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

English Keywords:Degeneration of the intervertebral disc  Rat  Nucleus pulposus cells  X-Ray  Cell senescence

English Abstract:

  【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.