电离辐射对MC3T3-E1细胞诱导的成骨细胞增殖分化及Notch信号通路的影响

发布时间：2018-06-25 01:01

本文选题：Notch信号通路 + 放射治疗　；参考：《北京协和医学院》2013年硕士论文

【摘要】：目的：放射治疗是一种常用的治疗癌症的方法,但是同时放射治疗也会对骨组织产生多种副作用。骨的重建是一个持续的动态过程,其中成骨细胞负责骨的生成,而破骨细胞进行骨吸收。成骨细胞和破骨细胞在数量和功能上的平衡对维持正常的骨代谢具有重要作用。目前,放射治疗对成骨细胞的分化和功能影响的分子机制尚不清楚。Notch信号通路是一种高度保守的信号通路,其对成骨细胞的增殖和分化有重要影响。研究辐射对成骨细胞中Notch信号通路的影响对于有效的预防和治疗由放射治疗引起的骨折和骨质疏松症有重要的意义。方法：本研究采用成骨细胞系MC3T3-E1为模型,在成骨细胞分化培养基(Osteoblast differentiation medium, OBDM)中培养8d后,分化为成骨细胞前体细胞；在成骨细胞分化培养基中培养18d后,分化成为成骨细胞。将MC3T3-E1细胞、其诱导形成的成骨细胞前体细胞和其诱导形成的成骨细胞经2和4Gy剂量的电离辐射后,通过MTT方法检测2和4Gy剂量的电离辐射对上述三组细胞增殖的影响,通过实时定量PCR和Western blot方法检测2和4Gy剂量的电离辐射对Notch信号通路及成骨细胞中重要的细胞因子的mRNA水平和蛋白表达水平的影响。结果：在2和4Gy剂量的电离辐射条件下,MC3T3-E1细胞和成骨细胞前体细胞的相对存活率降低,而成骨细胞的相对存活率仅在2Gy剂量的电离辐射条件下明显降低；Notch信号通路受体Notch1、Notch2、Notch3和Notch4在不同剂量的电离辐射条件下受到的影响不同；在4Gy的电离辐射的条件下Hes1在MC3T3-E1细胞和成骨细胞中的表达水平升高,而MC3T3-E1细胞和成骨细胞的增殖分化能力降低；4Gy剂量的电离辐射引起Runx2和ALP在成骨细胞的表达降低,而2Gy的电离辐射条件下,Runx2在成骨细胞中的表达水平降低,ALP的表达水平却略有升高；成骨细胞前体细胞细胞在接受4Gy剂量的电离辐射后,RANKL和M-CSF的基因表达水平升高,在成骨细胞中,RANKL和M-CSF在成骨细胞中的表达水平升高,OPG在成骨细胞中的表达水平降低。结论：电离辐射之所以能够抑制骨的发育和愈合,有可能是通过抑制成骨细胞及其前体细胞的增殖和分化造成的。电离辐射影响下,成骨细胞前体细胞和成骨细胞中Notch1的表达与成骨细胞分化的抑制之间没有直接联系。电离辐射所导致的骨损伤可能是由于Notch1-4这四种受体共同作用的结果。4Gy剂量的电离辐射通过激活Notch信号通路,诱导Hes1在成骨细胞中的表达升高,抑制成骨细胞分化。2Gy电离辐射条件下ALP在成骨细胞中的表达与Runx2无关。成骨细胞前体细胞和成骨细胞在接受4Gy电离辐射照射后对破骨细胞的增殖和分化具有促进作用,这也可能是电离辐射所导致的骨吸收增强的原因之一。目前为止,电离辐射对成骨细胞增殖和分化的影响尚未进行全面而深入的研究。Notch信号通路对多种细胞的增殖和分化具有调节作用,需要进一步的研究。
[Abstract]:Objective : Radiotherapy is a commonly used method for the treatment of cancer . However , the bone remodeling is a continuous dynamic process . Osteoblasts are responsible for the formation of bone . Osteoblasts are responsible for bone resorption . The molecular mechanism of bone resorption is not clear . Notch signaling pathway is a highly conserved signal pathway which has important influence on the proliferation and differentiation of osteoblasts . The effect of radiation on Notch signaling pathway in osteoblasts is of great significance for effective prevention and treatment of fractures and osteoporosis caused by radiotherapy .

Methods : Osteoblasts were cultured in osteoblast differentiation medium ( OBDM ) for 8 days and differentiated into osteoblast precursors .
MC3T3 - E1 cells were cultured for 18 days and differentiated into osteoblasts . MC3T3 - E1 cells were induced by 2 and 4 Gy doses of ionizing radiation . The effects of ionizing radiation doses of 2 and 4 Gy on the proliferation of the above three groups were detected by MTT assay . The effects of ionizing radiation doses of 2 and 4 Gy on the mRNA levels and protein expression levels of the important cytokines in the Notch signaling pathway and osteoblasts were detected by real - time quantitative PCR and Western blot .

Results : The relative survival rate of MC3T3 - E1 cells and osteoblasts decreased significantly at doses of 2 and 4 Gy , while the relative survival rate of osteoblasts decreased significantly at 2Gy dose of ionizing radiation .
Notch4 , Notch2 , Notch3 and Notch4 of Notch signaling pathway were affected by different doses of ionizing radiation .
The expression level of Hes1 in MC3T3 - E1 cells and osteoblasts was increased with 4 Gy of ionizing radiation , while the proliferation and differentiation of MC3T3 - E1 cells and osteoblasts decreased ;
The expression level of Runx2 and ALP in osteoblasts decreased and the level of expression of ALP decreased slightly .
After receiving the 4 Gy dose of ionizing radiation , the expression levels of RANKL and M - CSF increased , and in osteoblasts , the level of expression of RANKL and M - CSF in osteoblasts was increased , and OPG expression levels in osteoblasts were reduced .

Conclusion : The effect of ionizing radiation on the proliferation and differentiation of osteoblasts is not directly related to the proliferation and differentiation of osteoblasts . The effect of ionizing radiation on proliferation and differentiation of osteoblasts is not related to the proliferation and differentiation of osteoblasts .
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R730.55

【共引文献】