Hegyi Péter, Ordog Balázs, Rakonczai Zoltán, Takács Tamás, Lonovics János, Szabolcs Annamária, Sári Réka, Tóth András, Papp Julius-G, Varró András, Kovács Mária-K, Gray Mike-A, Argent Barry-E, Boldogköi Zsolt
Department of Biology, Faculty of Medicine, University of Szeged, Somogyi Bela str. 4, H-6720 Szeged, Hungary.
World J Gastroenterol. 2005 Oct 14;11(38):5997-6002. doi: 10.3748/wjg.v11.i38.5997.
To examine the effect of acute infection caused by herpesvirus (pseudorabies virus, PRV) on pancreatic ductal secretion.
The virulent Ba-DupGreen (BDG) and non-virulent Ka-RREp0lacgfp (KEG) genetically modified strains of PRV were used in this study and both of them contain the gene for green fluorescent protein (GFP). Small intra/interlobular ducts were infected with BDG virus (10(7) PFU/mL for 6 h) or with KEG virus (10(10) PFU/mL for 6 h), while non-infected ducts were incubated only with the culture media. The ducts were then cultured for a further 18 h. The rate of HCO(3)(-) secretion (base efflux -J(B-)) was determined from the buffering capacity of the cells and the initial rate of intracellular acidification (1) after sudden blockage of basolateral base loaders with dihydro-4,4-diisothiocyanatostilbene-2,2-disulfonic acid (500 micromol/L) and amiloride (200 micromol/L), and (2) after alkali loading the ducts by exposure to NH(4)Cl. All the experiments were performed in HCO(3)(-)-buffered Ringer solution at 37 degrees (n = 5 ducts for each experimental condition). Viral structural proteins were visualized by immunohistochemistry. Virally-encoded GFP and immunofluorescence signals were recorded by a confocal laser scanning microscope.
The BDG virus infected the majority of accessible cells of the duct as judged by the appearance of GFP and viral antigens in the ductal cells. KEG virus caused a similarly high efficiency of infection. After blockage of basolateral base loaders, BDG infection significantly elevated -J(B-) 24 h after the infection, compared to the non-infected group. However, KEG infection did not modify -J(B-). After alkali loading the ducts, -J(B-) was significantly elevated in the BDG group compared to the control group 24 h after the infection. As we found with the inhibitor stop method, no change was observed in the group KEG compared to the non-infected group.
Incubation with the BDG or KEG strains of PRV results in an effective infection of ductal epithelial cells. The BDG strain of PRV, which is able to initiate a lytic viral cycle, stimulates HCO(3)(-) secretion in guinea pig pancreatic duct by about four- to fivefold, 24 h after the infection. However, the KEG strain of PRV, which can infect, but fails to replicate, has no effect on HCO(3)(-) secretion. We suggest that this response of pancreatic ducts to virulent PRV infection may represent a defense mechanism against invasive pathogens to avoid pancreatic injury.
研究疱疹病毒（伪狂犬病病毒，PRV）引起的急性感染对胰腺导管分泌的影响。
本研究使用了PRV的强毒株Ba-DupGreen（BDG）和无毒株Ka-RREp0lacgfp（KEG）基因改造株，二者均含有绿色荧光蛋白（GFP）基因。小叶内/小叶间小导管分别用BDG病毒（10⁷ PFU/mL，作用6小时）或KEG病毒（10¹⁰ PFU/mL，作用6小时）感染，未感染的导管仅用培养基孵育。然后将导管再培养18小时。用二氢-4,4-二异硫氰酸芪-2,2-二磺酸（500 μmol/L）和阿米洛利（200 μmol/L）突然阻断基底外侧碱基转运体后，以及用氯化铵使导管碱化后，根据细胞的缓冲能力和细胞内酸化的初始速率（1）测定HCO₃⁻分泌速率（基础外流-J(B-)）。所有实验均在37℃的HCO₃⁻缓冲林格氏液中进行（每个实验条件n = 5根导管）。通过免疫组织化学观察病毒结构蛋白。用共聚焦激光扫描显微镜记录病毒编码的GFP和免疫荧光信号。
根据导管细胞中GFP和病毒抗原的出现判断，BDG病毒感染了导管中大多数可及细胞。KEG病毒也引起了同样高的感染效率。阻断基底外侧碱基转运体后，与未感染组相比，BDG感染在感染后24小时显著提高了-J(B-)。然而，KEG感染并未改变-J(B-)。导管碱化后，与对照组相比，BDG组在感染后24小时-J(B-)显著升高。正如我们用抑制剂阻断法所发现的，与未感染组相比，KEG组未观察到变化。
用PRV的BDG或KEG株孵育可有效感染导管上皮细胞。能够启动裂解性病毒周期的PRV的BDG株在感染后24小时可使豚鼠胰腺导管中的HCO₃⁻分泌增加约四至五倍。然而，能够感染但不能复制的PRV的KEG株对HCO₃⁻分泌没有影响。我们认为胰腺导管对强毒株PRV感染的这种反应可能代表了一种针对入侵病原体的防御机制，以避免胰腺损伤。
