| Squamous cell carcinoma is a common pathological type of oral and maxillofacial malignancies, accounting for90%, and tongue squamous cell carcinoma is one of the most common types of oral squamous cell carcinoma. To treat tongue squamous cell carcinoma, a sequential therapy program is commonly adopt, including the combination of surgery, radiotherapy, chemotherapy, biological therapy and other treatments. Some progresses are obtained, but the two main problems which are cancer recurrence and metastasis in clinical treatment of tumors have not been completely resolved. So, the long-term effects of the sequential therapy program are still not satisfied.In recent years, with the development of the studies of cancer, scholars have found some similar growth control mechanisms between some cancer cells and stem cells, so the theory of cancer stem cells (CSCs) was proposed. According to this theory, there were a small amount of stem cell-like cancer cell subsets which have the ability of self-renewal, differentiation and proliferation in the cancer tissues. The stem cell-like cancer cell subsets were called cancer stem cells (CSCs). They could differentiate into most of cancer cells which were constituted the cancer and enable the cancer have the characteristics of heterogeneity, proliferation, infiltration et al. Although account for a very small proportion of cancer tissue, CSCs were the root causes of cancer origin, growth, metastasis and recurrence.The theory of cancer stem cells breakthrough the concept of the traditional cancer treatment and open up a new era in the field of cancer treatment. The study of its biological characteristics, the establishment of some appropriate experimental models and the anti-cancer strategy for finding targeted cancer stem cells are the hotspots for curing the cancer recurrence and metastasis. The key point of research on cancer stem cells is to choose a cell surface marker, which can confirm the existence of cancer stem cells and be applied to extract and identify cancer stem cells.P75neurotrophin receptor (p75NTR) as a low affinity receptor is one of the neurotrophic factors, with molecular weight of75kDa. p75NTR is a member of the tumor necrosis factor receptor superfamily. In vivo, p75NTR can induce various biological behaviors including cell proliferation, differentiation, migration, apoptosis et al through different signaling pathways. It is shown that this complex biological activity is realized by two ways mainly:first, interacting with the Trks receptor; second, involving in the start-up and regulation of independent signals for promoting survival and apoptosis of cells. In recent years, it is confirmed that the expression of p75NTR is related to the occurrence and development of variety of cancers and p75NTR can be used as a surface marker of cancer stem cells.In this study, we investigated the expressions of p75neurotrophin receptor in tongue squamous cell carcinoma cell line Tca-8113and in Cal-27. Then, we used flow cytometry to sort tongue squamous cell carcinoma cell line with p75NTR as the mark. The biological characteristics of the p75NTR+tongue squamous cell carcinoma cells were researched specially. The significances of this study are to evaluate the possibility of p75NTR as a prognosis indicator of oral squamous cell carcinoma as well as a cancer stem cell marker and to provide clues for studying the pathogenesis of tongue squamous cell carcinoma and looking for new therapeutic targets from the perspective of cancer stem cells theory.Part1:The Single-cell Proliferation and the expression of p75NTR in tongue squamous cell carcinoma cell lineobjective:To determine the expression ofp75NTR in tongue squamous cell carcinoma cell line, and examine the proliferation ability of human tongue squamous carcinoma Cal-27cell line for finding the tumor stem cells and investigating the feasibility of p75NTR as the membrane mark of cancer stem cells in tongue squamous cell carcinoma cell line.Methods:Expression of p75NTR was detected by flow cytometry in tongue squamous cell carcinoma cell line Tca8113and Cal-27.And in vitro, single cell culture was employed to observe the proliferation of Cal-27cells. The data was analyzed by the χ2test via SPSS13.0software.Results:The percentages of the cells with p75NTR+in tongue squamous cell carcinoma cell lines Tca-8113and Cal-27are about3.1%and1.9%respectively. In vitro,8days after single cell culture, the cells with the ability of proliferation account for13.8%of the total cells in tongue squamous cell carcinoma cell line Cal-27;12days later, about4.4%of cultured Cal-27cells continued to proliferate;16days later, there were only about3.7%of the cells still went on.Conclusion:The cells of tongue squamous carcinoma cell line Cal-27have heterogeneity, and only a minority of cancer cells which may be cancer stem cells exhibited the ability of continuous proliferation. Simultaneously, there were a small number of cancer cells with positive expression ofp75NTR. The results suggest that the p75NTR can be taken as a marker of cancer stem cells. Further study should be taken to investigate the role and mechanism of p75NTR in the occurrence and development of cancer.Part2:Study of the biological characteristics of p75NTR+tongue squamous cell carcinoma cellsObject i ve:To study the biological characteristics of p75NTR+tongue squamous cell carcinoma cells which were separated by flow cytometry cell sorting, and to investigate whether p75NTR+cells have the characteristics of cancer stem cells.Methods:To determine the biological characteristics of p75NTR+cells which were separated from tongue squamous cell carcinoma cells by flow cytometry cell sorting, including proliferative capacity, self-renewal, multiple differentiation capacity and tumorigenicity:1. Expression of CD44was detected by immunocytochemistry experiments in p75NTR+cells;2. Study the capacity of cloning of p75NTR+and non-sorted cells by monoclonal cultivating;14days later, collect the progeny cells generated from monoclonal p75NTR+cells; and detect the percentage of p75NTR+cells;3. Compare the growth characteristics of p75NTRand non-sorted cells by MTT assay, and to draw cells growth curve;4. Analyze the metastasis ability of p75NTR+cells and non-sorted cells by wound healing assay;5. Compare the tumorigenicity of p75NTR+and non-sorted cells in vivo.Results:CD44is highly expressed in tongue squamous cell carcinoma cells with p75NTR+;the percentages are about50%both in Tca-8113and Cal-27. Comparing p p75NTR+cells with non-sorted cells from tongue squamous cell carcinoma cell lines(Tca-8113and Cal-27),we find that p75NTR+cells possess higher capacity of cloning than non-sorted cells (p<0.01). The percentage of p75NTR+cells of the progeny cells generated from monoclonal p75NTR+cells decreased to14.5%and5.8%during the first two weeks, a lot of p75NTR-cells generated by p75NTR+cells. p75NTR+cells possess higher proliferation ability than non-sorted cells by MTT assay (p<0.01), and higher metastasis ability than non-sorted cells by wound healing assay. The difference of tumorigenicity between p75NTR+cells and non-sorted cells has not statistical significance, but p75NTR+cells have higher speed of tumorigenicity.Conclusions:Comparing to non-sorted cells, p75NTR+cells possess higher proliferation ability, metastasis ability, self-renewal,varied differentiation capacity and higher speed of tumorigenicity. p75NTR+cells could generate both p75NTR+and p75NTR"progenies. These results suggest that p75NTR+cells isolated from tongue squamous cell carcinoma have the characteristics of cancer stem cells. This study provides an effective method for further studying the tongue squamous cell carcinoma stem cells and clues for studying the pathogenesis of tongue squamous cell carcinoma and looking for new therapeutic targets from the perspective of cancer stem cells theory. |
PDF Full Text Request