兔体外成骨细胞和脂肪细胞横向分化的初步研究
发布时间：2018-10-09 11:46
【摘要】：目的：探讨成骨细胞和脂肪细胞两者间横向分化的能力和方法,为进一步行缝隙连接通讯对其调控的实验做准备。 方法：选取3月龄新西兰大白兔腹股沟处脂肪组织进行分离培养脂肪细胞,天花板贴壁法对脂肪细胞进行细胞去分化培养,利用第3代去分化脂肪细胞进行成骨诱导,并设立对照组进行对比。成骨分化指标的检测：对培养3周后的两组细胞进行Ⅰ型胶原免疫组化染色；使用BCIP/NBT ALP显色试剂盒对两组细胞进行ALP染色；使用ALP活性检测试剂盒分别检测各组第7天、第14天、第21天细胞的ALP活性；使用茜素红对连续培养3周的两组细胞进行钙结节染色。取出生7天内的乳兔颅骨进行成骨细胞的获得及培养,利用第3代成骨细胞细胞进行成脂诱导,并设立对照组对比。脂肪细胞分化指标的检测：两组细胞培养3周后行油红0染色；RT-PCR检测两组细胞PPARγmRNA的表达。 结果：1、提取的成熟脂肪细胞经天花板贴壁法培养后形态为长梭形成纤维细胞状。2、成骨分化检测：Ⅰ型胶原免疫组化染色显示实验组细胞内表达出Ⅰ型胶原,与对照组比较有统计学意义(p0.05)；ALP活性检测试剂盒检测各组细胞不同时段的ALP活性发现实验组较对照组高(p0.05)；且随着培养时间的增长实验组ALP活性逐渐增强(组内p0.05),与对照组比较均有统计学意义(p0.05)；经BCIP/NBT ALP染色试剂盒检测,实验组细胞染色有棕黑色细微颗粒,多数分布在细胞膜上以及周围,对照组细胞染色无棕黑色颗粒出现;茜素红对连续培养3周的两组细胞进行钙结节染色发现实验组有较多橘红色结节,而对照组无明显发现。3、对提取的乳兔颅骨原代细胞茜素红染色后证实提取的细胞为成骨细胞。4、成脂检测指标：油红0染色显示实验组细胞内出现大量红染颗粒,对照组细胞未被红染; RT-PCR检测两组细胞PPARγmRNA的表达,实验组可见有PPARγmRNA表达,而对照组无PPARγmRNA的表达。 结论：成熟脂肪细胞可以通过体外天花板培养实现去分化,去分化的脂肪细胞在成骨诱导环境下可以向成骨细胞分化；成骨细胞在成脂诱导环境下可以分化为脂肪细胞；成骨细胞和脂肪细胞在一定条件下可以相互转化。
[Abstract]:Aim: to investigate the ability and method of lateral differentiation between osteoblasts and adipocytes in order to prepare for the regulation of gap junction communication. Methods: adipocytes were isolated from the inguinal adipocytes of New Zealand white rabbits at 3 months old. Adipocytes were dedifferentiated and induced by the third generation of dedifferentiated adipocytes. A control group was set up for comparison. Detection of osteogenic differentiation: type I collagen immunohistochemical staining was performed on two groups of cells after 3 weeks of culture; ALP staining was performed on both groups using BCIP/NBT ALP reagent kit; ALP activity assay kit was used to detect 7 days in each group, respectively. The activity of ALP was observed on day 14 and day 21, and calcium nodules were stained by alizarin red in two groups of cells cultured for 3 weeks. Osteoblasts were obtained and cultured from the calvaria of newborn rabbits within 7 days. The third generation of osteoblasts were used to induce adipogenesis and the control group was set up. Detection of adipocyte differentiation index: after 3 weeks of culture, the expression of PPAR 纬 mRNA in the two groups was detected by oil red 0 staining reverse transcription-polymerase chain reaction (RT-PCR). Results: the mature adipocytes cultured by ceiling adherent method showed long fusiform fibroblasts. The osteogenic differentiation was detected. The expression of type 鈪，
本文编号：2259220
[Abstract]:Aim: to investigate the ability and method of lateral differentiation between osteoblasts and adipocytes in order to prepare for the regulation of gap junction communication. Methods: adipocytes were isolated from the inguinal adipocytes of New Zealand white rabbits at 3 months old. Adipocytes were dedifferentiated and induced by the third generation of dedifferentiated adipocytes. A control group was set up for comparison. Detection of osteogenic differentiation: type I collagen immunohistochemical staining was performed on two groups of cells after 3 weeks of culture; ALP staining was performed on both groups using BCIP/NBT ALP reagent kit; ALP activity assay kit was used to detect 7 days in each group, respectively. The activity of ALP was observed on day 14 and day 21, and calcium nodules were stained by alizarin red in two groups of cells cultured for 3 weeks. Osteoblasts were obtained and cultured from the calvaria of newborn rabbits within 7 days. The third generation of osteoblasts were used to induce adipogenesis and the control group was set up. Detection of adipocyte differentiation index: after 3 weeks of culture, the expression of PPAR 纬 mRNA in the two groups was detected by oil red 0 staining reverse transcription-polymerase chain reaction (RT-PCR). Results: the mature adipocytes cultured by ceiling adherent method showed long fusiform fibroblasts. The osteogenic differentiation was detected. The expression of type 鈪，
本文编号：2259220
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