CAJ | 학술논문

目的研究放疗后海马区神经发生与小鼠放射性脑损伤认知损害的关系.方法 10 d龄C57BL/6J小鼠24只给予10 Gy 6MV X射线全脑照射,Morris水迷宫实验检测放射性脑损伤模型的建立.在照射后不同时间点(0.5、3、6、12、24、48、72 h,1、2、4、8、16、24周),取脑组织标本进行HE染色,观察海马区神经细胞的病理变化.免疫组织化学染色双皮质蛋白(DCX)、增殖细胞核抗原(PCNA)标记海马区神经发生水平,ED1标记小胶质细胞激活情况,TUNEL标记海马区神经细胞凋亡情况.采用Real-time PCR法检测脑组织TNF-α、IL-1β表达水平的变化；酶联免疫吸附法(ELISA)检测照射后不同时间点血浆TNF-α水平的变化.结果照射后急性期表现为间质水肿,炎性细胞浸润,海马区齿状回神经细胞逐渐出现变性、坏死、凋亡,慢性期间质水肿消退,炎性细胞减少,海马齿状回出现细胞再生呈极性分布.受照后各时间点,DCX阳性细胞和PCNA阳性细胞较对照组均有不同程度减少(F =4.9 ～12.5,5.2～15.7,P＜0.05),受照后ED1阳性细胞较对照组增多(F=20.8,P＜0.05).TUNEL标记凋亡神经元在放疗后6h可见,于48 h达到峰值(F=15.1,P＜0.05)；照射后0.5h,γ-H2AX焦点形成达到高峰(F=18.4,P＜0.05),后逐渐减少.照射后脑组织TNF-α和IL-1β表达较对照组均有所增加(t=16.3、12.7,P＜0.05)；血浆TNF-α照射后3h开始升高,至1周达到峰值(F=10.5,P＜0.05),随后逐渐降低.Morris水迷宫实验照射组和对照组相比,照后1、2、3d两者差异无统计学意义,但随着时间的延长,4、5、6d照射组躲避潜伏期较对照组延长(F=7.01、8.17、4.22,P＜0.05).结论 10 Gy全脑照射后,建立放射性脑损伤模型,发现海马区神经发生受到抑制和小胶质细胞激活可能是小鼠放射性脑损伤认知损害产生的原因之一.
목적 연구방료후해마구신경발생여소서방사성뇌손상인지손해적관계.방법 10 d령C57BL/6J소서24지급여10 Gy 6MV X사선전뇌조사,Morris수미궁실험검측방사성뇌손상모형적건립.재조사후불동시간점(0.5、3、6、12、24、48、72 h,1、2、4、8、16、24주),취뇌조직표본진행HE염색,관찰해마구신경세포적병리변화.면역조직화학염색쌍피질단백(DCX)、증식세포핵항원(PCNA)표기해마구신경발생수평,ED1표기소효질세포격활정황,TUNEL표기해마구신경세포조망정황.채용Real-time PCR법검측뇌조직TNF-α、IL-1β표체수평적변화；매련면역흡부법(ELISA)검측조사후불동시간점혈장TNF-α수평적변화.결과 조사후급성기표현위간질수종,염성세포침윤,해마구치상회신경세포축점출현변성、배사、조망,만성기간질수종소퇴,염성세포감소,해마치상회출현세포재생정겁성분포.수조후각시간점,DCX양성세포화PCNA양성세포교대조조균유불동정도감소(F =4.9 ～12.5,5.2～15.7,P＜0.05),수조후ED1양성세포교대조조증다(F=20.8,P＜0.05).TUNEL표기조망신경원재방료후6h가견,우48 h체도봉치(F=15.1,P＜0.05)；조사후0.5h,γ-H2AX초점형성체도고봉(F=18.4,P＜0.05),후축점감소.조사후뇌조직TNF-α화IL-1β표체교대조조균유소증가(t=16.3、12.7,P＜0.05)；혈장TNF-α조사후3h개시승고,지1주체도봉치(F=10.5,P＜0.05),수후축점강저.Morris수미궁실험조사조화대조조상비,조후1、2、3d량자차이무통계학의의,단수착시간적연장,4、5、6d조사조타피잠복기교대조조연장(F=7.01、8.17、4.22,P＜0.05).결론 10 Gy전뇌조사후,건립방사성뇌손상모형,발현해마구신경발생수도억제화소효질세포격활가능시소서방사성뇌손상인지손해산생적원인지일.
Objective To investigate the changes of hippocampal neurogenesis and cognitive dysfunction induced by cranial radiation therapy.Methods C57BL/6J mice aged 10 d were subjected to 10 Gy whole brain irradiation with 6 MV X-rays to develop irradiation-induced brain injury model.Morris water maze was designed to estimate spatial learning and memory.At different time post irradiation,brain tissue was removed to stain with hematoxylin-eosin for the pathological results.DCX and PCNA immunohistochemical staining was used to mark the level of neurogenesis in the hippocampus,and ED1immunohistochemical staining to mark the activation of microglia.The TUNEL assay was used to assess the apoptotic neuron death in situ in the hippocampus.Real-time PCR was supplied to inspect the expression of TNF-α and IL-1 β mRNA.Enzyme Linked Immunosorbent Assay (ELISA) was tested for the concentration of TNF-αt in the plasma.Results Pathological studies demonstrated that radiation could induce interstitial edema,inflammatory cell infiltration,cell degeneration,necrosis,apoptosis in the acute phase,edema subsiding,reduction of inflammatory cells,and cytothesis in the dentate gyrus of the hippocampus.IHC studies revealed that,at different time post irradiation,the number of DCX-positive cells and PCNA-positive cells decreased (F =4.9-12.5,5.2-15.7,P ＜ 0.05) but ED1-positive cells increased significantly (F =20.8,P ＜ 0.05).TUNEL-positive cells began to appear in the dentate gyrus of hippocampus 6 h post-irradiation,and its number reached to the highest level at 48 h post-irradiation (F =15.1,P ＜ 0.05).The formation of γ-H2AX foci got at the top 0.5 h post-irradiation (F =18.4,P ＜0.05) and then decreased.After irradiation,the expressions of TNF-α and IL-1β mRNA in the the irradiated group was higher than those of the control group (t =16.3,12.7,P ＜ 0.05).The concentration of TNF-α in the plasma of the irradiated group was higer than that in the control group 3 h post-irradiation,and maximized at 1 week post-irradiation (F =10.5,P ＜ 0.05).Morris water maze tests showed that the latency had no significant differences between the irradiated group and the control group at 1,2,3 d postirradiation,but the latency in the irradiated group was longer than that in the control group with a significant differences at 4,5,6 d post-irradiation (F =7.01,8.17,4.22,P ＜ 0.05).Conclusions Irradiation-induced cognitive dysfunction may be caused by microglial activation and suppression in hippocampal neurogenesis following cranial radiation therapy.