| Objective1. In this study,craniopharyngioma cells in vitro cultured have been as target cells, we investigate the effect of retinoic acid.2. By detecting the expression of CRABP-Ⅱ, FABP-5, RARαand PPARβ/δin craniopharyngioma cells and analyzing the correlation between the expression and effect of retinoic acid,we investigate the molecular mechanism of retinoic acid targeted treatment of craniopharyngioma,provide new ideas and means with the personalized treatment of craniopharyngioma.Methods1. Detect the effect of RA in craniopharyngioma cells in vitro cultured.1.1. Nuclear morphogical of apototid cells and dead cells were observed by fluorescence microscopy.1.2. The DNA ladder was revealed by agarose gel electrophoresis.1.3. Annexin-V-FITC/PI apoptosis was detected by flow cytometry.2. Analyze molecular mechanism of retinoic acid by detecting the expression of CRABP-Ⅱ, FABP-5, RARαand PPARβ/δin craniopharyngioma cells.2.1. Using fluorescence immunohistochemistry, we detected expression of CRABP-Ⅱ, FABP-5 protein in 49 cases of craniopharyngioma(AE 39 samples,SP 10 samples)and 20 cases of normal brain tissue due to cerebral hemorrhage and analyzed the difference of expression between two subtypes of craniopharyngioma and normal tissue.2.2. CRABP-Ⅱ, FABP-5 protein detection methods:CRABP-Ⅱ, FABP-5protein in cells in specific parts of the two indicators appear green fluorescence, staining intensity was higher than the background non-specific staining positive cells, positive cells reported in the literature to judge the standard reference to semi-quantitative method.2.3. The expression of RARαand PPARβ/δin31 cases of craniopharyngioma cells in vitro was quantified by reverse transcription-PCR, and analyze the relation between expression and RA.3. The proliferation and apoptosis on craniopharyngioma with different expression of RARαand PPARβ/δwere detected by using MTT assay.4. Analyzing the data accoding to statistical software SPSS 15.0Results1. The effect of retinoic acid in craniopharyngioma cells.1.1. Morphologic changes:Craniopharyngioma cells became sherican shape and detached. Apoptotic cells were identified by the presence of cells shrinkage,plasma membrance blebbing and final separation of apoptotic bodies.Apoptotic cells nuclear chromatin condensation and fragmentation shaped bright-pearl structure were observed by fluorescence microscopy.1.2. Agarose gel electrophoresis:Craniopharyngioma from RA-treated cells extract DNA electrophoresis,72h ladders began to clear and their intensity increased in a time-dependment manner.1.3. Detection of apoptosis Two-dimensional dot polt result from flow cytometry can be seen,apoptosis cells have significant increase in craniopharyngioma with RA 72hours.2. The result of the expression of CRABP-Ⅱ, FABP-5, RARαand PPARβ/δin craniopharyngioma cells.2.1. There were 39 cases with CRABP-Ⅱpositive expression in 49 cases of craniopahryngioma, the positive rate was 79.59%; Meanwhile, there were 16 cases with CRABP-Ⅱpositive expression in 20 cases of normal tissue, the positive rate was80.00%,the difference have no statistic meaning.(p>0.05).2.2. There were 35 cases with FABP-5 positive expression in 49 cases of craniopahryngioma, the positive rate was 71.42%; Meanwhile, there were 14 cases with FABP-5 positive expression in 20 cases of normal tissue, the positive rate was 70.00%,the difference have no statistic meaning. (p>0.05).2.3. In subtypes, there were 31 cases with CRABP-Ⅱpositive expression in 39 cases of AE, the positive rate was79.48%, there were 8 cases with CRABP-Ⅱpositive expression in 10 cases of SP, the positive rate was 80.00%,the difference have no statistic meaning.(p>0.05). There were 28 cases with FABP-5 positive expression in 39 cases of AE, the positive rate was71.97%,7 cases with FABP-5 positive expression in 10 cases of SP, the positive rate was 70.00%,the difference have no statistic meaning.(p>0.05)2.4. The RT-PCR result show that the expression of PPARβ/δ,RARαmRNA was different. 31 cases of craniopharyngioma cells in primary culture were divided into three groups according the expression of nuclear receptor: PPARβ/δ>RARα,RARα>PPARβ/δand RARα>>PPARβ/δ.3. The inhibition of retinoic acid in craniopharyngioma cells.MTT results showed that the inhibition of RARα>>PPARβ/δgroup was significantly higher than the other groups under same drug,the differences have statistical meaning(p<0.01).Conclusion1. RA can inhibit the in vitro primary cultured craniopharyngioma cells and induce apoptosis.2. CRABP-Ⅱ, FABP-5 can not be the target of RA role of craniopharyngioma cells, the steady-state CRABP-Ⅱ/FABP-5 does not exist in experiment, nor as a indicator to measure the degree of malignancy in craniopharyngioma.3. The expression of PPARβ/δRARαcan be used to evaluate the effect of RA in craniopharyngioma.The craniopharyngioma with low-expression PPARβ/δwas more sensitive to RA.4. Targeting higher RARαor targeting lower PPARβ/δis beneficial to the treatment of craniopharyngiomas,so we probably find new molecular targets.5. This study confirms the inhibition activity of RA in craniopharyngioma cultured in vitro,provide preliminary basis for future animal exeperiment and clinical studies.The result show that RA have potential applications in the treatment of craniopharyngiomas.
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