CN109294993B

CN109294993B - Tumor stem cell culture medium, sorting method and method for inhibiting tumor stem cell from differentiating into tumor-derived immune cells

Info

Publication number: CN109294993B
Application number: CN201811285910.3A
Authority: CN
Inventors: 丛斌; 贾娴娴; 贾苗苗
Original assignee: Hebei Medical University
Current assignee: Hebei Medical University
Priority date: 2018-10-31
Filing date: 2018-10-31
Publication date: 2020-04-14
Anticipated expiration: 2038-10-31
Also published as: CN109294993A

Abstract

The invention relates to the technical field of tumor stem cell sorting and differentiation, in particular to a method for inhibiting tumor stem cells from differentiating into tumor-derived immune cells. The tumor stem cell culture medium comprises a cytokine B27, a basic fibroblast growth factor, a transforming growth factor and an epidermal growth factor. The sorting method comprises the following steps: taking tumor cells in logarithmic growth phase, and digesting to prepare single cell suspension; culturing in a raft chip (microarray) until the tumor cells adhere to the wall; placing a micro raft (Microraft) only containing single cells in a cell culture plate, culturing until tumor cells form a full clone shape, and transferring the tumor cells to a tumor stem cell culture medium to obtain the tumor cell. Compared with the currently generally applied tumor stem cell sorting method, the method is quicker, more convenient and more accurate.

Description

Tumor stem cell culture medium, sorting method and method for inhibiting tumor stem cell from differentiating into tumor-derived immune cells

Technical Field

The invention relates to the technical field of tumor stem cell sorting and differentiation, in particular to a tumor stem cell culture medium, a sorting method and a method for inhibiting the differentiation of the tumor stem cell culture medium into tumor-derived immune cells.

Background

The tumor tissue is composed of parenchyma and stroma, and the tumor parenchyma is tumor cells, is a main component of the tumor and has tissue source specificity. The interstitial part comprises interstitial cells and acellular components, wherein the interstitial cells mainly comprise immune cells, endothelial cells, smooth muscle cells, fibroblasts and the like. The acellular component includes extracellular stroma, cytokines secreted to the outside of the cell, and the like. All of these components constitute a complex tumor microenvironment.

Tumor cells constituting tumor tissues, particularly malignant tumors, often escape from the immune system monitoring of the body through their specific mechanisms, and when tumors grow progressively, the immune function of tumor patients is suppressed, and the immune cells in the immune system hinder the recognition and removal of tumor cells and fail to exert normal function of removing abnormal self-life, thereby causing phenomena such as tumor metastasis and recurrence. The existing research and development and clinical treatment methods of new tumor medicines are based on the theory, including radiotherapy, chemotherapy, surgery, traditional Chinese medicine, immunotherapy and the like, but have little effect, the metastasis and recurrence of tumors cannot be avoided, and the death rate and five-year survival rate of human tumors are not obviously improved.

In recent years, the theory regarding tumor stem cells suggests that a small fraction of tumor cells have self-renewal, immortalization, multi-differentiation potential, and stem cell characteristics, which are the root causes of tumorigenesis, metastasis, and recurrence. It has been documented that blood vessels in tumor tissue are not formed by normal vascular migration and infiltration of the body, but are differentiated from tumor stem cells, and thus affect the effect of some antitumor drugs. In some cases, the tumor specific antibody can interfere the killing action of specific cellular immune response to tumor cells, and has the function of promoting tumor growth, and the mechanism is unknown. Therefore, the tumor stem cells with the multidirectional differentiation potential can be differentiated into tumor-derived immune cells for inducing tumor immune escape, so that the effective recognition and killing of the immune system on the tumor can be inhibited, and the occurrence and development of the tumor can be even promoted.

The precondition for the research of the tumor stem cells is to obtain the tumor stem cells, and the current methods for obtaining the stem cells include a single cell clone culture method, a magnetic bead sorting method, a flow sorting method and the like. The single cell clone culture adopts a limit dilution method, so that subjective artificial difference errors are large, time and labor are wasted, other methods need special instruments, special medicine antibodies or special medicines, and the like, for example, a flow type sorting method needs flow type machine sorting, numerous laboratories do not have the capacity of purchasing such large machines, and the special antibodies, magnetic beads or medicines and operation processes needed by other methods can damage cells to different degrees. After obtaining the tumor stem cells, the existing tumor stem cell culture medium is difficult to avoid the induced differentiation of the tumor stem cells while maintaining the physiological characteristics of the tumor stem cells, and the multidirectional differentiation potential of the tumor stem cells can interfere with the subsequent research on the tumor cells after the tumor stem cells are differentiated into different types of tumor cells.

Disclosure of Invention

The invention provides a method for inhibiting tumor stem cells from differentiating into tumor-derived immune cells.

In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:

a tumor stem cell culture medium comprises a cytokine B27, a basic fibroblast growth factor, a transforming growth factor and an epidermal growth factor.

The tumor stem cell culture medium provided by the invention can ensure that the tumor stem cells can keep the activity of the stem cells and do not grow adherently, thereby being capable of being cultured into tumor balls. The culture medium can also avoid the induction of the differentiation of the tumor stem cells and the interference of the tumor stem cells on the research of the tumor cells after the tumor stem cells are differentiated into different types of tumor cells. When experimenters need to obtain specific cells, the experimenters can obtain expected cell groups by performing directional induction through a specific induction method.

Preferably, the concentration of the cytokine B27 in the tumor stem cell culture medium is 1.8-2.2% wt; the concentration of the basic fibroblast growth factor is 18-22 ng/mL; the concentration of the transforming growth factor is 18-22 ng/mL; the concentration of the epidermal growth factor is 18-22 ng/mL. At the above concentrations, each cytokine is better able to keep the activity of tumor stem cells and avoid their differentiation.

Preferably, the tumor stem cell culture medium further comprises a double antibody, and the concentration of the double antibody in the serum-free culture medium is 0.1-1.0% v/v.

The embodiment of the invention also provides a method for sorting the tumor stem cells, which comprises the following steps:

step a, taking tumor cells in logarithmic growth phase, digesting the tumor cells by pancreatin, and adding a culture medium to prepare single cell suspension;

b, uniformly adding the single cell suspension into a micro raft chip, and culturing until the tumor cells adhere to the wall;

step c, taking a micro raft only containing single cells, placing the micro raft into a cell culture plate with a culture medium added in advance, enabling each culture hole of the cell culture plate to contain one micro raft, and continuing to culture until the tumor cells in the micro rafts form a full clone shape;

and d, taking the tumor cells with the full clone morphological characteristics formed in the step c, transferring the tumor cells into the tumor stem cell culture medium, and culturing to obtain the tumor stem cell suspension.

The micro raft chips (micro array) of the present invention are a series of microwells capable of providing cell sorting, cell isolation, long-term cell analysis, and clonal population propagation, each micro raft (micro) capable of holding a small number of individual cells. The chip can simultaneously carry out a large amount of tumor stem cell sorting work, so that the sorting operation of the tumor stem cells is simpler and more convenient, the balling time can be obviously shortened, the cost is lower, only one cell can be contained in the micro raft, the interference of other cells can be eliminated, and the sorting is more accurate.

Specifically, the culture medium in the step a and the step c is RMPI1640 culture medium. The medium can be adapted to the growth of a variety of cells, including tumor cells.

Preferably, the RMPI1640 culture medium further comprises 5-10% v/v fetal bovine serum.

Preferably, the RMPI1640 culture medium further comprises 0.1-1% v/v double antibody.

The specific operation of taking the micro-rafts containing only single cells in the step c is as follows: the micro rafts containing the single cells were punctured and released with a release needle and then aspirated with a magnetic rod.

The embodiment of the invention also provides a method for inhibiting the differentiation of tumor stem cells into tumor-derived immune cells, which is characterized in that at least one of cytokines IL-2, IL-3, TGF β or IL-6 is added into the tumor stem cell suspension obtained by the sorting method of claim 4, wherein the tumor-derived immune cells are cells which are obtained by the differentiation of the tumor stem cells, have the characteristics of immune cells and can enable the tumor cells to generate an immune escape mechanism.

Drawings

FIG. 1A 549 cell morphology routinely cultured in accordance with embodiments of the invention;

FIG. 2 STR typing test of DNA of A549 cells in example of the present invention;

FIG. 3A 549 cells by immunofluorescence according to an embodiment of the inventionCD133⁺CSCs；

FIG. 4 is a flow chart of detecting CD133 in A549 in an embodiment of the invention⁺(ii) CSCs content;

FIG. 5 tumor stem cells obtained by Microraft Arrays sorting according to an embodiment of the present invention;

FIG. 6 shows tumor stem cells obtained by immunomagnetic beads according to an embodiment of the present invention;

FIG. 7 shows tumor stem cells obtained by limiting dilution of a 96-well plate according to an embodiment of the present invention;

FIG. 8 shows the number of days required for culturing the tumor stem cells obtained by the immunomagnetic bead method, the 96-well plate limiting dilution method and the Microraft Arrays sorting method until the number of the spheroidised stem cells reaches 100;

FIG. 9 shows the results of spheronization experiments of tumor stem cells obtained by the method of Microraft Arrays sorting, immunomagnetic beads and 96-well plate limiting dilution according to the embodiment of the present invention;

FIG. 10 shows the measurement of the CD133 molecular expression of the tumor stem cells obtained by the Microraft Arrays sorting method, the immunomagnetic bead method and the 96-well plate limiting dilution method according to the embodiment of the present invention by immunofluorescence;

FIG. 11 is a flow cytometry method for measuring the expression level of CD133 molecules of tumor stem cells obtained by a Microraft Arrays sorting method, an immunomagnetic bead method and a 96-well plate limiting dilution method according to an embodiment of the present invention;

FIG. 12 is a comparison of the expression levels of CD133 molecules in tumor stem cells obtained by the method of Microraft Arrays sorting, immunomagnetic beads, and limiting dilution with 96-well plate according to an embodiment of the present invention;

FIG. 13 shows that Western Blot detects the expression of the transcription factors associated with A549 cells and tumor stem cells obtained by the Microraft Arrays sorting method in the embodiment of the present invention;

FIG. 14 shows the holoclonal morphology and paraclonal morphology that can be formed by single cells obtained by the Microraft Arrays sorting method according to an embodiment of the present invention;

FIG. 15 is a diagram showing the PCR Array of the present invention detecting the gene expression of 84 surface marker molecules of two types of cells with clonal morphology;

FIG. 16 shows the first 20 genes with the highest expression level of the whole clone morphology cells obtained by the Microraft Arrays sorting method in the embodiment of the present invention on the gene chip;

FIG. 17 shows the results of CD2 in qRT-PCR validation of two clonotype cells obtained by the Microraft Arrays sorting method according to the example of the present invention;

FIG. 18 PCR product specificity of agarose gel electrophoresis of two clonotype cells obtained by the Microraft Arrays sorting method of the example of the invention;

FIG. 19 sequencing of the PCR product CD2 from two clonal morphology cells obtained by the Microraft Arrays sorting method according to an embodiment of the present invention;

FIG. 20 shows flow cytometry analysis of CD2 expression in two clonotype cells obtained by Microraft Arrays sorting;

FIG. 21 Effect of different cytokine stimulation on the gene expression of CD2 molecules from cells obtained by the Microraft Arrays sorting method according to examples of the present invention;

FIG. 22 shows the expression positions of CD2 and CD133 molecules in cells obtained by the method of Microraft Arrays sorting according to the immunofluorescence technique of the present invention;

FIG. 23 shows the gene expression of CD4, CD8A and CD8B in two types of clonal morphology cells obtained by the Microraft Arrays sorting method of the example of the present invention;

FIG. 24 shows the gene expression of the specific transcription factors ROR γ t and IFR4 in two types of clonal morphology cells obtained by the Microraft Arrays sorting method according to the example of the present invention;

FIG. 25 shows the gene expression profiles of cytokines IL-17A and IL-23 in two types of clonotype cells obtained by the Microraft Arrays sorting method according to the embodiment of the present invention;

FIG. 26 expression of four chains of CD3 from two clonal morphology cells obtained by the Microraft Arrays sorting method according to an embodiment of the present invention;

FIG. 27 shows the ROR γ t, IL-17A and IL-23 gene expression profiles in cells derived from normal immune cells and tumor stem cells obtained by the Microraft Arrays sorting method according to the example of the present invention after induction with TFG β and IL-6;

FIG. 28 shows the IL-17A content in the supernatant of two clonal morphology cells obtained by the Microraft Arrays sorting method of the present invention after 12h, 24h, 36h, and 48h of culture;

FIG. 29 shows the IL-23 content in the supernatant of two clonotype cells obtained by the Microraft Arrays sorting method of the present invention at 12h, 24h, 36h and 48 h.

FIG. 30 Medium vs. tumor stem cells in comparative Medium 1 Sox2, Nanog, Oct-4 expression;

FIG. 31 medium vs. tumor stem cells in comparative medium 2 Sox2, Nanog, Oct-4 expression;

FIG. 32 Medium vs. tumor stem cells in comparative Medium 3 Sox2, Nanog, Oct-4 expression;

FIG. 33 expression of Sox2, Nanog, Oct-4 in tumor stem cells in the medium provided in example 2.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1

This example provides a tumor stem cell culture medium comprising 1.8% v/v cytokine B27, 18ng/mL basic fibroblast growth factor, 18ng/mL transforming growth factor, and 18ng/mL epidermal growth factor.

Example 2

This example provides a tumor stem cell culture medium comprising 2.0% v/v cytokine B27, 20ng/mL basic fibroblast growth factor, 20ng/mL transforming growth factor, and 20ng/mL epidermal growth factor.

Example 3

This example provides a tumor stem cell culture medium comprising 2.2% v/v cytokine B27, 22ng/mL basic fibroblast growth factor, 22ng/mL transforming growth factor, and 22ng/mL epidermal growth factor.

Example 4

This example provides a tumor stem cell culture medium comprising 2.0% v/v cytokine B27, 20ng/mL basic fibroblast growth factor, 20ng/mL transforming growth factor, 20ng/mL epidermal growth factor, and 0.1% v/v diabody.

Example 5

This example provides a tumor stem cell culture medium comprising 2.0% v/v cytokine B27, 20ng/mL basic fibroblast growth factor, 20ng/mL transforming growth factor, 20ng/mL epidermal growth factor, and 1.0% v/v diabody.

Example 6

This example provides a method for sorting tumor stem cells, and the obtained tumor stem cells are analyzed and tested. Wherein the tumor cells are from non-small cell lung cancer cell line A549 cells. The specific operation steps are as follows:

1. a549 cells were cultured routinely and when the cells were grown to 80% confluence, i.e., in log phase growth, the cells in log phase growth were collected for the following experiments.

The morphology of the normally growing A549 cells is shown in FIG. 1, and the cells are epithelioid polygonal and grow adherent to the single layer, and the state is good.

1.1STR genotyping for cell identification

Collecting the A549 cells in logarithmic growth phase, digesting with pancreatin, and collecting 1 × 10⁶Extracting cell DNA, carrying out composite PCR amplification by adopting a 21 identity identification system PCR amplification kit (Microreader 21ID system), and detecting STR loci and sex genes Amelogenin on an ABI3730xl type genetic analyzer. The typing results are shown in fig. 2, the DNA of the A549 cells in the logarithmic growth phase is subjected to PCR amplification, the map is clear, the typing results are good, three-allele genes do not appear, namely, the cross contamination of the cells does not appear, and hybrid immune cells or other cells do not appear. And the typing can be completely matched by comparison of ATCC and DSMZ databases.

1.2 immunofluorescence method for detecting expression of tumor stem cell marker molecule CD133

Collecting the A549 cells in logarithmic growth phase, digesting with pancreatin to obtain single cell suspension, adjusting cell concentration to 5 × 10⁴ Inoculating 1 mL/well into 24-well plate, after cell growth reaches 80% fusion, staining and sealing by conventional method, observing under laser confocal microscope, and observing CD133 moleculeThe results are shown in fig. 3, and it can be seen that a small number of cells express CD133 molecules, i.e., a few tumor stem cells are present in a549 cells in logarithmic growth phase.

1.3 flow cytometry quantitative determination of tumor Stem cell content

Collecting the A549 cells of the logarithmic growth phase, digesting with pancreatin to obtain single cell suspension, centrifuging, resuspending with RMPI1640 culture medium, adjusting cell concentration, collecting 1 × 10⁶Cells, add 1 × PBS 2mL, centrifuge, resuspend, repeat this step 2 times. Discarding supernatant, adding 10 μ L of CD133 antibody and isotype antibody, mixing, incubating at 4 deg.C for 10 min, repeating PBS washing step, detecting with BD flow detector, and finding that 1.24% of A549 cells are CD133⁺A tumor stem cell.

And (4) conclusion: the A549 cells in the logarithmic growth phase have no intercellular cross contamination and contain a very small amount of tumor stem cells.

2. Sorting tumor stem cells

2.1 sorting tumor Stem cells by Microraft Arrays sorting

5000A 549 cells in the logarithmic growth phase are taken and resuspended in 2mL of culture medium (10% v/v of serum and 1% v/v of double antibody are added into RMPI1640 culture medium) to prepare single cell suspension, and the single cell suspension is uniformly added into the whole array of the MicroraftArrays and then placed in an incubator to be cultured until the cells are settled and attached to the wall. After attachment, microscopically, the Microraft containing the single cells was punctured and released with a release needle and aspirated with a magnetic rod. A96-well plate containing 200. mu.L of medium per well (RMPI1640 medium with 10% v/v serum and 1% v/v diabody) was placed on a magnetic collection plate with one well aligned to the center of the plate. A magnetic rod with micro raft is inserted into the hole to transfer the released micro raft. This procedure was repeated until each well of the 96-well plate contained a single cell in each of the microwft wells. The results are shown in FIG. 5, where there is a single Microraft and a single cell per Microraft in a 96-well plate culture well.

2.2 sorting of tumor Stem cells by Immunomagnetic bead sorting (MACS)

Collecting the A549 cells in logarithmic growth phase, and pancreatin eliminatingCentrifuging to form single cell suspension, resuspending with 1 × PBS, centrifuging repeatedly for 3 times, and counting cells every 10 times⁷Each cell was resuspended in 60. mu.L buffer, and 20. mu.L of FcR Blocking Reagent and 20. mu.L of CD133 beads were added, incubated at 4 ℃ for 15 minutes, centrifuged with 2mL of buffer, and finally resuspended in 500. mu.L of buffer. The labeled cells were collected by sorting with a pretreated LS column. Cultured in the same serum-free medium as 2.1. The results are shown in FIG. 6, where freshly sorted tumor stem cells grew as single suspensions.

2.3 sorting tumor Stem cells by limiting dilution method with 96-well plate

Collecting A549 cells of the above logarithmic growth phase, digesting with pancreatin to obtain single cell suspension, centrifuging, resuspending with culture medium (RMPI1640 with 10% v/v serum and 1% v/v double antibody), counting cells, and collecting 1 × 10⁶The cells were resuspended in 10mL of this medium and diluted to 10 by dilution in multiples²Per 10mL, 100. mu.L/well in 96-well plates, and recording single-cell wells, eliminating multiple cells and cell-free wells, routinely cultured in incubators. As a result, as shown in FIG. 7, only a single cell was present in the wells of the 96-well plate.

3. Tumor stem cell analysis

3.1 comparing the tumor Stem cell Balling test time obtained by the three methods

The tumor stem cells obtained by the three methods of 2.1-2.3 are expanded and cultured in a serum-free culture medium (containing 2% v/v cytokine B27, 1% v/v double antibody, 20ng/mL basic fibroblast growth factor, 20ng/mL transforming growth factor and 20ng/mL epidermal growth factor), and when the number of the cells grown from a single cell reaches 100, the days are recorded, and as shown in FIG. 8, the balling time of the tumor stem cells obtained by the Microrafys sorting method is obviously shorter than that of the other two methods.

The tumor cell is round or elliptical, the cell combination is tight, the cell is full, the refractivity is strong, and the shape is more regular as shown in figure 9.

3.2 immunofluorescence method to compare the expression of the tumor stem cell CD133 obtained by the three methods

The experimental procedure is as in 1.2. The results are shown in FIG. 10, and the expression of CD133 of the tumor stem cells obtained by the three methods is not obviously different.

3.3 flow cytometry detection of expression of the tumor Stem cell surface molecular marker CD133 obtained by three methods

The experimental procedure is as in 1.3. As shown in FIGS. 11 and 12, the expression level of CD133 in the tumor stem cells obtained by the Microraft Arrays sorting method was higher than that of the tumor stem cells obtained by the other two methods.

3.4 Western Blot method (Western Blot) for detecting the expression of stem cell transcription factor in tumor stem cells obtained by Microraft Arrays sorting method

The A549 cells are used as a control to detect the expression of three proteins, namely Nanog, Oct-4 and Sox2 in the tumor stem cells obtained by the Microraft Arrays sorting method. The results are shown in fig. 13, and the expression level of the transcription factors of the three stem cells is obviously higher than that of the A549 adherent cells.

3.5 clonal morphological differences between Stem and non-Stem cells

As shown in FIG. 14, the single cells obtained by the Microraft Arrays method can form both full clone and paraclone clone morphologies. The full clonal morphology is the clonal morphological feature of stem cells, while the paraclonal morphology is the clonal morphological feature of differentiated non-stem cells.

As can be seen from FIG. 14, the cell colony in the fully cloned form has large volume, smooth boundary, compact structure, small cell volume and fast growth, and the cells grow in a nested shape. The paracloning morphological cell community has small volume, extremely irregular boundary and low cell proliferation activity. The difference between the two is obvious.

Example 7

This example provides a method for inhibiting the differentiation of tumor stem cells into tumor-derived immune cells, wherein the tumor stem cells used are two clonal morphology cells (i.e., stem cell population and non-stem cell population) obtained by the Microraft Arrays sorting method.

1. Immune characteristic analysis of tumor stem cells

1.1 Gene analysis

Two kinds of clone morphological cells (namely stem cell colony and non-stem cell colony) obtained by a Microraft Arrays sorting method are respectively taken) Each 1X 10⁶The gene expression levels of 84 cell surface markers were probed by steps of RNA extraction, reverse transcription, and detection by PCR Array (PCR Array) using cycle threshold (CT value) as an index. The results are shown in FIG. 15.

The PCR chip comprises 84 genes of 11 cells in total, namely T cells, B cells, NK cells, mononuclear macrophages, endothelial cells, smooth muscle cells, dendritic cells, fibroblasts, mast cells, epithelial cells and fat cells. After statistical analysis, 7 genes are only expressed in the side clone cells, 5 genes are only expressed in the whole clone cells, 6 genes are not expressed in the two cells, and 66 genes are jointly expressed.

The analysis of the first 20 genes of the 84 genes expressed by the cells in the full clone morphology on the gene chip revealed that 75% of the genes were immune system genes and were mainly T, B cells, as shown in FIG. 16.

1.2 Gene level verification of expression of the immune cell specific marker molecule CD2

CD2, also known as lymphocyte function-associated antigen 2 or sheep red blood cell receptor, is expressed on the surface of all T cells, binds to APC surface CD58, enhances intercellular adhesion, and directly mediates T cell activation.

After RNA extraction and reverse transcription are carried out on two clone morphological cells obtained by the Microraft Arrays sorting method, a real-time fluorescence quantification method is used, GAPDH is used as an internal reference, and the △△ CT method is used for comparing the expression difference of CD2 genes of two groups of cells, so that the result is shown in figure 17, the expression of a full-clone CD2 gene is obviously higher than that of a side-clone, the peak type of a dissolution curve is single, the specific amplification product is obtained, and no primer dimer is formed.

1.3 agarose gel electrophoresis to verify the specificity of the PCR products and sequencing

2g of agarose powder was dissolved in 100mL of 0.5 XTBE solution, and 7. mu.L of GelRedTM Nucleic acid gel Stain was added to prepare a 2% gel, which was electrophoresed at a constant pressure of 160V for 30 minutes. AzureSpot C500 acquires images. As a result, as shown in FIG. 18, the left side is 50bp DNA Ladder, the right side is listed as two columns as PCR product CD2, the size position of the target fragment is 161bp, and the target fragment is a specific CD2 target fragment.

In addition, capillary electrophoresis and dideoxy chain termination methods were used to sequence the PCR products using an ABI3730xl sequencer. The results are shown in FIG. 19, where the visible signal pattern is clear, no distinct double peaks appear, and the pattern is completely matched with the CD2 gene sequence.

1.4 flow cytometry detection of CD2 molecular expression

Two kinds of clone morphological cells obtained by the Microraft Arrays sorting method are respectively taken and are respectively 1 multiplied by 10⁶Flow cytometry was performed according to the above-mentioned conventional method. The results are shown in FIG. 20, in which CD2 was observed in the whole clone cells⁺The cell number was significantly higher than in the paracloning group.

1.5 immunofluorescence technique to observe the expression positions of CD2 and CD133 molecules

The whole cell clone obtained by the Microraft Arrays sorting method was subjected to immunofluorescence staining as described in 1.2 of example 6. The results of confocal laser scanning microscopic observation are shown in FIG. 22, and the four micrographs show, from left to right, the cell nucleus, the expression position of CD2 molecule, the expression position of CD133 molecule, and the co-expression of CD2 and CD133 molecule. As can be seen in FIG. 22, the tumor stem cells obtained by the Microraft Arrays sorting method expressed both the stem cell marker CD133 (red fluorescence) and the T cell marker CD2 (green fluorescence). From a more intuitive morphological perspective, it was demonstrated that tumor stem cells could differentiate into immune cells.

2. Inhibiting differentiation of tumor stem cells into tumor-derived immune cells

2.1 inhibiting differentiation of tumor Stem cells into tumor-derived immunocytes by use of IL-2 and IL-3

Taking the cells of the whole clone morphology obtained by the Microraft Arrays sorting method, adjusting the cell density to 1.5 × 10⁶The cells were subcultured in 10cm dishes at a rate of 10 mL/dish, and were divided into a control group, an IL-2 group and an IL-3 group, each group being 3 replicates. When the fusion degree reaches 70%, the culture medium is replaced by serum-free RMPI1640 basic culture medium, and starvation treatment is carried out for 6 hours. Adding 10 μ L of 0.1% BSA into the control group as control, adding IL-2 or IL-3 into IL-2 group and IL-3 group at a ratio of 10ng/mL, culturing for 24 hr, removing culture medium, collecting cells, extracting RNA by TRIZOL, reverse transcribing, and real-time fluorescenceThe results of comparing the differences in CD2 gene expression between the two groups of cells by △△ CT method using GAPDH as an internal reference are shown in FIG. 21, in which the CD2 gene was significantly reduced after IL-2 and IL-3 groups were induced by IL-2 and IL-3, compared with the control group cultured in the medium without the addition of the above-mentioned cytokine.

2.2 selecting two clone morphological cells obtained by Microraft Arrays sorting method, extracting RNA, reverse transcribing, using GAPDH as internal reference, comparing the relative gene expression difference of two groups of cell immune cells by △△ CT or △ CT method, the result is shown in figure 22, 23, 24, 27 and 28.

As shown in FIG. 23, CD4 in the Whole clone cell group⁺The expression quantity of the T cells is higher than that of the paraclone cell group, and the difference of CD8A is obvious, compared with a △ CT method after internal reference gene standardization, CD8A is expressed in the two groups but has no obvious difference, and CD8B is shown to be infinite, namely is not expressed.

As shown in FIG. 24, the genes of the specific transcription factors ROR γ t and IFR4 were expressed in both groups of cells, and the whole clone group was significantly higher than the side clone group.

As shown in FIG. 25, the genes for cytokines IL-17A and IL-23 were expressed in both groups of cells, and the whole clonal cell group was significantly higher than the paraclonal cell group.

As shown in FIG. 26, by examining the expression of four chains of the T cell receptor complex forming molecule CD3 in two groups of cells, it was found that CD3, which is indispensable for the function of the TCR complex, appeared to be infinite, i.e., not expressed in △ CT of the three genes by-cloned, whereas the complete clone group also expressed only the gene of one peptide chain of CD 3G.

After tumor stem cells obtained by a Microraft Arrays method are induced by TFG β and IL-6 (cytokines promoting Th17 cell production), compared with the result of culture in a culture medium without the cytokines, the ROR gamma t, IL-17A and IL-23 gene expression in the cells is reduced as shown in figure 27, the ROR gamma t, IL-17A and IL-23 are genes related to Th17 cells, and after the tumor stem cells are induced by TFG β and IL-6, the expression of the three genes is obviously reduced compared with the culture in the culture medium without the cytokines, which shows that the tumor stem cells can be inhibited from differentiating into tumor-derived immune cells after the tumor stem cells are induced by TFG β and IL-6.

Collecting culture supernatants of two clone morphology cells at different time, performing enzyme-linked immunosorbent assay (ELLISA) according to the kit, and detecting IL-17 and IL-23 secretion content of cells, with the results shown in FIGS. 26 and 27.

As shown in FIG. 28, the expression level of IL-17A was highest after 36h of culture, and the whole clonal cell group was significantly higher than the paraclonal cell group at 24h and 36 h.

As shown in FIG. 29, the IL-23 secretion was detected by ELLISA method in the two groups of cells cultured for 12h, 24h, 36h and 48h, but there was no significant difference in secretion amount at different time points.

As can be seen from the analysis of FIGS. 23-29, the tumor stem cells isolated from the Microraft Arrays can differentiate into tumor-derived immunocytes, which do not completely express the relevant genes expressed by the normal immunocytes of the body.

Media to ratio

This comparative example provides three tumor stem cell culture media, each of which consists of:

culture medium 1: 20ng/mL basic fibroblast growth factor, 20ng/mL transforming growth factor and 20ng/mL epidermal growth factor;

culture medium 2: 2.0% v/v cytokine B27, 20ng/mL basic fibroblast growth factor and 20ng/mL epidermal growth factor;

culture medium 3: 2.0% v/v cytokine B27, 20ng/mL basic fibroblast growth factor, 20ng/mL transforming growth factor, 20ng/mL epidermal growth factor and 500ng/mL insulin.

As shown in FIGS. 30 to 33, the results of culturing the tumor stem cells obtained by the Microraft Arrays sorting method using the above-mentioned

media

1, 2 and 3 and the medium provided in example 2 show that the expression levels of the three proteins (Sox2, Nanog and Oct-4) maintaining the stem cell dryness in each medium are different, and as can be seen from FIG. 33, the expression level in the medium provided in example 2 is almost 100% (red fluorescence) and is significantly higher than that in the other media.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A method for inhibiting tumor stem cell differentiation into tumor-derived immune cells is characterized in that at least one of cytokines IL-2, IL-3, TGF β or IL-6 is added into the tumor stem cell suspension obtained by sorting;

the sorting method of the tumor stem cells specifically comprises the following steps:

d, taking the tumor cells with the full clone morphological characteristics formed in the step c, transferring the tumor cells into a tumor stem cell culture medium, and culturing to obtain a tumor stem cell suspension; wherein the growth factors in the tumor stem cell culture medium only contain cytokine B27, basic fibroblast growth factor, transforming growth factor and epidermal growth factor; the volume percentage concentration of the cytokine B27 in the tumor stem cell culture medium is 1.8-2.2%; the concentration of the basic fibroblast growth factor is 18-22 ng/mL; the concentration of the transforming growth factor is 18-22 ng/mL; the concentration of the epidermal growth factor is 18-22 ng/mL.

2. The method for inhibiting the differentiation of tumor stem cells into tumor-derived immune cells according to claim 1, wherein the culture medium in step a and/or step c is RMPI1640 culture medium.

3. The method for inhibiting the differentiation of tumor stem cells into tumor-derived immune cells according to claim 2, wherein the RMPI1640 medium further comprises 5-10% v/v fetal bovine serum.

4. The method for inhibiting the differentiation of tumor stem cells into tumor-derived immune cells according to claim 3, wherein the RMPI1640 medium further comprises 0.1-1% v/v double antibody.

5. The method for inhibiting the differentiation of tumor stem cells into tumor-derived immune cells as claimed in claim 1, wherein the step c of taking the rafts containing only single cells comprises the following steps: the micro rafts containing the single cells were punctured and released with a release needle and then aspirated with a magnetic rod.

6. The method for inhibiting the differentiation of tumor stem cells into tumor-derived immune cells according to claim 1, wherein said tumor stem cell culture medium further comprises a diabody, and the concentration of said diabody in said culture medium is 0.1-1.0% v/v.