ECM组分和cAMP对大鼠前体脂肪细胞增殖与分化的调控
摘要
本文以生长发育正常、健康状况良好的20日龄雄性SD大鼠为实验动物，采用脂肪细胞离体培养技术系统地研究了ECM组分中的Ⅳ型胶原和层粘连蛋白(LN)、环腺苷酸(cAMP)以及胰岛素(INS)、肾上腺素对大鼠前体脂肪细胞增殖与分化的调控，并比较了ECM组分中的Ⅳ型胶原和LN对大鼠脂肪细胞中Ⅰ、Ⅲ、Ⅳ型三种胶原基因在转录水平上表达效果的影响。
经过反复试验，寻找到适合本实验室的大鼠前体脂肪细胞培养条件。用消化培养法时大鼠前体脂肪细胞数目增加快、生长旺盛，而组织块培养法细胞数目很少、生长一般；比较了四种底物对大鼠前体脂肪细胞培养的影响，发现细胞长势由好到差依次是加鼠尾胶原、不加底物、加盖玻片和加塑料膜：用HE染色、红油0染色和免疫组化染色等三种染色技术观察到大鼠前体脂肪细胞可转化成典型的成熟脂肪细胞：胞质中有许多数目不同、大小不等的脂滴，胞核在细胞边缘；并检测了大鼠前体脂肪细胞中不同培养时间下蛋白质、DNA、葡萄糖、甘油三酯等生化指标的含量，发现大鼠前体脂肪细胞在培养6d以前4种生化指标含量逐渐递增，而培养6d以后4种生化指标含量逐渐降低。
利用MTT比色、流式细胞仪分析、GPDH活性测定、透射电镜分析、斑点杂交等实验技术系统研究了ECM组分中的Ⅳ型胶原和LN对大鼠前体脂肪细胞增殖与分化的调控。结果表明：Ⅳ型胶原和LN均有利于大鼠前体脂肪细胞的增殖，100ul／ml Ⅳ型胶原促增殖作用比50、150、200ul／ml三种浓度的明显，10ul／ml LN促增殖作用比5、20、40ul／ml三种浓度的作用显著；两种ECM组分均可降低脂肪细胞中G1期的细胞比例，升高PrI期的细胞比例，经检验，100ul／ml Ⅳ型胶原处理的脂肪细胞中各细胞周期细胞比例与对照、50、150ul／ml三种处理相比差异显著(P＜0.05)，10ul／ml LN对各细胞周期细胞比例的影响与对照、5、20ul／ml三种处理相比差异显著(P＜0.05)，根据脂肪细胞中G2/G1期DNA含量的比值接近2，证明脂肪细胞分裂按二倍体进行；Ⅳ型胶原和LN对大鼠前体脂肪细胞中的蛋白质、DNA、葡萄糖、甘油三酯的含量有明显促进作用，其中100ul／ml的Ⅳ型胶原可使4种生化指标含量与对照、50、150ul／ml处理相比差异极显著(P＜0.01)，10ul／mlLN对4种生化指标的影响与对照、5、20ul／ml处理相比差异极显著(P＜0.01)：用LN和Ⅳ型胶原处理大鼠前体脂肪细胞后，其GPDH活性均极显著地高于对照组(P＜0.01)，说明LN和Ⅳ型胶原均能促进前体脂肪细胞的分化，其中10ul／ml LN和100ul／ml Ⅳ型胶原处理的前体脂肪细胞中GPDH活性极显著地高于相应的其它处理组(P＜0.01)；不同浓度的Ⅳ型胶原和LN对大鼠前体脂肪细胞形态及其超微结构的影响不同，其中100ul／mlⅣ型胶原和10ul／ml LN对其形态及其超微结构的改善最有利；并且Ⅳ型胶原和LN均可不同程度地增加Ⅰ、Ⅲ、Ⅳ型三种胶原cDNA探针与脂肪细胞中总RNA的阳性杂交信号，其中LN的作用效果比Ⅳ型胶原更强。
采用细胞计数、MTT比色、流式细胞仪分析、GPDH活性检测、透射电镜分析等技术探讨了直接用cAMP对大鼠前体脂肪细胞增殖与分化的调控。结果显示：不同浓度的cAMP溶液均可显著地
增加前体脂肪细胞数目（P＜0．05或P＜0．01）、缩短细胞倍增时间，其中10’PM的CAMP使前体脂肪
细胞数目的增加幅度最大、对减小细胞倍增时间最有利；CAMP处理的前体脂肪细胞生长曲线与对
照组相比左移，而且培养6d以前细胞数目递增，此后细胞数目递减；它可使G；期细胞比例明显
地低于对照组，Prl期细胞比例明显高于对照组，表明cAMP可促进大鼠前体脂肪细胞增殖；CAMP
处理的大鼠前体脂肪细胞中蛋白质、DNA、葡萄糖、甘油三酯含量明显高于对照，证明CAllP可提
高前体脂肪细胞中 4种生化指标含量，其中 10’PM的 CAMP提高 4种生化指标含量的效果最好；CAMP
溶液可使脂肪细胞中 GpDH活性极显著地高于对照组（p＜0．01），而且川加的 CAMp效果最佳；此
外，cMIP溶液对大鼠前体脂肪细胞的形态及超微结构具有明显影响，其中 10血的 CAMP对其影晌
最有利。
通过Mh比色、流式细胞仪分析、GPDH活性检测等技术研究了LS和肾上腺素对大鼠前体脂
肪细胞增殖与分化的调控。结果表明：INS可促进大鼠前体脂肪细胞的增殖，而肾上腺素则抑制
前体脂肪细胞的增殖，其中 25uU／。IINS（生理浓度）处理的前体脂肪细胞增殖效果好于 0．IU；’ml
INS（药理浓度），10y肾上腺素（药理浓度）抑制前体脂肪细胞增殖的效果高于川M肾上腺素
（生理浓度）；INS可降低G；期细胞比例，提高卜 期（细胞增殖指数）的细胞比例，肾上腺素提
高G；期细胞比例，降低 Prl期细胞比例，其中 25uU／ml INS促进增殖的效果最好，10一、肾上腺素
抑制增殖的程度最强；INS可使前体脂肪细胞中的 GPDH活性极显著地高于对照组（PCO，01）；肾
上腺素处理的前体脂肪细胞中GPDH活性极显著地低于对照组（P＜0．01），其中25uU／ml INS升高
细胞中 GPDH活性的幅度最大（约 4倍），10－初肾上腺素降低细胞中 GPDH活性的幅度最大（约 3
倍）。
Normal and healthy male SD mice were employed to systematically explore the regulation of ECM Component ?collagen-IV , LN, cAMP, INS and adrenal hormone, on the proliferation and differentiation of mice preadipocytes, and influences were compared between the effects of Collagen-IV and LN on the expression of type I, III, IV of collagen genes at transcriptional level in mice adipocytes.
Through repeated experiments, conditions required for the culture of mice preadipocytes were founded. In digestion culture, mice preadipocytes increase fast and vigorously, but in tissue culture, they increase slowly and evenly; when compared the influences of 4 substitutes on the culture of mice preadipocyte, it was found that growing conditions go down in the order of adding mice tale collagen, without substitutes, under glass slides and under plastic slides. By HE dyeing, Oil Red O dyeing and Immunohistochemical dyeing, it can all be found that mice preadiopocytes can be transformed to mature adipocytes with many lipid drops of various number and volume in cytoplasma, and the cellular nucleus by the side of the cells. Chemical indexes of protein, DNA, glucose, triglyceride were measured after mice preadipocytes were cultured for different times, it was found that the indexes increase gradually before the mice preadipocytes being cultured for 6 days, and they decline gradually after 6 days.
By MTT method, Flow cytometer, GPDH activity detection, TE analysis and dot hybridization, regulation were investigated of collagen-IV and LN on the proliferation and differentiation of mice preadipocytes. The results showed that collagen-IV and LN can enhance the proliferation of mice preadipocytes, especially with a concentration of 100 ul/ml and 10 ul/ml respectively; and the two ECM components can reduce the cellular ratio during Gl period of the adipocytes cell cycle, increase it during PrI period, both are remarkable. The DNA content ratio of G2 to G1 period is very close to 2, it can be proven that the cell mitosis is in the way of bisplit; collagen-IV and LN and remarkably improve the content of protein, DNA, glucose, triglyceride in the mice adipocytes, especially at the concentration of 100 ul/ml and 10 ul/ml respectively. GPDH activities were much high than the control after the mice preadipocytes were treated with collagen-IV and LN, especially at the concentration of 100 ul/ml and 10 ul/ml respectively, it shows that they can both enhance the differentiation of preadipocytes. Various concentrations of collagen-IV and LN can differently influence the morphology and super microstructure of mice adipocytes, of which 100 ul/ml of collagen-IV and 10 ul/ml of LN and mostly improve the morphology and super microstructure. Collagen-IV and LN both can increase the positive hybridization signals of cDNA probe of I, III, IV types of collagen with the total RNA in adipocytes, and LN acts more obviously than collagen-IV do.
By cellular numbering, MTT method, GPDH activity detection, TE analysis, the regulation of cAMP on the proliferation and differentiation of mice preadipocytes were investigated. The results are as follows: cAMPs of various concentrations can all remarkably increase the cell numbers of
preadipocytes, reduce the cell double time, especially at the concentration of 107PM. Growth curve of preadipocytes treated with cAMP moves to the left of the control, the cell number increases before being cultured for 6 days, decreases after that. The cellular ratio is remarkably lower than the control in Gl period, higher in PrI period, indicating that cAMP can enhance the proliferation of mice preadipocytes. The contents of proteins, DNA, glucose, triglyceride are obviously higher in mice preadipocytes treated with cAMP than the control, especially when cAMP is at the concentration of of 107PM, showing that cAMP can improve the contents of the 4 substances in preadipocytes. The GPDH activities of adipocytes treated with cAMP are much more active than the control, and cAMP can have obvious influences on the morphology and super mic
经过反复试验，寻找到适合本实验室的大鼠前体脂肪细胞培养条件。用消化培养法时大鼠前体脂肪细胞数目增加快、生长旺盛，而组织块培养法细胞数目很少、生长一般；比较了四种底物对大鼠前体脂肪细胞培养的影响，发现细胞长势由好到差依次是加鼠尾胶原、不加底物、加盖玻片和加塑料膜：用HE染色、红油0染色和免疫组化染色等三种染色技术观察到大鼠前体脂肪细胞可转化成典型的成熟脂肪细胞：胞质中有许多数目不同、大小不等的脂滴，胞核在细胞边缘；并检测了大鼠前体脂肪细胞中不同培养时间下蛋白质、DNA、葡萄糖、甘油三酯等生化指标的含量，发现大鼠前体脂肪细胞在培养6d以前4种生化指标含量逐渐递增，而培养6d以后4种生化指标含量逐渐降低。
利用MTT比色、流式细胞仪分析、GPDH活性测定、透射电镜分析、斑点杂交等实验技术系统研究了ECM组分中的Ⅳ型胶原和LN对大鼠前体脂肪细胞增殖与分化的调控。结果表明：Ⅳ型胶原和LN均有利于大鼠前体脂肪细胞的增殖，100ul／ml Ⅳ型胶原促增殖作用比50、150、200ul／ml三种浓度的明显，10ul／ml LN促增殖作用比5、20、40ul／ml三种浓度的作用显著；两种ECM组分均可降低脂肪细胞中G1期的细胞比例，升高PrI期的细胞比例，经检验，100ul／ml Ⅳ型胶原处理的脂肪细胞中各细胞周期细胞比例与对照、50、150ul／ml三种处理相比差异显著(P＜0.05)，10ul／ml LN对各细胞周期细胞比例的影响与对照、5、20ul／ml三种处理相比差异显著(P＜0.05)，根据脂肪细胞中G2/G1期DNA含量的比值接近2，证明脂肪细胞分裂按二倍体进行；Ⅳ型胶原和LN对大鼠前体脂肪细胞中的蛋白质、DNA、葡萄糖、甘油三酯的含量有明显促进作用，其中100ul／ml的Ⅳ型胶原可使4种生化指标含量与对照、50、150ul／ml处理相比差异极显著(P＜0.01)，10ul／mlLN对4种生化指标的影响与对照、5、20ul／ml处理相比差异极显著(P＜0.01)：用LN和Ⅳ型胶原处理大鼠前体脂肪细胞后，其GPDH活性均极显著地高于对照组(P＜0.01)，说明LN和Ⅳ型胶原均能促进前体脂肪细胞的分化，其中10ul／ml LN和100ul／ml Ⅳ型胶原处理的前体脂肪细胞中GPDH活性极显著地高于相应的其它处理组(P＜0.01)；不同浓度的Ⅳ型胶原和LN对大鼠前体脂肪细胞形态及其超微结构的影响不同，其中100ul／mlⅣ型胶原和10ul／ml LN对其形态及其超微结构的改善最有利；并且Ⅳ型胶原和LN均可不同程度地增加Ⅰ、Ⅲ、Ⅳ型三种胶原cDNA探针与脂肪细胞中总RNA的阳性杂交信号，其中LN的作用效果比Ⅳ型胶原更强。
采用细胞计数、MTT比色、流式细胞仪分析、GPDH活性检测、透射电镜分析等技术探讨了直接用cAMP对大鼠前体脂肪细胞增殖与分化的调控。结果显示：不同浓度的cAMP溶液均可显著地
增加前体脂肪细胞数目（P＜0．05或P＜0．01）、缩短细胞倍增时间，其中10’PM的CAMP使前体脂肪
细胞数目的增加幅度最大、对减小细胞倍增时间最有利；CAMP处理的前体脂肪细胞生长曲线与对
照组相比左移，而且培养6d以前细胞数目递增，此后细胞数目递减；它可使G；期细胞比例明显
地低于对照组，Prl期细胞比例明显高于对照组，表明cAMP可促进大鼠前体脂肪细胞增殖；CAMP
处理的大鼠前体脂肪细胞中蛋白质、DNA、葡萄糖、甘油三酯含量明显高于对照，证明CAllP可提
高前体脂肪细胞中 4种生化指标含量，其中 10’PM的 CAMP提高 4种生化指标含量的效果最好；CAMP
溶液可使脂肪细胞中 GpDH活性极显著地高于对照组（p＜0．01），而且川加的 CAMp效果最佳；此
外，cMIP溶液对大鼠前体脂肪细胞的形态及超微结构具有明显影响，其中 10血的 CAMP对其影晌
最有利。
通过Mh比色、流式细胞仪分析、GPDH活性检测等技术研究了LS和肾上腺素对大鼠前体脂
肪细胞增殖与分化的调控。结果表明：INS可促进大鼠前体脂肪细胞的增殖，而肾上腺素则抑制
前体脂肪细胞的增殖，其中 25uU／。IINS（生理浓度）处理的前体脂肪细胞增殖效果好于 0．IU；’ml
INS（药理浓度），10y肾上腺素（药理浓度）抑制前体脂肪细胞增殖的效果高于川M肾上腺素
（生理浓度）；INS可降低G；期细胞比例，提高卜 期（细胞增殖指数）的细胞比例，肾上腺素提
高G；期细胞比例，降低 Prl期细胞比例，其中 25uU／ml INS促进增殖的效果最好，10一、肾上腺素
抑制增殖的程度最强；INS可使前体脂肪细胞中的 GPDH活性极显著地高于对照组（PCO，01）；肾
上腺素处理的前体脂肪细胞中GPDH活性极显著地低于对照组（P＜0．01），其中25uU／ml INS升高
细胞中 GPDH活性的幅度最大（约 4倍），10－初肾上腺素降低细胞中 GPDH活性的幅度最大（约 3
倍）。
Normal and healthy male SD mice were employed to systematically explore the regulation of ECM Component ?collagen-IV , LN, cAMP, INS and adrenal hormone, on the proliferation and differentiation of mice preadipocytes, and influences were compared between the effects of Collagen-IV and LN on the expression of type I, III, IV of collagen genes at transcriptional level in mice adipocytes.
Through repeated experiments, conditions required for the culture of mice preadipocytes were founded. In digestion culture, mice preadipocytes increase fast and vigorously, but in tissue culture, they increase slowly and evenly; when compared the influences of 4 substitutes on the culture of mice preadipocyte, it was found that growing conditions go down in the order of adding mice tale collagen, without substitutes, under glass slides and under plastic slides. By HE dyeing, Oil Red O dyeing and Immunohistochemical dyeing, it can all be found that mice preadiopocytes can be transformed to mature adipocytes with many lipid drops of various number and volume in cytoplasma, and the cellular nucleus by the side of the cells. Chemical indexes of protein, DNA, glucose, triglyceride were measured after mice preadipocytes were cultured for different times, it was found that the indexes increase gradually before the mice preadipocytes being cultured for 6 days, and they decline gradually after 6 days.
By MTT method, Flow cytometer, GPDH activity detection, TE analysis and dot hybridization, regulation were investigated of collagen-IV and LN on the proliferation and differentiation of mice preadipocytes. The results showed that collagen-IV and LN can enhance the proliferation of mice preadipocytes, especially with a concentration of 100 ul/ml and 10 ul/ml respectively; and the two ECM components can reduce the cellular ratio during Gl period of the adipocytes cell cycle, increase it during PrI period, both are remarkable. The DNA content ratio of G2 to G1 period is very close to 2, it can be proven that the cell mitosis is in the way of bisplit; collagen-IV and LN and remarkably improve the content of protein, DNA, glucose, triglyceride in the mice adipocytes, especially at the concentration of 100 ul/ml and 10 ul/ml respectively. GPDH activities were much high than the control after the mice preadipocytes were treated with collagen-IV and LN, especially at the concentration of 100 ul/ml and 10 ul/ml respectively, it shows that they can both enhance the differentiation of preadipocytes. Various concentrations of collagen-IV and LN can differently influence the morphology and super microstructure of mice adipocytes, of which 100 ul/ml of collagen-IV and 10 ul/ml of LN and mostly improve the morphology and super microstructure. Collagen-IV and LN both can increase the positive hybridization signals of cDNA probe of I, III, IV types of collagen with the total RNA in adipocytes, and LN acts more obviously than collagen-IV do.
By cellular numbering, MTT method, GPDH activity detection, TE analysis, the regulation of cAMP on the proliferation and differentiation of mice preadipocytes were investigated. The results are as follows: cAMPs of various concentrations can all remarkably increase the cell numbers of
preadipocytes, reduce the cell double time, especially at the concentration of 107PM. Growth curve of preadipocytes treated with cAMP moves to the left of the control, the cell number increases before being cultured for 6 days, decreases after that. The cellular ratio is remarkably lower than the control in Gl period, higher in PrI period, indicating that cAMP can enhance the proliferation of mice preadipocytes. The contents of proteins, DNA, glucose, triglyceride are obviously higher in mice preadipocytes treated with cAMP than the control, especially when cAMP is at the concentration of of 107PM, showing that cAMP can improve the contents of the 4 substances in preadipocytes. The GPDH activities of adipocytes treated with cAMP are much more active than the control, and cAMP can have obvious influences on the morphology and super mic
引文
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