诱导新生小鼠对Graves病免疫耐受的研究

Study on induction of immunotolerance against Graves＇ disease in neonatal mice

目的研究TSH受体（TSHR）289基因诱导Graves病新生免疫耐受的可行性，并初步探索其可能机制。方法将出牛0～24hBALB／c雌性小鼠分为腹腔注射组、肌肉注射组、造模组及正常对照组，腹腔注射组或肌肉注射组分别设低剂量、中剂量、高剂量耐受组及相应对照组，将腹腔注射、肌肉注射不同剂量耐受组及各自对照组给予1×10。particles（低）、1×10^8particles（中）、1×10^10particles（高）的Ad—TSHR289或Ad—lacz进行预处理。6～7周后．除正常对照组肌肉注射Ad—lacz外，其余各组小鼠均给予Ad—TSHR289免疫，每3周1次．共3次，首次免疫后10d检测血清促甲状腺素受体抗体（TRAb），末次免疫后4周测血清TRAb、TT4、脾CD4＋CD25＋Foxp3／CD4＋比例，并行甲状腺组织学检查。结果首次注射后10d，腹腔注射及肌肉注射高剂量耐受组均未诱导Ⅲ针对TSHR的抗体反应，而对照组TRAb滴度明显升高（P〈0．05）。术次免疫后4周，腹腔及肌肉注射高剂量耐受组均只有1／10小鼠出现阳性的抗体反应，腹腔注射高剂量耐受组没有小鼠发生甲状腺功能亢进，而肌肉注射高剂耐受量组2／10小鼠出现轻微的TT4升高，其相应对照组却出现了强烈的抗体反应（P〈O．01），TT4水平明显升高（P〈0．05），所有TT4升高的小鼠均出现了甲状腺组织增生件改变。此外，腹腔注射及肌肉注射高剂量耐受组CD4＋CD25＋Foxp3／CD4＋比例与对照组比较明显增高（P〈O．01）。腹腔注射及肌肉注射其余组群，与其对照组和造模组比较Graves病发生率差异无统计学意义。结论TSHR289基因可以通过腺病毒为载体，腹腔和肌肉注射两种途径诱导新生小鼠成年后对Graves病免疫耐受，抑制Graves病的发生。而高剂量的抗原刺激容易导致耐受产生。CD4＋cD25＋Foxp3＋T细胞存免疫耐受的诱导和维持中可能起重要作用。

Objective To investigate the feasibility of inducing neonatal immunotolerance against Graves＇ disease by gene TSH receptor （TSHR） 289 and its possible mechanism. Methods Neonatal （ 0-24 h） female BALB/e mice were divided into intraperitoneal injection group, intramuscular injection group, model group, and normal control group. The intraperitoneal group and the intramuscular group were further divided into low-dosage, middle-dosage, high-dosage tolerance groups, and the corresponding control groups. The tolerance groups and the controls were intraperitoneally or intramuscularly pretreated with low-dosage （ 1× 10^6 particles ） , middle-dosage （ 1 × 10^8 particles）, high-dosage（ 1 ×10^10 particles） of Ad-TSHR 289 or Ad-laez respectively. 6 to 7 weeks later, the normal control group received intramuscular injection with Ad-lacz; the other groups were immunized with Ad-TSHR 289, three times at 3 weeks interval. 10 （lays afler the first immunization, serum TRAb was detected. 4 weeks after the last immunization, senlm TRAb, TT4, splenic CD4 ＋ CD25 ＋ Foxp3/CD4 ＋ were tested, and the thyroid tissues were examinated histologically. Results Ten days after the first immunization, no antibody response against TSHR was detected in the two high-dose tolerance groups, but the TRAb titer in respective controls was significantly higher（ P〈 0.05 ）. 4 weeks afler the last injection, in high-dose tolerance grnups, only 1/10 of mice immunized by intraperitoneal or intramuscular injection elicited anti-TSHR antibody, and no mice immunized intraperitoneally had elevated serum TT4. Two of ten mice challenged intramuscularly showed slightly increased TT4 levels, but the respective controls displayed a strong antibody response（ P〈0.01 ） and elevated TT4 level （P〈0.05）. The similar percentages of high TT4 and thyroid hyperplasia were found in all groups. Additionally, the frequencies of CD4 ＋CD25 ＋ Foxp3/CD4 ＋in two high-dose tolerance groups were significantly increased as compared to those in controls（P〈0.05）. The incidenc, e of Graves＇ disease in the other groups by intraperitoneal or intramuscular injections was not statistically different from those in the corresponding control groups and the model group. Conclusions The immune tolerance against Graves＇disease is induced in neonatal mice by either intraperitoneal or intramuscular pathway with specific auligen of TSHR 289, carried by adenovirus vector, and then inhibits Graves＇ disease in adults. Stimulation with the high-dosage antigen is liable to induce immune unresponsiveness. CD4＋CD25 ＋Foxp3 ＋T cells may play an important role in the induction and maintenance of toleranee .