- 學位論文
乾燥症和CREST症候群病人血清中自體抗體與蕈毒鹼型乙醯膽鹼接受器之影響
Effect of autoantibodies from patients with Sjogren's syndrome and CREST syndrome on muscarinic receptor 3
摘要
修格連氏症候群(Sjogren's syndrome)，俗稱乾燥症(sicca syndrome)，是一種慢性自體免疫疾病，其外分泌腺如唾液腺和淚腺等部位都會遭受免疫細胞浸潤，進而造成口乾舌燥(xerotomia)、乾性角膜結膜炎(keratoconjunctivities sicca)等症狀，血清學檢驗則是檢查是否有抗SSA/Ro及SSB/La抗體，這些抗體大多常見於乾燥症，但也會出現於其他自體免疫疾病，如紅斑性狼瘡(SLE)。先前文獻顯示有少部分乾燥症病人血清中有抗染色體中節抗體(anti-centromere antibody)，而無SSA/Ro和SSB/La。另外，有文獻也指出，乾燥症病人血清中含有抗M3R抗體，可以解釋乾燥症之致病機轉。所以我們除了想要探討血清中各種自體抗體對於M3R之影響，也以CREST症候群中的抗染色體中節抗體去觀察對M3R之效應，並試著解釋抗染色體中節抗體是否會造成乾燥的病徵。我們首先利用老鼠膀胱平滑肌肉，觀察正常血清以及病人血清對於肌肉收縮之影響。再利用molecular cloning的技術將M3R213-228 16個胺基酸序列大量複製並使用Ni-NTA親和性管柱純化蛋白。進一步利用酵素連結免疫分析法(ELISA)分析病人血清對於抗原之關連性。實驗結果顯示，在15位血清含有自體抗體的病人中有2位病人其血清有抑制老鼠膀胱平滑肌收縮之效應，而這兩位病人各帶有抗SSA/SSB及ACA抗體，分別為乾燥症和CREST症候群之病人。分別可抑制50%和36%乙醯膽鹼所刺激引起的正常收縮。進一步我們以M3R213-228等16個氨基酸序列為抗原以ELISA方法分析。總共有8位病人和2位正常捐血者(2/85)其吸光值(O.D. value)落在cut-off 0.78之上，為陽性。其中SSA+/SSB+病人有1位(1/56)，ACA+有3位(3/39)，RA病人也有4位(4/34)，其餘皆在cut-off之下。若以吸光值總平均比較，皆與正常組有統計上之意義(p<0.05)尤以SSA+/SSB+、SSA+、RA組更為顯著(p<0.001)。在本研究中我們觀察到乾燥症以及CREST症候群病人血清有抑制平滑肌收縮之效應，其血清中可能含有抗M3R的自體抗體；另外，在CREST症候群病人血清中大部分可檢驗出抗染色體中節抗體(ACA)，而此抑制效應是否為ACA在部分乾燥症病人身上所造成乾燥之原因，仍需進一步實驗證實。ELISA部分雖陽性率偏低，但以吸光值總平均比較，各組與正常組有統計上意義，我們推測可能這些病人其抗M3R213-228抗體處於low titer的情形。
並列摘要
Sjogren's syndrome (SS) is a systemic autoimmune connective tissue disease. It usually accompanies keratoconjunctivitis sicca and xerostomia. These symptoms are characterized by lymphocytic infiltration in exocrine glands and made exocrine glands destroyed, resulting in glandular dysfunction. It has been reported the presence of serum autoantibodies that target the Ro/La ribonucleoprotein for serological diagnosis in autoimmune diseases, such as SS and SLE. Anti-muscarinic receptor 3 antibody has been found in patients with Sjogren’s syndrome which might impair the fluid production and transport in salivary and lacrimal gland. However, many clinical findings indicated the anti-centromere antibody also present in the small part of patients of Sjogren’s syndrome without SSA/SSB antibodies. Therefore, we want to clarify whether the anti-centromere antibody in SS affect the salivation and result to sicca feature. We used functional assay to measure the contraction of bladder smooth muscle with serum from patients. Recombinant antigens of M3R213-228 peptide were tested with patients have autoantibodies in ELISA assay. The serum sample consisted of 56 patients with both of anti-SSA/Ro and anti-SSB/La antibody, 57 patients with anti-SSA/Ro antibody, 39 patients with anti-centromere antibody, 34 patients with Rheumatoid arthritis (RA), and 56 patients with systemic lupus erythmatosus (SLE). The 85 serum samples of health group were also obtained. Sera from 1of 4 patients (25%) with anti-SSA/SSB antibody and from 1 of 6 patients (16%) with anti-centromere antibody inhibited acetylcholine (Ach)-evoked bladder contraction by 50% and 36% , respectively. Sera from 3 of 3 healthy individuals had no effect. Analysis of ELISA with M3R213-228 peptide, we observed the positivity of anti-M3R213-228 antibody in 1 of 56 patients with anti-SSA/SSB antibody, 3 of 39 patients with anti-centromere antibody, 4 of 34 patients with RA and 3 of 85 normal individuals. The anti-M3R213-228 level (mean OD)is higher in each of comparison groups than normal group, except for SLE group. In this study, we observed serum from patients with SS and CREST syndrome has the inhibitory effect on bladder smooth muscle. We speculate the anti-M3R antibody presents in these serum and block the acetylcholine transmission. The ELISA data show anti-M3R213-228 positivity in a few patients, but the mean of OD has significant differences compared to normal groups. The serum maybe exist a low titer of anti-M3R213-228 antibody. The anti-centromere antibody whether affect the signal transduction by M3R mediated and how to play a pathological role in SS patients need to be elucidated in the future.