力生长因子对成骨细胞生理调控作用的研究

发布时间：2018-02-27 18:04

本文关键词： 成骨细胞力生长因子增殖和分化机械拉伸 SAGE　出处：《重庆大学》2010年硕士论文　论文类型：学位论文

【摘要】： 力生长因子(mechano-growth factor, MGF)是胰岛素样生长因子-1(insuin like growth factor-1, )基因的一种剪接变异体。该生长因子响应力学刺激而表达,现有研究表明其具有促进肌肉肥大、心肌保护以及神经修复等作用。在拉伸刺激成骨细胞时亦可发现MGF mRNA的高表达,这提示MGF也是骨组织的力效应分子,能对成骨细胞的功能产生影响。但是MGF对骨组织的影响机制还不是很清楚,所以,本研究构建了含有MGF基因的重组腺病毒载体,通过基因转染、抗体受体阻断研究MGF对成骨细胞增殖和分化行为的影响及可能的受体通道。另一方面,为了探究在基因水平上力刺激对成骨细胞的影响,本实验采用长标签基因表达系列分析法(long serial analysis of gene expression,LongSAGE)方法分析了拉伸刺激组与静止对照组的基因表达差异及差异基因的表达丰度,以寻找特殊应力环境下的差异表达基因。主要内容为: 1、构建含有MGF基因的重组腺病毒载体。 2、大鼠原代成骨细胞的培养、纯化、鉴定。 3、重组腺病毒载体感染3~6代成骨细胞,研究MGF对成骨细胞生理生化行为的影响。 4、介导MGF调控细胞生理功能的受体通道的研究。 5、对成骨细胞施以拉伸刺激,拉伸组施加应变为15%,频率为0.5Hz的周期性拉伸24h,对照组静止培养。收集细胞,提取RNA,用LongSAGE方法分析拉伸刺激对成骨细胞基因表达差异及丰度的影响。主要结果为: 1、构建了携带MGF基因的重组腺病毒载体,滴度可达8.5×108 pfu/mL。 2、培养了具有典型生物学行为的原代成骨细胞,经过纯化和鉴定,符合实验要求。取3~6代细胞用于后续实验。 3、重组腺病毒载体感染成骨细胞并培养一段时间,MTT比色法分析结果表明MGF对成骨细胞的增殖具有促进作用。碱性磷酸酶检测试剂盒(alkaline phosphatase,ALP)检测ALP活性结果表明MGF能抑制ALP的分泌,提示MGF能抑制成骨细胞的分化。 4、受体是介导MGF调控细胞生理功能的受体通道之一,此外,细胞中还存在其他介导MGF调控的受体通道。 5、构建LongSAGE标签库分析周期性拉伸刺激对成骨细胞基因表达差异的影响,发现172个标签在拉伸组和对照组细胞中的表达具有显著性差异。其中周期性拉伸刺激上调的标签有100个,下调的标签72个。
[Abstract]:MGF is a splicing variant of insulin-like growth factor like like growth factor-1, which is expressed in response to mechanical stimulation and has been shown to promote muscle hypertrophy. The high expression of MGF mRNA was also found in osteoblasts stimulated by tension, suggesting that MGF is also a force effector molecule in bone tissue. However, the mechanism of the effect of MGF on bone tissue is not clear. Therefore, a recombinant adenovirus vector containing MGF gene was constructed and transfected by gene. To investigate the effects of MGF on the proliferation and differentiation of osteoblasts and the possible receptor channels in order to investigate the effects of stress stimulation on osteoblasts at the gene level, In this experiment, the long serial analysis of gene expression long SAGEA method was used to analyze the difference of gene expression and the expression abundance of the differentially expressed genes between the tensile and static control groups, in order to find the differentially expressed genes under the special stress environment. The main contents are:. 1. The recombinant adenovirus vector containing MGF gene was constructed. 2. Culture, purification and identification of rat primary osteoblasts. 3. The effect of MGF on physiological and biochemical behavior of osteoblasts was studied. 4. The study of receptor channel mediated by MGF to regulate cell physiological function. 5. The osteoblasts were stimulated by stretching. The cells in the stretching group were subjected to 15 strain and 0.5 Hz periodic stretching for 24 h. The cells were collected, RNAs were extracted, and the effects of stretch stimulation on the gene expression and abundance of osteoblasts were analyzed by LongSAGE method. The main results are:. 1.Recombinant adenovirus vector carrying MGF gene was constructed and its titer was 8.5 脳 108pfu-mL. 2. Primary osteoblasts with typical biological behavior were cultured and purified and identified to meet the requirements of the experiment. 3. The recombinant adenovirus vector was infected with osteoblasts and cultured for a period of time. The results of MTT colorimetric analysis showed that MGF could promote the proliferation of osteoblasts, and alkaline phosphatase assay kit (ALP) showed that MGF could inhibit the secretion of ALP. These results suggest that MGF can inhibit the differentiation of osteoblasts. 4. Receptor is one of the receptor channels mediating the regulation of cellular physiological function by MGF. In addition, there are other receptor channels that mediate the regulation of MGF in cells. 5. LongSAGE tag library was constructed to analyze the effect of cyclic stretch stimulation on gene expression of osteoblasts. It was found that there were significant differences in the expression of 172 tags between stretch group and control group, among which 100 tags were up-regulated by cyclic stretch stimulation. The downgraded labels were 72.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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