| Congenital talipes equinovarus (CTEV, also called clubfoot) is one of the most common musculoskeletal anomalies. It's a type of congenital deformity of the foot and lower leg with an estimated incidence of 1/1,000 live births. Although it may not influence the survival and reproducibility of the patients, the CTEV deformity can severely affect their quality of life. The malformation typically manifest fixed in adduction, supination, and varus, with the addition of soft tissue abnormalities. There are much controversies on clubfoot, including its etiology, pathologic anatomy, and treatment. Previous epidemiological studies have attempted to reveal the etiology of CTEV but none has supported a significant association with any socioeconomic factors or teratogenic exposures. On the other hand, complex segregation analyses of CTEV pedigrees from different populations have strongly suggested a genetic etiology with Mendelian and/or multi-factorial inheritance. Exploration of pathogenetic mechanisms may provide important clues for the prediction, prevention and treatment of clubfoot.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 clubfoot - like deformity and analyze the variations in normal and abnormal rat pro-teomes by 2DE and mass spectrometry, in combination with bioinformatics, may help to delineate the roles of particular proteins in the pathogenesis of clubfoot -like deformities. Previous researches have found several proteins probably relate with clubfoot. One of them is slow skeletal muscle troponin T (sTnT) and itscoding gene is Tnntl .The objectives in the study are-.1) To construct a rat model for isolated - type talipes equinovarus with all -trans retinoic acid (ATRA);2) To compare the disease and normal proteomes by two - dimensional gel electrophoresis and mass spectrometry;3)To analyze the expression of Tnntl in disease and normal rat;4) To analyze the association between Tnntl and CTEV;Methods1. Construct rat model for isolated - type talipes equinovarus 1. 1 Pregnant Wistar rats were randomly assigned to control and experimental groups. On day 10 of pregnancy, 135 mg/kg of ATRA in mineral oil suspension was given intragestically to the experimental groups;corresponding volume of mineral oil was given to the controls.1. 2 On day 21 of gestation, rat fetus was removed through laparotomy. Spinal cord, ankle without musculatures and ankle without skeleton were removed.2. Proteomic analysis of spinal cord between disease and normal rat fetus 2. 1 Total protein of spinal cord from disease and normal rat fetus were removed .2.2 For isoelectric focusing, PROTEAN IEF cell was used. The IPG strip was bathed in balance solution I and II. Then transferred onto 12% SDS polyac-rylamide gel and electrophoresed. Following electrophoresis, the gels were stained, scanned and analyzed with the PDQuest 7. 1.0 software package.2. 3 Significant protein spots were selected for mass spectrometry analysis and bioinformatics analysis.3. The expression of Tnntl in disease and normal rat fetus3. 1 The total mRNA of spinal cord, ankle without musculatures and ankle without skeleton was refined, respectively.3. 2 To study the expression of Tnntl in disease rat fetus and the association between Tnntl and congenital talipes equinovarus deformation.Results1. Construct the rat model for clubfoot - like deformityTotally 344 rat fetus were gained. Among them, 112 for normal control and 232 for disease fetus. The incidence of deformity was 80. 98% and the incidence for isolated clubfoot -like deformity was 29. 27% .2. Analyze the 2DE image of spinal cord's proteome between disease and normal rat fetusThe total proteome of spinal cord of rat fetus were separated through pH3 -10 and pH5 -8 2DE, with good reproducibility and resolution achieved. Compared with those of the normal controls, spinal cord from disease rat fetus showed 7 ~9 and 10 ~ 13 gains, 9-11 and 11 ~ 13 loses, 13 ~ 15 and 8-10 up —regulated, 15 ~ 17 and 10 ~ 12 down —regulated, respectively.3. Analyze the spinal cords proteome between disease and normal rat fetus Compared with those of normal controls, samples from disease rat fetusshowed gains and loses, with the addition of a number of spots showing increase or decrease in expression for 5 times or more. Among them, one spots database analysis is netative, and two spots'database analysis is same, so the results are;enolase ( only seen in normal fetus) , ATP synthase mitochondrial Fl complex assembly factor 2 ( Atpl2p) ( only seen in disease fetus) , tubulin beta chain ( decreased expression for more than 5 times in disease fetus) and apolipoprotein A - I (increased expression for more than 5 times in disease fetus) .4. The expression of Tnntl in disease and normal rat fetusRT - PCR results find the expression of Tnntl down - regulated in 70. 0% (21/30) disease fetus'ankle without musculatures, down -regulated in 76.7% (23/30) disease fetus'ankle without skeleton. There is no expression of Tnntl in spinal cord.Conclusion1. The proteomic analysis of spinal cord in rat fetus with talipes equinovaruswas not reported before.2. Proteomic difference between disease fetus and normal controls can be well presented with 2 - D electrophoresis. Compared with normal control fetus, disease fetus gain ATP synthase mitochondrial Fl complex assembly factor 2 ( Atpl2p) , lose enolase, tubulin beta chain decreased expressed for more than 5 times and apolipoprotein A - I increased expressed for more than 5 times in disease fetus.3. RT - PCR results find the expression of Tnntl down - regulated in ankle without musculatures and ankle without skeleton of disease rat fetus, which indicated that the down — regulation of Tnntl might be one of the reasons in the development of talipes equinovarus. |
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