CN117805376B - Application of CD44 and Lgr5 as markers in screening gastric cancer tumor stem cells - Google Patents

Abstract

The invention belongs to the field of cell biology, and particularly relates to application of CD44 and Lgr5 serving as markers in screening of tumor stem cells. Experiments show that the sorting purity of the CD44+Lgr5-single positive mark subgroup and the CD44+Lgr5+ cell double positive mark subgroup is more than 90%, so that the two sorting antibodies have excellent sorting accuracy, the continuous culture state of cells of different mark subgroups is good, the cell damage in the sorting process is small, the cell integrity is good, and a feasibility foundation is provided for subsequent experimental research.

Description

Application of CD44 and Lgr5 as markers in screening gastric cancer tumor stem cells

Technical Field

The invention belongs to the field of cell biology, and particularly relates to application of CD44 and Lgr5 serving as markers in screening of tumor stem cells.

Background

Gastric cancer is the most common malignant tumor worldwide, about 95 ten thousand patients with gastric cancer are newly increased every year worldwide, and the fourth place of all tumor types is occupied; at the same time, gastric cancer patients die about 72 tens of thousands of people annually, locating the second place of all tumor types. Gastric cancer is a serious disease which seriously threatens the health of the people in China, and meanwhile, the disease burden brought to the whole society is gradually increased. With the rapid development of the current society economy, the quality of life of people is continuously improved, the knowledge of diseases is continuously deepened, and the demands for life health are also increased day by day. In recent decades, the overall prognosis of gastric cancer patients has improved, but admittedly the overall survival rate of gastric cancer remains wandering at lower levels worldwide, thanks to the increase in medical levels and the abundance of therapeutic means. This can be mainly analyzed from two aspects of disease diagnosis timing and therapeutic effect: 1. because the early detection rate of gastric cancer is very low, most gastric cancer patients are already in the advanced stage or the late stage when diagnosis is confirmed, tumors are mostly accompanied by local infiltration and lymph node metastasis, and the optimal treatment time window is missed; 2. gastric cancer has heterogeneity difference, and a single treatment mode or a single certain drug cannot achieve the aim of curing gastric cancer. Therefore, the treatment strategy of the gastric cancer at present is a comprehensive treatment mode taking multidisciplinary synergy as a framework, namely: in combination with surgery and chemotherapy (neo) adjuvant therapy, targeted therapy or immunotherapy can be performed in an individualized way according to the patient's condition. However, in reality, chemotherapy with strong specificity and good pertinence and poor targeted drugs are bottleneck problems limiting gastric cancer treatment.

The theory system of the 'tumor stem cells' is rich and perfect, and the deep knowledge of researchers on tumors is continuously improved. Tumor stem cells are considered to be "a small fraction of cells that have sustained self-renewal capacity and are capable of differentiating into differentiated tumor cells". The nature of such cells may be referred to simply as the "stem nature" of the tumour stem cells. Among them, gastric cancer tumor stem cells (GASTRIC CANCER STEM CELLS, GCSCs) are considered as the main culprit for the occurrence, development and metastasis of gastric cancer. The gastric cancer stem cells may be ^[6] transformed from normal gastric mucosal epithelial cells through a series of gene changes and environmental influences, or may be transformed from bone marrow-derived mesenchymal stem cells. GCSCs can generate offspring stem cells and differentiated cells with different genetic marks through asymmetric division, so that heterogeneity of gastric cancer is generated, and stronger proliferation and differentiation capability is maintained. At present, independent chemotherapy or targeted therapy can only play a role in killing a part of more sensitive gastric cancer cells, but the more immature and undifferentiated gastric cancer stem cells can still survive away from drug therapy, and re-proliferate and differentiate again under a certain condition, so that the drug resistance, recurrence and metastasis of gastric cancer are caused.

Therefore, in order to improve the clinical efficacy of gastric cancer, it is necessary to further explore the origin of gastric cancer tumor and the role of gastric cancer stem cells in the occurrence and development of tumor, and to explore the effective mechanism affecting GCSCs stem cell characteristics, to explore the key targets therein and to perform effective targeted intervention.

Disclosure of Invention

In view of the above problems, it is an object of the present invention to provide an application of CD44 and Lgr5 as markers in screening gastric cancer tumor stem cells.

In order to achieve the above purpose, the present invention may adopt the following technical scheme:

in one aspect, the invention provides an application of CD44 and Lgr5 as markers in screening gastric cancer tumor stem cells.

Further, the screening in the above application includes screening using a flow-sorting method in which anti-human Lgr5 mAb (Biolenged) is a flow-sorting-specific antibody of Lgr5 and APC-conjugated mouse anti-human CD44 mAb (Biolenged) is a flow-sorting-specific antibody of CD 44.

Further, the application comprises screening gastric cancer tumor stem cells from primary gastric cancer tissue cells.

Further, the application comprises screening gastric cancer tumor stem cells from an intestinal gastric cancer cell line HGC-27.

The invention also provides an application of the gastric cancer tumor stem cells screened by using the CD44 and the Lgr5 as markers in preparing medicines for treating gastric cancer.

Further, the application comprises the application of taking the gastric cancer tumor stem cells screened by taking CD44 and Lgr5 as markers as targets in preparing drugs for treating gastric cancer with low tolerance.

Further, the application comprises the application of taking gastric cancer tumor stem cells screened by taking CD44 and Lgr5 as markers as targets in preparing medicines for reducing the expression of Ki67 and Cyclin D1 proteins.

The beneficial effects of the invention at least comprise:

(1) The invention successfully separates two groups of sub-gastric cancer stem cells of CD44+Lgr5+ double positive markers and CD44+Lgr5-single positive markers from primary gastric cancer cells and gastric cancer cell line HGC-27 by a flow separation technology, and the two groups of sub-gastric cancer stem cells have good growth states and good purity, thereby proving that the flow separation technology is a feasible method for separating and enriching gastric cancer stem cells.

(2) The detection of a cell proliferation experiment, a suspension sphere formation experiment and the like proves that the CD44+Lgr5+ double-positive subgroup gastric cancer stem cells have stronger proliferation capacity and suspension sphere forming capacity than the CD44+Lgr5-single-positive subgroup, and reflects the 'dry' characteristic that the CD44+Lgr5+ double-positive marked gastric cancer stem cell subgroup has better self-renewal capacity.

(3) The invention determines the high expression phenomenon of OCT4, SOX2 and Nanog key stem transcription factors of CD44+Lgr5+double-positive subgroup gastric cancer stem cells compared with CD44+Lgr5-single positive subgroup at protein level and mRNA level, and further determines that the CD44+Lgr5+double-positive marked gastric cancer stem cell subgroup has stronger stem cell transcription activity.

(4) According to the invention, clinical common gastric cancer chemotherapeutics such as oxaliplatin, taxol, fluorouracil and the like with different concentrations are used for carrying out drug resistance intervention experiments on gastric cancer stem cells of CD44+Lgr5+ double positive and CD44+Lgr5-single positive sub-population, and the results show that CD44+Lgr5+ double positive sub-population cells have stronger drug resistance than CD44+Lgr5-single positive sub-population cells, and the drug resistance capability is weakened along with the reduction of the Lgr5 protein expression content, so that the Lgr5+ marked gastric cancer stem cells are one of possible reasons for resisting or resisting drugs in anti-tumor treatment.

(5) In the invention, different marked subgroup gastric cancer stem cells have different changes in protein level and mRNA level expression under different culture environments of suspension and adherence, and a definite result shows that the sorted subgroup gastric cancer stem cells have epithelial-mesenchymal transition capability, and the Lgr5+ marked gastric cancer stem cells can promote and strengthen the transition process.

(6) According to the invention, through in vivo tumor cell transplantation experiments and Transwell experiments, the CD44+Lgr5+ double-positive labeling subgroup stem cells are found to have higher in vivo tumor forming speed in nude mice, the expression of proliferation related proteins Ki67 and Cyclin D1 is up-regulated, and the number of cells penetrating through a small chamber is increased, so that the CD44+Lgr5+ double-positive subgroup cells have stronger in vivo tumor forming capacity, proliferation capacity and migration invasion capacity.

(7) Experiments show that the sorting purity of the CD44+Lgr5-single positive mark subgroup and the CD44+Lgr5+ cell double positive mark subgroup is more than 90%, so that the two sorting antibodies have excellent sorting accuracy, the continuous culture state of cells of different mark subgroups is good, the cell damage in the sorting process is small, the cell integrity is good, and a feasibility foundation is provided for subsequent experimental research.

Drawings

FIG. 1 is a diagram of the culture of gastric cancer cell line HGC-27 (scale length 50 μm);

FIG. 2 is a diagram of primary gastric cancer cell culture (scale length 50 μm);

FIG. 3 is a statistical plot of the sorting and enrichment of HGC-27 gastric cancer cell CD44+Lgr5-and CD44+Lgr5+ cell subsets;

FIG. 4 is a statistical plot of the sorting and enrichment of primary gastric cancer cell CD44+Lgr5-versus CD44+Lgr5+ cell subsets;

FIG. 5 is a graph of cell culture after sorting and enrichment of gastric cancer cell line HGC-27 (scale length 50 μm);

FIG. 6 is a graph of cell culture after primary gastric cancer cell sorting (scale length 50 μm);

FIG. 7 is a statistical chart of OD values of stem cell proliferation experiments of sorted subpopulations of gastric cancer;

FIG. 8 is a view showing the effect of secondary balling of gastric cancer stem cell suspension culture (scale length 50 μm);

FIG. 9 is a statistical chart of gastric cancer stem cell suspension culture secondary spherulitic cell count;

FIG. 10 is a chart showing the diameter statistics of gastric cancer stem cell suspension culture secondary spherulites;

FIG. 11 shows the detection of the expression of the cell stem transcription factor proteins of the sorted subpopulations by the WesternBlot test method;

FIG. 12 is a quantitative analysis and statistics of the expression of the cell stem transcription factor OCT4 protein of the sorting subpopulation;

FIG. 13 is a quantitative analysis and statistics of the expression of the cell stem transcription factor SOX2 protein of the sorting subpopulation;

FIG. 14 is a quantitative analysis and statistics of the expression of the sorted subset cell stem transcription factor Nanog protein;

FIG. 15 is a quantitative PCR assay to detect CD44+Lgr5+ dry transcription factor OCT4 expression;

FIG. 16 is a quantitative PCR assay for detecting CD44+Lgr5+ dry transcription factor SOX2 expression;

FIG. 17 is a quantitative PCR assay for detecting CD44+Lgr5+ dry transcription factor Nanog expression;

FIG. 18 shows the results of oxaliplatin (Oxaliplation) chemotherapy drug resistance experiments;

FIG. 19 shows the results of paclitaxel (Taxol) chemotherapy drug resistance experiments;

FIG. 20 shows the results of fluorouracil (5-Fu) chemotherapy drug resistance experiments;

FIG. 21 is a graph showing statistics of inhibition rate of oxaliplatin (Oxaliplation) on gastric cancer stem cells of a separate subpopulation;

FIG. 22 is a graph showing statistics of inhibition rate of paclitaxel (Taxol) on gastric cancer stem cells of the isolated subpopulation;

FIG. 23 is a graph showing the statistics of inhibition rate of fluorouracil (5-Fu) on gastric cancer stem cells of the subset;

FIG. 24 is a graph showing tumor-burden volume change in living mice;

FIG. 25 is a schematic representation of tumor-bearing mice;

FIG. 26 is a schematic representation of an isolated tumor (scale length 10 mm);

FIG. 27 is an in vitro tumor weight statistic;

FIG. 28 shows the body weight change of the living mice;

FIG. 29 shows Ki67 proliferation-associated protein expression (scale length 50 μm);

FIG. 30 is a statistical plot of Ki67 staining positive ratios;

FIG. 31 shows the expression of a cyclin D1 proliferation-related protein (scale length 50 μm);

FIG. 32 is a statistical chart of the proportion of staining positives of CyclinD 1;

FIG. 33 shows EMT regulatory factor expression of sorted subpopulations of cells in different culture environments;

FIG. 34 is a quantitative analysis of the expression of the sorting subpopulation E-cadherin protein;

FIG. 35 is a quantitative analysis of N-cadherein protein expression from a sorting subpopulation;

FIG. 36 is a quantitative analysis of sorted subpopulations of Twist protein expression;

FIG. 37 is a quantitative analysis of the expression of the sorted subpopulation of Snail proteins;

FIG. 38 is a quantitative analysis of the sorted subpopulation ZEB1 protein expression;

FIG. 39 is E-cadherein gene expression;

FIG. 40 is N-cadherein gene expression;

FIG. 41 is TWIST gene expression;

FIG. 42 is Snail gene expression;

FIG. 43 shows ZEB1 gene expression;

FIG. 44 is a comparison of cell through-hole status of sorted subpopulations;

FIG. 45 is a graph showing statistics of through-hole cell counts for Transwell experiments.

Detailed Description

The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context clearly differs, singular forms of expression include plural forms of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of a feature, number, operation, component, part, element, material, or combination. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, elements, materials or combinations thereof may be present or added. As used herein, "/" may be interpreted as "and" or "as appropriate.

An embodiment of the invention provides an application of CD44 and Lgr5 as markers in screening gastric cancer tumor stem cells.

It should be noted that CD44 is a single gene encoded transmembrane glycoprotein, whose encoding gene is located at 11p13 and is composed of at least 20 highly conserved exons. CD44 is generally considered a glass acid receptor and may act as a guanine nucleotide connecting protein with gtpase activity, so CD44 has a role in cell-to-cell adhesion, cell-to-outer matrix adhesion, and plays a role in cell differentiation, transformation, migration, and the like. CD44 is not expressed in normal gastric mucosa, and the strong expression of CD44 in focus tissues of gastric cancer patients often suggests poor prognosis; the research of CD44 as a gastric cancer tumor stem cell marker starts from Takaishi and the like, takaishi and a team apply a flow cell sorting technology to divide a gastric cancer cell line into two subgroups of CD44+ and CD44-, and prove that CD44+ cells have self-renewal capacity and stronger nude mice tumorigenicity capacity and can also play a role in resisting and resisting chemoradiotherapy. Thus, CD44 has become increasingly accepted as a surface marker. However, since CD44 is also highly expressed in tumor cells derived from other epithelia such as breast cancer stem cells, colorectal cancer, and prostate cancer, the specificity of identifying gastric cancer stem cells by CD44 alone has been questionable.

Lgr5 (Leucine-RICH REPEAT-containing G protein-coupled receptor 5), also known as G protein-coupled receptor 49 (GPR 49), was originally isolated and identified by a university of Stanford, university of America, university of medical college Sheau, et al, 1998. But not until 2007 have begun intensive studies by students. The Dutch scholars Barker et al [15] reported first an Lgr5 protein-related study and proved by experiments that it was a brand new marker present on intestinal mucosal surfaces. Immediately following, lineage follow-up studies further demonstrate that Lgr5 also has a distribution in the gastric mucosa. And the pylorus stem cells of the lgr5+ can be combined with the ligand R-spondin protein to mediate activation and strengthen Wnt signal paths, so that part of regulatory protein accumulation can not be normally degraded, and gastric cancer tumor generation is promoted. Recently, research on the relationship between Lgr5 and tumor has been attracting attention, and studies have revealed that abnormally expressed Lgr5 (which is expressed in most cases in an up-regulated manner) is highly correlated with malignant biological behaviors of tumors such as colorectal cancer, hepatocellular carcinoma, esophageal cancer, and the like.

In certain embodiments, the screening in the above application comprises screening using a flow-sorting method, wherein anti-human Lgr5 mAb (Lgr 5 antibody (Lgr 5-PE)) (Biolenged) is a flow-sorting-specific antibody for Lgr5 and APC-conjugated mouse anti-human CD44 mAb (CD 44 antibody (CD 44-APC)) (Biolenged) is a flow-sorting-specific antibody for CD 44.

In certain embodiments, the above-described uses include screening gastric cancer tumor stem cells from primary gastric cancer tissue cells.

In certain embodiments, the above-described use includes screening gastric cancer tumor stem cells from the intestinal gastric cancer cell line HGC-27.

The invention further provides an application of the gastric cancer tumor stem cells screened by taking CD44 and Lgr5 as markers as targets in preparing medicines for treating gastric cancer.

In certain embodiments, the above-described uses include the use of gastric cancer tumor stem cells screened with CD44 and Lgr5 as markers as targets in the preparation of a drug for treating gastric cancer with low tolerance.

In certain embodiments, the above uses include the use of gastric cancer tumor stem cells screened with CD44 and Lgr5 as markers as targets in the preparation of a medicament for reducing the expression of Ki67 and Cyclin D1 proteins.

For a better understanding of the present invention, the content of the present invention is further elucidated below in connection with the specific examples, but the content of the present invention is not limited to the examples below.

In the following examples, primary gastric cancer cells (PRIMARY GASTRIC CANCER CELL) were saved by the department of general surgical medicine, the general Hospital of the Lesion et al; the gastric cancer cell line HGC-27 is derived from the cell resource center of the national academy of medical science.

In the following examples, the target gene primer for real-time quantitative PCR was designed and then manufactured and synthesized by Life Technologies (China) Biotechnology Co.

1. Sorting preparation of CD44+Lgr5+double-positive marked gastric cancer stem cells

In the following examples, the components and preparation methods of the cell culture fluids used were as follows:

Gastric cancer cell general culture solution: 100mlFBS foetal calf serum solution (Gibco, 10270-106, total volume: 10%) was added to 400mlDMEM cell culture (Gibco, SH30022.01B, total volume: 90%) and mixed well to a total volume of 500ml. When in use, the penicillin/streptomycin double antibiotic solution is added, and the dosage is adjusted according to the total volume of the culture solution. So that the final penicillin concentration is 100U/ml and the final streptomycin concentration is 100 mug/ml.

Special culture solution for gastric cancer stem cells: 2mlB g of serum-free cell culture additive (total volume: 0.4%) was added to 500ml of DMEM/F12 (1:1) liquid medium and thoroughly mixed. When in use, the recombinant human basic fibroblast growth factor (basic fibroblast growth factor, bFGF) and the recombinant human epidermal growth factor (EPIDERMAL GROWTH FACTOR, EGF) are added, and the dosage is calculated and adjusted according to the total volume of the culture solution. So that the final concentration of bFGF was 10ng/ml and EGF was 20ng/ml.

In the following examples, in relative expression of western blotting, each group of data in the experiment is expressed as' x+ -s, the statistical treatment is analyzed by SPSS22.0 software, the comparison between the average values of each test group is t-test, and p < 0.05 has statistical significance.

In the following examples, the target gene and the internal reference of each sample were subjected to real-time PCR in real-time quantitative PCR. The data were analyzed for gene expression by relative quantification using the 2 ^{- △△ CT} method.

Culturing gastric cancer cell lines by the method

(1) Transferring the frozen state to recovery: and (3) clamping a cell freezing tube of a gastric cancer cell line HGC-27 and primary gastric cancer cells from a liquid nitrogen tank at the temperature of minus 190 ℃ by using long flat forceps, rapidly putting the cell freezing tube into a water bath kettle in the environment of 37 ℃ preheated in advance, and slightly shaking the cell freezing tube along a single direction by holding the long flat forceps so as to rapidly melt the cell freezing tube within 1 min. Then, the frozen cell liquid is sucked by using a sterile 10ml suction pipe to be transferred into a T25 cell culture dish after the tube is sterilized by the flame of the alcohol lamp, 7-10ml of cell culture liquid is added, and the cell culture dish is placed on the surface of an ultra-clean workbench to be gently shaken left and right, so that cells are uniformly dispersed.

(2) Culturing and changing liquid: the gastric cancer cell line HGC-27 and primary gastric cancer cells were grown in T25 cell culture dishes. The incubator was set to a constant temperature of 37℃and a concentration of 5% CO ₂.

(3) Sub-packaging: taking out the cell culture dish every 2-3 days, observing the growth state of experimental cells and the distribution density of the bottom of the dish under a microscope, if the confluence rate of the cells is more than 70% under the observation of the microscope, digesting the dispersed cells by using 0.25% trypsin-EDTA digestion solution, and adding the cell culture solution containing fetal bovine serum to be updated. The cell suspension density is properly adjusted according to the counting concentration of the cells and the experimental schedule, and then the cells are split into different cell culture dishes in equal proportion for continuous culture.

As seen by microscopic observation: the gastric cancer cell line HGC-27 and primary gastric cancer cells have good growth state, flat and stretched morphology, good purity, no impurity and no microbial pollution. The comparison document report and the previous research picture record have no abnormal cell interference, meet the cell requirement for experiments, and lay a foundation for the next step of gastric cancer stem cell sorting and enriching experiment. The culture effect is shown in FIGS. 1 and 2. (in the figure, the gastric cancer cell line HGC-27 is abbreviated as HGC-27, and the primary gastric cancer cell is abbreviated as PRIMARY CELL).

(II) sorting and enriching gastric cancer stem cells of different marked subgroups by using a flow cytometer

(1) Washing gastric cancer cell line HGC-27 and primary gastric cancer cells with staining solution for 1-2 times, and centrifuging at low speed and gravity center for 5 min;

(2) Adding humanized cell blocking antibodies into the staining solution according to a proportion;

(3) Resuspending the experimental cells with the staining solution, and incubating at low temperature for 30 min;

(4) Adding CD44 antibody and Lgr5 antibody and control antibody proportionally;

(5) Adding 50 mu l of the staining solution containing the specific antibody prepared in the step (4) into the incubated cells for full suspension;

(6) Washing the cells again with PBS phosphate buffer solution, and collecting the cells by low-speed centrifugation after washing for 3 times;

(7) The gastric cancer cell line HGC-27 to be sorted after specific antibody incubation treatment and primary gastric cancer cells are filtered and collected to a flow analysis tube, and the cell concentration is regulated to be1 multiplied by 10 ⁶/ml; finally, sorting is carried out on a flow cell sorting instrument, and two groups of cell subgroups of different surface molecular markers of the CD44+Lgr5-single positive marker and the CD44+Lgr5+ double positive marker are sorted;

(8) The sorted CD44+Lgr5-single positive marker and CD44+Lgr5+ double positive marker gastric cancer stem cell subgroup are collected and purified, and the purity is measured.

The sorting and enriching results are shown in detail in fig. 3-4, and the results show that: successfully separating and enriching gastric cancer cell lines HGC-27 and primary gastric cancer cells by a flow cell separation method to obtain different subpopulations of gastric cancer stem cells marked by CD44 and Lgr 5; in HGC-27 cells, the proportion of gastric cancer stem cells is 5.82%, the purity of the enrichment of CD44+Lgr5-single positive cell subset is 94.03%, and the purity of the enrichment of CD44+Lgr5+ double positive cell subset is 93.20%; of the primary gastric cancer cells, the proportion of gastric cancer stem cells was 29.08%, with purity enriched in the cd44+lgr5-single positive cell subset being 93.96% and purity enriched in the cd44+lgr5+ double positive cell subset being 93.10%. The purity of the gastric cancer stem cells of the two gastric cancer cell sorting and enriching subpopulations is more than 90%, the purity is higher, and the subsequent cell verification experiment and the functional experiment can be continued. The gastric cancer cell line HGC-27 obtained through enrichment by a flow cell sorting method, CD44+Lgr5-single positive and CD44+Lgr5+ double positive subgroup gastric cancer stem cells of primary gastric cancer cells are continuously cultured, and the growth state of the cells is recorded by photographing under a microscope (the culture of the gastric cancer cell line HGC-27 is shown in figure 5, and the culture of the primary gastric cancer cells is shown in figure 6).

In addition, the following qualitative determinations can be made by analyzing the growth characteristics of the cells: in the gastric cancer cell line HGC-27 and primary gastric cancer cells, on the premise of plating and planting at the same cell count concentration, the CD44+Lgr5-single positive cell subset grows sparsely, while the CD44+Lgr5+ double positive cell subset has relatively dense plating density. This demonstrates that the CD44+Lgr5+ double positive cell subset has a faster rate of division and proliferation.

In addition, among the primary gastric cancer cells, the gastric cancer stem cells of the CD44+Lgr5+ double positive subgroup and the single positive subgroup of the CD44+Lgr5-have certain differences in cell morphological characteristics, and are expressed as follows: the CD44+Lgr5+double positive subgroup cells grow in a multi-layer colony shape, are easy to accumulate into compact clusters, have no obvious cell limit and are shaped like bird nests as a whole.

(III) cell proliferation assay

(1) Collecting gastric cancer cell line HGC-27 in logarithmic growth phase, CD44+Lgr5-single positive and CD44+Lgr5+ double positive subgroup gastric cancer stem cells of primary gastric cancer cells;

(2) Cell density was counted and plated in 96-well plates. The number of cells per well was about 1X 10 ⁵ cells per well. Then, the 96-well plate is placed in a constant temperature incubator for culturing for 12 hours;

(3) After stationary culture for 12 hours, the cell state was observed under a mirror. If the cell growth state is good and all the cells are attached, the cell culture is continued until the total time is 48 hours.

(4) A pipetting gun is used for adhering 10 mu l of a special solution (Cell Counting Kit-8, CCK-8) for a cell viability detection kit to each hole, so that errors caused by the fact that bubbles generated by liquid adding affect absorbance (OD) are avoided.

(5) OD values were determined after an additional incubation of 2 h. The OD value can indirectly reflect the proliferation condition of the cells, and the higher the value, the more active the proliferation.

In the embodiment of the invention, the proliferation conditions of gastric cancer cell line HGC-27 and CD44+Lg5-single positive and CD44+Lg5+ double positive subgroup gastric cancer stem cells of primary gastric cancer cells after sorting are detected and compared by using a cell viability detection kit. The enzyme-linked immunosorbent assay (ELISA) measures the absorbance (OD) value. The OD value can indirectly reflect the proliferation condition of the cells, and the higher the value, the more active the proliferation. The statistical results are displayed in a bar graph (see fig. 7).

The results show that: the OD values of the CD44+Lgr5+ double positive subgroup gastric cancer stem cells are higher than those of the CD44+Lgr5-single positive subgroup gastric cancer stem cells (HGC-27,0.76 +/-0.12 VS.0.41+/-0.09, P < 0.05; PRIMARY CELL, 0.83+/-0.14 VS.0.61+/-0.07, P < 0.05). Therefore, the CD44+Lgr5+double positive subgroup gastric cancer stem cells have stronger proliferation capacity than CD44+Lgr5-single positive subgroup gastric cancer stem cells.

(IV) cell suspension balling culture

Primary balling cell culture:

(1) Collecting gastric cancer cell lines HGC-27 in logarithmic growth phase and CD44+Lgr5-single positive and CD44+Lgr5+double positive subgroup gastric cancer stem cells of primary gastric cancer cells, using trypsin solution to digest for 10-15min, fully digesting, presenting single cell suspension state, using cell culture solution containing fetal bovine serum to terminate digestion, transferring cell suspension into a centrifuge tube, centrifuging at low speed, discarding cell culture solution containing fetal bovine serum at the upper layer by a pipette, and retaining cell precipitation;

(2) Preparing a special gastric cancer stem cell culture solution prepared in advance, and adding additives such as B27, bFGF, EGF and the like and growth factors into the formula. Washing the centrifugally collected cells with PBS phosphate buffer solution for 3 times, and then re-suspending the cells by using a special culture solution for gastric cancer stem cells and accurately counting;

(3) Sub-packaging the cells in an ultralow adsorption cell culture 6-pore plate, wherein the number of cells in each pore is preferably 2000 (in principle, 500-5000 cells can be added in each pore, and the actual state and the balling capacity of the cells are properly adjusted), and finally, adding 4ml of special gastric cancer stem cell culture solution in a supplementary way;

(4) Different cell culture times are different, and the sorted gastric cancer stem cells used in the research can be subjected to balling culture generally for about 10 days, and are photographed and observed by using a research grade professional polarizing microscope.

Secondary balling cell culture:

(1) Collecting the cell spheres obtained in the above steps, filtering gastric cancer stem cell spheres by a cell sieve with the diameter of 70 mu m, centrifuging at a low speed of 1000rpm to collect cells, discarding the supernatant culture solution by a pipetting gun, and digesting for 10-15min by using a trypsin solution;

(2) Stopping digestion, centrifuging at a low speed, collecting cells, discarding the cell culture solution containing fetal bovine serum at the upper layer by a liquid transfer gun, washing the centrifugally collected cells with PBS phosphate buffer solution for 3 times, and then re-suspending the cells by using a special culture solution for gastric cancer stem cells and accurately counting;

(3) Sub-packaging the cells in an ultralow adsorption cell culture 6-well plate, wherein the number of the cells in each well is preferably 2000 (in principle, 500-5000 cells can be added in each well, and the actual state and the balling capacity of the cells are properly adjusted);

(4) The sorted subgroup cells can be subjected to balling culture for about 10 days, and a research grade professional polarizing microscope is used for photographing and observing, so that the balling state of the cells is recorded. Counting in visual field, and measuring the diameter of spherocyte.

The embodiment of the invention further detects the 'dryness' characteristics of the gastric cancer cell line HGC-27 and the CD44+Lgr5-single positive and CD44+Lgr5+ double positive subgroup gastric cancer stem cells of the primary gastric cancer cells after separation through a suspension balling capacity detection experiment. Selecting a cell trypsin solution with good growth state for digestion, and then re-suspending cells and accurately counting; the cell suspension concentration is adjusted according to the cell counting condition, and the cell suspension is subpackaged in an ultralow adsorption cell culture 6-hole plate for cell plating, wherein the number of cells in each hole is about 2000. After 10 days of culture, the primary balling is completed, the cell balls obtained by the primary balling are collected, digested again, plated and cultured, after 10 days, the inverted phase contrast microscope is used for observing the state of gastric cancer stem cells obtained by the secondary balling (figure 8), counting is carried out in the visual field (figure 9), and the diameter of the balled cells is measured (figure 10).

The results show that: in the gastric cancer cell line HGC-27 and primary gastric cancer cells, the cell balling morphology of the CD44+Lgr5+ double positive subgroup is more plump and concentrated than that of the CD44+Lgr5-single positive subgroup, and the three-dimensional suspension state is better; the CD44+Lgr5+ double positive cell population state of the gastric cancer cell line HGC-27 is better than that of the primary gastric cancer cell CD44+Lgr5+ double positive cell population in whole; the numbers of the balling cells of the CD44+Lgr5+ double positive subgroup of the gastric cancer cell line HGC-27 and the primary gastric cancer cells are more than those of the CD44+Lgr5-single positive subgroup (HGC-27, 113.56 +/-8.33 VS.70.23+/-5.37, P < 0.05; PRIMARY CELL,91.26 +/-4.95 VS.78.21+/-47.53, P < 0.05); gastric cancer cell line HGC-27 and CD44+Lgr5+ double positive subgroup of primary gastric cancer cells with larger spherical cell diameter than CD44+Lgr5-cell subgroup （HGC-27,310.47±20.18μm VS.218.81±8.63μm,P＜0.05; Primary cell,259.22±14.74μm VS.143.52±12.93μm,P＜0.05）.

(V) Western blotting experiment

(5.1) Collecting cells and extracting proteins

(1) Taking gastric cancer cell line HGC-27 and primary gastric cancer cell CD44+Lgr5-single positive and CD44+Lgr5+ double positive sorting subgroup cells as objects, and extracting cell total protein;

(2) Sucking and discarding the cell culture solution by using a pipette;

(3) Cells were washed 2 times by adding 2ml of pre-chilled PBS phosphate buffer solution to each well of cells, and blotted using a pipette. Then adding cell lysate, and fully lysing in an ice bath environment for 30min;

(4) Lysed cells on the surface of the dish were scraped using a small scraper and concentrated on one side of the culture well. The pipette draws the lysis mixture and transfers it to a centrifuge tube;

(5) Centrifuging at a low temperature and a high speed, and retaining the supernatant after centrifuging;

(6) Marking, packaging, and storing in a refrigerator at-70deg.C.

(5.2) Quantitative protein extraction

(1) And (5) gradient dilution. BSA (bovine serum albumin ) standards at a concentration of 2 μg/μl were compared and diluted to 6 gradients.

(2) Working fluid is prepared. According to the quantity of the BSA standard substance and the protein sample to be detected, preparing a working solution for measuring the protein concentration of the poly-n-butyl cyanoacrylate (Bicinchonininc Acid, BCA) and fully and uniformly mixing.

(3) And (5) detecting a sample.

① Respectively adding 25 mul diluted BSA standard substances into sample holes of an ELISA (enzyme-linked immunosorbent assay) by a pipette;

② Diluting a protein sample to be detected by 10 times, and adding the diluted protein sample into a sample hole of a detector;

③ Adding BCA solution into the sample hole, and placing the cells back into the incubator;

④ After 30min incubation, the sample plate is taken out of the incubator to be cooled for 10min, and then the absorbance value of A595 is measured;

⑤ And drawing a BSA standard absorbance curve. And (3) comparing the BSA standard substance absorbance curve, and substituting the absorbance of the protein sample to be detected, which is measured in the step ④, into the absorbance curve to obtain the protein concentration of the sample.

(5.3) Gel electrophoresis

(A) SDS-PAGE gels were prepared

(1) And (5) installing a glass plate. The clean and dry glass plates are fixed by using a fixing clamp, and the two glass plates are aligned up and down and left and right and vertically inserted on a glue making frame.

(2) Preparing a lower layer separation gel solution: the lower layer separator gum was prepared at a concentration of 10% in the formulation ratio shown in Table 1 below, and the total amount of the solution was 5ml. The volume of the formulation solution is doubled when the preparation is carried out according to the actual use quantity (block) of the separation gel. The reagents were added to a wide-mouth beaker using a 10ml sterile pipette or pipette and mixed well, and finally tetramethyl ethylenediamine (N, N' -TETRAMETHYLETHYLENEDIAMINE, TEMED) was added and mixed well.

Table 1 formulation of the separator gum solution

(3) Pouring lower layer separating glue: and (3) injecting the reagent solution uniformly mixed in the step (2) into the middle of the two layers of glass plates at a stable flow rate by using a 10ml sterile suction tube or a pipette, carefully observing the height of the solution, and stopping filling when the solution approaches to a green marking tape. Slowly replenishing deionized water to the highest position of the glass plate, and leveling the top end of the separation gel by using the deionized water. Standing at room temperature for about 15min, standing for solidification of the lower layer of separating gel, gently tilting to remove the upper layer of deionized water, and carefully sucking residual water droplets between the two layers of glass plates with water-absorbing paper.

(4) Preparing an upper concentrated gum solution: the 5% strength upper concentrated gel was prepared in the formulation ratio of Table 2 below, and the total amount of the solution was 2ml. According to the actual use quantity (block) of the concentrated glue, the total volume of the formula solution is doubled during preparation. The reagents were added to a wide-mouth beaker using a 10ml sterile pipette or pipette and mixed well, and finally TEMED was added and mixed well.

Table 2 upper concentrated gum solution formulation

(5) And (5) pouring upper concentrated glue: the reagent solution thoroughly mixed in step (4) was injected into the middle of the two glass plates (the remaining space above the separator gel) at a steady flow rate using a 10ml sterile pipette or pipette until the glass plate was the highest. Finally, a vertical sample adding comb is inserted until the upper concentrated glue is solidified. The sample-adding comb can be kept in the concentrated gel together for low-temperature preservation, and then taken out when electrophoresis is used for sample adding.

(B) Gel electrophoresis

(1) Instrument preparation: the glass plate was fixed on an electrophoresis apparatus, and an electrophoresis buffer solution prepared in advance was added.

(2) Protein sample denaturation:

① According to the protein quantitative result, sucking 30 mug of a protein sample to be detected, and adding the protein sample to be detected into a centrifuge tube;

② Adding SDS loading buffer solution according to the volume of the protein sample to be detected;

③ The protein sample and SDS loading buffer solution are fully mixed, sealed by using a sealing film and then placed in a water bath kettle at 95 ℃ for sample boiling for 5min, so that the protein sample is fully denatured.

(3) Protein sample loading: the denatured protein samples were centrifuged at high speed for 5min. And sucking the pre-dyed protein standard mark and the protein sample to be detected by using a pipetting gun, and sequentially adding the pre-dyed protein standard mark and the protein sample to be detected into an upper concentrated gel pore canal.

(4) Electrophoresis:

① And installing a power cover of the electrophoresis apparatus. Electrophoresis is carried out at 80V voltage for about 20min, and the advancing distance of the pre-dyed protein standard mark is observed;

② After the sample enters the separation gel, regulating the stable voltage to 120V for about 60min, and observing the advance distance of the pre-dyed protein standard mark;

③ Terminating electrophoresis when the blue dye is near the lower part of the gel;

④ The glass plate was removed and the gel was removed.

(5.4) Protein transfer Membrane

(1) The gel is placed in a flat tray completely, and is soaked in a transfer buffer solution.

(2) 6 Pieces of filter paper, 1 piece of PVDF membrane (polyvinylidene difluoride, polyvinylidene fluoride) and a gasket are prepared, and are placed in a membrane transfer buffer solution for soaking for standby.

(3) The 'sandwich' type is sequentially arranged for laying according to the sequence from outside to inside, two ends are provided with backing plates, filter paper is arranged inwards, and gel and PVDF film are arranged in the middle. After each layer was padded, the air bubbles were gently displaced using a plastic plate.

(4) And (3) vertically covering an instrument cover, adjusting the current to 80mA, and working for 3 hours.

(5) And (5) cutting off the power supply, taking out the PVDF film and making corresponding marks.

(5.5) Antibody binding reaction

(1) Preparing a solution. A blocking solution was prepared for use in advance with triethanolamine buffer salt-tween solution (Tris-Buffered SALINE AND TWEEN, TBST) 15min before the end of the protein transfer procedure.

(2) And (5) sealing treatment. Immersing the marked PVDF film into a sealing solution and completely overflowing, and slightly shaking the whole on a horizontal decolorizing shaking table, and sealing for 1h in a room temperature environment.

(3) And (5) incubating the anti-antibody.

① Diluting the primary antibody with TBST blocking solution according to the corresponding proportion of the product instruction or the prior literature report, such as diluting housekeeping gene GAPDH according to the proportion of the primary antibody to the diluted solution=1:3000-5000;

② PVDF membrane together with about 1ml of primary antibody dilution solution is put into an antibody incubation bag, and incubated for 8-12h at 4 ℃.

(4) And (5) incubating the secondary antibody.

① Recovering the primary antibody diluted solution in the antibody incubation bag;

② Soaking and washing the PVDF film by using TBST solution, slightly shaking on a horizontal decolorization shaking table, and replacing the TBST solution after 5-10min for total washing for 3 times;

③ The secondary antibody was diluted with TBST blocking solution in accordance with the instructions of the product or the corresponding proportions reported in the literature. For example, HRP-labeled IgG is diluted at a ratio of secondary antibody to dilution solution=1:3000-5000;

④ PVDF membrane together with about 1ml of secondary antibody diluted solution is put into an antibody incubation bag, and incubated for 60min at room temperature.

(5.6) Color development of markers

(1) Recovering the secondary antibody diluted solution in the antibody incubation bag;

(2) And soaking and washing the PVDF film by using TBST solution, slightly shaking on a horizontal decolorization table, and replacing the TBST solution after 5-10min for 3 times. Then placing the PVDF film on the surface of the preservative film, and sucking the liquid on the surface;

(3) Mixing ECL (enhanced chemiluminescence, enhanced chemiluminescent substrate solution) solution A and solution B in equal volumes;

(4) Covering the developing solution above the PVDF film;

(5) And (3) spreading the PVDF film wrapped with the preservative film in a film clamp after the color reaction, and tabletting in a darkroom. Timing for a certain time (generally 5-8 min), taking out film from darkroom, and developing in fixing solution;

(6) Finally, the film is soaked in clear water, ventilated and aired, and then the image is scanned. The scanned electronic image is used for subsequent qualitative analysis and relative expression quantitative analysis of proteins.

(5.7) Analysis of the Gray value and relative expression amount of target bands of protein samples Using software

In the embodiment of the invention, a Western Blot experiment method is used for detecting the differential expression of the dry transcription factor proteins of the gastric cancer stem cells of the two sub-populations of CD44+Lgr5-single positive and CD44+Lgr5+double positive. And extracting cell proteins from the cultured gastric cancer cell line HGC-27, primary gastric cancer cell CD44+Lgr5-single positive and CD44+Lgr5+ double positive subgroup gastric cancer stem cells. The method comprises the steps of respectively combining and incubating OCT4, SOX2, nanog and GAPDH primary antibodies, incubating the secondary antibodies, developing the markers, developing, scanning and the like to obtain a band image, and comparing the expression condition of dry transcription factors such as OCT4, SOX2, nanog and the like (see figure 11) and quantitative analysis results (see figures 12-14) by taking housekeeping gene GAPDH as an internal control.

The results show that: the OCT4, SOX2, nanog and other proteins of the CD44+Lgr5+ double positive subgroup gastric cancer stem cells of the gastric cancer cell line HGC-27 are obviously higher than those of the CD44+Lgr5+ double positive subgroup gastric cancer stem cells of the primary gastric cancer cells （OCT4,5.89±0.21 VS.0.74±0.09,P＜0.05; SOX2,4.33±0.62 VS.0.65±0.11,P＜0.05;Nanog,4.19±0.73 VS.0.53±0.08,P＜0.05）. of the CD44+Lgr5-, double positive subgroup gastric cancer stem cells, and the OCT4, SOX2, nanog and other proteins of the CD44+Lgr5+ double positive subgroup gastric cancer stem cells are obviously higher than those of the CD44+Lgr5-single positive subgroup gastric cancer stem cells （OCT4,9.17±0.21 VS.2.27±0.10,P＜0.01; SOX2,5.72±0.77 VS.1.14±0.16,P＜0.05;Nanog,7.91±0.82 VS.0.64±0.03,P＜0.05）., so that the CD44+Lgr5+ double positive subgroup gastric cancer stem cells have higher expression of the dry transcription factors than the CD44+Lgr5-single positive subgroup gastric cancer stem cells on the cellular protein level.

(Six) primer sequences of target genes

The invention refers to relevant literature and NCBI-Genome database, and specific sequences are shown in the following table 3.

TABLE 3 real-time quantitative PCR primers

(Seventh) real-time quantitative PCR experiment

(7.1) Extracting Total RNA from tissue samples

The research adopts a total RNA special extraction reagent to extract RNA of a cell tissue sample, and experimental operation is carried out by referring to a special extraction reagent instruction book. The specific method comprises the following steps:

(1) And collecting and washing the cell tissues. And (3) centrifuging the cultured gastric cancer cell line HGC-27 and the CD44+Lgr5-single positive and CD44+Lgr5+double positive four groups of subgroup gastric cancer stem cells of the primary gastric cancer cells respectively, removing the culture solution absorbed by a liquid-transfering gun, and respectively adding 5ml of PBS phosphate buffer solution with pre-cooling in advance at 4 ℃ and pH value of 7.2 into the reserved precipitated cells, and blowing and washing for 2 times by the liquid-transfering gun. Centrifuging again, sucking the PBS solution by a pipette, sucking the PBS solution as completely as possible, and standing the reserved cell tissue in an ice bath;

(2) Tissue was lysed. Adding 1ml of TRIzol total RNA extraction reagent into a cell tissue sample, and preserving for 5min at room temperature to fully separate nucleic acid-protein complexes in cells;

(3) The aqueous phase was separated and RNA was recovered. Adding 0.2ml of chloroform into the cracked tissue sample, shaking the centrifuge tube with force to fully mix the chloroform and standing for 5-10min at room temperature. Centrifuging at 12000rpm for 15min at low temperature of 4 ℃ under high speed, wherein the solution in the centrifuge tube presents three phases from top to bottom, which are respectively: an aqueous phase, an intermediate phase and an organic phase. The desired RNA was dissolved in a colorless transparent aqueous phase, which was about 70% by volume, and the upper aqueous phase was aspirated using a pipette and transferred to another new centrifuge tube. Adding isopropanol solution into a new centrifuge tube to reduce and recycle RNA;

(4) RNA pellet was obtained. Centrifuging at low temperature and high speed, retaining RNA precipitate, and washing the precipitate RNA with shaking. Centrifuging at 12000rpm for 5min at low temperature of 4deg.C, removing ethanol liquid by pipetting gun, opening centrifuge tube cap, standing at room temperature for 5min, naturally evaporating ethanol liquid, and air drying RNA precipitate;

(5) The RNA pellet was solubilized.

(7.2) Total RNA quality detection by extraction

(A) Ultraviolet absorbance determination to detect concentration and purity

In order to ensure the reliability of the follow-up real-time quantitative PCR experimental data, after extracting the total RNA of the cell tissue sample, the concentration and purity of the sample are detected and evaluated by using an ultraviolet absorption assay method, and the specific operation method is as follows:

(1) And (5) detecting concentration. If the reading value of the ultra-micro ultraviolet visible spectrophotometer is 0.5, the calculation formula of the sample RNA concentration is as follows: 0.5 X40 ng/μl=20: 20 ng/μl. The calculation method comprises the following steps:

① Dissolving RNA in 40 mul DEPC water, and fully and uniformly mixing;

② And 1 μl is taken from the uniformly mixed solution for detection. If the parameter a260=53.24 is measured, the RNA concentration=53.24x40 ng/μl= 2129.60 ng/μl;

③ After removal of the used liquid volume, the total amount of RNA remaining was: 39 [ mu ] l X2129.60 ng/[ mu ] l=83.05 [ mu ] g.

(2) And (5) detecting purity. Both the purine and pyrimidine rings of conjugated double bond systems have the property of absorbing ultraviolet light, so that the ultraviolet light absorption method can only be used to determine the concentration of RNA, and cannot be used to distinguish DNA from RNA. When the ratio is in the range of 1.8 to 2.1, the method can be used for carrying out subsequent experiments.

(B) Agarose gel electrophoresis detection method

(1) Agarose gel was prepared. Adding agarose powder into water according to a certain proportion, heating and dissolving, cooling and solidifying, and then adding electrophoresis buffer solution. The sugar gel plate was slid into the electrophoresis tank, and enough MOPS (3-Morpholinopropanesulfoinc Acid, 3-morpholinopropane sulfonic acid) running buffer was added, and the buffer was highly spread over the gel surface.

(2) RNA samples were prepared. And 1 mug of RNA is taken according to the calculated volume of the concentration of the RNA measured by an ultraviolet light absorption method, formaldehyde loading dye liquor with the volume equal to the volume is added, and ethidium bromide (Ethidium bromide, EB) fluorescent dye is added into the formaldehyde loading dye liquor to enable the final concentration to be 10 mug/ml. The RNA sample mixed with the loading dye solution and the fluorescent dye is heated and incubated, so that the sample is denatured.

(3) Loading and electrophoresis. And sucking the denatured RNA sample by using a pipette, injecting the RNA sample into a sample-adding hole reserved in agarose gel, adjusting the voltage intensity to 5-6V/cm, starting an electrophoresis process, and keeping the electrophoresis tank stable and free from shaking. Taking the travelling distance of the bromophenol blue indicator as an electrophoresis process judging point until the bromophenol blue indicator is seen to move into the gel to 2-3cm, turning off an electrophoresis power supply, and ending the electrophoresis process.

(4) And observing and photographing under ultraviolet transmission light. And opening ultraviolet transmitted light, observing a fluorescent strip on the gel, judging the purity of the extracted RNA sample, and photographing and recording.

Purity strip: depending on the species and type of RNA extracted, in general, the whole eukaryotic RNA has three bands after electrophoresis, representing ribosomes of different sizes, 28s, 18s and 5s, respectively, from top to bottom. Wherein the brightness of the upper two strips is slightly higher;

Degradation of the bands: if only the lowest 5s is displayed and the 28s and 18s bands disappear or darken in the three bands, the RNA sample is degraded in the extraction process and needs to be extracted again;

Pollution strips: if diffuse unclear staining material appears between the two upper bands, it is indicative of non-specific RNA contamination interference. If a band appears above the 28S band, suggesting DNA contamination during RNA extraction, in which case the solution needs to be re-extracted.

(7.3) Reverse transcription to cDNA

In the research, the special kit for reverse transcription is adopted to synthesize cDNA, and the kit has the advantages that the kit contains the components with stronger DNA decomposition activity, so that DNA pollution components in RNA samples can be further removed. In order to ensure the accuracy of the preparation of the reaction liquid and the loss in the pipetting process, the reaction liquid is uniformly mixed in a centrifuge tube with larger capacity and volume. And the total amount of premix is proportionally increased. Finally, the RNA samples are respectively packaged into the reaction tubes one by using a pipette, and finally, the RNA samples are added into the reaction tubes.

(7.4) Detection of samples Using real-time quantitative PCR

(1) A real-time quantitative PCR reaction system was prepared according to the following formulation volume ratios of Table 4, after the addition was completed, the reaction tube lid was closed, the solution was thoroughly mixed by flicking the bottom of the tube with a finger, and then a short centrifugation was performed using a speed of 5000 rpm. The concentration of the primer was 10. Mu.M.

TABLE 4 quantitative PCR reaction System

(2) CDNA product sample addition: transferring the reaction system mixed in the step ① into the corresponding hole of the reaction plate by using a liquid-transferring gun; each well contains 18ul of a reaction system and cDNA products obtained by 2 mu l of reverse transcription reaction; closing the tube cover of the reaction tube, packaging by using a sealing film, and placing the reaction tube in an ice bath environment to wait for the next experiment.

(3) And (3) PCR reaction: the instrument circulation procedure is set according to the following temperature and time to carry out the reaction, and the specific procedure is as follows: a:95 ℃ for 30 seconds;

B:40 PCR cycles (95 ℃,5 seconds; 60 ℃,40 seconds); c: after the amplification cycle is finished, a melting curve of the PCR product is established; d: the temperature was slowly heated to 99 ℃ (temperature ramp rate of 0.05 ℃/sec).

After the characteristic that the sorted subgroup cell stem transcription factors are differentially expressed at the protein level is clarified, a real-time quantitative PCR experimental method is used for detecting whether the expression of the two subgroup gastric cancer stem cell stem transcription factors of CD44+Lg5-single positive and CD44+Lg5+ double positive has the tendency of differential expression at the mRNA level. First, referring to NCBI-Genome database and references, sequences were designed and dry transcription factors OCT4, SOX2, nanog primers required for real-time quantitative PCR were synthesized (see chapter 2.2.3 for primer sequences). Real-time quantitative PCR sample detection and statistical analysis of data values were performed using the gastric cancer cell line HGC-27, CD44+Lgr5-single positive and CD44+Lgr5+ double positive sub-population gastric cancer stem cells of primary gastric cancer cells as subjects (see FIGS. 15-17).

The results show that: 1. the OCT4, SOX2, nanog and the like of the CD44+Lgr5+ double positive subgroup gastric cancer stem cells of the gastric cancer cell line HGC-27 are obviously higher than the CD44+Lgr5+ double positive subgroup gastric cancer stem cells of the primary gastric cancer cells （OCT4,11.58±1.03 VS.0.52±0.04,P＜0.05; SOX2,8.06±0.39 VS.0.74±0.08,P＜0.05;Nanog,11.89±1.25 VS.0.41±0.05,P＜0.05）.2. of the CD44+Lgr5-single positive subgroup gastric cancer stem cells in mRNA level, and the OCT4, SOX2, nanog and the like of the gastric cancer cell line HGC-27 are obviously higher than the CD44+Lgr5-single positive subgroup gastric cancer stem cells （OCT4,17.55±2.09 VS.1.45±0.46,P＜0.01; SOX2,13.37±2.13 VS.1.78±0.17,P＜0.01;Nanog,16.62±1.59 VS.0.84±0.03,P＜0.01）. in mRNA level, so that the CD44+Lgr5+ double positive subgroup gastric cancer stem cells also have higher expression of the dry transcription factors than the CD44+Lgr5-single positive subgroup gastric cancer stem cells in cell mRNA level.

2. Functional study of CD44+Lgr5+double-positive marked gastric cancer stem cells

In the following examples, the Lgr5 siRNA (h) plasmid (sc-62559) was used from Santa Cruz (U.S. Biotechnology), fluorouracil (5-Fu) (HY-90006) was used from MedChemExpress (U.S.), the cell proliferation and cytotoxicity test kit (C0009) was used from Biyun (Shanghai) Biotechnology institute, oxaliplatin (Oxaliplation) (B25381) was used from Source leaf (Shanghai) Biotechnology, inc., paclitaxel (taxol) (HY-B0015) was used from MedChemExpress (U.S.), and Lipofectamine ™ was used from Thermo Fisher (U.S.A.).

In the following examples, 6-week-old BALB/c Nude T cell immunodeficient Nude mice were used. 12 animals were purchased from Venlhua limited, line code 401, and bred in the general Hospital laboratory animal center of the free army.

In the following examples, the immunohistochemical kit (EnVision) was from Dako (denmark), ki67 primary antibody (ab 15580), cyclin D1 primary antibody (ab 16663), goat anti-rabbit IgG conjugate secondary antibody (ab 6721), E-cadherein antibody (ab 1416), twist antibody (ab 50887), snail antibody (ab 50887) and ZEB1 antibody (ab 203829) were all from Abcam (china), matrigel matrix gel (356234) from BD bi (united states), 1% crystal violet staining solution (G1062) from soriebao (north) limited, and insert Transwell cell culture chamber (3414) from Corning (china).

In the following examples, all values are expressed as' x+ -s in statistics and analysis of cell resistance experiments, and statistical analysis was performed using GRAPHPAD PRISM software, with significant differences in p <0.05, and very significant differences in p < 0.01. The measured OD value is taken into the following formula for calculation: inhibition ratio= (control-experimental)/control.

In the following examples, the tumor volume calculation formula was used in the statistics and analysis of tumor cell transplantation and neoplasia experiments: v (mm ³） = （W² XL)/2, W, L are the shorter and longer diameter lengths of the tumor, respectively.

Cell drug resistance test

(1) Collecting gastric cancer cell line HGC-27 in logarithmic growth phase, CD44+Lgr5-single positive and CD44+Lgr5+ double positive subgroup gastric cancer stem cells of primary gastric cancer cells;

(2) After trypsinization, the cell suspension density was adjusted, and the cell suspension was pipetted into 96-well plates using a pipette, the number of cells per well being approximately 1×10 ⁴/well. Then, the 96-well plate is placed in a constant temperature incubator for culturing for 24 hours;

(3) The gastric cancer cell line HGC-27 and primary gastric cancer cells are firstly divided into two groups of sub-populations of CD44+Lgr5-single positive and CD44+Lgr5+ double positive respectively, wherein the CD44+Lgr5+ double positive sub-population is subdivided into two groups of CD44+Lgr5+shRNA and CD44+Lgr5+siRNA (the two groups are respectively a transfected plasmid control group and an Lgr5 small interfering RNA plasmid transfected group, and the two plasmids are required to be used for transfecting CD44+Lgr5+ double positive sub-population cells 4-6 hours in advance). Up to this point, the gastric cancer cell line HGC-27 and primary gastric cancer cells respectively share three groups: a CD44+Lgr5-group, a CD44+Lgr5+shRNA group, a CD44+Lgr5+siRNA group;

(4) Fluorouracil (0 mu M, 50 mu M, 100 mu M, 150 mu M, 200 mu M), paclitaxel (0 mu M, 50 mu M, 100 mu M, 150 mu M, 200 mu M), oxaliplatin (0 mu M, 4 mu M, 8 mu M, 12 mu M, 16 mu M) chemotherapeutic drug solution with different concentrations are used for carrying out gradient pretreatment on the above grouping experiment cells, 3 compound holes are arranged in each concentration, the chemotherapeutic drug treatment time is 44h, and thiazole blue (Methylthiazolyldiphenyl-tetrazolium bromide, MTT) is used for dyeing detection. Simultaneously setting blank cell control holes which are not treated by the three chemotherapeutic drugs, setting 3 compound holes in each group, and culturing for the same time along with the same constant temperature environment of the experimental holes;

(5) Sucking the chemotherapeutic drug pretreatment solution in the cell culture hole by using a pipette, discarding the chemotherapeutic drug pretreatment solution, washing the cells by using PBS phosphate buffer solution, and repeating the washing for 3 times;

(6) Adding 180 mu l of culture medium and 20 mu l of MTT solution into each hole, and continuously incubating and culturing for 4 hours;

(7) Terminating the culture, adding dimethyl sulfoxide (Dimethyl sulfoxide, DMSO) into the well, and oscillating at low speed for 20min;

(8) Finally, OD value was measured and inhibition rate was calculated.

Gastric cancer cell line HGC-27, CD44+Lgr5-single positive and CD44+Lgr5+ double positive sub-population gastric cancer stem cells of primary gastric cancer cells were used as subjects. The experimental cells were subjected to gradient pretreatment with different concentrations of chemotherapeutic drug solutions such as oxaliplatin, paclitaxel, fluorouracil, etc., followed by staining with thiazole blue to determine OD (see fig. 18-20 for details of statistics). The results show that:

The cell absorbance values of the three experimental groups of the gastric cancer cell line HGC-27 show progressive decline trend along with the rise of the concentration of chemotherapeutic drug solutions such as oxaliplatin, paclitaxel, fluorouracil and the like, namely the number of the surviving gastric cancer stem cells is reflected to show progressive decline. And (1) when oxaliplatin and paclitaxel chemotherapeutics reach the highest concentration, the survival tumor stem cells of the CD44+Lgr5+shRNA group are more than that of the CD44+Lgr5+siRNA group （Oxaliplation,0.329±0.031 vs.0.215±0.023 p<0.05; Taxol,0.286±0.026 vs.0.203±0.022 p<0.05; 5-Fu,0.253±0.017 vs.0.221±0.009 p>0.05）;（2）, when oxaliplatin, paclitaxel and fluorouracil drugs reach the highest concentration, the survival of the CD44+Lgr5+shRNA group is more than that of the CD44+Lgr5-group （Oxaliplation,0.329±0.031 vs.0.123±0.013 p<0.01; Taxol,0.286±0.026 vs.0.128±0.005 p<0.05; 5-Fu,0.253±0.017 vs.0.098±0.004 p<0.05）;（3）CD44+Lgr5+siRNA group and the CD44+Lgr5-group have no statistical difference （Oxaliplation,0.215±0.023 vs. 0.123±0.013 p>0.05; Taxol,0.203±0.022 vs.0.128±0.005 p>0.05; 5-Fu,0.221±0.009 vs.0.098±0.004 p>0.05）.

The cell absorbance values of the three experimental groups of primary gastric cancer cells show progressive decline trend along with the rise of the concentration of chemotherapeutic drug solutions such as oxaliplatin, paclitaxel, fluorouracil and the like, namely the number of the surviving gastric cancer stem cells is reflected to show progressive decline. And (1) when oxaliplatin and paclitaxel chemotherapeutics reach the highest concentration, the survival tumor stem cells of the CD44+Lgr5+shRNA group are more than that of the CD44+Lgr5+siRNA group （Oxaliplation,0.373±0.028 vs.0.234±0.019 p<0.05; Taxol,0.325±0.022 vs.0.218±0.017 p<0.05; 5-Fu,0.289±0.016 vs.0.201±0.007 p>0.05）;（2）, when oxaliplatin, paclitaxel and fluorouracil drugs reach the highest concentration, the survival of the CD44+Lgr5+shRNA group is more than that of the CD44+Lgr5-group （Oxaliplation,0.373±0.028 vs.0.211±0.008 p<0.01; Taxol,0.325±0.022 vs.0.171±0.003 p<0.05; 5-Fu,0.289±0.016 vs.0.163±0.006 p<0.05）;（3）CD44+Lgr5+siRNA group and the CD44+Lgr5-group have no statistical difference （Oxaliplation,0.234±0.019 vs.0.211±0.008 p>0.05; Taxol,0.218±0.017 vs.0.171±0.003 p>0.05; 5-Fu,0.201±0.007 vs.0.163±0.006 p>0.05）.

OD values of control wells which are not treated by chemotherapeutic drugs such as oxaliplatin, paclitaxel and fluorouracil are tested, and the detection values are substituted into an inhibition rate calculation formula to calculate inhibition rates of the three chemotherapeutic drugs on gastric cancer stem cells of the sorting subpopulations (the results are shown in figures 21-23). The results show that:

The inhibition rate of the gastric cancer cell line HGC-27 in three experimental groups shows a progressive rising trend along with the rising of the concentration of chemotherapeutic drug solutions such as oxaliplatin, paclitaxel, fluorouracil and the like. And (1) when oxaliplatin and paclitaxel chemotherapeutics reach the highest concentration, the survival tumor stem cells of the CD44+Lgr5+shRNA group are more than that of the CD44+Lgr5+siRNA group （Oxaliplation,71.56±2.34 vs.84.96±4.53 p<0.05; Taxol,72.94±6.77 vs.88.63±2.57 p<0.05; 5-Fu,78.39±5.66 vs.90.23±4.68 p>0.05）;（2）, when oxaliplatin, paclitaxel and fluorouracil drugs reach the highest concentration, the survival of the CD44+Lgr5+shRNA group is more than that of the CD44+Lgr5-group （Oxaliplation,71.56±2.34 vs.90.21±4.38 p<0.05; Taxol,72.94±6.77 vs.97.23±2.65 p<0.01; 5-Fu,78.39±5.66 vs.91.56±3.65 p<0.05）;（3）CD44+Lgr5+siRNA group and the CD44+Lgr5-group have no statistical difference （Oxaliplation,84.96±4.53 vs.90.21±4.38 p>0.05; Taxol,88.63±2.57 vs.97.23±2.65 p>0.05; 5-Fu,90.23±4.68 vs.91.56±3.65 p>0.05）.

The cell inhibition rate of the three experimental groups of primary gastric cancer cells also shows a progressive trend along with the increase of the concentration of chemotherapeutic drug solutions such as oxaliplatin, paclitaxel, fluorouracil and the like. And (1) when oxaliplatin and paclitaxel chemotherapeutics reach the highest concentration, the survival tumor stem cells of the CD44+Lgr5+shRNA group are more than that of the CD44+Lgr5+siRNA group （Oxaliplation,62.79±3.53 vs.79.99±2.87 p<0.05; Taxol,68.94±2.15 vs.83.79±3.12 p<0.05; 5-Fu,70.24±3.09 vs.81.70±3.58 p>0.05）;（2）, when oxaliplatin, paclitaxel and fluorouracil drugs reach the highest concentration, the survival of the CD44+Lgr5+shRNA group is more than that of the CD44+Lgr5-group （Oxaliplation,62.79±3.53 vs.85.98±3.44 p<0.05; Taxol,68.94±2.15 vs.93.51±1.89 p<0.01; 5-Fu,70.24±3.09 vs.90.88±2.46 p<0.05）;（3）CD44+Lgr5+siRNA group and the CD44+Lgr5-group have no statistical difference （Oxaliplation,79.99±2.87 vs.85.98±3.44 p>0.05; Taxol,83.79±3.12 vs.93.51±1.89 p>0.05; 5-Fu,81.70±3.58 vs.90.88±2.46 p>0.05）.

From the above results, it can be seen that the chemotherapy drug has a lower inhibition rate on CD44+Lgr5+ double positive subset cells, and that CD44+Lgr5+ double positive subset cells have stronger resistance to the chemotherapy drug than CD44+Lgr5-single positive subset cells, and that when the CD44+Lgr5+ double positive subset cells are transfected with the small interfering RNA plasmid, the expression level of the protein of the CD44+Lgr5+ double positive subset cells is reduced, and the resistance to the chemotherapy drug is also affected. Therefore, whether or not gastric cancer stem cells are marked by lgr5+ has an important effect on the drug resistance of chemotherapeutic drugs, and therefore, it is inferred that lgr5+ marked gastric cancer stem cells are one of the possible causes of resistance or drug resistance in anti-tumor therapy.

(II) small interference RNA plasmid transfected cell experiment

Collecting CD44+Lgr5+ double positive subgroup external stem cells of gastric cancer cell line HGC-27 in logarithmic growth phase and primary gastric cancer cells, and making experimental cell density be up to above 60% 1 day before the quasi-transfection (the transfection reagent specification of Thermo Fisher (USA) suggests that cell density be 90-95%, and making improved regulation on cell density after searching experimental conditions). The cell culture solution is replaced 2-3 hours before transfection to be a cell culture solution without penicillin and streptomycin double antibiotic solution.

The transfected samples were prepared as follows:

(1) Diluting Lgr5 siRNA (plasmid information is shown in detail in 2.1.7) in a proper amount of DMEM/F12 (1:1) liquid medium, and lightly blowing the mixture by using a pipette to mix the mixture uniformly;

(2) Diluting the transfection reagent, and fully mixing by using a pipette;

(3) Mixing the mixture obtained in the steps ① and ②, lightly blowing the mixture by using a pipette to mix the mixture uniformly, and incubating the mixture at room temperature for 20min to wait for complex formation; ( Remarks: in this step, the mixed solution may be turbid, which is a normal phenomenon, without affecting the subsequent transfection effect. In addition, the total incubation time of step ② and step ③ is not longer than 30min, avoiding the decrease of transfection activity )

(4) Placing the culture bottle on the surface of an ultra-clean workbench, moving the mixed cell culture solution back and forth in parallel, and then culturing for 24 hours at constant temperature;

(5) After transfection for 4-6 hours, the cell culture solution needs to be replaced for 1 time, and penicillin and streptomycin double antibiotic solution can be added into the replaced cell culture solution.

(III) in vivo tumor burden experiment in nude mice

(1) Nude mice were prepared. 12 BALB/c Nude T cell immunodeficiency mice with 6 weeks of age are purchased as experimental subjects, and fed adaptively for 3-7 days after warehouse entry, and animal activity state and diet condition are observed.

(2) Tumor cell preparation.

① The CD44+Lgr5-single positive and CD44+Lgr5+ double positive sorting subpopulations of gastric cancer stem cells of primary gastric cancer cells in logarithmic growth phase. Wherein the CD44+Lgr5-single positive subgroup is a control group and the CD44+Lgr5+ double positive subgroup is an experimental group;

② Cells were counted after extensive trypsin digestion and cell suspension concentration was adjusted. The preferred cell concentration is 1X 10 ⁷/ml.

(3) And establishing a transplanted tumor model.

① Drawing the cells with the established densities in the control group and the experimental group in the step (2) by using a 1ml syringe;

② According to the experimental plan, 12 mice were randomly assigned to two groups of 6 numbers. The operator grabs and fixes the mice by left hand, and the right hand injector respectively inoculates the suspension of the gastric cancer stem cells of the CD44+Lgr5-single positive and CD44+Lgr5+double positive sorting subgroup of the primary gastric cancer cells into the mice. The cell suspension of each mouse is inoculated with 0.1ml, and the inoculation part is the left back subcutaneous part;

③ The procedure was strictly sterile and the mice were observed for their life status after inoculation.

(4) And (5) observing, calculating and sampling the loaded tumor.

① After the success of the transplanted tumor model was established, about 10d, the tumor growth volume was about 50mm ³, and the survival state of the mice was observed every 2 days and weighed. Measuring the longer diameter length and the shorter diameter length of the back-loaded tumor by using a vernier caliper, and calculating the tumor volume according to a calculation formula (the calculation formula is shown in 2.2.17.5 for details);

② After 44d, the mice were sacrificed harmlessly, tumor tissues were completely peeled off subcutaneously from the back and weighed, and the tumor tissues were stored at-20 ℃ and kept for further use in subsequent experiments.

The cd4+lgr5-single positive subpopulation and the cd4+lgr5+ double positive subpopulation of gastric cancer stem cells of the primary gastric cancer cells in the logarithmic growth phase were set as experimental groups, and the cd4+lgr5-single positive subpopulation of gastric cancer stem cells were set as control groups. About 10d after the model is built, the tumor-bearing volume of the mice can be up to about 50mm ³, and the length and the short diameter of the tumor body are measured by using a vernier caliper every 2 days from the moment. Tumor volume was estimated from the measured values and a volume increase curve was plotted (see fig. 24). Mice were sacrificed after 44d feeding, tumor tissue was extracted from the back subcutaneous intact dissection and weighed, the ex vivo tumor was ranked according to the experimental and control groups (see fig. 25-26) and statistically analyzed by weighing the ex vivo tumor (see fig. 27). The mice body weight change curve was recorded (see fig. 28).

The results show that: 4 mice in each of the experimental group and the control group survived until innocuous sacrifice; in addition, the tumor volume of the live load is observed, and the tumor volume of the experimental group CD44+Lgr5+ double positive cell subgroup load is gradually larger than that of the control group CD44+Lgr5-single positive cell subgroup (1202.96 +/-73.29 mm ³ VS.745.85±37.04mm³, P < 0.01) along with the extension of the growth time; 3. weighing the tumor after the vitro treatment, wherein the tumor weight of the CD44+Lgr5+ double positive cell subset of the experimental group is larger than that of the CD44+Lgr5-single positive cell subset of the control group, and the weight difference has statistical significance (437.79 +/-33.31mg VS.206.09 +/-17.07 mg, P < 0.05); 4. the difference in weight between the experimental and control mice was not statistically significant (23.17.+ -. 1.89g vs. 22.85.+ -. 1.77g, P > 0.05) during the animal model culture.

(IV) immunohistochemical experiments

The research adopts an immunohistochemical kit to carry out staining analysis on a tissue sample, and comprises the following specific steps:

(1) Dewaxing and hydrating: firstly, heating the tissue slices on a table top of a slice baking machine at a preheating temperature of 60 ℃ for 30 minutes, rapidly transferring the tissue slices into a xylene solution for dewaxing treatment for 10 minutes, and then carrying out hydration treatment on the slices;

(2) Placing the tissue slice into a steam engine for antigen retrieval, and transferring the tissue slice from the sodium citrate solution to distilled water for cleaning for 3 times after finishing;

(3) Polyethylene glycol octyl phenyl ether (Triton X-100) was diluted to a concentration of 0.5% using PBS phosphate buffer solution and the tissue sections were immersed for 20 minutes at room temperature;

(4) Covering 3 drops of endogenous peroxidase blocking solution on the tissue slice, standing at room temperature for 5 minutes, and then soaking the tissue slice in triethanolamine buffer solution (Tris buffered saline, TBS) for 3 times, wherein the soaking time is 5 minutes each time;

(5) Covering 2 drops of blood serum sealing liquid on the tissue slice, and sealing for 1 hour at room temperature;

(6) The dilution ratio of the Ki67 primary anti-antibody is 1:500, and the dilution ratio of the cyclin D1 primary anti-antibody is 1:250. The diluted antibody liquid is soaked and covered on the surface of a tissue slice carrying cells, and the tissue slice is incubated overnight in a light-proof moisture-preserving box at the temperature of 4 ℃;

(7) The TBS buffer solution was soaked 3 times for 5 minutes each. Dripping the secondary antibody of the kit to cover the tissue slice, standing for 30 minutes at room temperature, and then soaking in TBS buffer solution for 3 times, wherein the soaking time is 5 minutes each time;

(8) The DAB stock was diluted in proportion. The color development time is preferably 1 minute and 20 seconds, and finally, the slices are soaked in distilled water for 3 times;

(9) Firstly, counterstaining for 2 minutes by using hematoxylin dye, and then, staining cell nuclei for 2 minutes by using Prussian blue dye after soaking and cleaning;

(10) Dehydrating the tissue slice;

(11) After air-drying the sections, a few caplets were dropped onto the tissue sections, the staining effect was observed under a microscope and recorded by photographing.

After the tumor-loaded tissue of the mouse is stripped off, the immunohistochemical staining experiment is continued, and the proliferation capacity of the tumor-loaded tissue of the mouse is compared by detecting the positive expression proportion of the Ki67 and Cyclin D1 proliferation-related proteins. The staining effect was observed and the positive staining ratio was counted (staining effect image results are detailed at 29, 31, staining ratio statistics are detailed at 30, 32).

The results show that: the positive proportion of Ki67 protein expression, the tumor tissue of the experimental group CD44+Lgr5+ double positive cell subgroup after transplantation is higher than that of the control group CD44+Lgr5-single positive cell subgroup, and the proportion difference has statistical significance (53.12+/-11.31% of VS 36.79+/-5.06% and P < 0.05); the positive proportion of the expression of the cyclin D1 protein is that the tumor tissue of the experimental group CD44+Lgr5+ double positive cell subgroup after transplantation is higher than that of the control group CD44+Lgr5-single positive cell subgroup, and the proportion difference has statistical significance (71.83+/-9.25% of VS.39.44+/-7.85% and P < 0.05).

Studies have shown that the expression of Ki67 and Cyclin D1 proteins is positively correlated with gastric cancer proliferation potency, and that the higher the expression of Ki67 and Cyclin D1 proteins, the stronger the gastric cancer proliferation potency. From the above results, it can be seen that the CD44+Lgr5+ double positive subset cell transplantation tumor has higher expression and stronger proliferation capacity than the CD44+Lgr5-single positive subset proliferation-related protein, which is also the reason that CD44+Lgr5+ double positive subset cell transplantation mice model is followed by faster tumor formation.

(V) primer sequences of target genes

The design of the primer of the target gene of the real-time quantitative PCR is referred to the relevant literature and NCBI-Genome database, and the specific sequences are shown in the following Table 5.

TABLE 5 real-time quantitative PCR primers

In order to clearly determine whether the gastric cancer stem cells of the sub-population after sorting have the capability of epithelial-mesenchymal transformation, the gastric cancer cell line HGC-27, the CD44+Lgr5-single positive sub-population of the primary gastric cancer cells and the CD44+Lgr5+ double positive sub-population of the gastric cancer stem cells are respectively cultured in two different environments of suspension and adherence. And collecting the cultured cells, and extracting protein. E-cadherin, N-cadherin, twist, snail, ZEB1 and GAPDH primary antibody are respectively used for combined incubation, and then the corresponding secondary antibody is used for incubation, marker color development, scanning and other processes to obtain band imaging, and the housekeeping gene GAPDH is used as an internal control to compare the expression conditions of important regulatory factors in the processes of epithelial-mesenchymal transition (EMT) such as E-cadherin, N-cadherin, twist, snail, ZEB1 and the like (see figure 33) and quantitative analysis results (see figures 34-38).

The results show that the different marked subgroup cells E-cadherein, N-cadherin, twist, snail, ZEB1 and other EMT important regulatory factor proteins of the gastric cancer cell line HGC-27 and the primary gastric cancer cells have different expression intensities under different environments of suspension and adherence. The concrete steps are as follows:

In gastric cancer cell line HGC-27, (1) E-cadherein protein expression, CD44+Lgr5-single positive subset and CD44+Lgr5+ double positive subset of epithelial phenotype were weaker in suspension culture than in adherent culture (CD44+Lgr5-, 3.81+ -0.09 VS.5.47+ -0.12, P < 0.05; CD44+Lgr5+, 0.83+ -0.08 VS.2.45+ -0.13, P < 0.05); (2) N-cadherein protein expression, CD44+Lgr5-single positive and CD44+Lgr5+double positive sub-populations were stronger in suspension culture than in adherent culture (CD44+Lgr5-, 2.15+ -0.17 VS.1.37+ -0.10, P < 0.05; CD44+Lgr5+, 4.78+ -0.38 VS.3.05+ -0.19, P < 0.05); (3) The expression of the Twist protein with the mesenchymal phenotype characteristic is stronger in suspension culture of CD44+Lgr5+double-positive subgroup than in adherence culture (CD44+Lgr5-, 1.48+ -0.05 VS.1.02+ -0.04, P >0.05; CD44+Lgr5+, 2.63+ -0.08 VS.1.35+ -0.03, P < 0.05); (4) The suspension culture of the single positive subgroup of CD44+Lg5-and the double positive subgroup of CD44+Lg5+ was stronger than the adherent culture (CD44+Lg5-, 5.85+ -0.27 VS.0.87+ -0.09, P < 0.05; CD44+Lg5+, 8.02+ -0.44 VS.3.16+ -0.21, P < 0.05); (5) The ZEB1 protein expression of the mesenchymal phenotype was stronger for suspension cultures of CD44+Lgr5-single positive and CD44+Lgr5+ double positive sub-populations than for adherent cultures (CD44+Lgr5-, 2.15+ -0.31 VS.0.96+ -0.11, P < 0.05; CD44+Lgr5+, 3.47+ -0.29 VS.1.56+ -0.27, P < 0.05).

In primary gastric cancer cells, (1) E-cadherein protein expression of epithelial phenotype, suspension culture of CD44+Lgr5-single positive subset and CD44+Lgr5+ double positive subset is weaker than that of adherent culture (CD44+Lgr5-, 2.63+ -0.13 VS.3.91+ -0.15, P < 0.05; CD44+Lgr5+, 0.96+ -0.04 VS.1.92+ -0.09, P < 0.05); (2) N-cadherein protein expression of the mesenchymal phenotypic trait, suspension culture of CD44+Lgr5+ double positive subpopulation is stronger than adherent culture (CD44+Lgr5-, 1.91+ -0.22 VS.1.17+ -0.10, P >0.05; CD44+Lgr5+, 6.18+ -1.03 VS.3.97+ -0.62, P < 0.05); (3) The expression of the Twist protein with the mesenchymal phenotype characteristics, the suspension culture of the CD44+Lgr5-single positive subgroup and the CD44+Lgr5+ double positive subgroup is stronger than that of the adherence culture (CD44+Lgr5-, 4.18+ -0.15 VS.0.83+ -0.06, P < 0.05; CD44+Lgr5+, 6.09+ -0.31 VS.2.15+ -0.13, P < 0.05); (4) The suspension culture of the single positive subgroup of CD44+Lg5-and the double positive subgroup of CD44+Lg5+ was stronger than the adherent culture (CD44+Lg5-, 6.27+ -1.04 VS.0.88+ -0.07, P < 0.05; CD44+Lg5+, 9.30+ -0.65 VS.4.29+ -0.15, P < 0.05); (5) The ZEB1 protein expression of the mesenchymal phenotype was stronger for both CD44+Lgr5-single positive and CD44+Lgr5+ double positive sub-populations than for adherent cultures (CD44+Lgr5-, 3.23+ -0.75 VS.1.18+ -0.33, P < 0.05; CD44+Lgr5+, 4.76+ -0.39 VS.1.96+ -0.42, P < 0.05).

Therefore, the EMT regulatory factor proteins are different in different culture environments of suspension and adherence of the gastric cancer cell line HGC-27 and the subgroup cells after primary gastric cancer cell sorting: after suspension culture, the expression of the regulatory factor representing the epithelial phenotype of the cells becomes weak, the expression of the regulatory factor representing the mesenchymal phenotype of the cells becomes strong, and the transformation difference is more obvious in the subset of gastric cancer stem cells marked by CD44+Lgr5+ double cations. And therefore judge: the sorted subgroup gastric cancer stem cells have epithelial-mesenchymal transformation capacity, and the Lgr5+ marked gastric cancer stem cells can promote and strengthen the transformation process.

In addition, after the conclusion that the differential expression of the EMT regulatory factors exists at the protein level is clarified under different culture environments of suspension and adherence of the sorted subgroup cells, a real-time quantitative PCR experimental method is then used for detecting whether the differential expression trend of the EMT regulatory factors also exists at the mRNA level of the gastric cancer stem cells of the two subgroups of CD44+Lgr5-single positive and CD44+Lgr5+ double positive under different culture environments. First, referring to NCBI-Genome database and references, sequences were designed and EMT regulatory factors E-cadherein, N-cadherin, snail, ZEB1, TWIST primers required for real-time quantitative PCR were synthesized. Real-time quantitative PCR sample detection and statistical analysis of data values were performed using the gastric cancer cell line HGC-27, CD44+Lgr5-single positive and CD44+Lgr5+ double positive sub-population gastric cancer stem cells of primary gastric cancer cells as subjects (see FIGS. 39-43).

The results show that mRNA levels of EMT important regulatory factors such as E-cadherein, N-cadherin, twist, snail, ZEB and the like of different marked subgroup cells of the gastric cancer cell line HGC-27 and the primary gastric cancer cells have different expression intensities under different environments of suspension and adherence. The concrete steps are as follows:

In gastric cancer cell line HGC-27, (1) mRNA level expression of E-cadherein, an epithelial phenotypic trait, suspension culture of CD44+Lg5-single positive subset is weaker than adherent culture (CD44+Lg5-, 3.23+ -0.24 VS.5.67+ -0.23, P < 0.05; CD44+Lg5+, 1.08+ -0.03 VS.1.96+ -0.05, P > 0.05); (2) mRNA level expression of N-cadherein, a phenotypic trait of the stroma, suspension culture of CD44+Lgr5+ double positive subpopulation is stronger than adherent culture (CD44+Lgr5-, 1.86+ -0.04 VS.0.87+ -0.02, P >0.05; CD44+Lgr5+, 8.38+ -0.35 VS.4.28+ -0.12, P < 0.05); (3) mRNA level expression of Twist of the mesenchymal phenotypic trait, suspension culture of CD44+Lgr5-single positive subpopulation and CD44+Lgr5+ double positive subpopulation is stronger than that of adherent culture (CD44+Lgr5-, 3.78+ -0.23 VS.0.81+ -0.04, P < 0.05; CD44+Lgr5+, 6.35+ -0.11 VS.1.88+ -0.24, P < 0.01); (4) mRNA level expression of Snail for the mesenchymal phenotypic trait, suspension culture of CD44+Lgr5-single positive and CD44+Lgr5+ double positive sub-populations was stronger than that of adherent culture (CD44+Lgr5-, 3.82+ -0.03 VS.0.97+ -0.03, P < 0.05; CD44+Lgr5+, 5.78+ -0.69 VS.1.95+ -0.04, P < 0.01); (5) mRNA levels of ZEB1 expressed by the mesenchymal phenotypic trait, suspension cultures of CD44+Lgr5-single positive and CD44+Lgr5+ double positive sub-populations were stronger than adherent cultures (CD44+Lgr5-, 5.62+ -0.72 VS.0.85+ -0.03, P < 0.05; CD44+Lgr5+, 7.31+ -0.64 VS.1.89+ -0.03, P < 0.01).

In primary gastric cancer cells, (1) mRNA level expression of E-cadherein, an epithelial phenotypic trait, CD44+Lgr5-single positive subset suspension culture is weaker than adherent culture (CD44+Lgr5-, 2.74+ -0.12 VS.4.97+ -0.16, P < 0.05; CD44+Lgr5+, 0.92+ -0.15 VS.1.76+ -0.05, P > 0.05); (2) mRNA level expression of N-cadherein, a phenotypic trait of the stroma, suspension culture of CD44+Lgr5+ double positive subpopulation is stronger than adherent culture (CD44+Lgr5-, 2.08+ -0.12 VS.0.99+ -0.03, P >0.05; CD44+Lgr5+, 4.48+ -0.35 VS.2.95+ -0.03, P < 0.01); (3) mRNA level expression of Twist of the mesenchymal phenotypic trait, suspension culture of CD44+Lgr5-single positive subpopulation and CD44+Lgr5+ double positive subpopulation is stronger than that of adherent culture (CD44+Lgr5-, 3.08+ -0.24 VS.0.94+ -0.05, P < 0.05; CD44+Lgr5+, 4.52+ -0.12 VS.1.35+ -0.03, P < 0.01); (4) mRNA level expression of Snail for the mesenchymal phenotypic trait, suspension culture of CD44+Lgr5-single positive and CD44+Lgr5+ double positive sub-populations was stronger than that of adherent culture (CD44+Lgr5-, 2.73+ -0.34 VS.1.02+ -0.02, P < 0.05; CD44+Lgr5+, 3.84+ -0.39 VS.1.39+ -0.05, P < 0.05); (5) mRNA levels of ZEB1 expressed by the mesenchymal phenotypic trait, suspension cultures of CD44+Lgr5-single positive and CD44+Lgr5+ double positive sub-populations were stronger than adherent cultures (CD44+Lgr5-, 3.97+ -0.45 VS.1.02+ -0.02, P < 0.05; CD44+Lgr5+, 6.21+ -0.22 VS.2.18+ -0.08, P < 0.01).

Therefore, the EMT regulatory factor mRNA expression is also different in different culture environments of suspension and adherence of the gastric cancer cell line HGC-27 and the subgroup cells after primary gastric cancer cell sorting: after suspension culture, the expression of the regulatory factor representing the epithelial phenotype of the cells becomes weak, the expression of the regulatory factor representing the mesenchymal phenotype of the cells becomes strong, and particularly, the difference of the gastric cancer stem cells of the subgroup marked by CD44+Lgr5+ double positive on the mRNA expression of part of the regulatory factors is more obvious. And therefore judge: the sorted subgroup gastric cancer stem cells have differential expression in EMT regulatory factor mRNA and protein, and the gastric cancer stem cells marked by Lgr5+ promote the epithelial-mesenchymal transition process together by interfering two links of transcription and translation.

(Six) cell migration invasion experiment

(1) Cell starvation preparation. Conventional culturing gastric cancer cell line HGC-27, CD44+Lgr5-single positive and CD44+Lgr5+ double positive subgroup gastric cancer stem cells of primary gastric cancer cells, washing 3 times with PBS phosphate buffer solution or cell culture solution without fetal bovine serum which are sterilized in advance, and then adding conventional serum-free culture solution for starvation culture for 24 hours;

(2) And (5) spreading matrigel and moistening a culture dish. Taking out the inserted Transwell cell culture chamber on an ultra-clean workbench, placing the cell culture chamber in a 24-micropore cell culture plate, diluting matrix gel Matrigel proportionally, coating the bottom of the inner wall of the chamber, and standing in a constant temperature incubator at 37 ℃ to coagulate colloid. Then, a small amount of cell culture solution without fetal calf serum is added into the inner hole and the outer hole of the cell, and the cell culture solution is soaked and moistened for 5-15min, so that the inner side and the outer side of the substrate filter membrane of the Transwell cell culture cell are fully moistened;

(3) Cells were seeded into a Transwell chamber.

① The pipette aspirates the discarded serum-free medium. Cells were digested with trypsin solution, then the digested cells were blown apart into individual cells by adding serum-free cell culture medium, and collected into a 15ml centrifuge tube for low-speed centrifugation. In order to ensure the good state of cells, the culture solution and the centrifugal temperature environment are preferably 4 ℃;

② The cell culture solution supernatant without serum is sucked by a liquid-transferring gun and discarded, cells are resuspended and evenly mixed, and the cell density measured by a counter is adjusted to 1.25 multiplied by 10 ⁵/ml;

③ The cell counting solution 400 μl was slowly added to the chamber, i.e. the total number of cells was about 5×10 ⁴. In addition, 500 μl of culture broth containing 10% fetal bovine serum was aspirated and added along the gap between the outer side wall of the chamber and the inner wall of the 24-well plate. During operation, bubbles are prevented from being formed between the cell substrate filter membrane and the added culture solution, so that the cells in the cell are prevented from being fully contacted with the liquid level;

④ Cells were strictly static cultured for 48h after inoculation, cells were not observed during culture, and culture medium was not changed.

(4) Cells were stained and photographed.

① The cell culture plate was removed and the fluid in the chamber was discarded. Cleaning the inner wall of the cell by using cotton balls, washing the inner wall for 3 times by using PBS (phosphate buffer solution), inverting and airing;

② Fixing cells for 30min by using ice-ethanol, and cleaning the inner wall for 2 times by using PBS phosphate buffer solution after sucking out the ice-ethanol by using a liquid-transfering gun;

③ Finally, staining was performed for 30min with crystal violet dye, photographing under a microscope and recording the number of cells per well transmitted through the cell.

After the capability of epithelial-mesenchymal transition of gastric cancer stem cells of the sorted subgroup is clarified, intensive research is continued, and a Transwell experiment is designed to detect migration and invasion functions of gastric cancer stem cells of the sorted subgroup. Gastric cancer cell line HGC-27 and primary gastric cancer cell CD44+Lgr5-single positive and CD44+Lgr5+ double positive subgroup gastric cancer stem cells are subjected to starvation culture, washing, fixation and crystal violet staining, and then microscopic photographing counting is carried out (the result is shown in fig. 44-45).

The results show that: the cell number of gastric cancer stem cell perforations penetrating the cells of the CD44+Lgr5+ double positive sub-population is significantly greater than that of the CD44+Lgr5-single positive sub-population, and the difference is statistically significant (HGC-27, 117.33 + -6.37 VS.73.82+ -3.15, P < 0.01; PRIMARY CELL,141.26 + -5.79 VS.79.91+ -12.38, P < 0.01). From this, it is suggested that the cd44+lgr5+ double positive subset gastric cancer stem cells have a stronger migration invasion function than the cd44+lgr5-single positive subset gastric cancer stem cells.

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.

Claims (3)

1. Use of an antibody specific for binding CD44 and Lgr5 for the preparation of a product for screening gastric cancer tumor stem cells, characterized in that the screening comprises screening using a flow-sorting method, wherein the antibody specific for Lgr5 is anti-human Lgr5 mAb and the antibody specific for CD44 is APC-conjugated mouse anti-human CD44 mAb.

2. The use according to claim 1, comprising screening gastric cancer tumor stem cells from primary gastric cancer tissue cells.

3. The use according to claim 1, comprising screening gastric cancer tumor stem cells from the intestinal gastric cancer cell line HGC-27.