水生生物学报
水生生物學報
수생생물학보
ACTA HYDROBIOLOGICA SINICA
2010年
1期
29-34
,共6页
林旋%王寿昆%陈梅芳%林树根%黄玉章
林鏇%王壽昆%陳梅芳%林樹根%黃玉章
림선%왕수곤%진매방%림수근%황옥장
虎纹蛙%肥大细胞%类胰蛋白酶%免疫组化
虎紋蛙%肥大細胞%類胰蛋白酶%免疫組化
호문와%비대세포%류이단백매%면역조화
Indian bullfrog (Rana tigrina rugulosa)%Mast cell%Tryptase%Immunohistochemistry
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Elivision~(TM)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AA1可与中性缓冲福马林液同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同有层,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.
研究採用小鼠抗人肥大細胞類胰蛋白酶單剋隆抗體AA1,應用Elivision~(TM)plus免疫組化染色法對虎紋蛙(Rana tigrina rugulosa)消化道組織中類胰蛋白酶暘性肥大細胞存在的可能性進行研究.研究髮現單剋隆抗體AA1可與中性緩遲福馬林液同定的虎紋蛙組織的肥大細胞穫得良好的交扠反應,類胰蛋白酶暘性細胞胞漿染成棕黃色.證實虎紋蛙肥大細胞胞漿顆粒中也存在類胰蛋白酶.虎紋蛙組織中AA1免疫染色暘性細胞的分佈,與AB/SO和改良甲苯胺蘭染色暘性細胞的分佈存在較大的差異:虎紋蛙類胰蛋白酶暘性細胞數量很少,且暘性反應比人胃癌間質肥大細胞弱,主要見于黏膜型肥大細胞(MMC)分佈區域,如消化道黏膜上皮下方和同有層,少量分佈于腸絨毛基底部及食管腺和胃腺週圍.而在結締組織型肥大細胞(CTMC)分佈區域.如消化道黏膜下層結締組織中卻未見類胰蛋白酶暘性細胞.AB/SO和改良甲苯胺蘭染色暘性細胞數量多.廣汎分佈于消化道黏膜固有層、黏膜下層、腺體之間、肌間及外膜結締組織,說明併不是所有的虎紋蛙肥大細胞都含有類胰蛋白酶.很有可能是虎紋蛙 MMC 中含有類胰蛋白酶.而CTMC中不含類胰蛋白酶.虎紋蛙類胰蛋白酶暘性細胞數量很少.且暘性反應比人胃癌間質肥大細胞弱,說明虎紋蛙肥大細胞胞漿顆粒類胰蛋白酶含量較少,虎紋蛙屬于低等脊椎動物,可能與生物進化水平較低有關,有待進一步研究.
연구채용소서항인비대세포류이단백매단극륭항체AA1,응용Elivision~(TM)plus면역조화염색법대호문와(Rana tigrina rugulosa)소화도조직중류이단백매양성비대세포존재적가능성진행연구.연구발현단극륭항체AA1가여중성완충복마림액동정적호문와조직적비대세포획득량호적교차반응,류이단백매양성세포포장염성종황색.증실호문와비대세포포장과립중야존재류이단백매.호문와조직중AA1면역염색양성세포적분포,여AB/SO화개량갑분알란염색양성세포적분포존재교대적차이:호문와류이단백매양성세포수량흔소,차양성반응비인위암간질비대세포약,주요견우점막형비대세포(MMC)분포구역,여소화도점막상피하방화동유층,소량분포우장융모기저부급식관선화위선주위.이재결체조직형비대세포(CTMC)분포구역.여소화도점막하층결체조직중각미견류이단백매양성세포.AB/SO화개량갑분알란염색양성세포수량다.엄범분포우소화도점막고유층、점막하층、선체지간、기간급외막결체조직,설명병불시소유적호문와비대세포도함유류이단백매.흔유가능시호문와 MMC 중함유류이단백매.이CTMC중불함류이단백매.호문와류이단백매양성세포수량흔소.차양성반응비인위암간질비대세포약,설명호문와비대세포포장과립류이단백매함량교소,호문와속우저등척추동물,가능여생물진화수평교저유관,유대진일보연구.
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~(TM)plus 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, routine monoclonal antibody (AA1) raised against human mast cell tryptase, Elivision~(TM) 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 5OμL 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 bad 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.
