虎纹蛙消化道肥大细胞类胰蛋白酶免疫组化研究

STUDY ON TRYPTASE IN THE MAST CELL IN THE DIGESTIVE TRACT OF INDIAN BULLFROG(RANA TIGRINA RUGULOSA) BY AN IMMUNOHISTOCHEMICAL METHOD

研究采用小鼠抗人肥大细胞类胰蛋白酶单克隆抗体AA1,应用ElivisionTM plus免疫组化染色法对虎纹蛙(Rana tigrina rugulosa)消化道组织中类胰蛋白酶阳性肥大细胞存在的可能性进行研究。研究发现单克隆抗体AAl可与中性缓冲福马林液固定的虎纹蛙组织的肥大细胞获得良好的交叉反应,类胰蛋白酶阳性细胞胞浆染成棕黄色,证实虎纹蛙肥大细胞胞浆颗粒中也存在类胰蛋白酶。虎纹蛙组织中AA1免疫染色阳性细胞的分布,与AB/SO和改良甲苯胺兰染色阳性细胞的分布存在较大的差异:虎纹蛙类胰蛋白酶阳性细胞数量很少,且阳性反应比人胃癌间质肥大细胞弱,主要见于黏膜型肥大细胞(MMC)分布区域,如消化道黏膜上皮下方和固有层,少量分布于肠绒毛基底部及食管腺和胃腺周围。而在结缔组织型肥大细胞(CTMC)分布区域,如消化道黏膜下层结缔组织中却未见类胰蛋白酶阳性细胞。AB/SO和改良甲苯胺兰染色阳性细胞数量多,广泛分布于消化道黏膜固有层、黏膜下层、腺体之间、肌间及外膜结缔组织,说明并不是所有的虎纹蛙肥大细胞都含有类胰蛋白酶。很有可能是虎纹蛙MMC中含有类胰蛋白酶,而CTMC中不含类胰蛋白酶。虎纹蛙类胰蛋白酶阳性细胞数量很少,且阳性反应比人胃癌间质肥大细胞弱,说明虎纹蛙肥大细胞胞浆颗粒类胰蛋白酶含量较少,虎纹蛙属于低等脊椎动物,可能与生物进化水平较低有关,有待进一步研究。

A murine monoclonal antibody （AA1） raised against human mast cell tryptase was used in this experiment to investigate the possibility existence of tryptase-positive mast cells of the digestive tract tissue which collected from Indian bullfrog （Rana tigrina rugulosa） by Elivision ^TMplus immunohistochemical techniques. To prove its reliability, Alcian blue-Safranin O staining and modified toluidine blue staining was compared with this method when gastric cancer tissue from an adult man was used as a positive control. Four healthy Indian bullfrog weigh 150—200 g were used. After opening abdominal cavity, the esophagus, stomachus cardiacus, stomachus fundus, stomachus pylorus and small intestine （duodenum, jejunum and ileum） were fixed in the 10% neutral buffered formalin （10% NBF） and Carnoy solution respectively, dehydrated, transparentized, embedded and sliced into 6μm sections. Immunohistochemistry Kit, murine monoclonal antibody （AA1） raised against human mast cell tryptase, ElivisionTM plus Polyer HRP （Mouse/Rabbit） IHC Kit, Trypsin Kit, Poly-L-lysine, DAB Kit, and gastric cancer tissue from an adult man used as a positive control were bought from Fuzhou Maxim Biotech Inc. The experiment was conducted as following： （1） Washing the slices by PBS （pH7.4） （3 ×3min） after dewaxing and hydrating. （2） Hatching trypsinase 15—20min in incubator at 37℃ for repairing antigen. Rinsing in PBS （3 × 3min）. （3） Sections were incubated overnight at 4℃ with monoclonal antibody （AA1）. （4） Rinsing in PBS （3 × 3min）. （5） Removing PBS and adding 50 μL polymer intensifier to each slice, incubating at room temperature for 30min. （6） Rinsing in PBS （3 × 3min）. （7） Removing PBS and adding 50μL enzyme-mark polymer against murine, hatching at room temperature for 30min. （8） Rinsing in PBS （3 × 3min）. （9） Removing PBS and dropping 100μL fresh DAB Kit, studying it under a microscope for 3-10min, the positive showed brown. （10） Finishing coloration with the distilled water. Hematoxylin stained. Dehydration, clearing, and mounting with neutral gums. Group was carried out with the same steps murine monoclonal antibody （AA1）. The research demonstrated that the Indian bullfrog mast cells contained tryptase in their cytoplasm granules, and there was an excellent cross-reaction between monoclonal antibody AA1 and mast cells obtained from the Indian bullfrog tissues fixed by 10% neutral buffered formalin. The cytoplasm of tryptase-positive mast cells was dyed brown yellow. The research found that the distribution of positive cells of Indian bullfrog tissue dyed by AA1 were different from ones of Alcian blue-Safranin O and the modified toluidine blue staining. In Indian bullfrog, the number of tryptase-positive mast cells was small, and the positive reaction of them was weaker than gastric carcinoma interstitium in human. The tryptase-positive mast cells were mostly distributed in the base of enteron mucosa epithelium, lamina propria, base of intestinal villus, oesophagus gland and stomachus gland. The distributions of them were similar with mucosal mast cell （MMC）. While the tryptase-positive mast cells did not exist in the distributional range of connective tissue mast cell （CTMC） such as digestive tract mucosa connective tissues. A number of mast cells dyed by the modified toluidine blue staining and Alcian blue-Safranin O staining were mostly distributed in the digestive tract mucosa lamina propria, submucosa, gland interstitium, muscular layer and ectoblast connective tissues. It showed that not all Indian bullfrog mast cells had tryptase. Maybe the MMC of Indian bullfrog had tryptase but the CTMC did not. The number of tryptase-positive mast cells of Indian bullfrog was small and the positive reaction of tryptase-positive mast cells was weaker than gastric carcinoma interstitium of human. The study showed that the tryptase content in mast cells cytoplasm granules of Indian bullfrog was low. Indian bullfrog belonged to lower vertebrates, and these phenomenons possibly correlated with lower level of biological evolution. These problems remained to be further researched.