【作者】 朱峰；

【导师】 廖玉华；

【作者基本信息】 华中科技大学 ， 内科学， 2006， 博士

【摘要】 第一部分难治性高血压患者抗AT1受体自身抗体对VSMC胞浆游离钙水平的影响目的:观察难治性高血压患者血清中血管紧张素受体Ⅰ型(AT1受体)自身抗体对大鼠主动脉血管平滑肌细胞(Vascular smooth muscle cell,VSMC)胞浆游离钙水平的影响。方法:应用AT1受体的胞外第二环作为抗原,采用ELISA检测22例难治性高血压患者及24名非难治性高血压患者血清中AT1受体自身抗体。应用蛋白G亲和柱提纯难治性高血压患者血清中AT1受体自身抗体。用细胞免疫荧光染色方法检测该抗体与AT1受体的亲和性。培养大鼠主动脉VSMC,应用钙离子的荧光探针Fluo-3/AM负载原代培养VSMC,应用共聚焦显微镜检测细胞荧光强度变化来反映细胞内游离钙水平的变化。将患者血清、提纯AT1受体胞外自身抗体和血管紧张素Ⅱ(angiotoninⅡ,AngⅡ)分别作用于VSMC,观察荧光强度变化来检测VSMC胞浆游离钙水平变化。并观察AT1受体拮抗剂氯沙坦,α1肾上腺素受体拮抗剂哌唑嗪,AT1受体胞外第二环肽抗原表位对AT1受体自身抗体的拮抗作用。结果:ELISA检测显示22例难治性高血压患者中有10例患者AT1受体细胞外第二环自身抗体阳性,占45.5%,该抗体的检出率明显高于非难治高血压组(n=24,12.5%,P<0.05)。细胞免疫荧光染色显示提纯的AT1受体细胞外二环自身抗体能够与VSMC膜表面AT1受体特异性结合。并且该抗体能够刺激培养VSMC,使得胞内游离钙水平增高,呈现剂量依赖效应,说明该抗体具有与AngⅡ类似激动效应。该自身抗体的激动效应能够被AT,受体拮抗剂氯沙坦及ATl受体胞外第二环肽抗原表位所拮抗。结论:AT1受体胞外第二环肽是自身免疫重要的抗原表位,针对该表位的抗体能够与AT1受体特异结合并且模拟AngⅡ的激动作用,促进VSMC胞内游离钙水平升高,在难治性高血压患者高血压发病机制中起着重要作用。应用AT1受体拮抗剂氯沙坦能够有效的阻断该抗体的效应。第二部分主动免疫AT1受体胞外肽段对自发性高血压大鼠血压的影响目的观察血管紧张素Ⅱ的Ⅰ型(AT1)受体细胞外不同肽段主动免疫自发性高血压大鼠(SHR)对血压影响,探索针对AT1受体细胞外不同抗原表位的抗体对血压的效应,进一步筛选具有降压作用的肽段。方法选取大鼠AT1受体AT1A受体亚型,根据AT1A受体胞外肽段氨基酸序列设计肽段ATR11188、ATR12181、ATR13263、ATR13275,合成纯化并与破伤风类毒素载体蛋白耦联。SHR随机分组:(1).对照组(n=7),(2).ATR11188免疫组(n=7),(3).ATR12181免疫组(n=8),(4).ATR13263免疫组(n=6),(5).ATR13275免疫组(n=6)。免疫各组均给于肽一破伤风类毒素复合物加福氏佐剂皮下注射,主动免疫7次。对照组给予相同时间点生理盐水加福氏佐剂皮下注射。实验期间动态监测SHR的收缩压、心率及血清抗体滴度。亲和层析法提取各个抗肽抗体,刺激培养的大鼠血管平滑肌细胞(VSMC),观察抗体对VSMC细胞增殖的效应,判断各个抗体的对AT1受体效应。结果各组肽段免疫大鼠后都能产生高滴度抗体。ATR12181组收缩压(SBP)在第8周时,SBP为167.3±10.4mmHg较对照组180.6±8.0mmHg明显下降,P<0.05,该血压水平可以维持到实验结束。其他各免疫组血压与对照组比较SBP水平无显著差异。提取抗ATR12181抗体,免疫印迹结果与免疫荧光实验显示该抗体能与VSMC细胞膜上的AT1A受体特异性结合,并能够拮抗AngⅡ对VSMC细胞的增殖作用。结论我们再次验证了针对ATR12181肽段抗体可以使SHR血压下降,为降压疫苗的研究提供了实验依据,并且提示针对AT1受体细胞外不同抗原表位的抗体其生物效应是不同的。第三部分ATR12181疫苗对自发性高血压大鼠靶器官的保护作用目的观察具有降压作用的ATR12181肽段主动免疫自发性高血压大鼠对心肾两个重要的靶器官的保护作用。方法根据大鼠AT1A胞外肽段氨基酸序列设计ATR12181,合成纯化并与破伤风类毒素耦联加用福氏佐剂,制备ATR12181疫苗。分24周和64周两个时间点观察ATR12181疫苗主动免疫SHR后,对心肾器官的保护作用。SHR分组:(1).对照组24周(n=7),(2).ATR12181免疫组24周(n=7),(3).对照组64周,(4).ATR12181免疫组64周。免疫组均给于肽一破伤风类毒素复合物加福氏佐剂皮下注射,多次免疫后产生抗体后,鼠尾套管法测量各组大鼠血压,观察血压随时间变化。分别于24周及64周终止观察,处死动物,并测量大鼠体重、心脏重量,计算心脏体重比,留取动物心脏和肾脏进行Masson三色染色病理观察和透射电镜观察,评价心肾病理变化情况。并且测定64周左心室及肾脏皮质c-fos和c-jun的mRNA的水平。结果ATR12181肽段免疫大鼠后持续产生高滴度抗体。免疫组血压于实验第8周较对照组明显下降,并能持续到实验结束。24周时大体病理两组动物心肾没有明显变化,但免疫组心脏体重(3.70±0.13mg/g)比明显低于对照组(3.37±0.14mg/g,P<0.05),Masson染色结果也提示,免疫组心脏的心肌间纤维化及血管周的纤维化均明显轻于对照组,透射电镜观察对照组心室灶状肌纤维排列紊乱并有线粒体堆积,而免疫组病变明显减轻。64周大体病理观察对照组SHR左心室肥厚,而免疫组肥厚不明显,免疫组大鼠体重(538±36g)明显重于对照组(383±18,P<0.01),免疫组心脏重量(1.33±0.11g)明显轻于对照组(1.59±0.06g,P<0.01),免疫组从心脏的超声心动图上观察各个室壁厚度指标均明显低于对照组。64周时免疫组Masson三染色显示疫苗组肾脏肾小球及损伤不明显,间质纤维化也较对照组轻,对照组肾脏可见明显的肾小球损伤、间质纤维化和管型;透射电镜下,对照组肾脏肾小球基底膜广泛性增厚,间质纤维化,免疫组肾脏超微病变明显减轻,偶见局灶性基底膜增厚。两个时间点所有病理指标均显示ATR12181主动免疫后,SHR血压下降的同时心肾两个靶器官保护作用明显。进一步的对AP-1信号的探索表明,ATR12181主动免疫可以降低左心室及肾皮质组织c-fos和c-jun的mRNA水平。结论ATR12181主动免疫后降低SHR大鼠血压的同时,通过降低AT1受体下游信号原癌基因c-fos和cjun的表达减轻心肌重塑及肾脏的损伤。第四部分ATR12181疫苗安全性初步评价目的观察ATR12181疫苗多次免疫自发高血压大鼠(SHR),动物主要器官是否发生自身免疫损伤。方法留取该研究第三部分中64周时间点ATR12181免疫组动物及对照组动物的重要器官,包括心脏、肾脏、肝脏、脾脏、肺、主动脉和肠细膜动脉,进行HE病理染色,免疫组化染色观察抗ATR12181抗体IgG在器官沉积状况,并且对肾脏进行电镜观察,评价器官病理变化。结果HE染色结果显示,ATR12181免疫后,未在血管周及各个主要器官组织中发现淋巴细胞浸润;免疫组化染色结果显示,ATR12181免疫组大鼠靶器官的心肌、胸主动脉及肾脏组织细胞膜上染色阳性,表明大鼠免疫后,所形成的抗体可以结合于靶器官细胞膜上。肾脏透射电镜发现ATR12181组SHR大鼠肾脏的肾小球基底膜发生局灶性的增厚,但对照组病变更明显呈现广泛的肾小球基底膜增厚。并且,ATR12181组的肾小球基底膜未见任何电子致密物沉积。结论对于ATR12181疫苗的初步安全性评价,ATR12181作为短肽主动免疫SHR未发现针对主要器官的自身免疫损伤,但其安全性仍需要进一步更严格的实验进行评价。更多还原

【Abstract】 Part IAutoantibodies against the AT₁ receptor induce Ca²⁺ increase in cultured rat vascular smooth muscle cellsObjective: AT₁ receptor autoantibodies （AT₁-AAs） were described in the patients with malignant hypertension or preeclampia. We also found the autoantibodies were highly associated with refractory hypertension. Function of vascular smooth muscle cells （VSMC） is important in the regulation of blood pressure. We investigated and compared the ability of angiotensin II （Ang II） and AT₁-AA to stimulate the intracellular calcium mobilization and cellular proliferation of rat vascular smooth muscle cells （VSMC）.Methods: 22 patients with refractory hypertension, 24 patients with non-refractory hypertension and 37 normotensives were recruited randomly. The serum of each patient was detected for the presence of AT₁-AAs by ELISA. Ang II and AT1-AA from the sera of patients were used to stimulate rat VSMC in vitro, and celluar proliferation was evaluated by a Brdu ELISA kit.Results: AT1-AAs were detected in 45.5, 12.5 and 8.1% of patients with refractory hypertension, non-refractory hypertension normotensives, respectively. AT1-AAs led the increase intracellular Ca2+ mobilization in a dose-dependent manner and cellular proliferation of VSMC just as Ang II. Both of these effects caused by AT1-AAs were blocked by losartan and by a peptide corresponding to a part of the second extracellular loop of the AT1 receptor. In contrast, control IgG had no effects. Conclusion: Since AT1-AAs exhibited pharmacological activity in VSMC just as Ang II, they may play a role in the elevation of peripheral vascular resistance and in vascular remodeling. AT1-AAs were suggested to involve in resistance to antihypertensive therapy.Part II Influence of active immunizations against AT₁ receptor on blood pressure in spontaneously hypertensive rats.Objective: We designed four different peptides according to the extracellular parts of the rat AT₁A-receptor and used as antigens. We observed the influence of four different antibodies on blood pressure of SHRs to select peptides which can reduce the blood pressure.Methods: the four kinds of peptides （coded ATR11188, ATR12181, ATR13263 and ATR13275） were designed based on the amino acid sequences of rat AT_1A-receptor extracellular parts. The peptides were synthesized, purified, conjugated to tetanus toxoid （TT） and used as antigens. SHRs were divided into 5 groups: （1） Control group （n=7） were 0.9% sodium chloride hypodermically. （2） ATR11188 group （n=7）. （3） ATR12181 group （n=8）. （4） ATR13263 group（n=6）. （4）ATR13275 group （n=6）. All peptide groups were repeatedly immunized with peptide-TT conplex hypodermically. The systolic blood pressure （SBP） was recorded and the titres of specific antibodies were measured by EL1SA. And the immunogobulin （Ig）G was obtained from the SHRs’ serum, and used as antibodies to prove the affinity with AT₁-receptor in the cultured vascular smooth muscles （VSMC） by western blotting and immunofluorescence. IgG was used to stimulate VSMC, and celluar prolifreation was evaluated by a Brdu ELISA kit.Results: the SHRs in all peptide groups developed positive antibodies after repeated immunizations. Western blotting with IgG from ATR12181 produced bands of about 41KD molecular weight. Furthermore, fluorescence microscopy of vascular smooth muscle cells showed colocalization of IgG against ATR12181. In ATR12181 group, the SBP was found to be significantly decreased （SBP of ATR12181, 167.3 ± 10.4 mmHg, SBP of control group 180.6 ± 8.0 mmHg, n=7 per group, P < 0.05） in 8^th week. Other peptides did not reduce the SBP. In addition, proliferation of vascular smooth muscle cells, in response to angiotensin II, could be inhibited by antibodies against peptide ATR12181 in vitro.Conclusion: The results indicated that long-term immunized with ATR12181 effectively reduced blood pressure of SHRs. And active immunization with different peptides from the AT₁-receptor extracellular parts may have different effects on AT₁ -receptor.Part III Target organ protection from an angiotensin II receptor （AT₁） vaccine ATR12181 in spontaneously hypertensive ratsObjective: We have previously established that an angiotensin II receptor （AT₁） vaccine-ATR12181 attenuated the development of high blood pressure （BP） inspontaneously hypertensive rat （SHR） model of human essential hypertension. Ourobjective was to determine whether this attenuation of high BP is associated withprevention of target organ damages induced by hypertensive state.Methods: SHRs were immunized against a peptide （coded ATR12181） from theextracelluar portion of the AT_1A receptor by repeated subcutaneous injections ofpeptide- tetanus-toxoid complex in combination with Freund’s adjuvant. A 24 weekshort-term and a 64 weeks long-term observation were performed. At the end ofobsevations, all SHRs were sacrificed for morphological and ultrastructureexamination of the heart and kidneys. At last, c-fos and c-jun mRNA level of leftventricular muscles and renal cortex were detected by RT-PCR.Results: At the end of observations, vaccinated SHRs expressed lower BP, decreasedcardiac hypertrophy and attenuation of kidney injures. mRNA levels of c-fos andc-jun in heart and kidneys had decrease in vaccinated SHRs.Conclusion: These data demonstrate that repeated immunization against a domain ofthe extracellular portion of the AT₁ receptor is able to cause a target organ protectionagainst hypertension. Active immunization against the AT₁ receptor may beconsidered a promising new strategy in the treatment of hypertension.Part IV Safty of vaccine-ATR12181Objective: since a self-antigen (peptide ATR12181 from the rat AT_1A receptor) wasused, safety of vaccine was concerned. Our objective was to evalaute the safty ofATR12181-vaccine.Methods: SHRs were immunized against a peptide （coded ATR12181） from theextracelluar portion of the AT_1A receptor by repeated subcutaneous injections of peptide- tetanus-toxoid complex in combination with Freund’s adjuvant. A 64 weeks long-term observation was performed. In 64^th weeks, all SHRs were weighed and sacrificed for morphological and ultrastructure examination of important organs including heart, kidneys, liver, spleen, aorta and artery.Results: Body weight （BW） of SHR in ATR12181 group was significant heavier than control group（BW of control group 383±18g, BW of ATR12181 group 538±36g）, and behaviour of ATR12181 group was normal. Morphological examination of important organs did not find any signs of autoimmune damage. Kidney was has abundant arterioles and capillary, where the AT_1A receptors are abundantly expressed. There were no signs of perivascular cellular inflammation. Although the thickening basement membrane of glomcruluses was found under TEM, control group also developed similar ultrastructure changes that were more serious. In addition, there were no signs of depositions in basement membrane or other places of glomcruluses in vaccinated group.Conclusion: Preliminary evaluation found that the vaccine-ATR12181 was safe. Since a self-antigen was used, the more experiment needed be done to evaluate the safty of vaccine.更多还原