| The recurrence and metastasis is one of the primary causes for the death of patients with the malignant tumor, which is the biggest challenge for the tumor treatment. Until now the fundamental causes for the genesis, recurrence and metastasis of the tumor have not been identified, although some progress has been made in the study of biology.The conception of the tumor stem cell (TSC) has supplied a new thread for the study of the biological properties of the tumor, which make it possible for targeting the TSCs to kill them and preventing the recurrence and metastasis of the tumor. The conception has become the hot topic, but because most of the specific makers of the cells are unknown, the isolation and identification of the TSCs is quite difficult. The side population (SP)is a special type of the cell subpopulation, for which the isolation method is easy but mature. Therefore this method may bring a convenient way for the study of the TSCs.Many researchers have checked out the SP cells from various normal and tumor tissues. It was confirmed that compared with the non-SP (NSP)cells, SP cells from much tumor tissues possessed faster proliferation and self-renewal properties, they were more easier to form tumor in immunodeficient mice, and had stronger drug resistance, for which the stem cell characteristic cells were considered to be enriched in the SP cells. At present, the study of TSCs in laryngeal carcinoma is still in the primary stage. Our study investigated the biological properties of the SP cells from the laryngeal carcinoma cell line Hep-2 and the primary cultured laryngeal carcinoma cells, desiring to support a new approach and some experimental reference for the study of laryngeal TSCs.Part I:Detection and isolation of the SP cells from the laryngeal carcinoma cell line Hep-2Obsjective To detect and isolate the SP cells from laryngeal carcinoma cell line Hep-2, and characterize the generation mechanism of the phenotype of the SP cells. Methods The Hep-2 cells were cultured in vitro and observed under fluorescence microscope after dyed with Hoechst 33342.The SP cells and NSP cells were sorted by fluorescence-activated cell sorting(FACS).The SP and NSP cells were planted in DMEM with 10% FBS to observe the cell morphology. To detect and compare the expression of the mRNA and protein of ABCG2 between the SP and NSP cells, the assays of RT-PCR,cellular immunofluorescence,Western blot were performed. Results The SP cells existed in the laryngeal carcinoma cell line Hep-2,and the percentage of the Hoechst33342 negtive cells was (5.1±0.2)%.The proportion of the SP cells was (4.4±0.85)% sorted by FACS, which decreased to (0.63±0.31)% after the verapamil added. The two group cells showed the similar characteristics with Hep-2 cells after cultured in DMEM with 10% FBS. The expression of ABCG2 in SP cells was significantly higher than NSP cells(P<0.05).Conclusions SP cells do exist in laryngeal carcinoma cell line Hep-2, and ABCG2 is associated with the SP phenotype.Part II:Identification of the biological characteristics of cancer stem cell-like SP cells from laryngeal carcinoma cell line Hep-2Objective To explore the biological characteristics of cancer stem cell-like SP cells from laryngeal carcinoma cell line Hep-2. Methods The SP and NSP cells were cultured in vitro and observed under the inverted phase contrast microscope and the transmission electron microscope, the morphologic characteristics were compared between the two group cells. CCK-8 method was used to detect the proliferation capacity of the SP and NSP cells. The clone formation assay was carried out in the flat plates and the soft agar to observe the clone forming capacity of the SP and NSP cells. The two group cells were analysised by FASC to compare the differentiation potency and the cell cycle. To examine the invasion ability of SP and NSP cells, the matrigel invasion assay was performed. The transwell assay and the scarification test were used to evaluate the migration ability of the SP and NSP cells. The adherence property of the SP and NSP cells was detected by the method of CCK-8. To compare the tumorigenic activity, three numbers of SP and NSP cells were injected into the nude mice. CCK-8 assay was used to assess the proliferation inhibition of chemotherapy drug 5-FU for the two group cells. Results The SP cells depicted float colonies as proliferating, but the NSP cells did not generate the typical cell spheres observed under the inverted phase contrast microscope. Observed under the transmission electron microscope, the SP cells were integrated with big nucleolus and little cytoplasm, while the NSP cells showed the small pyknosis of the nucleolus, with much expanded endoplasmic reticulum and heterochromatin. In the CCK-8 proliferation assay, the absorbance the SP cells were higher than that of the NSP cells from the third cultured day. The ratio of clone formation of SP and NSP cells is (47.47±10.20)% and (4.98±1.41)% in the flat plates, and in the soft agar the ratio is (46.82±5.67)% and(12.53±3.51)% respectively, which demonstrated that the SP cells displayed the higher clone formation ratio than the NSP cells. In the differentiation potency assay by the FACS, the proportion of the sorted SP cells (4.2±0.47)% in the 14th day was similar to that of the first time sorting(4.4±0.85)%. Both the SP and NSP cells displayed a similar cell cycle distribution in the cell cycle assay. The SP cells depicted the higher migrating potency than the NSP cells in both the transwell assay and scarification test, of which the difference was significant(P< 0.05). The matrigel invasion assay showed that the SP cells that penetrated artificial basement membrane (39.04±4.78)% was higher than that of the NSP cells(25.16±4.63)% with significant differences(P<0.05). The adherence property assay of the SP and NSP cells in the CCK-8 test demonstrated that the SP cells owned the higher adherence property (P<0.05). Subcutaneous tumor formation needed as low as 1×103 SP cells inoculation in 3 of 5 mice, while as high as 1×105 NSP cells inoculation in 4 of 5 mice, which indicated that the tumorigenic activity of the SP cells were stronger than the NSP cells(P<0.05).The result of CCK-8 assay of proliferation inhibition showed that the chemotherapy drug 5-FU inhibited the proliferation of SP cells more obviously than the NSP cells. Conclusions The SP cells sorted from the laryngeal cancer cell line Hep-2 is a tumor stem cell-like subpopulation which displayed characteristics of high proliferative,clone formation,differentiation capacities, strong tumorigenic ability, and resistance of chemotherapy drugs.PartⅢ:Analysis of the SP cells in the primary laryngeal carcinoma cellsObjective To detect the SP cells in the primary laryngeal carcinoma cells, and explore the associativity of the SP cells and the clinical pathological characteristics of the laryngeal carcinoma. Methods The primary laryngeal cancer cells from 40 cases of clinical specimens were harvested by the collagenase digestive method, and detected by the FACS for the side population. The associativity between the SP cells proportion of the primary laryngeal carcinoma cultured cells and the prognostic factors of the laryngeal carcinoma was analysised. Results The SP cells existed in most of the primary laryngeal carcinoma cells, and the average proportion of the SP cells from the 40 cases of laryngeal carcinoma specimens is (3.1±2.36)%.The SP cells were correlated with the clinical staging,pathological grading,metastasis of lymph node of the laryngeal carcinoma.Conclusion The SP cells exist in most primary laryngeal carcinoma specimen cultured cells. The SP cells were correlated with clinical pathological characteristics of the laryngeal carcinoma.
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