蔡嘉哲Gregory J Tsay徐再靜Tsai-Ching Hsu醫學研究所2026-10-282007-07-312004https://handle.ncl.edu.tw/11296/vyzbpzhttps://scholars.csmu.edu.tw/handle/123456789/35037於1998年3月，在中山醫學大學附設醫院免疫風濕科門診發現有合併皮膚紅斑及關節炎現象的病人求診，經由parvovirus B19血清檢驗,發現四位病人有B19 IgM 和IgG 抗體，而且藉由nest PCR檢驗亦發現其中三個病人的 B19 DNA呈現陽性反應，證實這些病人是為B19感染。這些病人亦發現在自體免疫檢查中之抗嗜中性白血球細胞質抗體(ANCA)和抗牛心脂抗體(aCL)亦呈現陽性反應,這些資料顯示B19感染與自體免疫疾病有密切之關係。為了更近一步了解人類微小病毒B19(B19)感染與自體免疫疾病之關係，我們便展開一系列相關的研究。結果發現人類微小病毒B19 DNA僅在72位全身性紅斑性狼瘡病人中的17 位呈現陽性反應，而這17位患者中僅一位有B19 IgG 抗體，二位有B19 IgM抗體。然而沒有B19 DNA的55位全身性紅斑性狼瘡病人，發現有27位有B19 IgG 抗體(49.1%)，有21位有B19 IgM抗體(38.2%)。研究結果顯示沒有B19抗體的病人比有B19抗體的病人較常發現具有B19 DNA (p<0.05)。另外，在其它自體免疫疾病的病人身上，我們則並未發現有B19 DNA的存在。研究中也發現這些全身性紅斑性狼瘡病人之所以沒有表現B19抗體，可能的原因是因為這些宿主的免疫力低下或者使用了免疫抑制劑所造成的結果。同時也發現這些病人也往往伴隨著有補體低下和雷諾氏症候群的情形。此外，已知在急性或猛暴型肝炎的病人也有B19感染的情形，但是慢性肝炎病人感染的狀況並不清楚。在我們的研究結果發現，慢性B型肝炎或C型肝炎的病人亦常發生B19感染。同時我們也發現B19感染並未影響到慢性B型肝炎或C型肝炎病人肝功能缺失的情形(p>0.05)。經由DNA定序後，我們發現所有20位慢性B型肝炎的病人皆顯示序列為TW-3 基因型，而所有12位慢性C型肝炎的病人顯示其序列為TW-9基因型。此外亦發現感染B19的慢性B型肝炎病人，其B19抗體呈現為IgG(+)IgM(-)的情形，B19 DNA檢測也較常呈現陽性 (P<0.01)。這可能代表著B19感染在這些病人身上並不能完全被清除而造成持續感染。此外，B19病毒感染對宿主的傷害，已知主要為B19的非結構蛋白NS1會對紅血球系細胞造成毒性並引起細胞凋亡。最近，又有文獻指出B19 DNA也會存在於非紅血球系的器官或組織。因此我們便試圖分析NS1對於非紅血球系細胞之影響。結果發現在轉染NS1(EGFP-NS1)的COS-7細胞(非紅血球系細胞)會產生細胞凋亡的情形，而且是經由粒線體路徑所誘發的。NS1的表現不但造成p53和Bax的表現增加並且也促使下游的caspase 9和caspase 3 的活化而非caspase 8或Fas的活化，進而使細胞產生凋亡。另外，NS1的表現也影響細胞週期的進行促使p16INK4 和 p21WAF1/CIP1 的表現增加而使細胞週期停留在G1 phase。最近，我們更進一步利用DNA array分析轉染NS1的COS-7細胞之基因表現，資料中顯示像p53，Caspase 3等基因表現有增加的情形。這和我們目前的研究結果是相似的。另外我們目前利用RT-PCR方法分析CPR2基因，結果顯示EGFP-NS1轉染細胞之CPR2表現具有統計學上意義(p<0.05)。這些結果資料可以提供我們一些線索，以更加了解人類微小病毒B19感染及其非結構蛋白NS1對非紅血球系細胞之影響，並作為未來人類微小病毒B19致病機轉相關研究的參考。Previously, we described four patients who had clinical diagnosis of erythema infectiosum and presented with skin rash, polyarthralgia, and polyarthritis. Anticardiolipin antibody (aCL) and anti-neutrophil cytoplasmic antibody (ANCA) were also positive in these patients. These data indicate parvovirus B19 may be linked to the induction of an autoimmune response. In order to understand the relationship between B19 infection and autoimmune diseases, we then launch a series of research on them. Parvovirus B19 DNA was detected in 17 of 72 patients with SLE, but not in patients with other systemic rheumatic diseases. Of the 17 patients with B19 DNA, only one had B19 IgG antibody and two had B19 IgM antibodies. B19 DNA was found more commonly in sera from SLE patients without anti-B19 antibodies than in those with anti-B19 antibodies (P<0.05). B19 infection in patients with SLE may due to lack of anti-B19 antibodies because of either the immunocompromised nature of the host or the use of immunosuppressive drugs. There was a higher prevalence of hypocomplementaemia and RP in patients with parvovirus B19 viraemia than in those without parvovirus B19 viraemia. Moreover, our data herein revealed that human parvovirus B19 infection was frequently found in patients with chronic hepatitis B or hepatitis C infection. The occurrence of liver dysfunction was not affected by B19 infection in patients with HBV and HCV infection (p>0.05). All of the 20 serum samples with B19 DNA from patients with chronic HBV infection and all of the 12 serum samples with B19 DNA from patients with chronic HCV infection exhibited TW-3 genotype and TW-9 genotype, respectively. The frequency of B19 IgG(+)IgM(-) in HBV patients with B19 DNA PCR positivity was higher than in patients with PCR negativity (p<0.01). The results indicate that B19 may not be eradiated in these patients with HBV and HCV infection, causing persistent infection. Human parvovirus B19 has been found in various tissues in addition to erythroid lineage cells and nonstructural protein (NS1) is reported to induce cytotoxicity and apoptosis in erythroid lineage cells, but the mechanism in non-permissive cells is still not clear. So we attempted to elucidate the possible molecular mechanism of NS1-induced apoptosis in non-permissive cells and identified the expression of EGFP-NS1 fusion protein in COS-7 cells. The results suggest that the cell death of the NS1-transfected cells is associated with the mitochondria related apoptosis. The expression of p53, an important molecule in apoptosis and cell cycle regulation, and its downstream cell cycle kinase inhibitors p16INK4 and p21WAF1/CIP1 were up-regulated in the NS1-transfected cells. Also, increased expression of the pro-apoptotic Bcl-2 members Bax, Bad and activation of caspase 3 and caspase 9, but not the activation of caspase 8 nor Fas were detected in the NS1-transfected cells. Recently, the DNA array analysis revealed that p53, caspase 3 gene were up-regulation in NS-1 transfected cells. This is similar to our present findings. Moreover, we analyzed the CPR2 gene by using the RT-PCR method and demonstrated the difference was statistically significant between EGFP and EGFP-NS1 transfection in COS-7 cells (p<0.05). These findings might provide alternative information for further study in B19 infection and characterization of B19 NS1 protein in B19 non-permissive cells, and these will be used as reference to further study B19 pathogenesis.人類微小病毒B19自體免疫疾病細胞凋亡Human Parvovirus B19Autoimmune DiseasesApoptosisthesis