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甲状腺功能减退症(简称甲减)是放射性131I治疗甲状腺功能亢进症(简称甲亢)时一个最主要的并发症。目前,131I治疗甲亢已经有60多年的历史,在欧美国家已成为了治疗成人甲亢的首选治疗方法,我国自1958年开始应用131I治疗,近几年,131I在治疗甲亢和甲状腺恶性肿瘤方面的优势逐渐被国内医师和患者所认可,较多医院选择将131I治疗作为甲亢的首先治疗方式[1]。131I治疗后容易发生甲减[2],其中131I治疗甲亢后的甲减占了较大的比重,在青少年和儿童中的发生率甚至高达95%[3-5]。越来越多的证据表明,甲减对于心血管系统有严重影响,大大增加了心血管疾病的发生率。亚临床与明显的甲减都会引起心输出量降低、外周阻力增高和血管内皮功能受损[6],甲减与动脉粥样硬化关系密切[7],对心血管系统有重要影响。一氧化氮(nitric oxide,NO)作为一种能引起平滑肌细胞舒张的化合物,从多个方面参与调节心血管系统的功能。NO主要由一氧化氮合酶(nitric oxide synthases,NOS)以L-精氨酸(L-arginine)为底物合成。NOS主要由内皮性一氧化氮合酶(eNOS)、诱生性一氧化氮合酶(iNOS)及神经性一氧化氮合酶(nNOS)3种亚型构成,且均在心血管系统中扮演着重要的角色。因此,随着放射性131I治疗甲亢的逐渐普及,131I治疗后甲减发生率的逐渐增加,其对于心血管系统的影响不容忽视,所以对NO的研究也进入了一个新时代[8]。
本实验用131I诱导的大鼠甲减模型,研究甲减以及辐射对大鼠主动脉内皮功能异常中NO的作用及其机制,以期寻找一个较为安全的131I治疗剂量,为临床上预防131I治疗甲亢所导致的甲减性心脏病提供新的思路。
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由表1可知,正常对照组与2.775 MBq剂量组间甲状腺功能差异无统计学意义(FT3:t=1.547~1.615,均P>0.05;FT4:t=2.014~2.103,均P>0.05;TSH:t=1.254~1.528,均P>0.05)。在其他组,与正常对照组比较随着剂量的增加,血清FT3、FT4水平逐渐下降,差异均有统计学意义(FT3:t=5.324~14.210,均P<0.05;FT4:t=5.017~13.587,均P<0.05);另外,随着剂量的增加血清TSH浓度在各时间点逐渐升高,且差异均具有统计学意义(t=5.214~20.369,P<0.05)。同时,结合各剂量组的不同时间点大鼠的体质量数据(表2)可知,除2.775 MBq剂量组外,与正常对照组相比,其余3组大鼠的体质量变化均有统计学意义(t=1.987~13.542,均P<0.05),说明甲减模型构建成功,且5.550 MBq组作为放射性131I治疗的剂量是较为合适的,故选取5.550 MBq组大鼠进行接下来的研究。
组别 FT3(ng/mL) FT4(pmol/L) TSH(mIU/L) 正常对照组 4周 1.17±0.11 13.36±0.28 7.65±0.32 8周 1.22±0.11 13.36±0.50 8.03±0.83 16周 1.17±0.12 13.47±0.54 8.31±0.23 2.775 MBq组 4周 1.02±0.30 12.55±1.01 9.02±0.58 8周 1.25±0.82 12.41±0.98 8.43±1.56 16周 1.10±0.14 12.52±0.83 8.56±0.23 5.550 MBq组 4周 0.79±0.03* 9.87±2.21* 16.01±2.02* 8周 0.64±0.15* 6.99±1.05* 19.02±2.25* 16周 0.58±0.15* 6.05±0.44* 20.61±0.79* 11.100 MBq组 4周 0.53±0.70* 7.77±0.93* 19.08±4.01* 8周 0.31±0.09* 6.25±1.31* 21.87±0.56* 16周 0.30±0.01* 5.29±0.42* 22.52±2.58* 16.650 MBq组 4周 0.42±0.04* 5.54±1.22* 21.51±2.20* 8周 0.30±0.02* 4.87±0.10* 23.46±1.22* 16周 0.26±0.05* 5.25±0.16* 23.82±4.25* 注:表中,*:与正常对照组比较,差异均有统计学意义(t=5.017~20.369,均P<0.05)。FT3:游离三碘甲腺原氨酸;FT4:游离甲状腺素;TSH:促甲状腺激素。 表 1 不同剂量131I诱导的SD大鼠模型在不同时间的甲状腺功能检测结果(每组n=3,
$ \bar x \pm s$ Table 1. Thyroid function of treated rats at different doses of 131I (each group n=3,
$ \bar x \pm s$ 组别 大鼠体质量(g) 4周 8周 16周 正常对照组 211.33±10.21 325.23±15.07 304.76±19.01 2.775 MBq组 218.67±11.03 331.33±16.20 404.00±17.28 5.550 MBq组 304.76±19.01* 415.25±22.01* 513.31±9.89* 11.100 MBq组 189.67±11.58* 207.67±16.91* 209.04±6.55* 16.650 MBq组 192.54±12.48* 217.67±16.07* 211.96±10.04* 注:表中,*:与正常对照组比较,差异均有统计学意义(t=1.987~13.542,均P<0.05)。 表 2 不同剂量组各个时间点131I诱导的SD大鼠体质量(每组n=3,
$\bar x \pm s$ Table 2. Body weight of treated rats at different doses of 131I at different time (each group n=3,
$\bar x \pm s$ -
图1和图2显示甲减大鼠在使用5.550 MBq 131I治疗时血清中的TNF-α和IL-6的表达水平。TNF-α水平在第4、8、16周时分别为1441.23、1601.85、1521.51 pg/mL,与正常对照组相比,差异均有统计学意义(t=21.021、17.578、14.498,均P<0.05),其中在第8、16周时的TNF-α水平高于第4周。血清中IL-6水平在第4、8、16周时分别为209.23、291.87、302.97 pg/mL,与正常对照组比较,IL-6水平在第4周时差异无统计学意义(t=2.825,P>0.05),在第8、16周时,差异均有统计学意义(t=8.841、14.224,均P<0.05)。图3显示,与正常对照组相比,5.550 MBq剂量组血清NO水平在第4周时水平高于正常对照组,差异有统计学意义(t=31.014,P<0.05),而在第8周(t=4.214,P>0.05)和第16周(t=7.014,P<0.05)时均有所下降。
图 1 5.550 MBq剂量组甲减SD大鼠与正常对照组SD大鼠在不同时间的TNF-α水平
Figure 1. The content of TNF-α in serumat at different time in hypothyroid rats treated with 5.550 MBq 131I versus normal group
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由图4可知,5.550 MBq剂量组甲减大鼠的主动脉内皮总NOS活性随时间的延迟持续升高,与正常组相比,差异均有统计学意义(t4w=5.089、t8w=9.672、t16w=13.394,均P<0.05)。
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5.550 MBq剂量组甲减大鼠的Western-blotting分析结果见图5。eNOS在不同时间点的表达量为25 985、16 306、6248,与正常对照组相比,eNOS蛋白水平明显降低,但是在第4周的差异无统计学意义(t4w=3.546,P>0.05;t8w=8.841,t16w=14.224,均P<0.05)。nNOS表达量为24 562、36 114、58 211,nNOS蛋白水平在第4周时下降,但在第8、16周时逐渐增加,差异有统计学意义(t=5.751、7.251,均P<0.05)。在各个时间点,iNOS表达量为55 973、50 575、62 364,蛋白表达水平均较正常对照组明显增加,差异均有统计学意义(t4w=21.017、t8w=16.412、t16w=24.981,均P<0.05)。
131I诱导甲状腺功能减退大鼠主动脉内皮功能异常相关机制的研究
Effect and mechanism of aortic endothelium of hypothyroidism rats induced by 131I
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摘要:
目的 探究放射性131I诱导大鼠甲状腺功能减退(简称甲减)模型中血清一氧化氮(NO)、炎症介质肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)的表达水平及在大鼠甲减模型中主动脉内皮细胞中一氧化氮合酶(NOS)蛋白的表达情况,以期寻找一个较为安全的131I治疗剂量。 方法 健康雄性SD大鼠45只,其中36只分别腹腔注射2.775、5.550、11.100、16.650 MBq 131I+0.5 mL生理盐水,构建大鼠甲减模型,分为4组,每组9只;剩余9只作为正常对照组,只注射0.5 mL生理盐水。分别于注射后第4、8、16周时每组各处死大鼠3只,用ELISA法检测大鼠血清中NO、TNF-α和IL-6水平,用化学发光免疫分析法检测血清中游离三碘甲状腺原氨酸(FT3)、游离甲状腺素(FT4)、促甲状腺激素(TSH)水平,提取主动脉标本检测总的NOS活性,应用Western-blotting分析大鼠主动脉标本中主动脉内皮型NOS(eNOS)、神经型NOS (nNOS)与诱导型NOS(iNOS)蛋白表达水平。组间两两比较采用t检验。 结果 血清中FT3、FT4、TSH水平检测结果发现除2.775 MBq剂量组外的其他3组大鼠均甲减模型构建成功,且5.550 MBq组作为放射性131I治疗的剂量是较为合适的。5.550 MBq组大鼠血清NO水平在注射131I后第4周升高至24.01 µmol/L后逐渐减少;血清中IL-6水平在第4、8、16周时分别为209.23、291.87、302.97 pg/mL,与正常对照组相比,第8、16周差异均有统计学意义(t=8.841、14.224,均P<0.05)。TNF-α水平在第4、8、16周时分别为1441.23、1601.85、1521.51 pg/mL,与正常对照组相比,差异均有统计学意义(t=21.021、17.578、14.498,均P<0.05)。eNOS在不同时间点的表达量为25 985、16 306、6248,各组蛋白水平明显降低,与正常对照组相比,除第4周时蛋白水平差异无统计学意义(t=3.546,P>0.05)外,第8、16周时差异均有统计学意义(t=8.841、14.224,均P<0.05)。nNOS表达量为24 562、36 114、58 211,第4周时下降,第8、16周时逐渐升高,与正常对照组相比,第4、16周时的差异均有统计学意义(t=5.751、7.251,均P<0.05)。iNOS在不同时间点的表达量为55 973、50 575、62 364,与正常对照组相比,各组均明显增加,差异均有统计学意义(t=21.017、16.412、24.981,均P<0.05)。 结论 放射性131I所致甲减对于心脏具有一定的影响,大剂量的131I可能会刺激合成甲状腺激素减少,从而导致甲减,最终影响其血管内皮NOS基因及蛋白表达,对心血管系统产生影响。 Abstract:Objective To investigate the expression of nitric oxide(NO), inflammatory mediators(TNF-a, IL-6) in serum and nitric oxide synthase(NOS) protein in aortic endothelial cells of rats with hypothyroidism induced by radioactive 131I, in order to find a safe dose of 131I for the prevention of hypothyroidism heart disease. Methods Forty-five male rats were selected. The hypothyroidism model was established by intraperitoneal injection of 2.775, 5.550, 11.100, 16.650 MBq 131I + 0.5 mL saline into 36 male rats, with 9 rats in each group. The remaining 9 rats were taken as normal control group and only 0.5 mL saline was injected to each rat. Three rats were sacrificed in each group at 4, 8, and 16 weeks after 131I administration. The serum levels of IL-6, TNF-α and the aortal content of total-NOS activity were determined with chemiluminescence immunoassay. Western blotting was used to analyze the expression of rat aortic specimens of eNOS, nNOS, and iNOS, and count data were analyzed by the t test. A value of P<0.05 was considered statistically significant. Results The serum levels of FT3, FT4 and TSH showed that all the groups of hypothyroidism model of rats were successfully constructed except 2.775 MBq group, and 5.550 MBq was regarded as an optimum dosage. The serum level of NO in rats(5.550 MBq) at 4 week increased to 24.01 mol/L and then reduced gradually; the level of serum IL-6 in 4, 8, and 16 were 209.23, 291.87, and 302.97 pg/mL, respectively. Compared with nomal control, statistical differences were observed at 8 and 16 week(t=8.841, 14.224, both P<0.05). The levels of TNF- alpha were 1441.23, 1601.85, and 1521.51 pg/mL, respectively, with statistical differences at different time points(t=21.021, 17.578, 14.498, all P<0.05). The expression of aortic endothelial NOS(eNOS) at different time points was 25 985, 16 306, and 6248. Compared with nomal control, the levels of protein in each group decreased, no statistical significance was observed at 4 week(t=3.546, P>0.05) and statistical differences were observed at 8 and 16 week(t=8.841, 14.224, both P<0.05). The expression of NOS(nNOS) was 24 562, 36 114 and 58 211, down at 4 week(t=3.546, P>0.05), and gradually increased at 8 and 16 week(t=5.751, 7.251, both P>0.05). The expression of inducible nitric oxide synthase(iNOS) was 55 973, 50 575 and 62 364, and all groups increased statistically(t=21.017, 16.412, 24.981, all P<0.05). Conclusion The hypothyroidism induced by 131I has certain effect on the heart, and our study can provide a new idea for prevention of hypothyroid heart disease for clinical practice and reduction of cardiovascular disease. -
Key words:
- Iodine radioisotopes /
- Hypothyroidism /
- Aorta endothelium /
- Nitric oxide /
- Nitric oxide synthase /
- Aorta endothelium
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表 1 不同剂量131I诱导的SD大鼠模型在不同时间的甲状腺功能检测结果(每组n=3,
$ \bar x \pm s$ Table 1. Thyroid function of treated rats at different doses of 131I (each group n=3,
$ \bar x \pm s$ 组别 FT3(ng/mL) FT4(pmol/L) TSH(mIU/L) 正常对照组 4周 1.17±0.11 13.36±0.28 7.65±0.32 8周 1.22±0.11 13.36±0.50 8.03±0.83 16周 1.17±0.12 13.47±0.54 8.31±0.23 2.775 MBq组 4周 1.02±0.30 12.55±1.01 9.02±0.58 8周 1.25±0.82 12.41±0.98 8.43±1.56 16周 1.10±0.14 12.52±0.83 8.56±0.23 5.550 MBq组 4周 0.79±0.03* 9.87±2.21* 16.01±2.02* 8周 0.64±0.15* 6.99±1.05* 19.02±2.25* 16周 0.58±0.15* 6.05±0.44* 20.61±0.79* 11.100 MBq组 4周 0.53±0.70* 7.77±0.93* 19.08±4.01* 8周 0.31±0.09* 6.25±1.31* 21.87±0.56* 16周 0.30±0.01* 5.29±0.42* 22.52±2.58* 16.650 MBq组 4周 0.42±0.04* 5.54±1.22* 21.51±2.20* 8周 0.30±0.02* 4.87±0.10* 23.46±1.22* 16周 0.26±0.05* 5.25±0.16* 23.82±4.25* 注:表中,*:与正常对照组比较,差异均有统计学意义(t=5.017~20.369,均P<0.05)。FT3:游离三碘甲腺原氨酸;FT4:游离甲状腺素;TSH:促甲状腺激素。 表 2 不同剂量组各个时间点131I诱导的SD大鼠体质量(每组n=3,
$\bar x \pm s$ Table 2. Body weight of treated rats at different doses of 131I at different time (each group n=3,
$\bar x \pm s$ 组别 大鼠体质量(g) 4周 8周 16周 正常对照组 211.33±10.21 325.23±15.07 304.76±19.01 2.775 MBq组 218.67±11.03 331.33±16.20 404.00±17.28 5.550 MBq组 304.76±19.01* 415.25±22.01* 513.31±9.89* 11.100 MBq组 189.67±11.58* 207.67±16.91* 209.04±6.55* 16.650 MBq组 192.54±12.48* 217.67±16.07* 211.96±10.04* 注:表中,*:与正常对照组比较,差异均有统计学意义(t=1.987~13.542,均P<0.05)。 -
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