Zhang L F, Wang C
College of Veterinary Medicine, Mississippi State University, MS 39762-9825, USA.
Am J Vet Res. 1998 Jun;59(6):717-21.
To determine whether differences exist in induction and quantity of tumor necrosis factor alpha (TNF-alpha), interleukin (IL)1 beta, and IL-10 mRNA transcripts when mouse J774A.1 macrophages are infected with Listeria monocytogenes, including 2 isolates originating from channel catfish, the wild-type virulent (EGD) strain, and a nonhemolytic strain (ATCC 15313).
Listeria monocytogenes isolates from kidneys or fillets of channel catfish were used to stimulate cytokine production from mouse macrophages. The RNA from the infected macrophages was collected.
Four hours after infection with L monocytogenes, total cellular RNA was extracted from the J774A.1 cells and reversed transcribed to cDNA, which was amplified, using specific primers for TNF-alpha, IL-1 beta, or IL-10. The specific amplified DNA fragments were detected on polyacrylamide gels and quantified, using a reverse transcription polymerase chain reaction (PCR)-mediated ELISA.
The wild-type hemolytic EGD strain and the 2 hemolytic catfish isolates of L monocytogenes induced higher amounts of TNF-alpha-, IL-1 beta-, and IL-10-specific mRNA in J774A.1 cells than did the nonhemolytic strain.
Hemolysin-associated induction of TNF-alpha, IL-1 beta, and IL-10 cytokines may be related to survival and replication of L monocytogenes in macrophages. It also suggests that the PCR-mediated ELISA procedure is a sensitive test to quantify cytokines from cell cultures.
确定当小鼠J774A.1巨噬细胞感染单核细胞增生李斯特菌时，肿瘤坏死因子α（TNF-α）、白细胞介素（IL）1β和IL-10 mRNA转录本的诱导情况及数量是否存在差异，其中包括源自斑点叉尾鮰的2株分离株、野生型强毒株（EGD）和非溶血菌株（ATCC 15313）。
使用源自斑点叉尾鮰肾脏或鱼片的单核细胞增生李斯特菌分离株刺激小鼠巨噬细胞产生细胞因子。收集感染巨噬细胞的RNA。
用单核细胞增生李斯特菌感染4小时后，从J774A.1细胞中提取总细胞RNA并逆转录为cDNA，使用针对TNF-α、IL-1β或IL-10的特异性引物进行扩增。在聚丙烯酰胺凝胶上检测特异性扩增的DNA片段，并使用逆转录聚合酶链反应（PCR）介导的酶联免疫吸附测定（ELISA）进行定量。
与非溶血菌株相比，野生型溶血EGD菌株和2株单核细胞增生李斯特菌溶血斑点叉尾鮰分离株在J774A.1细胞中诱导产生的TNF-α、IL-1β和IL-10特异性mRNA量更高。
溶血素相关的TNF-α、IL-1β和IL-10细胞因子诱导可能与单核细胞增生李斯特菌在巨噬细胞中的存活和复制有关。这也表明PCR介导的ELISA程序是一种定量细胞培养物中细胞因子的灵敏检测方法。
