【作者】 余毅恺；

【导师】 张木勋；

【作者基本信息】 华中科技大学 ， 内科学， 2008， 博士

【摘要】 目的:探讨可能影响甲亢患者服药后再次复发的多种危险因素。方法:选取96例初诊Graves病患者,正规抗甲亢药物治疗(ATD)一年,然后继续进行为期半年的随访观察,评价治疗效果。将分为治疗缓解组和复发组。ELISA法测定血清TRAb浓度,荧光定量PCR测定外周血单个核细胞表面共刺激分子CD80 mRNA含量,温和酸消化法测定血清总碘含量。以血清总碘含量、TRAb浓度、外周单核细胞(PBMC)膜上CD80 mRNA含量等可能影响甲亢治疗预后的危险因素为自变量,以ATD一年半后缓解或复发为应变量,结合其他临床参数进行病例对照研究,做多因素条件logistic回归分析。直线相关分析TRAb和CD80 mRNA以及血清总碘含量的相关性。结果:多因素logistic回归模型确定系数R2=0.783(p=0.007),进入回归模型的危险因素有:TRAb浓度(βj=0.565,OR=5.506),家族史(βj=0.528,OR=3.401), CD80mRNA含量(βj=0.453,OR=3.089),血清总碘含量(βj=0.389,OR=1.497),发病年龄(βj=0.272,OR=1.301)。相关性分析显示:血清TRAb浓度(r=0.79)和CD80含量之间的相关性大于血清总碘( r=0.45)。结论:CD80含量和血清TRAb浓度相关性高于血清总碘含量。患者TRAb浓度增高、具有甲亢阳性家族史、PBMC表面CD80mRNA表达量增高、血清总碘含量增高、发病年龄早可能是导致ATD复发的重要危险因素。目的:构建表达mCD80基因真核质粒载体。大量扩增并提纯pcDNA3.0-hTSHR质粒和pcDNA3.0-mCD80质粒。获得瞬时表达hTHR的CHO细胞,为测定第二信使cAMP提供检测载体。方法:用RT-PCR方法获得Balb/C鼠CD80基因全长序列,插入真核表达质粒pcDNA3.0。降重组表达质粒pcDNA3.0-mCD80,转化大肠杆菌DH5α筛选阳性克隆,双酶切和基因测序鉴定。脂质体介导pcDNA3.0-hTSHR转染CHO细胞并用Western blot免疫印记法检测转染产物的表达。结果:重组质粒双酶切后凝胶电泳产生两条大小分别为5.4kb和2.7kb的片断,大小和预期相同。基因公司测序结果与Genebank中的全长序列一致。在700ug/ml的G418浓度压力下,获得瞬时转染CHO细胞,Western blot结果表明该蛋白具有明显的免疫增强作用。结论:用基因工程技术获得mCD80基因真核表达质粒,为进一步研究CD80分子的对甲亢免疫应答过程的影响奠定了基础。目的:探讨基因枪注射和普通质粒肌肉注射两种不同方法对甲亢动物模型的差异。方法:对45只小鼠进行随机分成四组,分别用基因枪金颗粒包裹质粒免疫、普通质粒肌肉注射免疫。加强免疫后第4周后处死小鼠RIA法测定血清FT3、FT4、TSH水平。RIA法测定血清与hTSHR-CHO孵育后上清cAMP浓度以计算TRAb活性,RIA法测定脾脏培养后IL-4的浓度,ELISA法测定血清TSAb浓度,取甲状腺做石蜡HE切片观察组织学变化。结果:基因枪免疫组(GGI)动物4周后血清FT4(0.34±0.11)pg/ml高于质粒肌肉注射组(PLS)(0.21±0.06)pg/ml和基因枪免疫空质粒组(GG0)(0.17±0.08)pg/ml(F=14.34,p=0.002)。GGI组TRAb活性(167.27±37.37)%高于PLS组(113.81±25.50)%和GG0组(98.27±14.80)%(Hc=35.198,p=0.007)。GGI组脾脏培养上清液IL-4产量(0.244±0.092)pmol/L明显高于PLS组(0.173±0.039)pmol/L和GG0组(0.151±0.041)pmol/L(F=7.0403,p=0.005)。GGI组TRAb浓度(29.3±1.2)IU/L明显高于GG0组(13.3±0.2)IU/L(F=9.02,p<0.001),但和PLS组(24.3±0.9)IU/L比无显著性差异(q=-1.32,p=0.423)。HE切片观察造模成功小鼠甲状腺滤泡细胞高柱状排列,未出现甲状腺细胞破坏甲状腺炎的嗜酸性变。结论:基因枪免疫雌性Balb/c鼠是一种可行的复制Graves动物模型,在免疫第2-4周可以观察到FT4升高和自身抗体改变等理想的免疫效果。目的:为了进一步明确共刺激分子CD80在甲状腺疾病中的作用和明确Graves’病甲亢自身免疫疾病的病理生理机制。方法: 40只远交系Balb/c小鼠随机分成A,B,C,D四组,分别为用表达pCDNA3.1-mCD80和pCDNA3.1-hTSHR的质粒用基因枪免疫。两周后处死小鼠,留取血清测定游离甲状腺素(FT4)水平以确定是否造模成功,ELISA法测定血清TRAb浓度,脾脏原代培养7天后放射免疫法测定培养上清液INF-γ和IL-4浓度,离心柱粗提血清IgG后和转染人hTSHR-CHO细胞共同孵育测定培养液释放cAMP浓度。HE染色切片观察甲状腺形态学变化。结果:同时注射两种质粒的小鼠B组FT4水平高于其他三组(0.40±0.13)pg/ml (F=57.02, p<0.0001),小鼠血清TRAb浓度(31.6±2.7)IU/L(F=11.90,p<0.0001)和TRAb活性(179.14±44.96)%也高于对照组。B组小鼠脾脏原代培养上清液IL-4的产量(0.226±0.029) pmol/L (F=13.21, p<0.0001)pmol/L都高于A、C、D三组,但INF-γ(1.86±0.24)pmol/L含量仅高于C和D组,和A组比无显著性差异(F=6.37, P=0.078)。HE切片观察造模成功小鼠甲状腺滤泡细胞高柱状排列,未出现甲状腺细胞破坏甲状腺炎的嗜酸性变。结论:基因枪注射pCDNA3.1-hTHSR质粒可以成功诱导出甲亢的动物模型,共刺激分子CD80可以上调这种动物模型产生的免疫应答过程。更多还原

【Abstract】 Objective: To explore various possible factors that maybe influence the prognosis of anti-thyroidism treatment. Method: 96 GD patients were followed by one and half year with regular ATD and then were reexamined for the treatment effect. Finally all patients were classified as remission or relapse group. All patient’serum were collected and TRAb was measured by ELISA, the co-stimulator molecular CD80 mRNA on peripheral blood mononuclear cell membrane was detected by real time PCR and serum total iodine concentration was measured with mild acid digestion method. Those risk factors that may influence the prognosis of GD were selected as independent variable and GD therapeutic effect was selected as dependent variable. Multiple conditional logistics regression study was conducted with the combination of clinical parameters such as serum iodine, TRAb, CD86 mRNA. Outcome: The logistic model determination coefficient R2=0.783(p=0.007) and the risk factors entered into the model were TRAb concentration(βj=0.565,OR=5.506), positive family history(βj=0.528,OR=3.401), CD80 mRNA content(βj=0.453,OR=3.089),serum total iodine concentration(βj=0.389,OR=1.497),age of onset(βj=0.272,OR=1.301)。Conclusion: Those patients that have high level of TRAb, positive family history of GD, increase of expression of CD80 mRNA on PBMC membrane and early onset age were more inclined to relapse after ATD. Abstract Objective: To construct the mouse CD80 expression plasmid vector and express construction of CD80 gene expression in E. coli and DH5αexpression of CD80 protein. Methods: RT-PCR method was Balb/C mice CD80 full length, the recombinant expression plasmid pcDNA3.0-CD80, and transformed into E. coli DH5α, a double digestion and gene sequencing methods screening positive clones, Western blotting to detect expression products. Results: The positive recombinant DH5αin temperature in the CD80 protein expression can be induced by molecular weight consistent with the theoretical value. Lymphocyte transformation test results show that the protein has significant immune-enhancing role. Conclusion: we constructed the CD80 gene expression vector plasmid by genetic engineering technology was in mouse protein for further research on the biological function of mouse CD80 laid the foundation. Purpose: To explore the difference in establishment of Graves disease animal model between gene gun injection and normal plasmid muscle injection. Methods: 45 mice were randomized into four groups and then were immunized with either gene gun gold particles or muscle plasmid. After 4 weeks mice were killed and blood sample were taken to determine FT3、FT4、TSH with RIA, TSAb concentration with Elisa, Spleen was cultured to determine IL-4 concentration with RIA. TRAb activity was calculated with production of cAMP after serum was cultured with hTSHR-CHO cell. Thyroid HE section was applied to observe the thyroid gland histomology. Outcome: Mice immunized with gene gun gold particles compared with other groups after 4th week showed a significant increase in FT4 (0.34±0.11)pg/ml (F=14.34,p=0.002), TRAb activity(167.27±37.37)%(Hc=35.198, p=0.007)and IL-4 production in spleen cell(s0.244±0.092)pmol/L (F=7.0403,p=0.005) than GG0 group in which FT4(0.17±0.08)pg/ml, TRAb activity(98.27±14.80)%and IL-4 production(0.151±0.041)pmol/L and PLS group in which FT4(0.21±0.06)pg/ml, TRAb activity(113.81±65.50)%and IL-4 production(0.173±0.039)pmol/L . TRAb concentration (29.3±1.2)IU/L in gene gun group was higher than GG0 (13.3±0.2)IU/L group(F=9.02,p<0.001)but no difference between PLS (24.3±0.9)IU/L group(q=-1.32,p=0.423). HE sections showed that mice immunized successfully with gene gun had thyroid gland follicular cells hyperplasic ,however, they didn’t had any signs of cellular destruction or lymphocyte infiltration. Conclusion: Female Balb/c mice immunized with gene gun gold particles was an feasible way to establish Graves disease animal model, from the second to fourth week expected immunized response could be observed including increase of FT4 and autoimmune antibody.更多还原