全淋巴区电子线照射治疗MRL／lpr狼疮小鼠疗效观察与机制探讨

Therapeutic efficacy and mechanism of total lymphoid irradiation with electron beam in MRL/Ipr mouse model of lupus

目的 观察全淋巴区电子线照射治疗MRL/lpr狼疮小鼠的疗效，并探讨其与调节性T细胞的关系。 方法 将18只4月龄的MRL/lpr小鼠随机分为阴性对照组、放射治疗组、地塞米松治疗组，每组6只，雌雄各半，阴性对照组予生理氯化钠溶液0.5 ml腹腔注射每日1次，连续2周；放射治疗组全身淋巴结予电子线照射，2 Gy/d，每周5次，连续2周；地塞米松治疗组予地塞米松1 mg/kg腹腔注射每日1次，连续2周，实验结束后2周测各项指标。考马斯亮蓝法测24 h尿蛋白，ELISA法检测各组小鼠ANA及抗dsDNA抗体水平，直接免疫荧光法观察各组小鼠IgG抗体在肾脏的沉积情况，PAS染色法观察各组小鼠的肾脏情况，流式细胞仪测定各组小鼠脾脏细胞中调节性T细胞所占比例。用SPSS11.5统计软件进行多样本组间单因素方差。 结果 放射治疗组小鼠24 h尿蛋白含量为（3.33 ± 0.17） mg，阴性对照组为（3.97 ± 0.33） mg，两组比较，P 〈 0.05。放射治疗组小鼠血清抗核抗体ANA水平为（275.12 ± 30.18） U/ml，抗小鼠dsDNA抗体为（455.74 ± 43.38） IU/ml；阴性对照组小鼠分别为（434.82 ± 36.63） U/ml和（956.78 ± 37.69） IU/ml，两组比较，均为P 〈 0.05。放射治疗组与地塞米松治疗组小鼠肾小球内IgG沉积较阴性对照组减少，强度较弱，放射治疗组、地塞米松治疗组两组无明显差异。PAS染色可见放射治疗组与地塞米松治疗组肾脏病变较阴性对照组减轻，放射治疗组、地塞米松治疗组两组无明显差异。流式细胞仪检测显示，放射治疗组小鼠调节性T细胞所占比例为（1.08 ± 0.52）%，地塞米松治疗组为（1.18 ± 0.60）%，阴性对照组为（0.38 ± 0.14）%，放射治疗组、地塞米松治疗组两组的调节性T细胞所占比例较阴性对照组升高（P 〈 0.05）。 结论 全淋巴区电子线照射能降低MRL/lpr狼疮小鼠尿蛋白、血清抗核抗体、双链DNA抗体水平，对肾脏病理改变亦有恢复作用，其机制可能与提高体内调节性T细胞的比例有关。

Objective To evaluate the therapeutic efficacy of total lymphoid irradiation with electronic beam in MRL/lpr mouse model of lupus, and to assess the relationship between total lymphoid irradiation and regulatory T （Treg） cells. Methods Eighteen four-month-old MRL/lpr lupus mice were randomly divided into three groups with three female mice and three male mice in each group： negative control group treated with intraperitoneal sodium chloride physiological solution 0.5 ml once daily, radiotherapy group receiving total lymphoid irradiation with the electronic beam （2 Gy per day） five times a week, and dexamethasone group treated with intraperitoneal dexamethasone 1 mg/kg once daily for two consecutive weeks. Then, Coomassie brilliant blue method was used to quantify the 24-hour urinary protein, and enzyme-linked immunosorbent assay （ELISA） to measure the serum levels of antinuclear antibody （ANA） and anti-dsDNA antibody. All the mice were sacrificed, renal tissue was obtained and subjected to direct immunofluorescence assay for the observation of IgG deposits as well as periodic acid-Schiff staining for the evaluation of renal injury, and spleen tissue was isolated and subjected to flow cytometry for the determination of proportion of Treg cells in spleen cells. Data were processed using the SPSS 11.5 software. Statistical analysis was done with one-way analysis of variance. Results Significant differences were observed between the radiotherapy group and negative control group in 24-hour urinary protein level （（3.33 ± 0.17） vs. （3.97 ± 0.33） mg, P 〈 0.05）, serum ANA antibody level （（275.12 ± 30.18） vs. （434.82 ± 36.63） U/ml, P 〈 0.05） and anti-dsDNA antibody level （（455.74 ± 43.38） vs.（956.78 ± 37.69） IU/ml, P 〈 0.05）. The glomerular deposition of IgG antibody was obviously decreased with attenuated renal injury in the radiotherapy group and dexamethasone group compared with the negative control group, but no significant difference was noted between the radiotherapy group and dexamethasone group. The proportion of Treg cells in the radiotherapy group and dexamethasone group was significantly higher than that in the negative control group （（1.08 ± 0.52）% and （1.18 ± 0.60）% vs. （0.38 ± 0.14）%, both P 〈 0.05）. Conclusions Total lymphoid irradiation with electronic beam can effectively reduce urine protein level, serum ANA and anti-dsDNA antibody level in MRL/lpr lupus mice, alleviate renal injury, likely by up-regulating the proportion of Treg cells in vivo.