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目的研究严重急性呼吸综合征冠状病毒(SARS-CoV)通过微血管内皮细胞释放细胞因子/趋化因子,介导急性肺损伤的可能机制。方法广州呼吸疾病研究所2003年2月至5月确诊为 SARS 的住院患者23例,男15例,女8例,年龄27～55岁,平均(36±6)岁。用液相蛋白芯片系统测定 SARS 患者不同阶段血中的细胞因子/趋化因子水平,免疫组化方法检测 SARS 患者肺组织中γ干扰素诱导蛋白10(interferon-γ inducible protein 10,IP-10)的表达;通过昆虫-杆状病毒表达系统表达 SARS-CoV 的 S 蛋白,并经镍亲和磁珠纯化,将重组的 S 蛋白作用于人脐静脉内皮细胞(HUVEC)观察其形态学变化并检测相应细胞因子/趋化因子的变化。结果 IP-10在健康对照组水平较低[(1200±500)ng/L],疾病早期就显著增高[(7600±2400)ng/L,P<0.01],并且在进展期[(8100±2300)ng/L,P<0.01]和疾病晚期[(8000±2800)ng/L,P<0.01]都保持显著增高的水平,直至恢复期[(1 250±450)ng/L,P>0.05]才正常,其在 SARS 患者肺组织显著表达。S 蛋白作用于人血管内皮细胞可引起细胞内出现空泡,细胞变圆有脱落,随时间延长细胞破碎溶解。基础状态下内皮细胞并不生成 IP-10,S 蛋白(5、20、40 mg/L)分别作用后可见 IP-10生成呈显著量效反应,如12 h分别为[(179±34)、(889±212)、(1676±199)ng/L,P 均<0.05]。结论 (1)SARS-CoV 感染的患者血中 IP-10活性显著增高,一直持续到恢复期,肺中 IP-10的表达增加。(2)SARS-CoV 可通过其 S蛋白诱导血管内皮细胞合成释放 IP-10,损害内皮细胞。(3)SARS-CoV 的 S 蛋白诱导 IP-10在宿主细胞的生成不依赖 IFN-γ。
Objective To study the possible mechanism of acute lung injury induced by SARS-CoV releasing cytokines / chemokines through microvascular endothelial cells. Methods Twenty-three inpatients were diagnosed as SARS from February to May 2003 in Guangzhou Respiratory Disease Institute. There were 15 males and 8 females, aged from 27 to 55 years (mean, 36 ± 6 years). The levels of cytokines / chemokines in different stages of SARS patients were measured by liquid protein chip system, and the expression of interferon-γ inducible protein 10 (IP-10) in lung tissue of SARS patients was detected by immunohistochemistry. The S protein of SARS-CoV was expressed by the insect-baculovirus expression system and purified by nickel affinity magnetic beads. The recombinant S protein was applied to human umbilical vein endothelial cells (HUVECs) to observe its morphological changes and detected Corresponding cytokine / chemokine changes. Results The level of IP-10 was significantly lower in the healthy control group [(1200 ± 500) ng / L] [7600 ± 2400 ng / L, P <0.01] (1 250 ± 450) ng / L, 2300 ng / L, P <0.01] and advanced stage of disease [(8000 ± 2800) ng / L, P <0.01] respectively. 0.05] was normal, which was significantly expressed in lung tissue of SARS patients. S protein acting on human vascular endothelial cells can cause vacuoles in the cells, rounded cells fall off, with the passage of time broken cell lysis. Under the basal conditions, IP-10 production was not significantly affected by endothelial cells producing IP-10 and S protein (5, 20, and 40 mg / L) (889 ± 212) and (1676 ± 199) ng / L respectively, all P <0.05]. Conclusions (1) The IP-10 activity in the blood of patients with SARS-CoV infection was significantly increased until the convalescence, and the expression of IP-10 in the lung increased. (2) SARS-CoV can induce the synthesis and release of IP-10 by vascular endothelial cells through its S-protein and damage the endothelial cells. (3) The S protein of SARS-CoV induces IP-10 production in host cells independent of IFN-γ.