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Background Efficient and lowcost way to isolate keratocytes is helpful for research on cornea.Either relatively expensive or inefficient is the shortage of those means now applied,while raising the keratocytes through passage will change the phenotype of them quickly.Our aim is to approach the way getting keratocytes effectively utilizing modified two step enzymatic digestion by type I collagenase. Objective To evaluate the effect of isolating the bovine keratocytes utilizing two step enzymatic digestion and observe the morphological changes of the keratocytes during cultivation in vitro. Methods Keratocytes were isolated from bovine corneas using 0.5 mg/mL and 1 mg/mL type I collagenase digestion.The harvesting rate and viability rate of the primary keratocytes were evaluated.During the primary cultivation in vitro,the morphological changes of the keratocytes and their F-action distribution were observed.Results(1)The extracellular matrix of the bovine corneas were almost dissolved by the two step enzymatic digestion,followed the keratocytes completely isolated from the solid matrix.The amount of the harvested keratocytes from each cornea was(2.11±0.15)X106 on average while the viability rate was(91.69±3.73)% and the inoculation rate Was(81.20±1.25)%.(2)The primary keratocytes attached and spreaded out with dendritic and stellate morphology.After 3 days cultured,the branches of the keratocytes were contacting and formed networks.The F-actin detected by phalloidin binding showed a limited cortical localization. Conclusion (1)The method of two step enzymatic digestion can make the extracellular matrix of bovine cornea stroma completely degraded with the advantages in high efficiency of harvesting keratocytes and high cell viability and relatively simple manipulation. (2) The primary bovine keratocytes have dendritic morphology and with limited F-action distribute in cellular cortex.
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Objective To explore the diagnostic value of endoscopic retrograde cholangio-pancreatiography (ERCP),ultrasonography (US) and CT scanning on cholagio-pancreatic diseases caused by clonorchis sinensis infection.Methods The results of US,CF and ERCP examination in 65 cases of obstructive jaundice caused by clonorchiasis and confirmed by presence of imagoes or eggs in feces or bile juice were analyzed and compared retrospectively.Results US examination was the simplest and most convenient which was characterized by diffuse even dilatation,thickening of walls,strengthened echo with the shape of“equal sign”intrahepatic bile ducts.The characteristic findings of CT included saccular dilatation of bile ducts of the periphery of the liver,thickening of the walls of bile ducts.Under duodenoscopy,32.3% (21/65) of cases presented as abnormal papilla such as small opening,mucosa outward turned,trapping or stiffness.Alterations in ERCP were characterized by shm or oval filling defect,diffuse saccular dilatation of terminal intrahepatic bile ducts.The most common complications included cholangiolithiasis (40%,26/65),carcinoma of bile duct on papilla (9.8%,6/65),and pancreatitis (1.5%,1/ 65).Conclusions Three methods were all useful for diagnosis of cholangio-pancreatic diseases caused by clonorchis sinensis infection,which were identically characterized by diffuse saccular dilatation of terminal intrahepatic bile ducts.The gold standard of diagnosis was the presence of imagoes or eggs in bile juice aspirated by the route of ERCP.Endoscopic sphincterectomy with postoperative vermifugal was the first choice of the treatment.