| BACKGROUD AND OBJECTNasopharyngeal carcinoma (NPC) is a kind of epithelial origin in nasopharygneal mucosa malignant tumors and a common cancer in southern China and Southeast Asia, especially in Guangdong Province. Epidemiology studies suggest the occurrence of NPC is influenced by both environmental and genetic factors. Epstein-Barr virus (EBV), as well as environmental factors and genetic components are involved in the etiology of NPC. The undifferentiated carcinoma of nasopharyngeal(UCNT,WHOType III) is dominant in high-risk areas and is consistently associated with EBV infection. EBV latent infection maybe the first step of NPC.EBV mainly infects B lymphocytes and epithelium in vivo. It is clearly that EBV infection is initiated by specific attachment to human complement receptor type 2 (hCD21), a cell surface protein that is highly expressed on B lymphocytes. The major EBV glycoprotein gp350/220 attaches to the two most membrane distal CD21 repeats SCR1 and SCR2, and soluble forms of CD21 containing only these two repeats can prevent virus infection. The CD21 molecule contains 15 or 16 short consensus repeats (SCR1 to SCR16), domains of 60 residues that are found in a large number of cell surface receptors, many with complement regulatory function.EBV can infect B lymphocytes effectively and CD21 play determination role during EBV infection of B-Lymphoblastic cells. The expression of CD21 is the perhaps earliest markers of the B cell lineage. Such cells may represent B progenitor cells preceding classical pre-B-lymphocytes in pathways of B cell differentiation. Fetal marrow B lineage cells show higher susceptibility to transformation than is typical for adult cells. Maintaining the EBV infecting needs undifferentiated environment and lead to the expression of LMP1 which will prevent differentiation. EBV infection only occurs at base of epithelium. All of these suggest the environment for EBV infection similar to stem cells. CD21 express in some progenitor and tumor stem cells and also coexpress with SOX2. So we ask whether CD21 express in progenitor/stem cells of epithelium.EBV infection efficiency on epithelium is very low and the exact mechanism remains to be further studied. Up to now, some study found EBV infection was higher may through 2 independent pathways:1) by direct cell-to-cell contact (primary mode) of apical cell membranes with EBV-infected lymphocytes.2) by entry of cell-free visions through basolateral membranes. But some study proved EBV infection efficiency was higher when CD21 was over expressed on epithelial cells.However CD21 expresses on epithelium is controversial. Whether cell-free EBV can infect epithelium cells by CD21 is not clearly. The patients of NPC often accompanied by nasopharyngitis with mass B lymphocytes, it interferes with the detection the expression of CD21. So we detect the expression of CD21 of fetuses (different ages) nasopharyngeal epithelial tissue and analyze CD 133, CD21 and CK5 expression by Immunohistochemical staining. In vitro flow cytometry detected the percentage of CD 133+ and CD21+ cells in CNE1 CNE2 and NP69.We also confirm CD21 maybe the one stem cell marker using cell and tumor sphere Immunofluorescence and 5-bromo-2-deoxyuridine(Brdu) to label stem cells which is also called label-retaining cells(LRCs).Cell-free EBV infected NPC cell lines and NP69, EBNA1 was detected after EBV infection. To further verify whether cell-free EBV infect epithelium through CD21, first we detect the coexpression of EBNA1 and CD21 after EBV infection, then CD21 was closed, after that we again detect the expression of EBV and observe the morphological changes and markers of malignant phenotype of cells.Methods1. Detection and analyze the expression of CD21 CD133 and CK5 of fetuses nasopharyngeal epithelial tissueExpression of CD21 CD 133 and CK5 were detected by immunohistochemistry in specimens of fetuses (different ages) nasopharyngeal epithelial tissue. We infer position of stem cells of nasopharyngeal epithelial tissue. We confirm the relationship between CD21 and stem cells and differentiation.2. Exploration of the possibility of CD21 expressing in nasopharyngeal epithelial and NPC stem cells1)Flow cytometry detected percentage of CD21+ and CD133+ cellsFlow cytometry detected and analyzed percentage of CD21+ and CD 133+ cells in NP69,CNE1 and CNE2.2)immunofluorescenceCD21 and CD 133 were detected by immunofluorescence in NP69,CNE1 and CNE2 cell lines and detected whether CD21 and CD 133 were coexpressed.3) LRCs in vitroThe CNE1,CNE2 and NP69 cells were grown in standerd medium, and pushed with 10ng/ml Brdu for 7 days, and then washed out Brdu for 14 days. Brdu immunofluorescence were was proceeded at 2h after Brdu pushed in and on the 14d after Brdu washed out,respectively. LRCs were numbered under microscope.4) Detection whether LRCs express CD21 and CD133LRCs and CD21 were detected together by immunofluorescence, LRCs and CD133 were detected together by immunofluorescence.5) tumor sphere immunofluorescenceNPC tumor spheres were formed and the expression of CD21,CD133 and CK5 were detected by immunofluorescence. The coexpression of CD21 and CD 133 were also detected by immunofluorescence.6) tumor sphere differentiationThe expression of CD21,CD133 and CK5 were detected when tumor sphere differentiated.3.Cell-free EBV infects nasopharyngeal epithelial cells1) extraction of cell-free EBVB95-8 cells were cultured for some time and the cell culture medium was collected and then centrifuged at 4℃, we filtered the medium with 0.22μm filtrator.2) the infection of EBVThe same viral titer of cell-free EBV infected NP69,CNE1 and CNE2.3) Detection of EBNA1 after EBV infectionThe expression of EBNA1 in NP69,CNE1 and CNE2 was detected by immunofluorescence after 10 days of EBV infection.4) The morphological observation of cells after EBV infectionObservation was made on the cell morphology of the cells after EBV infection and compared with the control group.5) Detection of cytoskeleton after EBV infectionCytoskeleton was detection and observation was made on the cell invadopodia.4. Exploration of the possibility of cell-free EBV infection depending on CD211) Double immunofluorescent stainingEBNA1 and CD21 was detected by double immunofluorescent staining after 10 days of EBV infection.2) closed CD21The CD21 of cell lines was closed by CD21 antibody and then the cells were infected by EBV.3) Observation was made on the EBNA1, cell morphology and cytoskeleton after CD21 closed.Results1) Expression of CD21 was detected by immunohistochemistry in specimens of fetuses (different ages) nasopharyngeal epithelial tissueCD21 was positively expressed in cell nucleus and cytoplasm of nasopharyngeal epithelial tissue of the small fetal month fetus. At 14w,20w,26w, the expression of CD21 respectively was 32.15±27.06%,16.96±16.96%,7.74±8.32% and all the ages have significantly difference (F=607.296,P=0.000). the expression of CD21 in the top wall, side wall and bottom of front end, top wall, side wall and bottom of midpiece and top wall, side wall and bottom of back end respectively was 10.00±5.43%,0.78±0.97%,0.00±0.00%,39.00±20.04%,24.56±14.83%, 3.89±2.98%,51.22±23.82%,25.22±16.02%,15.55±15.06% and all the parts have significantly difference (F=604.844,P=0.000). All the cells in the bottom of fold were positive expression.2)Detection the expression of CD 133 in fetuses nasopharyngeal epithelial tissueCD 133 was positively expressed in cell nucleus and cytoplasm of nasopharyngeal epithelial tissue of the small fetal month fetus. At 14w,20w,26w, the expression of CD133 respectively was 31.15±26.48%,16.96±17.12%,7.52±8.17% and all the ages have significantly difference (F=480.029,P=0.000). The expression of CD133 in the top wall, side wall and bottom of front end, top wall, side wall and bottom of midpiece and top wall, side wall and bottom of back end respectively was 9.67±5.70%,0.78±1.09%,0.00±0.00%,39.00±20.00%,25.22±14.29%,3.78±2.82%, 50.78±24.00%,23.22±13.92%,14.44±13.35%, and all the parts have significantly difference (F=637.724,P=0.000).The top wall of back end and top wall of midpiece have significantly difference (P=0.021).The top wall of midpiece and side wall of back end have significantly difference (P=0.008).The side wall of back end and side wall of midpiece have no significantly difference (P=0.230).The side wall of midpiece and bottom of back end have significantly difference (P=0.002). All the cells in the bottom of fold were positive expression.3) Detection the expression of CK5 in fetuses nasopharyngeal epithelial tissueAt 14w,20w and 26w the expression of CK5 in back end of nasopharyngeal epithelial was respectively 29.22±8.67%,70.33±16.71%,49.22±33.38% and all the ages have significantly difference (F=151.252,P=0.000). The expression of CK5 in the top wall, side wall and bottom of back end was respectively 35.44±16.93%, 46.67±18.87%,66.67±34.85% and all the parts have significantly difference (F=114.898,P=0.000). All the cells in the bottom of fold were negtive expression.The expression of CD 133 in fetuses nasopharyngeal epithelial tissue was same with CD21. CD 133 may be a broad-spectrum marker of cancer stem cells. Recently some study showed CD 133 can serve as a specific surface marker for nasopharyngeal cancer stem cells. Our study showed the expression of CD 133 was high in younger gestational age fetuses and decrease with gestational age. There was on CK5 expression where expressed CD 133. CK5 is a marker of early differentiation. So the cells expressing CD133 maybe undifferentiated cells. These suggest CD 133 maybe as a specific surface marker for nasopharyngeal epithelial tissue. Because the expression of CD21 in fetuses nasopharyngeal epithelial tissue was same with CD 133, CD21 may also serve as a specific surface marker for nasopharyngeal epithelial tissue. However whether CD21 disappears or remains as a specific surface marker with age needs further study.2. Exploration of the possibility of CD21 expressing in nasopharyngeal epithelial and NPC stem cells in vitro.1) Flow cytometry detected percentage of CD21+ and CD 133+ cellsWe detected the percentage of CD21 positive cells in NP69, CNE1 and CNE2 cell lines respectively using flow cytometry analysis. The percent of CD21 positive cells was very low and respectively 0.53±0.06%,0.17±0.06% and 1.03±0.15% . The percent of CD133 positive cells was respectively 0.5±0.1%,0.23±0.06% and 1.30±0.2%. There was no obvious difference between the percentage of CD12 and CD133 in CNE1, CNE2 and NP69 cell lines.2) Cell immunofluorescence assayWe examined the coexpression of CD21 and CD133 by immunofluorescence assay. CD21 and CD133 distributed predominantly in the cytosol and membrane of cells and CD21 and CD133 co-expressed in CNE2 and NP69 cell lines.3) Labeling LRCs in vitroThe cells retaining the BrdU labels in NP69 and CNE2 cell lines were very few and the nucleus was much smaller than other cell nucleus. The percent of LRCs was respectively only 0.50±0.08% and 1.10±0.15%. There was no significant differences among the percent of CD21, CD 133 positive cells and LRCs e also expressed CD21 and CD133.4) Tumor sphere immunofluorescence assayCD21 and CD133 co-expressed in the tumor sphere but CK5 was abcent.5) Tumor sphere differentiation assayWhile CD21 and CD 133 decreased and CK5 increased with differentiation of tumor sphere.These data suggest CD21 maybe a stem marker of nasopharyngeal epithelia cells3. Cell-free EBV infects nasopharyngeal epithelia cells1) The detection of cell-free EBV infectionThe percent of EBNA1 expression in NP69 and CNE2 was respectively 0.40±0.08% and 0.67±0.09%, CNE1 cell line appeared lytic death.2)) The detection of cell morphology after cell-free EBV infectionWe found that very few cells shape went from round to long fusiform after cell-free EBV infection in CNE2 and increased pseudopodia in NP69 cell line.3) The detection of cytoskeleton after cell-free EBV infectionA significant increase in the formation of microvillus and pseudopodia was observed in cell-free EBV infection cells.4) Exploration of the possibility of cell-free EBV infection depending on CD21When CD21 was blocked EBNA1 could not been detected and there was no obvious change of cell morphology and cytoskeleton after cell-free EBV infection, but CNE1 cell line also appeared lytic death.EBV infection includes latent infection and lytic infection, the infection in NPC is mainly Ⅱ latent infection and expressing the EBV nuclear antigen 1 (EBNA1).Our data suggest cell-free EBV latently infected nasopharyngeal epithelia cells depending on CD21 and contribute to the malignant phenotype. However lytic infection did not depending on CD21. We proved CD21 may only express in stem cells, so we infer cell-free EBV latently infected the stem cells of nasopharyngeal epithelia depending on CD21 and contribute to NPC. |
PDF Full Text Request