力信号调控的大鼠成骨细胞力生长因子表达及其对成骨细胞生理行为的影响
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摘要
本文采用了生物力学、细胞生物学、生物化学及分子生物学等方法,研究机械拉伸对成骨细胞力生长因子表达的影响和力生长因子对成骨细胞增殖和分化以及相关基因的影响。
主要内容和结果如下:
改进了周期性基底膜拉伸应变装置。改进后该装置接种细胞量大,避免了以往实验中多次收集细胞的麻烦。该装置组装简便,应变大小可由注入储液囊的流体体积决定,频率由控制系统的开关频率调节,操作方便。
运用组织块法培养了满足实验需求的大鼠颅骨成骨细胞,并利用差时贴壁法对其进行了纯化。通过形态观察、Von Kossa染色和碱性磷酸酶(ALP)染色等方法进行了鉴定。结果表明所培养细胞具有成骨细胞的典型生物学行为,可以用于后续实验。第二代至第六代的细胞用于后续实验。
对细胞施加应变量为15%周期性拉伸刺激,作用6h、12h、24h、36h后,分别收集细胞,提取蛋白质,利用western blot法检测是否有力生长因子(MGF)表达。结果显示,在6h、24h时有少量MGF表达,12h时较多,对照组和36h未观察到。
以成骨细胞株MC3T3为对象,研究了力生长因子对细胞增殖和分化的影响。将力生长因子配成一定浓度的溶液,分别加入到接种细胞的96孔板中,用MTT法检测其对细胞增殖的影响。利用碱性磷酸酶检测试剂盒检测ALP活性,分析其对细胞分化的影响。结果显示,10ng/ml MGF明显促进成骨细胞的增殖,抑制ALP分泌。
利用RT-PCR方法分析了加入MGF后细胞中BMP-2、Cbfα-1、Col I、OCN mRNA表达的变化。结果显示,10 ng/ml MGF促进BMP-2、Cbfα-1、Col I mRNA的表达,抑制OCN mRNA的表达。
The effects of mechanical stretching on the expression of mechano growth factor and the effects of mechano growth factor on proliferation and differentiation of osteoblasts were studied through biomechanical, cell biological and molecular biology methods. The main work and conclusions were as follows:
Cyclic mechanical strain unit was improved. The improved unit can seed more cells, avoiding the trouble of gathering cells repeatedly during experiment. The assembly and disassembly of the flow chamber is very easy. The peak value and frequency could be changed according to experimental requirements. Expansion of the membrane was controlled by frequency of the electromagnetic valve and speed of the constant-flow pump.
The primary Wistar rats osteoblasts were cultured from the calvaria of new-born rats by the tissue piece culture method. The osteoblasts were purified by the difference of the adhibition time with other type of cells. It was identified by the morphology, alkaline phosphatase (ALP) staining and Von Kossa staining, and the results showed that the culture osteoblasts had typical characteristics of osteoblasts. Cells from the second to sixth passages were used for the mechanical experiments.
Cells were stretched using the cyclic-strain unit and static-strain unit at 15% and gathered 12h、24h、36h and 48h later, then the protein was isolated and analyzed by western blot to make sure whether the mechano growth factor(MGF) was expressed. The results showed that MGF was expressed at 12h and 24h.
In order to study the effect of mechano growth factor on the proliferation and differentiation of osteoblasts, the MGF was confected solution and used to cell plate seeded cells. The proliferation was measured by MTT essay and the alkaline phosphatase (ALP) activity measured by ALP kit. The results showed that 10 ng/ml MGF increased cell proliferation and inhibited differentiation.
The changes of BMP-2、cbfα-1、Col I、OCN mRNA were detected by RT-PCR. The results demonstrated that10 ng/ml MGF increased the expression of BMP-2、cbfα-1、Col I mRNA and inhibited OCN mRNA.
主要内容和结果如下:
改进了周期性基底膜拉伸应变装置。改进后该装置接种细胞量大,避免了以往实验中多次收集细胞的麻烦。该装置组装简便,应变大小可由注入储液囊的流体体积决定,频率由控制系统的开关频率调节,操作方便。
运用组织块法培养了满足实验需求的大鼠颅骨成骨细胞,并利用差时贴壁法对其进行了纯化。通过形态观察、Von Kossa染色和碱性磷酸酶(ALP)染色等方法进行了鉴定。结果表明所培养细胞具有成骨细胞的典型生物学行为,可以用于后续实验。第二代至第六代的细胞用于后续实验。
对细胞施加应变量为15%周期性拉伸刺激,作用6h、12h、24h、36h后,分别收集细胞,提取蛋白质,利用western blot法检测是否有力生长因子(MGF)表达。结果显示,在6h、24h时有少量MGF表达,12h时较多,对照组和36h未观察到。
以成骨细胞株MC3T3为对象,研究了力生长因子对细胞增殖和分化的影响。将力生长因子配成一定浓度的溶液,分别加入到接种细胞的96孔板中,用MTT法检测其对细胞增殖的影响。利用碱性磷酸酶检测试剂盒检测ALP活性,分析其对细胞分化的影响。结果显示,10ng/ml MGF明显促进成骨细胞的增殖,抑制ALP分泌。
利用RT-PCR方法分析了加入MGF后细胞中BMP-2、Cbfα-1、Col I、OCN mRNA表达的变化。结果显示,10 ng/ml MGF促进BMP-2、Cbfα-1、Col I mRNA的表达,抑制OCN mRNA的表达。
The effects of mechanical stretching on the expression of mechano growth factor and the effects of mechano growth factor on proliferation and differentiation of osteoblasts were studied through biomechanical, cell biological and molecular biology methods. The main work and conclusions were as follows:
Cyclic mechanical strain unit was improved. The improved unit can seed more cells, avoiding the trouble of gathering cells repeatedly during experiment. The assembly and disassembly of the flow chamber is very easy. The peak value and frequency could be changed according to experimental requirements. Expansion of the membrane was controlled by frequency of the electromagnetic valve and speed of the constant-flow pump.
The primary Wistar rats osteoblasts were cultured from the calvaria of new-born rats by the tissue piece culture method. The osteoblasts were purified by the difference of the adhibition time with other type of cells. It was identified by the morphology, alkaline phosphatase (ALP) staining and Von Kossa staining, and the results showed that the culture osteoblasts had typical characteristics of osteoblasts. Cells from the second to sixth passages were used for the mechanical experiments.
Cells were stretched using the cyclic-strain unit and static-strain unit at 15% and gathered 12h、24h、36h and 48h later, then the protein was isolated and analyzed by western blot to make sure whether the mechano growth factor(MGF) was expressed. The results showed that MGF was expressed at 12h and 24h.
In order to study the effect of mechano growth factor on the proliferation and differentiation of osteoblasts, the MGF was confected solution and used to cell plate seeded cells. The proliferation was measured by MTT essay and the alkaline phosphatase (ALP) activity measured by ALP kit. The results showed that 10 ng/ml MGF increased cell proliferation and inhibited differentiation.
The changes of BMP-2、cbfα-1、Col I、OCN mRNA were detected by RT-PCR. The results demonstrated that10 ng/ml MGF increased the expression of BMP-2、cbfα-1、Col I mRNA and inhibited OCN mRNA.
引文
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[3] Goldspink G. Gene expression in muscle in response to exercise [J]. Muscle Res Cell Motil, 2003, 24(2-3): 121-126.
[4] Goldspink G. Changes in muscle mass and phenotype and the expression of autocrine and systemic growth factors by muscle in response to stretch and overload [J]. Amat, 1999, 194: 323-334.
[5] Hameed M, Orrell RW, Cobbold M, et al. Expression of IGF-I splice variants in young and old human skeletal muscle after high resistance exercise [J]. Physiol, 2003, 547: 247-254.
[6] Kim SW, Lajara R, Rotwein P. Structure and function of a human insulin-like growth factor-I gene promoter [J]. Mol Endocrinol, 1991, 5(12): 1964-1972.
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