【作者】 张晴；

【导师】 董静；

【作者基本信息】 青岛大学 ， 特种医学， 2023， 硕士

【摘要】 目的:(1)建立四种不同体重指数(Lee’s指数)小鼠模型:依次为正常(对照组,Wide type,WT)、偏瘦(Thin)、微胖(Overweight,OW)、肥胖(Obesity,Ob);(2)探究头颈部放射对不同体重指数小鼠血浆甘油三酯及游离脂肪酸含量的影响;(3)探究头颈部放射对不同体重指数小鼠下丘脑室旁核中促食欲神经肽刺鼠相关蛋白表达量的影响;(4)探究头颈部放射对不同体重指数小鼠糖代谢的影响;(5)探究头颈部放射对不同体重指数小鼠脂代谢的影响。方法:(1)4周龄的雄性C57BL/6小鼠适应环境一周后建立四组不同体重指数的小鼠:对照组喂食普通饮食(normal diet,ND)——WT,饮食时间不受限制;偏瘦组喂食普通饮食(ND)——Thin,饮食时间受限,采取八小时饮食法(晚上十时到次日早上六时进食);微胖组喂食高脂饮食(high fat diet,HFD-D12492)——OW,饮食时间受限,采取八小时饮食法(晚上十时到次日早上六时进食);肥胖组喂食高脂饮食(HFD-D12492)——Ob,饮食时间不受限制。(2)造模成功后,于第43天上午九时从每组中选取相应数量小鼠进行一次性6Gy(其光子能量为6MV,剂量率为2.3Gy/min)的头颈部放射(Irradiation,IR)干预。(3)通过酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测小鼠血浆中甘油三酯(Triglyceride,TG)和游离脂肪酸(free fatty acids,FFA)水平。(4)通过免疫荧光技术检测小鼠下丘脑室旁核(Paraventricular nucleus,PVN)中促食欲神经肽刺鼠相关蛋白(Agouti-related peptide,Ag RP)表达量。(5)糖代谢相关检测:通过尾静脉采血测空腹血糖,腹腔注射葡萄糖耐量实验(Intraperitoneal Glucose Tolerance Test,IPGTT)评估小鼠的糖耐量。(6)脂代谢相关检测:通过油红O(Oil red O)染色实验观察小鼠肝脏组织中脂滴的含量;通过苏木素-伊红(Hematoxylin-eosin staining,HE)染色统计小鼠棕色脂肪组织(brown adipose tissue,BAT)和白色脂肪组织(white adipose tissue,WAT)的细胞数目;通过免疫蛋白印记法检测小鼠棕色脂肪和白色脂肪中UCP1的表达水平。结果:1.四种不同Lee’s指数小鼠模型造模成功:(1)Lee’s指数(3√[(体重(g)×1000)÷鼻肛门长度(cm)]):对照组(13.72±0.25)、偏瘦组(13.45±0.35)、微胖组(13.99±0.17)、肥胖组(14.26±0.12),四组之间均出现明显统计学差异。(2)体重(g):对照组(23.93±1.37)、偏瘦组(22.25±2.11)、微胖组(24.60±1.39)、肥胖组(26.49±0.69),与对照组相比,偏瘦组和肥胖组出现明显统计学差异。2.头颈部放射使偏瘦组和肥胖组小鼠血浆中甘油三酯含量升高:(1)头颈部放射干预后,与接受放射干预对照组相比,偏瘦组(P<0.01)和肥胖组(P<0.05)甘油三酯(ng/L)含量升高;微胖组无差异(P>0.05)。(2)头颈部放射干预后,四组游离脂肪酸(umol/L)含量升高。3.头颈部放射使肥胖组小鼠下丘脑PVN核团中Ag RP表达量升高:与接受放射干预对照组小鼠相比,接受放射肥胖组PVN核团中Ag RP表达量较高(P<0.05);而偏瘦组和微胖组与其没有统计学差异(P>0.05)。4.关于糖代谢,头颈部放射使偏瘦组和肥胖组小鼠空腹血糖升高并且引起摄食量变化:(1)与接受放射干预对照组(IR-WT)相比,头颈部放射使接受放射干预的肥胖组(IR-Ob)空腹血糖(mmol/L)升高(P<0.05),偏瘦组(IR-Thin)有升高的趋势,而接受放射干预的微胖组(IR-OW)空腹血糖与其无统计学差异。(2)与接受放射干预的对照组(IR-WT)相比,头颈部放射使接受放射干预偏瘦组(IR-Thin)、微胖组(IR-OW)和肥胖组(IR-Ob)夜间累积摄食的质量(g)减少。5.关于脂代谢,头颈部放射使肥胖组小鼠肝脏脂滴增多,使偏瘦组小鼠单位面积内白色脂肪细胞数目减少,使四组小鼠棕色脂肪和白色脂肪中UCP1表达量下降:(1)肝脏油红O:接受放射干预后,肥胖组与对照组、微胖组相比,小鼠肝脏中脂滴较多(P<0.05)。(2)头颈部放射对棕色脂肪的影响:头颈部放射干预后,微胖组和肥胖组相较于对照组UCP1表达量较高(P<0.05)。与配对喂养的未放射组相比,接受放射的四组UCP1表达量都有下降趋势,但无统计学意义(P>0.05)。(3)头颈部放射对白色脂肪的影响:与接受放射干预对照组相比,头颈部放射使偏瘦组小鼠单位面积内细胞数目减少(P<0.05),细胞体积增大;微胖组和肥胖组与其未有明显统计学差异(P>0.05)。头颈部放射干预后,四组小鼠UCP1表达量无统计学差异(P>0.05)。但在未接受放射组别中,微胖组中的UCP1表达量较高(P<0.05)。结论:本研究表明,不同体重指数小鼠受头颈部放射后所引起机体的糖脂代谢变化不同。相对于对照组小鼠,肥胖组和偏瘦组小鼠受电离辐射后空腹血糖升高、糖耐量受损,而微胖组小鼠未出现上述变化。且电离辐射会使肥胖组和偏瘦组小鼠血浆中甘油三酯水平升高,使肥胖组小鼠肝脏中脂滴增多,而微胖组皆未出现差异。白色脂肪UCP1在未受电离辐射的微胖组小鼠中表达增加,进而促进褐变,提高机体代谢能力。综上推测,稍多脂肪含量的机体可通过增加解偶联蛋白1的表达增强代谢平衡能力,继而抵御电离辐射带来的代谢紊乱危害。更多还原

【Abstract】 Objective:(1)To establish mouse model with four different Lee’s index:Thin,WT(wide type),OW(overweight),and Ob(obesity);(2)To investigate the effects of head and neck radiation on the content of triglycerides and free fatty acids in the plasma of mice with different Lee’s index;(3)To investigate the effect of head and neck radiation on the expression of appetite-promoting neuropeptide agouti-associated protein in the paraventricular nucleus of mice with different Lee’s index;(4)To investigate the effects of head and neck radiation on glucose metabolism in mice with different Lee’s index;(5)To investigate the effects of head and neck radiation on lipid metabolism in mice with different Lee’s index.Methods:(1)4-week-old male C57BL/6 mice were randomly divided into four groups after one week of normal diet acclimatization,and the following methods were used:the Thin group was fed with a normal diet but with restricted dietary time and an eight-hour diet(eating from 10 pm to 6 am);the WT group was fed with a normal diet with free eating time;the OW group was fed with a high-fat diet(D12492)with an eight-hour diet(eating from 10 pm to 6 am);the Ob group was fed with a high-fat diet(D12492)with free eating time.(2)After successful modeling,at 9 am on the 43rd day,we selected a certain number of mice from each of the four groups for disposable 6Gy(Its photon energy is 6MV,and its dose rate is 2.3Gy/min)head and neck radiation.(3)The levels of plasma triglycerides(TG)and free fatty acids(FFA)were detected by enzyme-linked immunosorbent assay(ELISA).(4)The effect of head and neck radiation on the expression of AgRP in the hypothalamic PVN nuclei of mice with different Lee’s index was detected by immunofluorescence technology.(5)Glucose metabolism-related detection:fasting blood glucose was measured by tail vein blood collection,and IPGTT was injected to evaluate the glucose tolerance of mice.(6)Lipid metabolism-related detection:the number of lipid droplets in liver tissue was observed by oil red O staining experiment;the number of cells in brown adipose tissue(BAT)and white adipose tissue(WAT)was counted by Hematoxylin-eosin staining(HE)staining;the expression of UCP1 in brown fat and white fat was detected by western blot.Results:1.Modeling of mice with four different Lee’s index:(1)Lee’s index(3(?)((body weight g*1000)/length cm)):Control group(13.72 ± 0.25),Thin group(13.45 ±0.35),OW group(13.99 ± 0.17),Ob group(14.26 ± 0.12).(2)Weight(g):control group(23.93 ± 1.37),Thin group(22.25 ± 2.11),OW group(24.60 ± 1.39),and Ob group(26.49 ± 0.69).2.Head and neck radiation increased the plasma triglyceride content of mice in the Thin group and the Ob group:(1)After head and neck radiation intervention,TG(ng/L)levels were elevated in the Thin(P<0.01)and Ob(P<0.05)groups compared with the control group receiving radiation,and there was no significant difference in the IR-OW group(P>0.05).(2)FFA(umol/L)levels were relatively elevated in four groups following head and neck radiation intervention.3.Expression of AgRP in PVN in hypothalamus:the expression of AgRP was higher in the PVN in IR-Ob group,(P<0.05),compared with IR-WT group.While the IR-Thin and IR-OW groups were not statistically different from them(P>0.05).4.Regarding glucose metabolism,head and neck radiation increased fasting blood glucose in mice in the Thin and Ob groups and caused changes in food intake:(1)Compared with control mice receiving radiation intervention(IR-WT),head and neck radiation increased fasting blood glucose(mmol/L)in the IR-Ob group(P<0.05).At the same time,there was no significant difference in the IR-OW group.(2)Compared with the control mice receiving radiation intervention(IR-WT),the head and neck radiation intervention reduced the mass(g)of cumulative nocturnal food intake in the IR-Thin,IR-OW,and IR-Ob groups.5.Regarding lipid metabolism,head and neck radiation increased liver lipid droplets in mice in the Ob group,decreased the number of white adipocytes per unit area in the Thin group,and decreased the expression of UCP1 in brown fat and white fat in the four groups:(1)Liver oil red O:lipid droplets were more in the liver of the IR-Ob group receiving radiation relative to the control and IR-OW groups(P<0.05).(2)Effect of head and neck radiation on brown adipose tissue:after head and neck radiation intervention,UCP1 expression was higher in the IR-OW and IR-Ob groups compared with the IR-WT group(P<0.05).Relative to the paired-fed un-radiated group,all four groups that received radiation showed a trend of decreased UCP1 expression,but it was not statistically significant(P>0.05).(3)Effect of head and neck radiation on white adipose tissue:compared with the IR-WT group,head and neck radiation caused a decrease in the number of cells and an increase in cell volume per unit area in the IR-Thin group(P<0.05);there was no statistical difference between the IR-OW and IR-Ob groups(P>0.05).There was no statistical difference in UCP1 expression between the four groups after the head and neck radiation intervention(P>0.05).However,among the groups that did not receive radiation,the UCP1 expression in the Ctrl-OW group was highest(P<0.05).Conclusions:This study showed that the changes in energy metabolism caused by ionizing radiation in mice with various Lee’s index are different.After being stimulated by ionizing radiation,relative to the control group of mice,fasting blood glucose was increased,and glucose tolerance was impaired in obese and thin mice.Still,these changes did not occur in mice of the IR-OW group.Ionizing radiation increased triglyceride levels in the plasma of the IR-Ob group and IR-Thin group of mice and increased lipid droplets in the liver of the IR-Ob group of mice.The expression of UCP1 in white adipose tissue was increased in mice without ionizing radiation,promoting browning and improving body metabolism.In conclusion,it is hypothesized that the body with a little more fat content can resist the hazards of ionizing radiation by enhancing the metabolic balance of UCP1.更多还原