【作者】 涂晓坤；

【导师】 陈如泉；

【作者基本信息】 湖北中医药大学 ， 中医内科学， 2013， 博士

【摘要】 目的探索实验性大鼠结节性甲状腺肿模型的建立；探讨大鼠结节性甲状腺肿的发病机制及观察活血消瘿方对实验性大鼠结节性甲状腺肿的作用；分别从活血消瘿方对实验性结节性甲状腺肿大鼠甲状腺细胞凋亡、甲状腺细胞增殖、甲状腺血管生成、甲状腺细胞趋化的影响等角度,探讨活血消瘿方对实验性大鼠结节性甲状腺肿的作用机制。方法1实验性大鼠结节性甲状腺肿模型的制备和实验分组处理：SD雄性大鼠60只,体重120-140g。大鼠编号后随机分为6组,即正常组、模型对照组、L-T4组、低剂量活血消瘿方组、中剂量活血消瘿方组、高剂量活血消瘿方组,每组10只。正常组(按1m1/100g体重)灌服给予生理盐水,其余各组均分别(按1m1/100g体重)灌服给予浓度为0.1%的丙基硫氧嘧啶(PTU)溶液,各组连续灌服8周。第9周起,L-T4组(按1m1/100g体重)灌服给予浓度为0.1mg/L的左甲状腺素钠(L-T4)溶液,低、中、高剂量活血消瘿方组分别(按1m1/100g体重)灌服给予浓度为44g/L、88g/L、176g/L的活血消瘿方溶液,模型对照组(按1m1/100g体重)灌服给予蒸馏水,各组连续灌服8周,于造模结束后利用彩色多普勒判断模型均制备成功。于第17周将大鼠麻醉后处死,颈动脉采血2m1,分离血清以备ELISA检测；完整分离两侧甲状腺,分别将各只大鼠左侧甲状腺分成两部分：一部分用4%多聚甲醛固定,制作组织切片,用于免疫组化分析；一部分用2.5%戊二醛固定,制作超薄组织切片,用于电镜观察；分别将各只大鼠右侧甲状腺切开分成两部分,均-20℃冻存,一部分用于蛋白印迹分析,一部分用于流式细胞仪检测。2形态学观察：超薄组织切片标本经染色后采用电镜观察。3蛋白印迹法：检测大鼠甲状腺组织中PCNA的表达。4Annexin-V法结合流式细胞仪：检测大鼠甲状腺细胞的凋亡。5ELISA法：检测大鼠血清中VEGF的浓度。6免疫组织化学法：检测大鼠甲状腺组织中FGF-2、CXCR-4的表达,并用Image-Pro Plus6.0软件对结果进行定量分析。结果1电镜下观察超薄组织切片的结果表明：正常组大鼠的甲状腺组织细胞质内有较多的溶酶体,各处可见粗面内质网,粗面内质网表面可见密布的核糖体小颗粒,细胞间的交界复合体清晰可见,滤泡腔内充满大小较为均匀一致的细小颗粒状物质,滤泡腔边缘存在较多的微绒毛。模型对照组大鼠甲状腺组织的滤泡上皮细胞高度肿胀,内质网扩张,内质网的表面核糖体分布密集。与正常组相比,L-T4组及低、中、高剂量活血消瘿方组溶酶体较少；与模型对照组相比,L-T4组及低、中、高剂量活血消瘿方组大鼠甲状腺组织的滤泡腔可见,内质网的扩张较小,细胞质内多见游离的核糖体小颗粒。2蛋白印迹法检测PCNA表达的结果显示：与正常组相比,模型组大鼠甲状腺组织PCNA的表达显著增多(P<0.01)；与模型对照组相比,L-T4及低、中、高剂量活血消瘿方组大鼠甲状腺细胞PCNA的表达均显著减少(P<0.01)；与L-T4组相比,中、低剂量活血消瘿方组大鼠甲状腺细胞PCNA的表达均有显著差异(P<0.01),而高剂量活血消瘿方组大鼠甲状腺细胞PCNA的表达无显著差异；与低剂量活血消瘿方组相比,中、高剂量活血消瘿方组大鼠甲状腺细胞PCNA的表达均有显著差异(P<0.01)；与低剂量活血消瘿方组相比,高剂量活血消瘿方组大鼠甲状腺细胞PCNA的表达有显著差异(P<0.01)。随活血消瘿方的浓度增大而PCNA的表达降低更为明显,即高剂量活血消瘿方对大鼠甲状腺组织PCNA表达的抑制作用最为显著。3Annexin-V法结合流式细胞仪检测细胞凋亡的结果显示：与正常组相比,模型对照组大鼠甲状腺细胞的凋亡率显著减少(P<0.01)与模型对照组相比,L-T4及低、中、高剂量活血消瘿方组大鼠甲状腺细胞的凋亡率均显著增大(P<0.01)；与低剂量活血消瘿方组相比,中剂量活血消瘿方组大鼠甲状腺细胞的凋亡率增大(P<0.05),高剂量活血消瘿方组大鼠甲状腺细胞的凋亡率显著增大(P<0.01)；与中剂量活血消瘿方组相比,高剂量活血消瘿方组大鼠甲状腺细胞的凋亡率有显著差异(P<0.01)。4ELISA法检测血清中VEGF浓度的结果显示：与正常组相比,模型对照组大鼠血清中VEGF的浓度显著增大(P<0.01)；与模型对照组相比,低剂量活血消瘿方组大鼠血清中VEGF的浓度降低(P<0.05),L-T4及中、高剂量活血消瘿方组大鼠血清中VEGF的浓度均显著降低(P<0.01)；与低剂量活血消瘿方组相比,高剂量活血消瘿方组大鼠血清中VEGF的浓度有明显差异(P<0.05),而中剂量活血消瘿方组无显著差异；与中剂量活血消瘿方组相比,高剂量活血消瘿方组大鼠血清中VEGF的浓度无明显差异。5免疫组织化学法检测FGF-2表达的结果表明：与正常组相比,模型对照组大鼠的甲状腺组织可见到FGF-2表达呈强阳性；与模型对照组相比,L-T4组及低、中、高剂量活血消瘿方组大鼠甲状腺组织FGF-2表达较少。定量的形态学分析结果显示,与正常组相比,模型对照组大鼠甲状腺组织的FGF-2阳性表达面积和累积光密度显著增大(P<0.01)；与模型对照组相比,L-T4组及低、中、高剂量活血消瘿方组大鼠甲状腺组织的FGF-2阳性表达面积和累积光密度均显著降低(P<0.01)；与L-T4组相比,低剂量活血消瘿方组大鼠甲状腺组织的FGF-2阳性表达的面积和累积光密度有显著差异(P<0.01),中、高剂量活血消瘿方组无显著差异；与低剂量活血消瘿方组相比,中、高剂量活血消瘿方组大鼠甲状腺组织的FGF-2阳性表达面积和累积光密度均显著降低(P<0.01)；与中剂量活血消瘿方组相比,高剂量活血消瘿方组大鼠甲状腺组织的FGF-2阳性表达面积和累积光密度无显著差异。6免疫组化三步法检测在各组大鼠甲状腺组织中CXCR-4表达水平的结果表明：与正常组相比,模型对照组大鼠的甲状腺组织可见到CXCR-4表达呈强阳性；与模型对照组相比,L-T4组及低、中、高剂量活血消瘿方组大鼠甲状腺组织CXCR-4表达较少。定量的形态学分析结果显示,与正常组相比,模型对照组大鼠甲状腺组织的CXCR-4阳性表达面积和累积光密度显著增大(P<0.01)；与模型对照组相比,L-T4组及低、中、高剂量活血消瘿方组大鼠甲状腺组织的CXCR-4阳性表达面积和累积光密度均明显降低(P<0.01)；与L-T4组相比,低、中剂量活血消瘿方组CXCR-4阳性表达的面积和累积光密度有显著差异(P<0.01),高剂量活血消瘿方组CXCR-4阳性表达的面积和累积光密度无显著差异；与低剂量活血消瘿方组相比,中、高剂量活血消瘿方组CXCR-4阳性表达的面积和累积光密度有显著差异(P<0.01)；与中剂量活血消瘿方组相比,高剂量活血消瘿方组大鼠甲状腺组织的CXCR-4阳性表达面积和累积光密度无显著变化。结论1活血消瘿方可改善结节性甲状腺肿模型大鼠甲状腺的微观形态及结构。2活血消瘿方可通过抑制结节性甲状腺肿模型大鼠甲状腺细胞的增殖、促进结节性甲状腺肿模型大鼠甲状腺细胞的凋亡、抑制结节性甲状腺肿模型大鼠甲状腺血管生成、抑制结节性甲状腺肿模型大鼠甲状腺细胞的趋化等途径对大鼠结节性甲状腺肿发挥作用。更多还原

【Abstract】 Objective:To explore the establishment of the experimental model of nodular goiter on rats. To investigate the pathogeny of nodular goiter in rats. To observe the effect of "Huo-Xue-Xiao-Ying" on nodular goiter model rats. To observed the effect of "Huo-Xue-Xiao-Ying" on form of thyroid in nodular goiter model rats; to approach the effect of "Huo-Xue-Xiao-Ying" on apoptosis of thyrocyte in nodular goiter model rats; to approach the effect of "Huo-Xue-Xiao-Ying" on cell proliferation of thyrocyte in nodular goiter model rats; to approach the effect of "Huo-Xue-Xiao-Ying" on vascularization of thyroid in nodular goiter model rats; to approached the effect of "Huo-Xue-Xiao-Ying" on chemotaxis of thyrocyte in nodular goiter model rats.Methods:1To prepare the model of nodular goiter in rats and grouping:60SD male rats,weighing120-140g, were separated into six groups at random, scilicet, normal control group.model control group,L-T4 group, low dose " Huo-Xue-Xiao-Ying " group,medium dose " Huo-Xue-Xiao-Ying" group,high dose " Huo-Xue-Xiao-Ying " group. The subsequence five groups had been drenched0.1%PTU solution and normal the control group drenched equivalent physiological saline until the end of experiment. After8weeks, the L-T4group was drenched0.1mg/l L-T4solution;low、medium、 high dose "Huo-Xue-Xiao-Ying" groups each were drenched44g/l、88g/l、176g/1"Huo-Xue-Xiao-Ying" solution; the model control group was drenched equivalent distilled water. Which continue after8weeks, the rats were sacrificed under pentobarbital anaesthesia.2ml blood were collected. The thyroid glands were devided inio three parts:one was fixed with4%paraf ormaidehyde; another was fixed with3%glutar aldehyde for electron mieroscopy;the third was preserved in-20℃for Western blot analysis or flow cytometer detecting.2Morphological observation:section preparations were stained and electron microscope observed.3Western blotting:To detect the expression of PCNA in thyrocyte of rats.4Flow cytometer detecting:apoptosis of thyrocyte in rats.5ELISA method:to detect density of VEGF in serous of rats.6Immunohistochemical method:To detect the expression of FGF-2and CXCR-4in thyrocyte of rats. The results were analyzed quantified with the softwire Image-Pro Plus6.0.Result:1To observe ultrathin tissue slice on electron microscope indicated that.follicular cavity,the smaller expansion of endoplasmic reticulum.and the small particles of the common free ribosomes in the cytoplasm can be seen within the thyroid tissue of "Huo-Xue-Xiao-Ying" group rats compared with model group; there were more lysosomes compared with normal group.2The result of the expression of PCNA detected by Western blotting showed that,compared with model group,the expression of PCNA significantly decrease in normal group (P<0.01);L-T4and low,medium and high dose "Huo-Xue-Xiao-Ying" can decrease the expression of PCNA in thyroid cell of nodular goiter rats and it was reduced more significantly with the increased concentration of "Huo-Xue-Xiao-Ying", so the expression of PCNA in thyroid cell was the most significantly inhibited by high dose of "Huo-Xue-Xiao-Ying"(P<0.01).3Flow cytometer detecting apoptosis showed that,compared with model group, apoptosis rate significantly increased in thyroid cell of the normal group rat (P<0.01). L-T4and low, medium and high dose "Huo-Xue-Xiao-Ying" can promote apoptosis of thyroid cells in the model rat,and apoptosis is more obvious with the increase of the concentration of of " Huo-Xue-Xiao-Ying ", so high dose "Huo-Xue-Xiao-Ying" played the most significant role in promoting apoptosis in thyroid cells of rats (P<0.01).4The results of concentration of VEGF in serum detected by ELISA showed that, compared with the model group, the concentration of VEGF reduced in the serum of normal control rats (P<0.05); L-T4and low, medium and high dose can reduce the concentration of VEGF in the serum of rats, concentration of VEGF was reduced more significantly with the increase of the concentration of "Huo-Xue-Xiao-Ying", so the expression of VEGF in thyroid cell was the most significantly inhibited by high dose "Huo-Xue-Xiao-Ying"(P<0.01). 5The result of the expression of FGF-2detected by the immunohistochemistry assay show that, compared with normal control group, expression of FGF-2was strongly positive in thyroid tissue of model group rat; compared with the model group,The expression of FGF-2decreased in thyroid tissue of L-T4, low,medium and high dose "Huo-Xue-Xiao-Ying" group rats. Quantitative morphological analysis showed that the area and integrated optical density of positive expression of FGF-2in the model group was significantly larger than the normal group, L-T4group and group (P<0.01); the area and integrated optical density of positive expression of FGF-2in the low dose "Huo-Xue-Xiao-Ying" group was significantly larger than high dose "Huo-Xue-Xiao-Ying" group (P<0.01).6The result of the expression of CXCR-4detected by the immunohistochemistry assay show that, compared with normal control group, expression of CXCR-4was strongly positive in thyroid tissue of model group rat; compared with the model group,The expression of CXCR-4decreased in thyroid tissue of L-T4, low,medium and high dose "Huo-Xue-Xiao-Ying" group rats. Quantitative morphological analysis showed that the area and integrated optical density of positive expression of CXCR-4in the model group was significantly larger than the normal group, L-T4group and group (P<0.01); the area and integrated optical density of positive expression of CXCR-4in the low dose "Huo-Xue-Xiao-Ying" group was significantly larger than high dose "Huo-Xue-Xiao-Ying" group(P<0.01).Conclusion:1"Huo-Xue-Xiao-Ying" can improve the morphology of thyreoid in nodular goiter model rats. 2"Huo-Xue-Xiao-Ying" can inhibit cell proliferation of thyrocyte in nodular goiter model rats.3"Huo-Xue-Xiao-Ying" can promote apoptosis of thyrocyte in nodular goiter model rats,which takes on dose-effect relationship.4"Huo-Xue-Xiao-Ying" can inhibit vascularization of thyroid in nodular goiter model rats. Which takes on dose-effect relationship.5"Huo-Xue-Xiao-Ying" can inhibit chemotaxis of thyrocyte in nodular goiter model rats.更多还原