Effects of endothelial cells in apical periodontitis on nitric oxide production by polymorphonuclear leukocytes

1)标本：手术切除的根尖周围病变行苏木精-伊红染色检查。根尖囊肿16例，根尖肉芽肿14例。2) iNOS免疫组化：用合成的iNOS特异性肽段免疫兔，纯化人特异性iNOS抗血清。应用iNOS抗血清对根尖囊肿冰冻切片进行免疫组化处理。内皮细胞、上皮细胞、成纤维细胞和巨噬细胞iNOS阳性。淋巴细胞和多形核白细胞也呈iNOS阳性。3) iNOS和IL-1β双免疫组化：部分iNOS阳性细胞IL-1β阳性，部分细胞IL-1β不阳性。另一方面，部分il -1β阳性细胞显示iNOS阳性，部分未显示iNOS阳性。这些数据揭示了IL-1β对iNOS产生的自分泌和旁分泌作用。此外，il -1β阳性细胞与inos阳性细胞相邻。4) iNOS mRNA的检测：将人脐静脉内皮细胞（HUVEC）与脂多糖和IL-1β刺激的外周血多形核白细胞（PB-PMNs）共培养。收获HUVEC，用TRIsol提取RNA。将RNA逆转录成互补DNA （cDNA）。利用cDNA、人iNOS特异性PCR引物和DNA聚合酶，通过聚合酶链反应扩增人iNOS mRNA的表达。扩增的PCR产物用1.7%琼脂糖凝胶与溴化乙啶分离，并在紫外光下观察。5) Southern杂交：将PCR产物负载在琼脂糖凝胶上，将分离后的信息表达转移到尼龙膜上。将地高辛标记的inos特异性探针与DNA杂交。在柯达x射线分析胶片上发展了放射自显影术。用密度计记录强度。然后确认RT-PCR的特异性。

1) Samples:Surgically removed periapical lesions were examined by hematoxylin-eosin stains. Sixteen of 30 samples were apical cyst, whereas 14 were apical glanuloma.2) iNOS immunohistochemistry:Human specific iNOS anti-sera were purified from rabbits immunized with iNOS specific synthesized peptide. Immunohistochemistry using iNOS anti-sera was performed for cryostat sections of apical cyst. Endothelial cells, epithelial cells, fibroblasts and macrophages were iNOS positive. Lymphocytes and polymorphonuclear leukocytes also showed iNOS positive.3) Double immunohistochemistry of iNOS and IL-1β:Some iNOS-positive cells showed IL-1β positive, and some did not. On the other hand, some IL-1β-positive cells showed iNOS positive, and some did not. The data revealed that autocrine and paracrine effects of IL-1β on iNOS production. In addition, IL-1β-positive cells were present adjacent to iNOS-positive cells.4) Detection of iNOS mRNA:Human umbilical vein endothelial cells (HUVEC) were co-cultured with peripheral blood polymorphonuclear leukocytes (PB-PMNs) stimulated with lipopolysaccharide and IL-1β. HUVEC were harvested, and RNA was extracted using TRIsol. The RNA was reverse-transcribed into complementary DNA (cDNA). Human iNOS mRNA expression was then amplified by polymerase chain reaction using cDNA, human iNOS specific PCR primers and DNA polymerase. Amplified PCR products were fractionated using 1.7% agarose gel with ethidium bromide and were visualized on UV light.5) Southern hybridization:After the PCR products were loaded on agarose gels, the fractionated message expression was transferred to nylon membrane. Digoxigenin-labeled iNOS-specific probes were hybridized to the DNA. Autoradiography was developed on Kodak x-ray analysis films. The intensity was recorded using a densitometer. Specificity of RT-PCR was then confirmed.