| Preface5 - hydroxytryptamine, also called serotonin, an important bio-genie amine, is both a central nervous neurotransmitter and a peripherally neuromodulator. It involves in many important physiological and pathological courses in human and exerts these various functions by specifically binding to its receptors on cell surface. To date, 14 mammalian 5 - HT receptor subtypes have been found, grouped into 7 distinct subfamilies ( designated 5-HT1 to 5-HT7 ). 5 - HT2 receptor has been grouped into 3 subtypes, 2A, 2B and 2C. Among them, 5 -HT2B receptor is the one which has the highest affinity with 5 - HT. 5 -HT2B receptor is found to be expressed in many tissues of central nervous system and peripheral tissues. But till now, there are no reports about its expression on bladder cells. In order to decide that there are 5 - HT2B receptors expressions on bladder cells, we use semi - quantitive RT - PCR to detect the bladder cells of rats. At the same time we detect whether praziquantel, melatonin, dexfenfluramine and benzidine have effect on this receptor expression level on bladder cells. We also detect the 5 - HT2B receptor expression on the bladder cancer cells of patients and corresponding healthy bladder cells beside the cancer tissue to determine whether the expression level of this receptor on bladder cells is changed when bladder cancer happens.Materials and MethodsMaterialsAnimals: 28 male Wistar rats,weight 200 ?10 g.Clinical samples; 32 bladder cancer samples of final diagnosis patients. 8 healthy bladder samples beside the cancer tissues.Reagents: PBS, RNeasy Mini Kit, oligo dT(16), Nuclease free water, RNase inhibitor, dATP, dGTP, dCTP, dTTP, Taq DNA poly-merase, M - MuLV Reverse Transcriptase, benzidine, praziquantel, melatonin, dexfenfluramine, PBS buffer(pH7.4).Apparatus; UV300 ultraviolet spectrophotometer, HH -4 digital homoeothermic water bath, HH . W21 . 600 electro thermal water bath, HHW21 Cu600 electro thermal water bath, PTC -200 Peltier Thermal Cycler, GDS8000 gel analyzer.Methods1. Group the animals and treated with drugs: 28 male Wistar rats are divided into 7 groups randomly, 4 in each group. Treat the rats in each group with praziquantel, melatonin, dexfenfluramine and benzidine separately, corn oil, normal saline and 20% ethanol of same capacity acting as control groups. 2 hours later kill the rats, get the bladder sterile, prepare the liquid of single cell with the cell number 5+106 cells/ml.2. Treat the clinical samples; bladder cancer samples and healthy bladder tissue samples beside the cancer tissues are all brought back to the lab on ice immediately, prepare the liquid of single cell with the cell number 5 x 106 cells/ml.3. Extract the total RNA of bladder cells; get 1ml liquid of bladder cells, extract the total RNA according to the directions of the kit.Use UV300 ultraviolet spectrophotometer to estimate the purity and concentration of total UNA.4. RT - PCR : use total RNA as template to synthesis the first strand cDNA. Get each kind of cDNA as template, housekeeping gene P - actin acting as inner control, then amplificate the 5 - HT2B receptor and b-actin gene.5. Detection and analysis the amplification products of PCR; Detect the amplification products with 1.7% agarose gel electrophoresis, dyed with ethidium bromide, take pictures with GDS8000 gel analyzer. Analysis the bands of the electrophoresis with Gelworks 1D software , the volume below the apex acting as the density of the bands, 5- HT2B receptor expression level is represented by the ratio of density of the bands between 5 - HT2B receptor and b- actin.6. Statistical analysis: Statistical evaluation is done by t - test with SPSS 10.0 for windows software, data are showed in x+ s.ResultsWhen detect the 5 - HT2B receptor expression level on the bladder cells of the animals, it shows that all the animal bladder cells, including the groups given drugs and their control groups, express 5 - HT2B receptor. The data suggest that the 5 -...
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