Proliferation In Brain Gliomas And Expression Of Apoptosis-related Genes P53,IASPP

Objective:The brain glioma originates from neural epithe lium,that is the most commom intracranial tumor, consisting of about 35.2%~60.9% in all intracranial tumors.The unbounded proliferation of tumor cells is the major characteristic, the more malignancy, the more proliferation, the more DNA error in the DNA duplication and cell division,the more damaged DNA apoptosis.Obviously,tumor completed growth is the proliferati on and apoptosis.p53 is one of the most important inhibitory genes.It lies in the short arm of the seventeenth chromosome e,and translates a 53kd intranuclear phosphoprotein.Because of its expression and demiperiod character , the Streptavidin- Peroxidase Coiugate Method(s-p) method can't check the wild p53 protein ,however could check the mutant p53.The positive and mutant expression of p53 protein have the same meaning.The new discovered ASPP (Apoptosis stimulating protein of p53)family is a protein which can exceptionally regulate the cancer inhibiting function of p53.The function is implemented by specifically impairing cell apoptosis function. When cells incur gene damage,the expression of p53 gene augment greatly,forming p53 protein.The protein could stop cell duplication to strive for repairing,or don't repair in order to cell death,so it could inhibit abnormal cellular proliferation, nevertheless by what mechanisms,how to determine why stop duplication or into apoptosis procedure. The discovery of ASPP satisfactorily explain that how p53 determine stopping duplica- tion or to apoptosis procedure.Being insufficient ASPP,p53 incline to combine with genes stopping promotor,while ASPP are existent,ASPP can combine with p53 firstly , causing changes of p53 with promoter affinity,ASPP-p53 compound trends to combine with genes apoptosis promotor,so gene damaging cells apoptosis. IASPP(inhibitory member of the ASPP family) could compete to combine with p53 binding site from ASPP1 and ASPP2,accordingly impact the combining ASPP and p53,so it inhibits to induce cell apoptosis capability of p53.The investigation deploy SP method to detect PCNA and deploy TUNEL method to detect apoptosis,approaching the relation between proliferation,apoptosis with malignancy degrees.As well as the significance of IASPP,p53 in apoptosis,and the relationship with tumor malignancy.Method : 40 neurospongioma ( Astrocyte tumors ) examples, fixed by 10% formalin, mineral wax enveloped, serial sections 4μm.By WHO nervous system tumor separation criteria (2000 year) :Ⅰ～Ⅱgrades 19,Ⅲgrade 11,Ⅳgrade 10. Anti-rabbit IASPP antibody from Beijing BOAOSEN company limited, working concentration 1:200 attenuated; p53 antibody from Santa Cruz company, working concentration 1 : 75 attenuated; Anti- rabbit PCNA antibody from Beijing Zhong shan jin qiao company limited;biotinylation IgG kit and (diam inobezidine, DAB) from Beijing zhong shan company limited.Immunohistochemistry:applying SP method (Streptavidin- Peroxidase Coiugate Method )and PBS(phosphate buffered solution, PBS),coordination deliquation normal rabbit serum substitution one antibody blank.Procedures are as follows:①paraffin section deparaffinage hydration,at 3% H2O2–pyroli- gneous alcohol block endogenous peroxydase 20min;②By microwave repair antigen 18min cool at ambient temperature;③normal goat serum at ambient brood temperature 60min, to remove;④dropwising one antigen at 4℃to stay overnight, 37℃incubation 30 min;⑤dropwising biotinylated goat anti rabbit IgG 37℃incubation 60min;⑥dropwising 3 antibody at 37℃incubation 50min; except③step every step in 0.01mol/L (pH7.4)PBSwashing3times,5min every time;⑦DAB colouration hematoxylin to dye, deaquation transparence, neutro-balsam mounting.PCNA detection:PCNA detection index , routine sp method;buffer instead of microtome section is negative control. TUNEL detection procedure:according to description,no TUNEL microtome section is negative control.Judgement standard:cell nucleus of apoptosis and PCNA is Buffy.Observation in 400 times microscope,every micro- tome section select six field visions.Calculating proportion positive tumor cells in all tumor cells every example ,as apoptosis index(AI) and proliferative index( PI ).p53 protein lies in nuclear in major, expressing buffy-granula.IASPP protein lies in nuclear in major, expressing buffy-granula .Expression results deploy semiquantitative method analyse.Positive tumor cells percentage decided by 400 times high power lens more than six fields, counting scores by five ranges: 0:<1%; 1score: 1%~25%; 2scores:25%~50%;3scores:50%~75%;4scores:>75%.Tumor cells immunostaining intension are as follows:1,Buff;2,yellow;3,deep buff.Because immunostaining isn't uniform, scoring at major format.weighting score is positive tumor cell propotion by immunostaining intension.immunostaining inten- sion zero is recognized negative,others is positive.Results①AI determination Apoptosis cells are expressed in all 40 gliomas,AI range is between 6.67%~ 27.40% in 40 gliomas,Median and quartile are(17.07,7.54)%.AI is expressed highly with the malignancy level of gliomas increasing.②PI determination proliferation cells are expressed in all 40 gliomas, PI range is 10.22%~50.00%.Median and quartile are (27.05,3.52 )%.PI is expressed highly with the malignancy level of gliomas increasing.AI/PI is expressed lowly with the malignancy level of gliomas increasing, which have negative dependablity(rs=-0.36 p<0.05).PI and AI have obvious positive dependablity(rs=0.90 p<0.05).③IASPP,p53 expression and tumor cells apoptosis dependablity IASPP is expressed in all brain glioma tissues,while positive expression rate was 70%(30/40) in 40 of gliomas.IASPP is expressed highly with the malignancy level of gliomas increasing.p53 expression rate was 60%(24/40) in 40 of gliomas.p53 is expressed highly with the malignancy level of gliomas increasing.The AI median scores of positive IASPP class and negative IASPP class have obvious difference (P<0.05).The AI median scores of positive p53 class and negative p53 class have obvious difference(p<0.05).they have positive dependability between p53 weighting scores and AI (rs =0.55, P<0.01).p53 protein and IASPP protein have obvious positive dependablity(rs=0.53 p<0.05).Conclusion:1,Apoptosis reveal the difference between tumor actual growth and predictive growth.2,AI/PI can guide to determine the biological behaviors of glioma and histological grading, AI/PI>0.6 has lower malignancy, AI/PI <0.6 have higher malignancy.3,IASPP is expressed highly with the malignancy level of gliomas increasing.IASPP could compete with ASPP1 or ASPP2 to combine with p53.4,To inhibit IASPP expression which may be the new target to promote tumor cells apoptosis.