【作者】 孙红梅；

【导师】 孙超； 杨公社；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2007， 硕士

【摘要】 脂肪组织与肌肉组织通过旁分泌、内分泌产生相互作用，参与机体脂肪沉积与肌肉发育过程，在物质与能量代谢过程中具有重要调节功能，建立脂肪细胞与肌细胞的联合培养体系，有助于研究两类细胞间的作用关系。本实验以初生健康长白仔猪为实验动物，分离、纯化前体脂肪细胞与骨骼肌肌卫星细胞，采用MTT比色、油红O染色、Desmin免疫组化、HE染色和半定量RT-PCR等方法，建立了一种猪前体脂肪细胞与肌卫星细胞体外直接联合培养的适宜体系，并对联合培养细胞的增殖分化等生长特性进行了初步研究，得出如下研究结果：1．比较了接种浓度比对联合培养细胞增殖率的影响。在13个接种浓度比中，MTT比色结果显示，当前体脂肪细胞与肌卫星细胞以2.5×10⁷个／L与1×10⁸个／L接种时，联合培养细胞在测定第9天的增殖率可达到最大。2．分析了贴壁时间对联合培养细胞存活率的影响。随培养时间的延长，联合培养细胞贴壁率增大，但在48～66 h间趋于稳定，66 h后部分细胞进入增殖分裂期。3．研究了培养基及血清浓度对联合培养细胞存活率的影响。细胞贴壁率结果显示，有血清培养基较无血清培养基有利于细胞的存活，其中DMEM／F12（1：1）培养基较DMEM、RPMI及M199培养效果好，联合培养细胞存活率在10～200 mL／L的范围内呈浓度依赖性。4．分析了联合培养细胞生长形态与增殖特性的变化。形态学动态观察、2～9天MTT比色结果显示，联合培养细胞贴附后呈纤维样伸展延伸，增殖率高于单独两类细胞，但接触汇合后交织重叠生长，7～9天维持缓慢的增殖过程，进入平台期较单独两类细胞晚。5．形态学观察联合培养对脂肪细胞与肌细胞分化过程的影响。油红O染色、HE染色结果显示，联合培养细胞中仅观察到极少量的小脂滴，且呈现较晚；多核肌细胞形成与肌管的融合在联合培养中显著，但没有单独肌细胞明显，且形成较晚；6．分子水平RT-PCR检测联合培养对脂肪细胞与肌细胞分化过程的影响。第5、10天的联合培养细胞皆表达LPL、Myf₅ mRNA，并与单独细胞差异不显著OP＜0.05)；但PPARr₂、MyoG mRNA在第5天表达丰度较单独细胞低，且差异显著（P＜0.05），第10天表达量有所提高。更多还原

【Abstract】 There is a close interaction between fat and muscle tissue by autocrine and paracrine,which plays a significant role in fatty deposition and muscle development, and have important regulated function on the establishment substance and energy metabolism. So establishment of adipocytes and myocytes co-culture system will help to study on the interaction between them.The present research established a eligible system for co-culture ofpreadipocytes and myogenic satellite cells, on the base, and methods of MTT spectrophotometry,Oil Red O staining, Desmin immunohislochemistry staining, HE staining and semiquantitative RT-PCR were used to study on the growth characteristics of cocultures in porcine adipose and muscle tissue by distracting and purifying preadipocytes and myogenic satellite cells from nascent pigs.This research includes following several aspects:1 The effect of different concentration rates on the proliferation of cocultures in Seeding were tested by the method of MTT colorimetry. The results show that growth rate of cocultures on 9th day in culture is more than other 12 rates as preadipocytes and myogenic satellite cells were seeded in the rate of 2.5×10⁷/L: 1×10⁸/L.2 The effect of adherenced time after seeding on the proliferation of cocultures were tested by the method of blood cell counting. The results show that the rate of adherenced cells is increased following time, but get to stabilization from 48 to 66 hours, and a part of cocultures in culture begins to proliferating after 66 hours.3 The effect of medium types and concentrations of blood serum contained in medium on survival rates of cocultures were tested by the rate of coherenced cells. The results show that using of serum is more useful for culture than a serum-free medium,DMEM/F12（1:1） medium is more benefit for co-culture than DMEM, RPMI and M199 medium, and the surivival rates of cocultures is increased as the concentration of serum contained in medium.4 To study on the growth morphology and characteristics of cocultures, Morphologic survey from 2nd～15th day and MTT spectrophotometry in 2nd～9th day were adopted. The results show that the morphology of cocultures was fusiform shape extending before confluence, but after confluence it changed into irregular with coinciding and interlace with each other. The growth rate is more than adipocytes and myocytes in culture, a rapid growth until 7th day,but keep on slow later growth to 9th day,which is different from adipocytes and myocytes.5 To study the effect of co-culture on the differentiation of preadipocytes and myogenic satellites cells, the methods of Oil Red O staining, HE staining were applied. The results show that the cells in coculture filled lipid appeared later and fewer than adipocytes as the control group. Polykaryocytes fuse into myotubes was visible by HE stanining but also later and less standing than myocytes as the control group.6 To study the effect of co-culture on the differentiation of preadipocytes and myogenic satellites cells, the methods of semiquantitatives RT-PCR also was applied. The results show that the mRNA levels of LPL and Myf₅ were tested by semiquantitative RT-PCR on both 5th and 10th day in co-culture, but it is difficulted to detected the mRNA of PPARr₂ and MyoG on 5th day, and until 10th day low levels were detected.更多还原