| Glioma originated in brain glial cells, it is the most common tumor in the central nervous system, according to reseach gliomas account for40-50%of the central nervous system primary tumors. Compared with other tumor, glioma incidence is low. However, glioma have extremely disproportionate morbidity and mortality, the prognosis overall totally poor. With the development of immunology, a lot of research has found that there is a close relationship between the occurrence and development of tumor and the human immune system. NLR(Nod-like receptor) is a kind of intracellular pattern recognition receptor, which plays an important role in recognition of the invading pathogens and activation of innate immune responses, and it is a class of intracellular sensing molecules. As a member of NLR family, NLRP3plays an important role in prevention and pathogenesis of varieties of diseases. NLRP3regulates the maturation and secretion of IL-1(3and IL-18, they both are members of IL-1family and closely related to each other, as key regulatory factors in the host immune response, they also play an important role in the occurrence and development of glioma. At present, there is few reseaches of the expression of NLRP3in gliomas and prognosis Significance.Objective1> To research the expression of Nod-like receptor pyrin domain-containing protein3(NLRP3) in Glioma. 2、Discuss the role of NLRP3in the glioma genesis and improvement. To provide a theoretical basis for the finding of new targets to treat glioma.Materials Method1. MaterialsThe49cases of human glioma were graded according to World Health Organization (WHO)2007criteria, and were divided into low grade group(WHO Ⅰ-Ⅱ,20cases) and high grade group(WHO III-IV,29cases). And collect10cases of normal brain tissue of the brain trauma patients by resection of intracranial decompression operation as control.2. Method2.1The presence and tissue localization of NLRP3was measured by immunohistochemistry.Cut the paraffin embedded tissues into serial sections of3μm, Dewaxing by xylene for two times,5～10min per time, hydrate by gradient alcoholic,and high-pressure repair by antigen retrieval buffer of EDTA(pH9.0) for2min.Wash the tissues by PBS after natural cooling for one time.Add the monoclonal antibody NLRP3(antibody Ⅰ) of the concentration of1:100, then incubation in4℃Overnight.Wash the tissues by the PBST for three times,5min per time.Add antibody Ⅱ (sheep anti-mouse) to the tissues,then incubate them in37℃for45min, Wash the tissues by the PBST for three times.Colorate the tissues by DAB (1:50),the recolorate them by haematoxylin for30s, Encapsulate the tissues by neutral gum.Take the normal brain tissues as control.2.2The expressions of NLRP3protein were detected by western blot technology.Take the glioma and normal tissues from the-80℃fridge and add Lysis buffer with protease inhibitors into them,pestle the tissues and centrifugate the Ultrasonic shattered tissues(15000×g/min Centrifugal radius13.5cm) in4℃, shift the supernatant liquid into a new EP tube,and mark it, mensurate it by bicinchonininc acid method,and adjust the concentration of protein. Add1/6Volume of protein sample buffer into protein samples,and boil it for10min.The well prepared samples are added to the SDS gel,spacer gel90V,and separation gel110V.Then cut the gel and transfer it to membranes.Fix the membranes by1%BSA in room temperature for1.5h.then add the monoclonal antibody NLRP3(antibody I) of the concentration of1:100, then incubation in4℃Overnight.Wash the membranes by1X TBST for10min,repeat for three times. Add antibody II (sheep anti-mouse) to the membranes,then incubate them in37℃for1h, Wash the membranes by1X TBST for1Omin,repeat for three times.At last colorate them.2.3The expressions of NLRP3mRNA were detected by real-time PCR technology.According to the Total RNA kit extraction specification of Invitrogen company,We complete the experimentation.We determined The RNA concentration of each sample by ultraviolet spectrophotometer. According to the Ferments reverse transcription kit, reverse transcription system is20μL, reverse each sample RNA for1μg. Primer selection:oligo(dT)1μL,5×Reactin Buffer4μL,10Mm dNTP Mix2uL,RibolockTM Rnase Inhibitor1μL,RevertAiTMM-MuLV Reverse Transcriptase1μL,RNase-free water(0～11μL). The reaction conditions:65℃incubation,5min,42℃,60min,70℃5min.Detect the objective gene and reference by QRT-PCR.The reaction system is25μL:(2x) SYBR Green qPCR master mix12.5μL,Template cDNA2μL,Forward Primer1μL,Reverse Primer1μL, nuclease-free Water8.5μL. The reaction conditions:predenaturing in95℃for5min,annealing in55℃for lmin,rolling out in95℃for30s, repeat the40cycle.ResultsNLRP3had negative staining in10normal brain tissues, there is a low level expression of NLRP3in low grade group, and there were obvious expression of NLRP3in the high grade glioma group. The level of NLRP3mRNA and protein in normal group, low grade group and high grade group was1.04±0.52、1.85±1.02、 3.36±1.38respectively, there were significant statistical differences in Comparisons between groups(P<0.01). Compared with normal brain tissue, the expression of NLRP3in human glioma in both mRNA and protein levels are higher(P<0.01).Conclusion1、There is unusual expression of NLRP3in glioma, the level of NLRP3has a positive correlation with malignancy of human glioma.2、NLRP3may play an important role in the genesis and invasion of glioma.3、NLRP3may helps to estimate the malignancy of glioma.Innovation and significances1、This study is the first to examine the expression of NLRP3in human glioma tissues, We found that NLRP3increased with increasing pathological grade. So we predict that it may play an important role in the occurrence and development of glioma.2、We can conjecture the existence of glioma and its malignant degree by detecting the expression quantity of NLRP3, which has certain directive significance to the clinical diagnosis and prognosis.3、NLRP3may become a new target for the treatment of glioma to delay the progress of the disease and even as far as possible to cure patients with brain glioma. |
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