponectin and its receptor gene expression.Insulin markedly decreased adiponectin and AdipoR2 mRNA expression in a dose- dependent fashion and time-dependent fashion(0, 2, 4, 12 and 24 h) in 9-day-differentiated bovine adipocytes, but various concentrations of insulin and insulin treatment them for different times did not effect the expression of AdipoR1 mRNA. Moreover, 100 nmol/L insulin completely inhibits adiponectin and AdipoR2 mRNA expression compared with other insulin concentrations.4. Insulin at least partially suppressed the expression of adiponectin and AdipoR2 mRNA via the PI3K/Akt pathway in bovine adipocytes, the inhibitory effect of insulin on adiponectin and AdipoR2 expression was reversible.更多还原