| Idiopathic talipes equinovarus (ITEV, also called clubfoot) is a common type of congenital deformity of the foot and lower leg with an estimated incidence of 1/1,000 live births. The malformation typically manifests fixed in adduction, supination, and varus, in addition with soft tissue abnormalities. Previous epi-demiological studies have attempted to reveal the aetiology of ITEV but none has supported a significant association with any socioeconomic factors or teratogenic exposures. On the other hand, complex segregation analyses of ITEV pedigrees from different populations have strongly suggested a genetic etiology with Mende-lian and/or multifactorial inheritance. Although it may not influence the survival and reproductivity of patients, the ITEV deformity can severely affect their quality of life. Exploring its pathogenetic mechanisms may.provide important clues for its prediction, prevention and treatment.Proteins play a dominant role in all life activities and proteomics research has attracted much attention in recent years. Since it is difficult to perform biopsy for talipes equinovarus in human subjects, to construct a rat model for clut-foot-like deformity and analyze the variations in normal and abnormal rat pro-teomes by 2D gel electrophoresis and mass spectrometry, in combination with bioinformatics, may help to delineate roles of particular proteins in the pathogen-esis of clubfoot-like deformities. The objectives of this research include:(1) To construct a rat model for isolated-type clubfoot-like deformities with all-trans retinoic acid ( ATRA) and to determine the optimum dosage of ATRA.(2) To explore the method for analyzing the proteomes obtained from tibia-fibulae musculature of rat fetus.(3 ) To compare the disease and normal proteomes by 2D gel electrophore-sis and mass spectrometry, and to obtain some clue for further study of the basic molecular mechanisms for talipse equinovarus.MethodsPregnant Wistar rats were randomly assigned to one control and four experimental groups. On day 10 of pregnancy, variable doses (120 mg/kg, 130 mg/ kg, 135 mg/kg and 140 mg/kg, respectively) of ATRA in mineral oil suspension were given intragestically to the experimental groups, a volume of mineral oil equivalent to the 140 mg/kg group was given to the controls.On day 21 of gestation, rat fetus was removed through laparotomy and carefully examined under a stereomicroscope for structural abnormalities including clubfoot-like deformities ( Adduction/inner rotation of anterior portion of the feet, introversion of heels, dropping of ankle, with the angle between longitude axis of the foot and leg being greater than 130). Optimum dosage of ATRA was determined through statistical analysis.Hind legs of model rat fetus and normal controls were removed and made into serial sec tions along three anatomical planes of the foot, and analyzed under a light microscope.Sections of spinal cords, vertebra and musculatures from lower hind legs of model rat fetus and normal controls were made for apoptosis analysis by TUNEL labelled with DeadEnd?Fluorometric TUNEL System kit .Musculatures from hind lower legs of model rat fetus and normal controls were removed and dried, and then thoroughly mashed within ice-bath following addition of lysis solution (1 ml/50mg). The mash was left at room temperature for approximately 1 hour and then centrifuged. The supernatant was retained, with concentration determined.For 2D gel electrophoresis, immobilized pH gradient gel (IPG, pH 3-10 and pH 5-8) isoelectric focusing was set as the first dimension, and 12% SDS polyacrylamide gel electrophoresis was set as the second dimension. For isoelectric focusing, PROTEAN IEF cell was used. Loaded sample sizes were: 0. 5 mg (350ul) for pH 3-10 and 1.0 mg for pH 5-8, respectively. Isoelectric focus wasperformed at 50v for 13 hour, 250v for 30 min, 1000v for 1 hour,and finally focused under 10 000v for 60 000 vh.The IPG was bathed in balance solution I and II 15 minutes, respectively, then transferred onto...
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