IGF1R基因沉默对梅花鹿鹿茸软骨细胞增殖周期凋亡等作用研究
发布时间：2019-01-07 08:38
【摘要】：鹿茸是梅花鹿第二显著性征,具有周期性再生的特性。在它生长最旺盛时期,其生长速度可达到2cm/d。已有的研究表明:鹿茸的再生是一个基于鹿茸干细胞代谢且受多种因子调控的复杂过程,此外有大量研究报道IGF1R基因参与多种细胞的增殖与代谢。然而,IGF1R基因在鹿茸软骨细胞的研究鲜有报道,因此本试验旨在从鹿茸软骨细胞生长、增殖、周期和凋亡的角度探究IGF1R的功能,为进一步深入研究鹿茸生长发育分子机理提供新的观点。本试验采用RNAi技术干扰鹿茸软骨细胞IGF1R m RNA的表达,从不同角度分析其对软骨细胞的影响。本研究以RNAi-Ready p SIREN-Retro QZs Green plasmid为载体,以IGF1R作为候选基因研究其对鹿茸软骨细胞生长的调控机制,成功构建了2个RNAi重组质粒,分别命名为psh RNA-1和psh RNA-2,用于转染软骨细胞。利用Real-Time PCR和Western blot技术,对转染48h后的RNAi效果进行分析。研究IGF1R对鹿茸软骨细胞增殖、凋亡及自身m RNA和蛋白表达的调控,进一步探讨鹿茸生长调节机制。研究内容和结果如下:(1)转染软骨细胞48h,相比阴性对照组,软骨细胞经psh RNA-1和psh RNA-2处理,IGF1R基因m RNA表达量分别为51%和75%;蛋白质的表达量也有明显下降。表明干扰细胞效果明显。(2)采用CCK-8方法检测转染24h、48h和72h后细胞增殖情况。相比于阴性对照组,试验组的细胞增殖呈下降趋势,尤其是psh RNA-1质粒干扰效果明显((1.09±0.10 vs 1.16±0.09(P0.01),1.04±0.02 vs 1.10±0.06(P0.05),0.79±0.06 vs1.01±0.07(P0.01))。(3)转染软骨细胞48h,相比于阴性对照组psh RNA-negative,试验组细胞更多的处于G1期(79.47±0.11vs 82.27±0.35(P0.01)),而S期细胞数量显著下降(16.56±0.10 vs 14.82±0.014(P0.05))。(4)转染软骨细胞48h后,试验组psh RNA-1和阴性对照组psh RNA-negative的早期凋亡水平分别为(22.12±2.72%vs 8.56±0.16%,P0.01);正常活细胞分别为(62.85±5.97 vs 77.39±4.46,P0.05)。相关凋亡基因p53(P0.01)、bcl-2(P0.05)和bax的表达水平显著下降(P0.01)。表明转染psh RNA-1可促进软骨细胞的凋亡。结果表明,IGFR对鹿茸的生长可能具有一定的调控作用,特别是对鹿茸软骨细胞的增殖和凋亡具有显著的调控作用,此外IGFR还可能通过调控软骨细胞的细胞周期调节其生长发育。
[Abstract]:Velvet antler is the second significant sexual characteristic of sika deer and has the characteristic of periodic regeneration. At its peak, it can grow at a rate of 2 cm / d. Previous studies have shown that the regeneration of velvet antler is a complex process based on the metabolism of pilose antler stem cells and regulated by many factors. In addition, a large number of studies have reported that IGF1R gene is involved in the proliferation and metabolism of many kinds of cells. However, the study of IGF1R gene in antler chondrocytes is rarely reported. Therefore, the purpose of this study was to investigate the function of IGF1R from the perspectives of growth, proliferation, cycle and apoptosis of antler chondrocytes. It provides a new viewpoint for further studying the molecular mechanism of velvet antler growth and development. In this experiment, RNAi technique was used to interfere the expression of IGF1R m RNA in velvet antler chondrocytes, and the effects of IGF1R m RNA on chondrocytes were analyzed from different angles. In this study, RNAi-Ready p SIREN-Retro QZs Green plasmid was used as vector and IGF1R as candidate gene to study its regulatory mechanism on the growth of velvet antler chondrocytes. Two RNAi recombinant plasmids named psh RNA-1 and psh RNA-2, were successfully constructed. It is used to transfect chondrocytes. Real-Time PCR and Western blot techniques were used to analyze the effect of RNAi transfection for 48 h. To study the regulation of IGF1R on the proliferation, apoptosis and the expression of m RNA and protein of antler chondrocytes, and to further explore the mechanism of antler growth regulation. The results were as follows: (1) the expression of m RNA of IGF1R gene in chondrocytes treated with psh RNA-1 and psh RNA-2 was 51% and 75%, respectively. The results showed that the effect of interfering cells was obvious. (2) CCK-8 method was used to detect the proliferation of cells after transfection for 48 h and 72 h. Compared with the negative control group, the cell proliferation of the test group showed a downward trend, especially the effect of psh RNA-1 plasmid interference was obvious (1.09 卤0.10 vs 1.16 卤0.09 (P0.01), 1.04 卤0.02 vs 1.10 卤0.06 (P0.05). Chondrocytes were transfected with 0.79 卤0.06 vs1.01 卤0.07 (P0.01). (3) for 48h. Compared with the negative control group, the cells were in G1 phase (79.47 卤0.11vs 82.27 卤0.35, P0.01),). The number of S phase cells decreased significantly (16.56 卤0.10 vs 14.82 卤0.014 (P0.05). (4) 48 h after transfection of chondrocytes. The early apoptotic level of psh RNA-1 in the experimental group and psh RNA-negative in the negative control group was (22.12 卤2.72%vs 8.56 卤0.16); The normal living cells were (62.85 卤5.97 vs, 77.39 卤4.46, P 0.05). The expression of p53 (P0.01), bcl-2 (P0.05) and bax decreased significantly (P0.01). The results showed that psh RNA-1 transfection could promote the apoptosis of chondrocytes. The results showed that IGFR could regulate the growth of velvet antler, especially the proliferation and apoptosis of antler chondrocytes. In addition, IGFR might regulate the growth and development of chondrocytes by regulating the cell cycle of chondrocytes.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S825
本文编号：2403458
[Abstract]:Velvet antler is the second significant sexual characteristic of sika deer and has the characteristic of periodic regeneration. At its peak, it can grow at a rate of 2 cm / d. Previous studies have shown that the regeneration of velvet antler is a complex process based on the metabolism of pilose antler stem cells and regulated by many factors. In addition, a large number of studies have reported that IGF1R gene is involved in the proliferation and metabolism of many kinds of cells. However, the study of IGF1R gene in antler chondrocytes is rarely reported. Therefore, the purpose of this study was to investigate the function of IGF1R from the perspectives of growth, proliferation, cycle and apoptosis of antler chondrocytes. It provides a new viewpoint for further studying the molecular mechanism of velvet antler growth and development. In this experiment, RNAi technique was used to interfere the expression of IGF1R m RNA in velvet antler chondrocytes, and the effects of IGF1R m RNA on chondrocytes were analyzed from different angles. In this study, RNAi-Ready p SIREN-Retro QZs Green plasmid was used as vector and IGF1R as candidate gene to study its regulatory mechanism on the growth of velvet antler chondrocytes. Two RNAi recombinant plasmids named psh RNA-1 and psh RNA-2, were successfully constructed. It is used to transfect chondrocytes. Real-Time PCR and Western blot techniques were used to analyze the effect of RNAi transfection for 48 h. To study the regulation of IGF1R on the proliferation, apoptosis and the expression of m RNA and protein of antler chondrocytes, and to further explore the mechanism of antler growth regulation. The results were as follows: (1) the expression of m RNA of IGF1R gene in chondrocytes treated with psh RNA-1 and psh RNA-2 was 51% and 75%, respectively. The results showed that the effect of interfering cells was obvious. (2) CCK-8 method was used to detect the proliferation of cells after transfection for 48 h and 72 h. Compared with the negative control group, the cell proliferation of the test group showed a downward trend, especially the effect of psh RNA-1 plasmid interference was obvious (1.09 卤0.10 vs 1.16 卤0.09 (P0.01), 1.04 卤0.02 vs 1.10 卤0.06 (P0.05). Chondrocytes were transfected with 0.79 卤0.06 vs1.01 卤0.07 (P0.01). (3) for 48h. Compared with the negative control group, the cells were in G1 phase (79.47 卤0.11vs 82.27 卤0.35, P0.01),). The number of S phase cells decreased significantly (16.56 卤0.10 vs 14.82 卤0.014 (P0.05). (4) 48 h after transfection of chondrocytes. The early apoptotic level of psh RNA-1 in the experimental group and psh RNA-negative in the negative control group was (22.12 卤2.72%vs 8.56 卤0.16); The normal living cells were (62.85 卤5.97 vs, 77.39 卤4.46, P 0.05). The expression of p53 (P0.01), bcl-2 (P0.05) and bax decreased significantly (P0.01). The results showed that psh RNA-1 transfection could promote the apoptosis of chondrocytes. The results showed that IGFR could regulate the growth of velvet antler, especially the proliferation and apoptosis of antler chondrocytes. In addition, IGFR might regulate the growth and development of chondrocytes by regulating the cell cycle of chondrocytes.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S825
【引证文献】
相关会议论文 前1条
1 王欣;李光玉;李春义;;IGF1与睾酮激素对鹿角柄发生影响的研究[A];2011中国鹿业进展[C];2011年
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