【作者】 杨沫毅；

【导师】 向旭东；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 第一部分急性哮喘小鼠模型的建立与评价目的通过补充完善急性哮喘动物模型评价标准,探讨建立的急性哮喘小鼠模型是否成功。方法35只SPF级雌性BALB/c小鼠随机分为两组：哮喘组(20只)以OVA致敏、激发；正常对照组(15只)以等体积的PBS代替OVA致敏、激发,末次激发24 h后处理小鼠。查看小鼠的一般行为活动；雾化吸入Ach行支气管激发试验,有创肺阻抗法测定小鼠的气道反应性；BALF行细胞学分类、计数；HE染色观察肺组织的病理变化。研磨小鼠脾脏制成单个核细胞悬液,裂解红细胞,免疫磁珠分离小鼠脾源性CD4+T细胞,悬于加有胎牛血清的RPMI 1640培养液,ConA和PMA刺激培养24小时后,计算脾源性CD4+T细胞总数；流式仪检测Th17细胞的阳性率；ELISA测定BALF和脾源性CD4+T细胞培养上清中IL-4和IL-17的浓度。结果(1)哮喘组小鼠出现类似于人的哮喘发作症状,而正常对照组小鼠表现正常。(2)与正常对照组相比,哮喘组小鼠对Ach的刺激反应明显,浓度反应曲线上移(均P<0.01)。(3)与正常对照组相比,哮喘组小鼠肺组织支气管及血管周围大量炎性细胞(包括中性粒细胞、嗜酸性粒细胞、淋巴细胞和单核细胞)浸润(P<0.01)。(4)与正常对照组相比,哮喘组小鼠BALF中白细胞总数、Neu(％)、Eos(％)和Lym(%)显著增多(均P<0.01)。(5)与正常对照组相比,哮喘组小鼠脾源性CD4+T细胞总数和Th17细胞阳性率显著增高(均尸<0.01)。(6)与正常对照组相比,哮喘组小鼠BALF和脾源性CD4+Υ细胞培养上清中IL-4和IL-17的浓度显著增高(均P<0.01)。(7)急性哮喘小鼠BALF中IL-17的浓度与Neu(%)显著正相关(r=0.903,P<0.01)。结论1.对脾源性CD4+T细胞总数、Th17细胞阳性率和细胞培养上清中IL-17浓度的检测,可做为急性哮喘动物模型的评价标准。2.本实验中建立的急性哮喘小鼠模型是成功的。第二部分IL-23受体对急性哮喘小鼠脾源性Th17细胞增殖和致炎机能的影响目的通过检测IL-23受体的表达、Th17细胞阳性率和IL-17的浓度,探讨IL-23受体对急性哮喘小鼠脾源性Th17细胞增殖和致炎机能的影响。方法Th17细胞的阳性率反应其增殖情况,细胞培养上清中1L-17的浓度反应Th17细胞的致炎机能。免疫磁珠分离的小鼠脾源性CD4-T细胞悬于加有胎牛血清的RPMI-1640培基、ConA和PMA刺激培养24小时,real-time PCR和WB测定小鼠脾源性CD4+T细胞亚群Th17细胞中1L-23受体的表达,流式仪测定Th17细胞的阳性率,ELISA测定脾源性CD4+T细胞培养上清中IL-17的浓度,并分析哮喘组小鼠IL-23受体的表达和Th17细胞阳性率、IL-17浓度的相关性。结果(1)与正常对照组相比,哮喘组小鼠脾源性CD4+T细胞中IL-23受体mRNA的表达显著增高(P<0.01)。(2)与正常对照组相比,哮喘组小鼠脾源性CD4+T细胞中IL-23受体蛋白的表达显著增高(P<0.01)。(3)与正常对照组相比,哮喘组小鼠脾源性CD4+T细胞中Th17细胞的阳性率显著增高(P<0.01)。(4)与正常对照组相比,哮喘组小鼠脾源性CD4+T细胞培养上清中IL-17的浓度显著增高于(P<0.01)。(5)哮喘组小鼠脾源性CD4+T细胞中IL-23受体mRNA的表达与Th17细胞的阳性率显著正相关(Υ=-0.789,P<0.05)；与IL-17的浓度显著正相关(Υ=0.788,P<0.05)。(6)哮喘组小鼠脾源性CD4+T细胞中IL-23受体蛋白的表达与Th17细胞的阳性率显著正相关(r=0.828,P<0.01)；与IL-17的浓度显著正相关(r=0.802,P<0.05)。结论1.急性哮喘小鼠脾源性CD4+T细胞中IL-23受体的表达、Th17细胞的阳性率和IL-17的浓度均显著增高。2.急性哮喘小鼠中IL-23受体的表达可能影响Th17细胞的增值和致炎机能。第三部分下调T-bet表达通过影响1L-23受体促进Th17细胞的增殖和致炎机能目的通过抑制T-bet的表达,观察对急性哮喘小鼠脾源性Th17细胞增殖和致炎机能的影响。方法T-bet为观察对象,IL-23受体为联系T-bet和Th17细胞的桥梁,Th17细胞的阳性率反应其增殖情况,细胞培养上清中IL-17的浓度反应Th17细胞的致炎机能。三对siRNA-T-bet片断和阴性对照,分别瞬时转染哮喘组小鼠脾源性CD4+T细胞后,Western Blot和real-timePCR检测T-bet的表达,筛选出具有最佳沉默效果的片断,此片断转染哮喘组小鼠脾源性CD4+T细胞。3组细胞：正常对照组、未转染哮喘组和转染最佳沉默siRNA片断哮喘组的脾源性CD4-T细胞,悬于加有胎牛血清的RPMI-1640培基、ConA和PMA刺激培养24小时,检测各组细胞中T-bet的表达、IL-23受体的表达、Thl7阳性率和细胞培养上清中IL-17浓度,并分析转染最佳沉默siRNA片断哮喘组的脾源性CD4-T细胞中T-bet的表达与IL-23受体的表达、Th17阳性率、IL-17浓度的相关性。结果(1)三对siRNA片断中以siRNA-T-bet-429沉默效果最为显著(P<0.01)。(2)与正常对照组相比,未转染哮喘组和转染siRNA-T-bet-429哮喘组的脾源性CD4+T细胞中T-bet mRNA的表达显著降低(均P<0.01)；与未转染哮喘组相比,转染siRNA-T-bet-429哮喘组的脾源性CD4+T细胞中T-bet mRNA的表达显著降低(P<0.01)。(3)与正常对照组相比,未转染哮喘组和转染siRNA-T-bet-429哮喘组的脾源性CD4-T细胞中T-bet蛋白的表达显著降低(均P<0.01)；与未转染哮喘组相比,转染siRNA-T-bet-429哮喘组的脾源性CD4+T细胞中T-bet蛋白的表达显著降低(P<0.01)(4)与正常对照组相比,未转染哮喘组和转染siRNA-T-bet-429哮喘组的脾源性CD4+T细胞中IL-23受体mRNA的表达显著增高(均P<0.01)；与未转染哮喘组相比,转染siRNA-T-bet-429哮喘组的脾源性CD4+T细胞中IL-23受体：mRNA的表达显著增高(P<0.01)。(5)与正常对照组相比,未转染哮喘组和转染siRNA-T-bet-429哮喘组的脾源性CD4+T细胞中IL-23受体蛋白的表达显著增高(均P<0.01)；与未转染哮喘组相比,转染siRNA-T-bet-429哮喘组的脾源性CD4+T细胞中IL-23受体蛋白的表达显著增高(P<0.01)。(6)与正常对照组相比,未转染哮喘组和转染siRNA-T-bet-429哮喘组的脾源性CD4+T细胞中Th17的阳性率显著增高(均P<0.01)；与未转染哮喘组相比,转染siRNA-T-bet-429哮喘组的脾源性CD4+T细胞中Th17的阳性率显著增高(P<0.01)(7)与正常对照组相比,未转染哮喘组和转染siRNA-T-bet-429哮喘组的脾源性CD4+T细胞培养上清中IL-17的浓度显著增高(均P<0.01)；与未转染哮喘组相比,转染siRNA-T-bet-429哮喘组的脾源性CD4+T细胞培养上清中IL-17的浓度显著增高(P<0.01)(8)转染siRNA-T-bet-429哮喘组的脾源性CD4+T细胞中T-bet mRNA的表达与IL-23受体mRNA的表达显著负相关(Υ=-0.786,P<0.05),与Thl7阳性率显著负相关(Υ=-0.882,P<0.01),与IL-17的浓度显著负相关(Υ=-0.893,P<0.01)。(9)转染siRNA-T-bet-429哮喘组的脾源性CD4+T细胞中T-bet蛋白的表达与IL-23受体蛋白的表达显著负相关(Υ=-0.873,P<0.01),与Th17阳性率显著负相关(Υ=-0.786,P<0.05),与IL-17的浓度显著负相关(r=-0.939,P<0.01)。结论1.急性哮喘中下调T-bet的表达可促进Th17细胞的增值和IL-17的分泌。2.急性哮喘中T-bet可能通过IL-23受体影响Th17细胞的增值和致炎机能。更多还原

【Abstract】 Part I Establishment of Acute Asthmatic Mouse Model and Confirmation of its ReliabilityObjective:To confirm the reliability of established acute asthmatic mouse model with modified evaluation criterion.Methods:35 SPF female BALB/c mice were randomly divided into control group and asthmatic group. The asthmatic model was reproduced by sensitization with intraperitoneal injection of OVA, followed by repeated inhalation of OVA. The control group received only PBS in the same manner. After 24 hours of the last inhalation, asthmatic symptoms were observed. The changes in airway responseveness were determined by lung resistance(RL) stimulated by acetylcholine(Ach); the white cell count, neutrophils(%), eosinophils(%), lymphocytes(%) were measured from bronchoalveolar lavage fluid (BALF); Lung tissue sections were stained for general pathology. Spleenic mononuclear cells from acute asthmatic mouse model were removed red blood cells, followed by being isolated CD4+T cells using immuno-magnetic beads. Spleenic CD4+T cells were cultivated in RPMI-1640 with 10% Fetal Bovine Serum, stimulated by ConA and PMA. After 24 hours, the total spleenic CD4+T cells and ratio of positive Thl7 cells were detected; the levels of IL-4 and IL-17 concentration obtained from BALF and spleenic CD4+T culture supernatant were examined.Results:(1) Asthmatic symptoms were more severe in asthmatic group as compared with controls.(2) Total Lung Resistance was obviously increased in asthmatic group as compared with controls (p<0.01).(3) More extensive inflammatory cells infiltrated around the bronchi and mucus deposition in airway lumen were found in asthmatic group as compared with controls (p<0.01).(4) White cell count, Neu(%), Eos (%) and Lym(%) in the BALF of asthmatic group were obviously increased in sthmatic group as compared with controls (respectively p<0.01).(5) Spleenic CD4+T cell count and the ratio of positive Th17 cells were obviously increased in asthmatic group as compared with controls (p<0.01).(6) IL-4 and IL-17 concentration were significantly elevated in BALF and spleenic CD4+T culture supernatant from asthmmtic group as compared with controls (respectively p<0.01).(7) IL-17 concentration in BALF form asthmatic group positively correlated with Neu(%) (r=0.903,P<0.01).Conclusions:Acute asthmatic mouse model reproduces successfully according to modified evaluation criteria. PartⅡEffect of IL-23 Receptor on Proliferation and Inflammation of Th17 Cells from Acute Asthmatic Mouse ModelObjective:To detect the effect of interleukin-23 receptor(IL-23 Receptor) on proliferation and inflammation of Th17 cells, a subset of spleenic CD4+T cells from acute asthma mouse model.Methods:Spleenic CD4+T cells isolated using immuno-magnetic beads and cultured in RPMI-1640 with 10% Fetal Bovine Serum, stimulated by ConA and PMA. After 24 hours, the expression of IL-23 receptor mRNA and IL-23 receptor protein were detected by real-time PCR and Western blotting respectively; the ratio of positive Th17 cells was detected by flow cytometer (FCM); IL-17 concentration was detected by ELISA. Finally, the correlation between IL-23 receptor expression with the ratio of positive Th17 cells and IL-17 concentration were analyzed.Results:(1) The expression of IL-23 receptor mRNA in spleenic CD4+T cells from asthmatic group was significantly elevated as compared with controls (p<0.01).(2) The expression of IL-23 receptor protein in spleenic CD4+T cells from asthmatic group was significantly elevated as compared with controls (p<0.01).(3) The ratio of positive Thl7 cells in asthmagc group was obviously higher than that of controls (p<0.01).(4) The more extensive IL-17 concentration was found in asthmtic group as compared with controls (p<0.01)(5) The IL-23 receptor mRNA expression in asthmatic group positively correlated with the ratio of positive Th17 cells(r=0.789, P<0.05) and the IL-17 concentration (r=0.788, P<0.05)。(6) The IL-23 receptor protein expression in asthmatic group positively correlated with the ratio of positive Th17 cells (r=0.828, p<0.01) and the IL-17 concentration (r=0.802, P<0.05)。Conclusions:IL-23 receptor expression is significantly increased in spleenic CD4+T cells from acute asthmatic mouse model and associates with proliferation and inflammation of Th17 cells.PartⅢDown-regulating of T-bet Affects the Promotion the Poliferation and Inflammation of Th17 Cells througth IL-23 Receptor in acute asthmaObjective:To investigate the effect of down-regulating T-bet on proliferation and inflammation of Th17 cells from acute asthmatic mouse model.Methods:Spleenic CD4+T cells from acute asthmatic mouse model were transient transfected with three chemosynthesis siRNA sequences by Lipofectamine2000. After culture and stimulation with cytokine(ConA and PMA), the best efficient siRNA-T-bet choosn by detection of T-bet expression, was trasnsfected into spleenic CD4+T cells from asthmatic group. Cells divided into three groups:control group, untransfected asthmatic group and transfected asthmatic group. The expression of IL-23 receptor and T-bet mRNA were analyzed by real-time PCR. The expression of IL-23 receptor and T-bet protein were analyzed by Western blotting. The ratio of positive Th17 cells was measured by FCM. The IL-17 concentration was detected by ELISA. Finally, the correlation between T-bet expression with IL-23 receptor expression, the ratio of positive Th17 cells and IL-17 concentration were analyzed.Results:(1) siRNA-T-bet-429 had better silencing effect than other siRNA sequences (p<0.01). (2) The expression of T-bet mRNA in spleenic CD4+T cells from transfected asthmatic and transfected siRNA-T-bet-429 group were lower than that of controls (p<0.01). The expression of T-bet mRNA in transfected siRNA-T-bet-429 group was lower than that of transfected asthmatic group (p<0.01).(3) The expression of T-bet protein in spleenic CD4+T cells from transfected asthmatic and transfected siRNA-T-bet-429 group were lower than that of controls (p<0.01). The expression of T-bet protein in transfected siRNA-T-bet-429 group was lower than that of transfected asthmatic group (p<0.01).(4) The expression of IL-23 receptor mRNA in spleenic CD4+T cells from transfected asthmatic and transfected siRNA-T-bet-429 group were lower than that of controls (p<0.01). The expression of IL-23 receptor mRNA in transfected siRNA-T-bet-429 group was lower than that of transfected asthmatic group (p<0.01).(5) The expression of IL-23 receptor protein in spleenic CD4+T cells from transfected asthmatic and transfected siRNA-T-bet-429 group were lower than that of controls (p<0.01). The expression of IL-23 receptor protein in transfected siRNA-T-bet-429 group was lower than that of transfected asthmatic group (p<0.01).(6) The ratio of positive Th17 cells in transfected asthmatic and transfected siRNA-T-bet-429 group significantly elevated as compared with controls (p<0.01). The ratio of positive Th17 cells in transfected siRNA-T-bet-429 group significantly elevated as compared with transfected asthmatic group (p<0.01).(7) The IL-17 concentration of spleenic CD4+T culture supernatant in transfected asthmatic and transfected siRNA-T-bet-429 group significantly elevated as compared with controls (p<0.01). The IL-17 concentration of spleenic CD4+T culture supernatant in transfected siRNA-T-bet-429 group significantly elevated as compared with transfected asthmatic group (p<0.01).(8) The T-bet mRNA expression in transfected siRNA-T-bet-429 group negatively correlated with IL-23 receptor mRNA expression (r=-0.786,p<0.05), and the ratio of positive Thl7 cells (r=-0.882, P<O.01), and the concentration (r=-0.893,P<O.01).(9) The T-bet protein expression in transfected siRNA-T-bet-429 group negatively correlated with IL-23 receptor protein expression (r=-0.873,P<0.01), and with the ratio of positive Thl7 cells (r=-0.786,P<0.05), and the IL-17 concentration (r=-0.939,p<0.01).Conclusions:Down-regulation of T-bet can promote the proliferation and inflammation of Th17 cells through IL-23 receptor in acute asthma.更多还原