低剂量X线对钛颗粒干预成骨样细胞后的影响

The effects of low dose X - ray irradiation on the MC3T3 - E1 cells co - cultured with titanium particles

目的观察低剂量电离辐射（LDI）对钛（Ti）颗粒干预成骨样细胞MC3T3-El后的影响。方法不同浓度Ti颗粒与MC3T3-E1细胞共同培养后，接受LDI干预，采用细胞计数试剂盒（CCK-8）、酶联免疫吸附试验法（ELISA）、碱性磷酸酶（ALP）试剂盒、实用荧光定量聚合酶链反应（Real—timePCR）及茜素红染色法检测MC3T3-El细胞的增殖、分化、矿化及炎性因子白细胞介素-6（IL-6）的表达。结果照射后24h，Ti颗粒各组细胞活性（0．78±0．03、0．61±0．04、0．63±0．07）与对照组（1．00±0．10）比较显著下降（P〈0．01），但LDI对细胞活性没有显著影响。LDI对MC3T3-El细胞的活性在照射48h和72h表现出抑制效应（P〈0．01）。照射后7d，照射各组Ⅰ型前胶原羧基端肽（PICP）的浓度[（3．97±1．00）、（3．70±2．00）、（3．78±0．39）、（2．22±1．53）ng／g]与假照射各组[（3．32±0．84）、（2．47±0．94）、（2．51±0．50）、（1．39±0．40）．g／g]比较均升高，差异有统计学意义（P〈0．05）；ALP结果显示照射后14d，各个Ti颗粒浓度组ALP活性[（0．0123±0．0002）、（0．0117±0．0006）、（0．0115±0．0006）、（0．0120±0．0002）μmol／（min·μg）]均高于似照射绢[（0．0108±0．0005）、（0．0091±0．0005）、（0．0095±0．0006）、（0．0101±0．0002）μmol／（min·μg）]，差异均有统计学意义（P〈0．05）。Real—time PCR结果显示LDI可有效降低Ti颗粒刺激后MC3T3-E1细胞IL-6的表达（0．86±0．12，P〈0．05）。茜素红染色结果显示在Ti颗粒存在的情况下LDI仍具有促进矿化的效应。结论LDI早期会加重Ti颗粒对成骨样细胞增殖的影响，但在Ti颗粒存在的情况下仍具有促进成骨样细胞分化、矿化的作用，同时可下调Ti颗粒刺激后成骨样细胞IL-6的表达。

Objective To test low dose irradiation （LDI） effect nn MC3T3 -E1 cells co-cul- tured with different concentration of titanium （Ti） particles. Methods MC3T3 - E1 cell was cultured with different coneentrations of Ti particles, then were irradiated with 0 and 0. 5 Gy X - rays. Cell viability was determined with the cell counting kit - 8 （ CCK - 8 ） assay. The procollagen 1C - terminal propeptide （PICP） concentration in the supernatant was determined by enzyme -linked immuno sorbent assay （ELISA） kit. Assessed alkaline phosphatase （ALP） activity by the ALP kit. The interleukin -6 （IL -6） expression were compared by real - time quantitative polymerase chain reaction （ Real - time PCR） and on day 14 assessed matrix mineralization by alizarin red stain. Results The viability of MC3T3 - E1 cell in the grous cultured with Ti patricles 24 h after irradiation （0. 78 ±0. 03, 0. 61 ±0. 04, 0. 63 ±0. 07） all were significantly inhibited compared with 0 Gy- Ti-0 group （1.00 ± 0. 10, P 〈 0. 01 ）. After 48 h and 72 h, the cell viability was also significantly inhibited by LDI. The concentration of PICP concentration in the 0Gy-Ti-100 group [（1.39±0.40） ng/g] was still lower than that of the 0 Gy-Ti-0 group [ （ 3.32 ± 0. 84 ） ng/g, P 〈 0. 05 ] , and X - ray irradiation significantly elevated the PICP concentration [（3.97±1.00）, （3.70±_2.00）, （3.78±0.39）, （2.22±1.53） ng/glcommapredwith0 Gy groups [（3.32±0.84）, （2.47±0.94）, （2.51±0.50）, （1.39±0.40） ng/g]at 7 dpost-irradiation （P〈 0. 05 ）. X -ray irradiation significantly elevated the ALP activity of each groups [ （0. 012 3 ± 0. 000 2 ）, （0.011 7±0.0006）, （0.011 5±0.0006）, （0.0120±0.0002） p.mol/（min·μg）]compared with 0 Gy groups [ （0.0108± 0.0005）, （0.009 1± 0.0005）, （0.0095± 0.0006）, （0.010 1± 0. 000 2 ）μmol/（min. μg） ] at 14 d post - irradiation （ P 〈 0. 05 ）. The expression of IL - 6 was significantly inhibited by X -ray irradiation （0. 86 ± 0. 12, P 〈 0. 05 ）. The alizarin red staining and semi - quantitative analysis showed that in the presence of Ti particles, LDI can still promote calcification of MC3T3 - E1 cells. Conclusion LDI significantly promote the functions of MC3T3 - E1 including increased the ALP activity and de novo synthesis of type I collagen in the presence of Ti particles. Further- more, LDI can significantly inhibit the IL - 6 expression in the presence of Ti particles.