| The incidence rate of malignant tumor had leapt to the second place in deaseses spectrum.Moreover,it would be increasing with the environment contaminated aggravate and the coming of aging population. Mechanisms of malignant tumor initiate and progress still unclear completely so far.With the research go further,people have realized that the properties of malignant tumor, such as growth, metastasis and relapse were amazingly similar to what of the normal tissue, according to which, people put forward the hypothesis of tumor stem cells(TSCs),which state that there is a small subset of cancer cells within the tumor, named as cancer stem cells(also known as tumor-initiating cells or tumorigenic cancer cells ), with the exclusive ability to self-renew and differentiate, is origination of a tumor and maintain it. The TSCs hypothesis cognized the origination and progress of malignant tumor from a newly perspective and shed a new light to malignant tumor therapy by targeting and eradicating .However,despite existence of TSCs have been proven by more and more evidences, special morphology and biomarkers of any malignant tumor stem cells have never been proven, the field of TSCs theory applied to clinical cannot made a breakthrough as well.The reasons of what above were largly because research of TSCs was still at an initial stage, methods and techniques used for TSCs studying still not well developed , our knowledge of TSCs specific biological and molecular mechanisms were scarcely as well.Therefore,further study on the biological behaviours and relative mechanisms of TSCs were very importiant and meaningful to clarify the mechanisms of tumor initiation and progression, also contributing to the diagnosis,prognosis and targeting therpy of malignant tumors. Breast cancer stem cells was the first proven TSCs of solid tumors, CD44+/CD24-/low subset has been reported to be the enrichment of TSCs in breast cancer.But the biomarkers expression of TSCs maybe have a variation when they growing in the complex microenvironment.The reliability of defining a stem cell based solely on surface markers is debatable.Therefore, the gold standard of TSCs identification is by serial transplantation in animal models to forming tumor,which is regarded as the best functional assay for detecting those two abilities: self-renewal and differentiation.However, result of xenotransplant assays would seriously influented by the microenvironment and residual innate immunity of the recipient mouse.To those TSCs in cancer cell line,which have adapted to microenvironment of cultrue in vitro,the more reliable identification assay is combine with both in vitro and in vivo. This study aimed to creating a newly identification assay to identify the TSCs in MCF7 cell line,by which,we would investigated the proliferate characteristics and the influence factors of breast cancer stem cells. Dfferential proteomics techniques would be applied to analysis differentially expressed proteins relative to TSCs proliferation.All these work would forming the foundation for the further studying on the biological behaviours of TSCs.Obvjectives:To study the identification model suitable for identification of TSCs in cancer cell line.By the identification assay to investigate proliferation characteristics of breast cancer stem cells in vitro and the influence factors,analysis the relative expressed proteins by differential proteomics techniques.Methods:1.Single-cell isolation and transplanting assay:improvement on the basis of micromanipulate separate method to creat a equipment which make up of micropipettor and the smallest intravenous needle and fumble the most available program in practice to isolate and transplant single-cell. 2. Tumorigenic-colony identification assay: By the single-cell isolation and transplanting techniques , single cells of MCF7 were transplanted to the wells (one cell per well)of 96-well plate,then observed the colony froming,those colonies derive from single cell would be identified as tumorigenic-colony when they were amplified successfully and form a tumor at the orthotopic site of nudemice by xenotransplantation assay.According to the characteristics of single cell colony,a criteria of tumorigenic-colony identification would be verified and applied to the subsequent research.3.TSCs content in MCF7 cell line were detected by single-cell-transplant-tumorigenic-colony forming assay.This method was applied to investigate the variation of TCSs content in MCF7 cell line at series timing after passaged culture, and the factors which influence the content of TSCs would be analysized.The proportion of the CD44+/CD24-/low subset were detected by flow cytometry at the same time.Then the correlation of CD44+/CD24-/low subset with TSCs was analysized according their proportion variation.4.To further investigated the proteins which relative to the TSCs proliferation,we analysized differentially expressed proteins of MCF7 cell by two-dimensional chromatography liquid-phase separation and MALDI-TOF/ MS.Results:1. Equipment and the operate program used for single-cell isolation and transplanting assay has shown that success rate of single-cell per well higher than 90%,3 to 6 single-cell were isolated and transplanted per minute.2.The"single-cell-transplant-tumorigenic-colony"model for TSCs identifi- cation have been set up successfully. The identification criteria of a tumorigenic-colony wasâ‘ the single cell devided rapidly and formed a colony comprise more than 50 daughter cells in a week after transplanted, morphology of most of the cells were in natural and growing well andâ‘¡, the cells number of colony rapidly reach 300-1000 in another week,most of the cells growing well,sometimes with (or without )some apoptosis cells on the colony centre.3.The proportion of TCSs and the CD44+/CD24-/low subset in MCF7cell line both variety in a regulation after passaged culture. But the variation of CD44+/CD24-/low subset was more drastic (from 36.84% to 81.95)than that of TCSs(only fluctuated between 38.54% and 47.39%). The proportion of CD44+/CD24-/low subset was not always greater than that of TSCs.But correlation analysis showed that proportion of CD44+/CD24-/low subset and content of TSCs present a relationship of positive correlation in some degree, the coefficient was 0.748(P<0.001).Fuether analysis sugguested that the CD44+/CD24-/low subset maily relate to the division of TSCs ,that was said, phenotype of the offspring of TSCs were both showed as CD44+/CD24-/low,this subset would entried the next round of proliferation or turn into the CD44+CD24+ phenotype with the culture time going no matter they are TSCs or not.Content of TSCs was influenced by the culture medium with low concentration of FBS,which not only contributed to enhance the content of TSCs in MCF7 cell line(from 27.6% to 49.3%,P<0.01)but also boost the pattern of symmetry division of TSCs(from 17.1% to 39.7%, P<0.05) .4.There were some proteins related to the proliferation of TSCs by analysized the differentially expressed proteins of MCF7 cell line culture in condition with the 2%FBS and the routine concentration (10%FBS) respectively.In all of the proteins have been identified,those contribute the cancer cells to proliferate and infiltrate such as amyloid beta A4 protein(SAA), growth-regulated alpha protein(GRO-1), desmoplakin III(DPIII) and nuclease-sensitive element-binding protein 1(NSEP1 or YB-1) were all significant upregulated expressed in MCF7 cells cultrue with 2%FBS,but other proteins which restrain the cancer cells from infiltrating and metastasis such as Inter-alpha-trypsin inhibitor heavy chain H4(ITIH4) was downregulated expressed in MCF7 cells cultrue with 2%FBS.Conclusions:1. Equipment and the operate program used for single-cell isolation and transplanting assay have many advantages, material make up of it was economical and easily to find.Manipulation of it was simple and easy to popularization and application.The efficiency was credible by use it to isolate and transplant single-cell,which was important to ensure the result and meet the request of cells number in TSCs identification assay.2.The model of"single-cell-transplan-tumorigenic-colony"was suitable for identification of TSCs in MCF7 cell line in vitro.3.The CD44+/CD24-/low subset maily relate to the division of TSCs, but neither representative nor enrichment of TSCs in MCF7 cell line.4.TSCs propagating in vitro have the property to modulate and balance the content by itself.5.Culture condition with low concentration of FBS contributed to enhance the content of TSCs in MCF7 cell line and the proportion of symmetry division of TSCs.6. Some proteins expression related to the tumor biological behaviors such as progress, infiltrative and metastasis maybe induced or suppressed by the culture condition with low concentration of FBS to contributing to the TSCs proliferation,and TSCs proliferation maybe the way by which, the breast cancer stem cells evolved to the more malignancy phenotype.7. Studying on the rule of TSCs proliferation and the influence factors was helpful for us to understand the initiation and progression of tumor in essencial,which would help us to raise and investigate the newly strategies for tumor therpy. |
