The Research Of Expression Of ADAM10 And Significance In Giloma

Intracranial glioma is the most common primary malignant tumor,which is characterized as invasive growth.Because there are no obvious boundaries between tumor and normal brain tissue so surgery can not be completely cured.And all the patients with giloma died of tumor recurrence finally. The tumor in general bear a grim cure due to diffuse infiltration of tumor cells into the surrounding brain tissue, preventing complete surgical removal of the tumor. Studies have shown that a variety of biological macromolecules involved in proliferation and invasion of glioma cells in the process of growth. These molecules are zinc-dependent protein with proteolytic enzymes function,including MMPs family and ADAMs family.There are some similarities with functions and structures in the two families. ADAMs family have caused widely concern in recent years,and some researchers have found that there were some differents among the ADAM12 expression in different grades of giloma.The higher level of grades,the higher level of expression.Further study have shown that ADAM12 can produce EGF by hydrolysising HB-EGF,which involving the signaling process.Then EGF conbined with EGFR so that the extracellular signal delivered to cell to activate the genes associated with the proliferation of glioma cells. Some studies have proven that AD AM10 involved the epithelial update of cells,the tissue reconstruction,and cell adhesion.Foreign researchers have found that ADAM10 was overexpressed in gastric cells in vitro and confirmed that ADAM10 promotes cancer cells proliferation by shedding gametes. It was also found to be hydrolysis of extracellular collagen typeⅣ, which was thought to promote the growth of cancer cell invasion to the distance.In our study we divided the sample into two groups of low-level and high-level according to the WHO standards with meningioma as the control group.The three groups gene are detected through RT-PCR and the results were compared with each other. We applied immunohistochemistry to show the protein of ADAM10 in the cell to explore the mechanism.All we done in the experiments are to find the the target for targeted therapy and provide the experimental basis.Materials and Methods1. Obtain experimental specimens43casesof giloma samples are collected in our hospital from 2007-2010,including 22 cases of low-grade and 21 cases of high-grade.10 cases of meningioma are served as control group.The samples were from the core of the tumor and were saved at-70℃immediately.The information of the patients are collected and the pathological results were confirmed.2. Experimental Methods(1) Extract RNAAccording to the TaKaRa description we extract the RNA from the tumor samples.The 100mg tumor tissue were made into homogenates and was put into 1.5ml free RNA enzymes EP tube and was mingled with 1ml RNAiso Plus. Put it aside for 5 minutes at room temperature. Add 0.2ml chloroform to Supernatant obtained, put it aside, after centrifugation, then the supernatant was added to isopropyl alcohol, white flakes can be seen after centrifugation and then put it aside, adding 40ul water-soluble enzymes to the RNA after dried,then RNA purity and content was measured by UV spectrophotometer.(2) Two-step RT-PCR:Reference primers synthesized by TaKaRa Company, upstream and downstream sequences:5'-TGGGTCAAAAAGAAAATGGC-3'和5'-CCCAGGTTTCAGTTTGCA TT-3'.Amplified fragment length is 281bp, usingβ-actin as an internal control. upstream and downstream respective is as follw:5'-AAATCGTGCGTGACATTAA-3' ,5'-CTCGTCATACTCCTGCCTG-3', the size of amplify fragment is 474bp,the whole system is 10ul,take 2.5ul template (cDNA)to amplify,RT system denature at 94℃for 2 minutes,cycle condition is at 94℃for 30secend, at 55℃for 30 secend,at 72℃for 2 minute,add up to 30 cycles,extension for 10 minute. The products were separated through electrophoresis using agarose gel,photographs were taken in the UV lamp camera,statistical analysis was made by gray gel analyzer. (3) ImmunohistochemistryOperation is made according to instructions of SABC staining kit.In the light microscope, there are brown-yellow granules reaction appears on the cell membrane,in the cytoplasm,which was regarded as positive reaction.500 cells were counted in 5 different high power field and the rate of positive cell less than 5% is marked(-),5%-50% marked (+),50%-75% marked (++),>75% marked (+++)(4) StatisticsThe results of data was analysised using SPSS 14.0 software. Different groups parameters were compared using One-way ANOVA analysis, p<0.05 means significant statistically.Results1. The expression of AD AM10 gene63 grayscale strip was scaned and the results showed that the most expression was in high-grade group, little expression was in control group.Single-factor analysis of the average gray showed that the different between high-grade group and low-grade group was statistically significant (f=13.31, p<0.05), Low-grade group and control group differences are significant (t=22.83, p<0.01)2. The expression of AD AM10 proteinThere are 47 positive cells in the control group in the high-powered microscope(9.4%),269 postive cells in the low-grade group(53.8%),382 positive cells in the:high-grade group(76.4%).Many tumor cells had been destroied in the low-grade,tumor cell membrane and cytoplasm was sporadic positive staining. circular positive staining was showed in the blood vessel walls.Conclusion1. ADAM 10 is expressed in meningiomas and gliomas and the expression was positively correlated with the level of glioma. Glioma and meningioma was statistically significant differences in the expression.2. ADAM 10 expression are shown in the vassel of the glioma,which may be one of the mechanisms that increased permeability of glioma blood vessel wall that caused peritumoral edema.