| BackgroundThe traditional view is that tumorigenesis is a multi-step process that involves the external factors stimulation, activation of oncogenes, tumor suppressor gene inactivation process. But found the people to study colorectal cancer, intestinal epithelial survival period is short, not enough time for the accumulation of gene mutations cause tumors. In this regard, the researchers pay attention to the undifferentiated stem cells, which can long-term presence rather than the differentiated tumor cells.1997Bonnet isolated human acute myeloid leukemia stem cells [8] found that CD34+cd38-phenotype of the tumor cells which are able to make the NOD/SCID mice in vivo tumorigenicity only accounted for0.2%, while most of the other phenotypic cells can not form a tumor.2001, Reya et al [9] first proposed the theory of cancer stem cells (TSC),which believe that there are a small amount of self-renewal, unlimited proliferation and differentiation potential cancer stem cells in tumor tissue. Proportion of these stem cells in the tumor tissue, although rarely, but with tumor origin, development, transfer of radiotherapy and chemotherapy resistance are closely related, and most other tumor cells after a short-lived generations of differentiation eventual death. In subsequent srudies,a variety of tumor stem cells of colorectal cancer, breast cancer, liver cancer, pancreatic cancer and other solid tumors were cultured one after another, and their cell markers, biological characteristics were studied.So far, CD133is the generally accepted cell surface markers of stem feature cells in brain tumors.Singh et al cultured neural stem cells, inoculated100CD133positive cells in NOD/SCID mice cause tumor, the formation of tumors is consistent with the original tumor phenotype. However, with the progress of the cancer stem cell research, the researchers found that CD133positive tumor cell as a cancer stem cells have certain limitations. Myung and Woolard et al found that about40%of freshly isolated glioblastoma specimens did not express CD133positive tumor cells, Wang, etc.found in gliomas there is part of the CD133-negative tumor cells still tumorigenic,CD133-negative cells have the ability to differentiate into the CD133-positive cells,so that such CD133-negative cells can be considered brain glialtumor stem cells (BTSCs).2011Rath, P. cultured and identified meningioma stem cells, and also studied the surface markers, study suggest that CD133is not the only brain tumor stem cell markers, cell leukocyte adhesion factor CD166/CD166also a kind of valuable stem cell markers.Meningioma is a common intracranial tumor, accounting for about20%of intracranial tumors, the main treatment is surgery,90%of meningiomas WHO Ⅰ level, WHO Ⅱ Meningiomas account for about4.6%-7.2%, WHO Ⅲ meningiomas about accounting for1%to3%.Recurrence and metastasis of meningioma after surgery is key to influencing prognosis. Markers CD166and CD133of brain tumor stem cells provides new ideas and possible therapeutic target spot for antitumor therapy. The early literature confirmed the level of expression of CD133and CD166is closely related to the prognosis of a variety of tumors, which is one of the prognostic factors.This study was using immunohistochemical method to detect the expression of the brain tumor stem cell markers CD133and CD166,explore the distribution and clinical significance of CD133and CD166positive cells in tumor tissue, as well as the feasibility of CD166stem cell marker in meningioma.ObjectiveThis study aims to find a better target for meningioma diagnosis, treatment, investigate the expression of CD166and CD133in different grade of meningiomas with immunohistochemistry Chemical Technology, and the correlation between CD166and CD133,and the relationship between brain tumors and the brain tumor stem cells.Methods1. Materials and methods1.1Specimen collection80cases of paraffin specimens were obtained from archival meningiomas from2006to2009surgical specimens in pathology department of Southern Medical Uiversity affiliated Zhujiang Hospital. All the specimens were reviewed and diagnosed by well-experienced pathologist.Classificated and graded strictly according to the World Health Organization (WHO) guidelines2007of the nervous system, grade Ⅰ33cases, grade Ⅱ44cases, among them atypical meningeal21cases, other11cases; grade Ⅲ3cases, all belong to anaplastic brain tumors. Tumors of grade I are low-level meningiomas, tumors of grade Ⅱ-Ⅲare high-grade meningiomas.1.2Reagents and immunohistochemical methods CD166rabbit anti-human polyclonal antibody (Beijing Biosynthesis company), CD133rabbit anti-human polyclonal antibody (Beijing Zhongshan Golden Bridge), Immunohistochemistry kit (Biogenex). Specimens fixed in10%formalin, embedded in paraffin, sliced thick5um dewaxing hydration, repair antigen.Operating in strict accordance with the instructions. Colon cancer specimens stained biopsy as CD133and CD166positive control, PBS solution, instead of an antibody, as a negative control.1.3results criteria CD133and CD166positive expression refers to the cell membrane and cytoplasm appeared brown and higher than the background color positive. The overall degree of positive expression of the tumor tissue with brownish yellow substance Integrated optical density (IOD) value. Quantitative image analysis process: Random selection of10high-powered microscope (400×) field of view, using the Image-Pro Plus6.0pathology image analysis software calculated the IOD of positive cells.2. Experimental proceduresAfter the dewaxing and hydration of paraffin sections, performed the repairment of the tissue with pressure cooker. Natural state cooling,then flushed twice with distilled water, each time for5min. Dipped in PBS three times, each time for5min. Absorbent paper adsorption of residual on slides in PBS buffer,added50-100μl3%peroxide blockers in each slice to block endogenous peroxidase, incubated at room temperature for10minutes. Flushed in PBS3times, each time for3min. Removed the PBS, added power blocker:5%bovine serum,incubated at room temperature for10minutes. Removed the non-immune bovine serum blocking solution. Added anti-CD133/anti-CD166(use PBS buffer in place of anti-CD166/anti-CD133as negative control), incubated at4℃in the refrigerator overnight. Flushed in PBS3times, each time for5min. Removed the PBS, added Super Enhancer, incubated at room temperature for10-15minutes. Flushed in PBS3times, each time for3min.; Removed the PBS, added Polymer HRP reagent, incubated at room temperature for30minutes. Flushed in PBS3times, each time for3min.Removed the PBS solution, added2drops of fresh preparated DAB solution to each slice (preparated according to the instruction),3min later,observed the stainning under the microscope. Stopped the reaction when the stainning just right.. Flushed15min in flowwing water, hematoxylin redyed1min, ethylalcohol differentiated2seconds, flushed more than10min in flowwing water, dryed, dimethylbenzene hyalined, sealed section with neutral gum.3. Statistics analysisData were analyzed using SPSS13.0software. The values given was median(M).Used the two independent sample compared with the rank and inspection (Wilcoxon W rank sum test) to compare the IOD between CD166+cells and CD133+cells in different grade of meningiomas in the same age and gender. Used multiple samples compared with the rank and inspection (Kruskal-Wallis H test) to compare the IOD between CD166+cells and CD133+cells in different grade of meningiomas in different parts. Correlation between the IOD of CD166+cells and CD133+cells was analyzed by the Spearman method, P<0.05indicated statistically significant.ResultsCD133/CD166expressed different shapes in meningiomas,the basic form were mainly as follows:CD133+cell morphology had dotted positive, gathered positive around vascular exist;CD166+cell morphology scattered and positive gathered positive;both antibodies were expressed on the cell membrane and cytoplasm.CD133+/CD166+were expressed at different grades meningioma tissue distribution characteristics as follows:; CD133and CD166expressed in different grade meningioma;CD133+cells and CD166+cells distributed by regularity, CD133+and CD166+cells expression levels changed with meningioma pathological grading higher, and in low-level group,CD133+and CD166+cells were lower, mostly scattered,;in high-level group of CD133+and CD166+cells were relatively more able to be observed and more specimens positive aggregated visible around the wall of the blood vessel visible.in CD staining, CD133+blood vessels, these CD133+cells were considered to be the vascular endothelial precursor cells, CD166staining was negative.The CD133+cells and CD166+cells IOD valuesof different grades meningiomas;The CD133+cells IOD value was2466.59(a high-level group) and578.56(a low-level group), rank sum test two groups had a significant difference (Z=-4.080, P=0.000), the CD166+cells IOD value was12,919.51(a high-level group) and4678.79(a low-level group),rank sum test two groups existed significant difference (Z=-4.657, P=0.000).The relationship and co-expression between CD133and CD166,we found CD166protein expression increased with CD133protein expression Spearman correlation test performed CD133and CD166IOD values in meningiomas showed a significant positive correlation (R=0.706, P=0.000). In microscope,we observed sections organizations of the existence of the phenomenon of co-expressed in the same area of the two antibodies, the negative surrounded tissue expression, as an internal control.The relationship in CD133, CD166and meningioma prognosis. In three years of the high and low-level group meningioma recurrence rate were CD133weak expression group recurrence rate was15.38%, and the strong expression group28.57%, chi-square test had not significant difference(p=0.16), suggested that CD133expression was no significant correlation with meningioma recurrence.CD166weak expression group recurrence rate was10.86%, the strong expression group32.35%, both the existence had a significant difference (P=0.018). CD166high expression group had a higher recurrence rate than low expression group.ConclusionCD133and CD166were expressed in different grades glioma.The expression of CD133+and CD166+are increased with the increasing of meningioma pathological expression. And CD133+cells (p=0.001) and CD166+cells (p=0.000) IOD value at different grades of meningioma are significant. Difference.The CD133and CD166expressed as a positive correlation. By Spearman linear correlation analysis to CD133+cells and CD166+cells IOD value was significantly positively correlation (p=0.000).Slice staining for CD133and CD166expression; CD166can be one of the brain tumor stem cell markers together with CD133which has been known to us.The recurrence rate was higher in the CD166high expression group,,CD166is related to meningioma recurrence. |
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