X线诱导大鼠椎间盘髓核细胞体外衰老模型的构建及评价

Construction and evaluation of X-ray induced senescence model of rat intervertebral disc nucleus pulposus cells in vitro

中文关键词:  椎间盘退变  大鼠  髓核细胞  X线  细胞衰老

英文关键词:Degeneration of the intervertebral disc  Rat  Nucleus pulposus cells  X-Ray  Cell senescence

中文摘要:

  【摘要】 目的：建立一种由X线诱导的大鼠椎间盘髓核细胞体外衰老模型并对其进行验证评价。方法：选取12只清洁级成熟SD大鼠，取其腰段椎间盘髓核做体外细胞培养，将培养好的髓核细胞按照接受X线照射剂量随机分为0Gy组（对照组）、5Gy组、10Gy组、15Gy组。随后通过细胞形态学观察、β-半乳糖苷酶（SA-β-Gal）染色、细胞增殖检测（cell counting kit-8，CCK-8）、流式细胞术（flow cytometry，FCM）、聚合酶链式反应（polymerase chain reaction，PCR）检测、免疫荧光（immunofluorescence，IF）检测、蛋白印迹实验（Western blot，WB）、1，9-二甲基亚甲基蓝（1，9-dirnethyl-methylene blue，DMMB）比色法检测等实验方法，观察比较不同组别髓核细胞衰老程度和增殖情况，检测衰老相关基因、蛋白和相关因子的表达情况。结果：随着接受X线辐射剂量的增大，髓核细胞在形态学上逐渐表现出典型的衰老特征，并且衰老程度与射线剂量呈正相关，SA-β-Gal测定显示对照组、5Gy组、10Gy组、15Gy组髓核细胞阳性百分比分别是15％、41％、88％和77％。CCK-8实验显示随着辐射剂量的增加，髓核细胞增殖率逐渐降低，第5天10Gy组髓核细胞恢复增殖，而15Gy组髓核细胞仍无增殖表现，FCM证实其细胞仍存在活性，而当辐射剂量提升至20Gy，髓核细胞早期凋亡率骤增。IF及PCR检测均显示衰老基因p16、p21的表达随着髓核细胞接受辐射剂量的增加而逐渐增强，同时聚集蛋白聚糖碎片（aggrecan fragmentation）在WB实验中的表达也逐渐增强。以上实验结果提示15Gy为适宜辐射剂量，再次将15Gy组与对照组比较，CCK-8显示第9天15Gy组髓核细胞依然无增殖表现，PCR检测aggrecan fragmentation的表达15Gy组明显高于对照组（P<0.05）。基质金属蛋白酶MMP-1、MMP-3以及炎症因子IL-6、IL-8在PCR及IF中的表达，15Gy组也明显高于对照组（P<0.05）。结论：X线能诱导体外平面培养的大鼠髓核细胞衰老，接受15Gy辐射量的髓核细胞衰老程度明显且稳定，可作为理想的髓核细胞衰老模型。

英文摘要:

  【Abstract】 Objectives: To establish an X-ray induced senescence model of rat intervertebral disc nucleus pulposus cells in vitro and to evaluate it. Methods: 12 clean-grade mature SD rats were selected to take the lumbar intervertebral disc nucleus pulposus for cell culture in vitro, and the cultured nucleus pulposus cells were randomly divided into 0 Gy group(Control group), 5Gy group, 10Gy group and 15Gy group on the basis of different X-ray doses of irridiation. Cell morphology observation, SA-β-Gal staining, cell counting kit-8(CCK-8), flow cytometry(FCM), polymerase chain reaction(PCR), immunofluorescence(IF) technique, Western blot(WB) and 1, 9-dirnethyl-methylene blue(DMMB) colorimetric method were used to observe and compare the senescence and proliferation of different groups of nucleus pulposum cells. The expression of age-related genes, proteins and related factors were detected. Results: The nucleus pulposus cells gradually showed typical characteristics of senescence in morphology with the increase of X-ray radiation dose, and the degree of aging was positively correlated with radiation dose. SA-β-Gal assay showed that the positive percentages of nucleus pulposus cells in 0Gy, 5Gy, 10Gy and 15Gy groups were 15%, 41%, 88% and 77%, respectively. CCK-8 experiment showed on the 5th day, the proliferation of nucleus pulposus cells in the 10Gy group resumed, while that in the 15Gy group still showed no proliferation. FCM confirmed that the cells were still active, and when the radiation dose was increased to 20Gy, the early apoptosis rate of nucleus pulposus cells increased sharply. Both IF and PCR showed that the expression of aging genes p16 and p21 increased with the increase of radiation dose, and the expression of aggrecan fragmentation was also increased in WB assay. The above experimental results suggested that 15Gy was the appropriate radiation dose. When the 15Gy group was compared with the control group again, CCK-8 showed no proliferation of nucleus pulposus cells in the 15Gy group on day 9, and the PCR showed significantly higher expression of aggrecan fragmentation in the 15Gy group than the control group(P<0.05). The expression of MMP-1, MMP-3 and inflammatory factors IL-6 and IL-8 in PCR and IF were also significantly higher in 15Gy group than in the control group(P<0.05). Conclusions: X-ray can induce senescence of the rat nucleus pulposus cells cultured in vitro, and the senescence degree of nucleus pulposus cells receiving 15Gy radiation is obvious and stable, which can be used as an ideal model of nucleus pulposus cell senescence.