γ电离辐射对破骨细胞代谢的效应及机理研究

发布时间：2018-03-16 15:16

本文选题：电离辐射　切入点：破骨细胞　出处：《苏州大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：研究γ电离辐射对破骨细胞代谢的效应，并掌握相应的辐照剂量，初步探究其效应的机制。有利于进一步了解电离辐射改变骨代谢的途径，为防治电离辐射所致的骨损害提供新的思路。方法：本研究主要内容包括四部分。第一部分研究2Gy γ射线全身单次辐照对小鼠体内破骨细胞代谢和骨吸收的影响。C57BL/6雄性小鼠随机分为对照组和辐照组。双抗体夹心ELISA法测定辐照后不同时间点小鼠血清中骨代谢相关指标TRAP-5b、sRANKL、CTX-1和TNF-α；采用甲基百里香酚蓝比色法测定血清中Ca~(2+)浓度；TRAP染色骨病理切片观察破骨细胞形态变化。第二部分建立体外破骨细胞培养模型。通过使用小鼠重组sRANKL和转移骨髓基质细胞条件培养液两种方法分别诱导破骨前体细胞株RAW264.7和骨髓单核细胞为破骨细胞。第三部分研究γ射线对RAW264.7细胞分化为破骨细胞的影响。在sRANKL诱导RAW264.7细胞分化为破骨细胞的过程中给予γ射线辐照，计数形成的破骨细胞数量和检测破骨细胞代谢指标TRAP-5b，并用流式细胞术检测辐照后细胞内ROS和Ca~(2+)含量；第四部分研究γ射线对骨髓基质细胞调控破骨细胞代谢的影响。不同剂量的γ射线全身单次照射C57BL/6雄性小鼠后第1、2、3和7天，使用一步法RT-PCR检测骨髓细胞中RANKL mRNA和OPG mRNA。给予体外培养的骨髓基质细胞γ射线照射，ELISA法和共聚焦定量法检测其RANKL蛋白的表达，并使用其条件培养液诱导骨髓单核细胞分化为破骨细胞。结果：各部分对应的试验结果如下：（1）辐照后第4天，与对照组相比，辐照组小鼠血清中TRAP-5b、CTX-1、sRANKL和TNF-α含量分别升高了37.30%、47.14%、19.55%和17.67%（P＜0.05），辐照组小鼠骨内破骨细胞呈代谢活化相。辐照后第90天，辐照组小鼠血清TRAP-5b和CTX-1水平仍然要高于对照组，差异具有统计学意义（P0.01）。（2）两种方法均成功地诱导出由多个细胞互相融合形成的TRAP阳性多核破骨细胞。加入外源性的sRANKL刺激RAW264.7诱导出的破骨细胞比骨髓基质细胞条件培养液刺激骨髓单核细胞形成的破骨细胞形态较大，数量也较多。（3）1、2、4Gy辐照组破骨细胞数量与对照组相比增加，差异具有统计学意义（P0.001）；破骨细胞培养液中TRAP-5b的含量随着辐照剂量的增加而升高，两者呈正向直线线性相关（r~2=0.919，P=0.041）。辐照后，细胞内ROS水平和Ca~(2+)水平均升高。（4）辐照小鼠后3天，2、4和6Gy组的骨髓细胞RANKL/OPG mRNA比值与对照组相比明显升高，差异具有统计学意义（P0.01），RANKL/OPG mRNA比值的改变依赖RANKL mRNA的变化。γ射线辐照后，骨髓基质细胞的RANKL蛋白表达在辐照后早期增加。RANKL的表达升高具有剂量依赖性和时间依赖性，辐照剂量越大，RANKL升高得越早。辐照组骨髓基质细胞条件培养液诱导形成的破骨细胞数量和TRAP-5b水平比对照组高，辐照剂量为2Gy时尤为明显。结论：2Gy的γ射线全身单次辐照可促进小鼠破骨细胞代谢，骨吸收增加，破骨细胞的代谢增强是电离辐照导致的骨损害的重要原因之一。γ射线促进破骨细胞代谢活化的机制包括如下： 1. γ射线通过升高破骨细胞活化的信号分子ROS和Ca~(2+)，以剂量依赖的方式促进破骨前体细胞分化形成破骨细胞； 2. γ射线以剂量和时间依赖的方式早期促进骨髓基质细胞表达RANKL，辐照剂量越大，RANKL升高越早，，进而间接促进破骨细胞代谢活化，体外2Gy辐照时促进作用最为明显； 3. γ射线辐照增加的TNF-α等炎症因子，可能协同RANKL或直接促进破骨细胞代谢活化。
[Abstract]:Objective: To study the effect of gamma irradiation on the metabolism of osteoclasts, and to grasp the corresponding radiation dose, and preliminarily explore the mechanism of its effect. It is helpful for us to further understand the way of ionizing radiation to alter bone metabolism and provide new ideas for preventing and treating bone damage caused by ionizing radiation.
Methods: the main content of this study includes four parts. The first part of the study of 2Gy gamma ray irradiation effects on systemic single mice bone breaking metabolism and cellular uptake of.C57BL/6 male mice were randomly divided into control group and irradiation group. The determination of TRAP-5b serum markers of bone metabolism in mice at different time points after irradiation by double antibody sandwich ELISA sRANKL TNF-, CTX-1 and alpha; using methylthymol blue colorimetric method for the determination of Ca~ in serum (2+) concentration; bone pathological section to observe osteoclasts changes with TRAP staining. The second part is the establishment of in vitro osteoclast culture model. Through the use of recombinant mouse sRANKL and metastasis of bone marrow stromal cell conditioned medium induced by two methods were broken bone marrow mononuclear cells and RAW264.7 cells into osteoclasts. The third part of the study of gamma ray irradiation on the differentiation of RAW264.7 cells into osteoclast induced by RAW in sRANKL. 264.7 cell differentiation during osteoclast to gamma ray irradiation, the number of osteoclasts and counting the formation of osteoclast metabolism index of TRAP-5b were detected by flow cytometry after irradiation of intracellular ROS and Ca~ (2+) content; the fourth part is the study of gamma ray irradiation on bone marrow stromal cells regulate osteoclast metabolism. Different doses of gamma ray whole body single irradiation C57BL/6 male mice after 1,2,3 and 7 days, using one step RT-PCR for detection of RANKL in bone marrow cells of mRNA and OPG mRNA. with bone marrow stromal cells irradiated in vitro, the protein expression of RANKL was detected by ELISA method and confocal quantitative method, and its use conditions culture medium induced bone marrow mononuclear cells differentiated into osteoclasts.
Results: the results of the corresponding tests were as follows:
(1) fourth days after irradiation, compared with the control group, serum TRAP-5b, irradiated group mice CTX-1, sRANKL and TNF- were increased by 37.30%, 47.14%, 19.55% and 17.67% (P < 0.05), irradiated group mice bone osteoclasts showed metabolic activation. Ninetieth days after irradiation, irradiation in serum of mice TRAP-5b and CTX-1 levels are higher than the control group, the difference was statistically significant (P0.01).
(2) the two methods were successfully induced by multiple cells fused with each other to form TRAP positive multinucleated cells. Exogenous sRANKL stimulation of cultured bone marrow mononuclear cells to stimulate the formation of osteoclasts is larger than the conditions of bone marrow stromal cells of osteoclasts induced by RAW264.7, the number is more.
(3) 1,2,4Gy irradiation group the number of osteoclasts increased compared with the control group, the difference was statistically significant (P0.001); osteoclasts cultured in liquid TRAP-5b increased with the increase of radiation dose, showed a positive linear correlation between them (r~2=0.919, P=0.041). After irradiation, the level of ROS and Ca~ cells (2+) levels were elevated.
(4) 3 days after irradiation in mice, 2,4 and 6Gy group the bone marrow cells of RANKL/OPG mRNA ratio increased significantly compared with the control group, the difference was statistically significant (P0.01). The change of RANKL/OPG mRNA ratio change on RANKL mRNA. After irradiation, the expression of bone marrow stromal cells of RANKL in the early stage after irradiation increased.RANKL the expression increased with dose and time dependent manner, the higher the irradiation dose, RANKL increased sooner. Fluid induced the formation of osteoclast number and the level of TRAP-5b was higher than that of the control group irradiated bone marrow stromal cells cultured, irradiation at the dose of 2Gy is obvious.
Conclusion: the whole body single irradiation of 2Gy can promote the metabolism of rat osteoclasts, increase bone resorption and enhance the metabolism of osteoclasts. It is one of the important reasons for bone damage induced by ionizing radiation. The mechanism of promoting the activation of osteoclasts by gamma rays is as follows:
1. gamma rays can promote osteoclast to form osteoclasts in a dose dependent manner by increasing the activation of osteoclasts ROS and Ca~ (2+).
2. gamma rays promoted RANKL expression in bone marrow stromal cells in a dose and time dependent manner. The higher the radiation dose, the earlier the RANKL increased, which indirectly promoted the metabolic activation of osteoclasts. The effect of 2Gy irradiation was most obvious in vitro.
3. gamma ray irradiation increased TNF- alpha and other inflammatory factors, which may be synergistic with RANKL or direct the activation of osteoclast metabolism.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R144

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