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Research of U87Cells Apoptosis Mechanism by CRF Intervention

Author: WuQiang
Tutor: WangLiQun
School: Hebei Medical University
Course: Surgery
Keywords: CRF U87cells Real time fluorescence quantitative PCR CCK8 Apoptosis
CLC: R739.41
Type: Master's thesis
Year: 2014
Downloads: 1
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Abstract

Backgroud: Glioma is one of the common tumors in the nervoussystem, it has the characteristics of high incidence rate and high fatality rate.With traditional treatment of surgical treatment, radiotherapy andchemotherapy, treatment effect is still not ideal. Especially for glioblastoma,the average survival period is less than14.6months by the application ofcomprehensive treatment. With the further understanding of the pathogenesisof glioma, the effect of neural peptides in the development of glioma isgradual attented. Corticotropin-releasing factor is a41-amino acid peptide,which is extracted and depurated from hypothalamus of goats by Vale and hiscolleagues. It mainly distributes in the human central nervoussystem,especially in hypothalamus, and peripheral systems are widelydistributed. Corticotropin-releasing factor has a major role in hypothalamus-pituitary-adrenal axis, regulates the body of endocrine, autonomic nerve,immune response in the stress state.In recent years, CRF as a kind ofimportant neuroendocrine peptides and its role in cancer is increasinglyvalued.The effect of CRF in other systems is: CRF inhibits the growth ofbreast cancer cells by CRFR1activation, CRF inhibits the growth of small celllung cancer cells by CRFR2activation, Urocortin inhibits the growth ofhepatoma carcinoma cell and vascularization by CRFR2activation. However,the function and mechanism of CRF on glioma is rarely reported in thedomestic and abroad.Objective: Find out the effect of CRF on U87cells apoptosis and itsinfluence on U87cells fas/fasl, Bcl-2/Bax gene expression andCRFR1,PCNA,caspase-3protein expression under different CRFconcentrations. Methods：1Cells proliferation-virulence detectionU87cells were grown in96-well plates, each group set up3repeatedsamples. After been cultivated in the incubator for24hours, they werereplaced the medium which had contained different concentration of CRF(0,10-9,10-8,10-7umol/L), set up blank control group(excluding the CRF andcells),continued to be cultivated for24hours. Then every samples addedreagent of cell-counting kit. The determination of optical density at450nmwas done three hours later.2Detecting CRFR1，PCNA，caspase-3，cytC protein expression bywestern-blot.Total proteins were extracted from the cells by CRF intervention for24hours, quantified protein concentration by Bradford. The proteins blendedwith sample buffer at4:1were boiled for5min and then separated by10%sodium dodeyl sulfate polyacrylamide gel electrophoresis. The proteinstransferred onto a nitrocellulose membrane in conditions of205mA and1.5hours. After blocking with5%skim milk for2hour at room temperature, themembrane was washed with Tris-buffer saline5minutes,hatched with primaryantibodies of CRFR1(1:500),PCNA(1:800), caspase-3(1:800)，cytC(1:800),β-actin(1:500)at4degree centigrade overnight. After washingwith Tris-buffer saline5minutes, the membrane was hatched secondaryantibody for1hour at37degree centigrade. The immunoprotein blots weredetected using chemiluminescence system. Density of resulting brands wasanalyzed by the Image J. The results were counted as the percentage of theimmunoprotein blots ofβ-actin.3Detecting fas/fasl， Bcl-2/Bax mRNA expression by Realtimefluorescence quantitative PCRThe total RNA was extracted from cells with Trizol reagents anddetected its integrity by1.2%agarose gel electrophoresis(AGE). Briefly, theRNA was converted to cDNA with cDNA synthesis kit. Designing fas/fasl,Bcl-2/Bax primer sequences by DNA man software and synthesizing fromSBS Genetech CO.Ltd. PCR was proceeded in the AB7300Real-Time PCR system. The amplication ramp included an initial hold step of5min, at95degree centigrade followed by a three-step cycle consisting of30sec at95degree centigrade,15sec at55degree centigrade, and30sec at72degreecentigrade,repeated40times. In the end, the product was extended5min at72degree centigrade. Then dissolve curve was analyzed at60to95degreecentigrade. The results were analyzed using the2-△△CT-method and countedas the percentage ofβ-actin mRNA.Results:1Detecting apoptosis of U87cells:In cells proliferation-virulencedetection, the apoptosis rates of U87cells, which are by CRF of differentconcentration invention are variant, with statistical significance (F=35.79，P<0.05).2Analysis of western-blotting results:U87cells CRFR1protein ofexperimental group is higher than that of control group, the difference isStatistically significant (F=83.32, P<0.05). U87cells PCNA protein ofexperimental group is higher than that of control group, the difference isStatistically significant (F=36.19, P<0.05). U87cells cytC protein ofexperimental group is higher than that of control group, the difference isStatistically significant (F=11.93, P<0.05). U87cells caspase-3protein ofexperimental group is higher than that of control group, the difference isStatistically significant (F=44.82, P<0.05).3Analysis of RTFQ-PCR results, U87cells Bcl-2mRNA ofexperimental group is lower than that of control group, the difference isStatistically significant (F=138.82, P <0.05). U87cells Bax mRNA ofexperimental group is higher than that of control group, the difference isStatistically significant (F=215.25, P <0.05).The expression of fas mRNAwhich are by CRF of different concentration invention are not all the same,with statistical significance (F=14.5, P <0.05). The expression of fasl mRNAwhich are by CRF of different concentration invention are not all the same,with statistical significance (F=54.14, P <0.05). Conclusions：1CCK8showed CRF plays a role in the promotion of U87cellsapoptosis.2Western-blot showed CRF can raise up the expression of CRFR1,theexpression of apoptosis-related protein caspase-3,cytC are consistent with thetrend of apoptosis. The expression of protein PCNA is showed that the cellcycle is stopped at the before of G2phase.3RTFQ-PCR showed the expression of mRNA fas/fasl，Bcl-2/Bax areconsistent with the trend of apoptosis.In summary, we deduced that CRF may play a role for the apoptosis ofU87cells on some degree and it may through the apoptosis factors and theapoptosis pathway above.

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