| Background Brain tumor stem cells (BTSCs) refers to a small population of cells in brain tumors, which has strong resistance to the conventional radiotherapy and chemotherapy, extensive self-renewed capacity and differentiation function, with a uniquely ability to regeneration into tumor, it is believed to contribute to tumor occurrence,metastasis and recurrence, its amount may relate to the severity and prognosis of the malignant tumor. At present, targeted therapy based on BTSCs has became one of the research hotspot. So far, CD133is the generally accepted cell surface markers of stem feature cells in brain tumors. CD133is originally reported by researchers as a pronoun of BTSCs, or at least, a prerequisite. However, recent reports showed that CD133may not be the absolute indicator. CD133is high selectivity rather than high specificity to BTSCs. It also presents in the embryo neural stem cells, adult neural stem cells and vascular endothelial progenitor cells(EPCs). And CD133is glycoconjugates, which are usually localized on the cell surface, and their expression pattern often changes drastically during development.Furthermore, if tumors originated from stem/progenitor cells at different points along the normal developmental pathway, tumor stem cells(TSCs) from different subsets of gliomas may have different immunophenotypes and characteristic stem cell markers. Similar to hematopoietic stem cells, a combination of markers will ultimately best define glioma tumor stem cells. So seeking for new markers of brain tumors stem cells are of great essential and emergency. In2009, scholars one after another reported that in mice medulloblastoma model found stem feature cells, which expressed CD15surface markers. The researchers through different experiments showed that CD15can be used as the markers of brain tumor stem-like cells (including stem cell, progenitor cells) from CD133-brain tumors. Markers CD15and CD133of brain tumor stem cells provides new ideas and possible therapeutic target spot for antitumor therapy.Objective To investigated the expression of CD15and CD133in different grade of gliomas, and its correlation between CD15and CD133,and the relationship between brain tumors and the brain tumor stem cells.Methods1. Materials and methodsSpecimen collection80cases of paraffin specimens were obtained from archival gliomas from2008to2010surgical specimens in pathology department of Southern Medical Uiversity affiliated Zhujiang Hospital. All the specimens were reviewed and diagnosed by well-experienced pathologist, and had detailed clinical, pathology and follow-up data. Classificated and graded strictly according to the World Health Organization (WHO) guidelines2007of the nervous system, grade Ⅰ10cases, all belong to neuronal and mixed neuronal-glial tumors; grade Ⅱ31cases, among them oligodendrocytes tumor13cases, neuronal and mixed neuronal-glial tumors18cases; grade Ⅲ9cases, among them anaplastic oligodendrocytes tumor7cases, neuronal and mixed neuronal-glial tumors2cases; gradeⅣ30cases, among them medulloblastoma10cases, glioblastoma multiforme20cases. Tumors of grade Ⅰ-Ⅱ are low-grade gliomas (41cases), tumors of grade Ⅲ-Ⅳ are high-grade gliomas (39cases).Abiotin immunohistochemistry stainning was used to detecte the expression of CD15and CD133in glioma. Used Image-Pro Plus6.0Image analysis software calculated the IOD of CD15+cells and CD133+cells in different grade of gliomas. Comparing the difference between the IOD of CD15+cells and CD133+cells in different grade of gliomas, and performed correlation analysis between the IOD of CD15+cells and CD133+cells.2. Experimental proceduresAfter the dewaxing and hydration of paraffin sections, performed the repairment of the tissue according to the antigen. Flushed twice with distilled water, each time for5min. Dipped in PBS twice, each time for5min. Added50-100μl 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,incubated at room temperature for10minutes. Flushed in PBS3times, each time for3min.Added anti-CD15/anti-CD133(use PBS buffer in place of anti-CD15/anti-CD133as negative control), incubated at room temperature for2hour. 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 for15-30minutes. Flushed in PBS3times, each time for3min.Removed the PBS solution, added2drops of fresh preparated DAB solution to each slice (preparated according to the instruction),3-5min 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.3. StatisticsResults were analyzed by SPSS13.0software. Selected the representative tumor area in each slice, choose5vision at high magnification (400x) at random.Used Image-Pro Plus6.0Image analysis software calculated the IOD of positive cells (among which the IOD of CD15positive cells excluded positive neutrophils, the IOD of CD133positive cells excluded CD133positive blood vessels). 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 CD15+cells and CD133+cells in different grade of gliomas; Correlation between the IOD of CD15+cells and CD133+cells was analyzed by the Spearman method, P<0.05 indicated statistically significant.Results:1. The basical morphology of expression of CD15and CD133mainly as follows: there were pointlike positive morphology, peri-nuclear positive morphology, short clumps positive and long clumps positive morphology of CD15positive cells in gliomas; there were cytoplasmicprocess-like positive morphology (unipolar, bipolar and multistage), peri-nuclear cytoplasm empty bubble positive morphology, short clumps positive and long clumps positive morphology of CD133positive cells in gliomas.2. The distribution characteristic of CD15+cells and CD133+cells positive expression in different grade gliomas:CD15and CD133were expressed in different grade of gliomas. And there was certain regularity of the distribution of CD15+cells and CD133+cells have, along with the tumor grading increased, both the CD15+and CD133+cells increased. There were less CD15and CD133positive cells in the low-grade gliomas, most were diffuse, and less CD15and CD133positive niche. There were more CD15and CD133positive cells in the high grade gliomas,gathered in nest-like distribution. And most CD15+cells and CD133+cells were distributed perivascularly, radial around the blood vessels,and there were more CD15and CD133positive niche. In stainning, CD133positive blood vessels was seen,those CD133positive cells of CD133positive blood vessels was considerd as endothelial progenitor cells(EPCs). What’s more, special stainning of CD133was seen,there were pictures of pseudopalisade-like necrosis and railway-like staining pattern,the former necrosis was located inside the pseudopalisade formed by CD133positive cells, generally, this kind of necrosis was incomplete necrosis, the letter was CD133positive cells around the blood vessels arranged in railway-like staining pattern.In adittional,there is lots of CD133positive fat cells in gliomas with fat cells component.3. The IOD of CD15+cells and CD133+cells in different grade gliomas.In different grade gliomas, the IOD of CD15+cells were respectively1879.96(high-grade gliomas) and430.43(low-grade gliomas), the IOD of CD133+cells were respectively3218.35(high-grade gliomas) and473.11(low-grade gliomas). And there was significant difference between the IOD value of CD15+cells and CD133+cells in different grade of gliomas.(z=-3.432, p=0.001; z=-4.192, p=0.000)4. The correlation between CD15and CD133in different grade gliomas.Along with CD133positive expression increased, CD15positive expression increased. The Spearman linear correlation analysis showed that, there was significant positive correlation between the IOD of CD15+cells and CD133+cells (r=0.535, p=0.000).Conclusion:1. CD15and CD133are expressed in different grade of gliomas. Along with the tumor grading increases, both the CD15+and CD133+cells and their niches increase significantly. And there is significant difference between the IOD of CD15+cells (p=0.001) and CD133+cells (p=0.000) in different grade of gliomas.2. CD15and CD133expression are positively correlated. The Spearman linear correlation analysis shows that there is significant positive correlation between the IOD of CD15+cells and CD133+cells (p=0.000).3. There is lots of CD133positive fat cells in gliomas with fat cells component, which conforms with the poor clinical prognosis of these tumors.4. CD15is one of the Brain stem cells(BTSCs) markers, compares with the currently accepted BTSCs markers CD133, CD15has relatively low sensitivity, however, may be higher specificity. |
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