CN1940062B - A kind of liver cancer stem cell and its separation method and application - Google Patents

Abstract

The invention relates to a liver cancer stem cell and its separation method and application, relating to stem cells, in particular to a human liver cancer stem cell. The purpose of the present invention is to provide a method for isolating liver cancer stem cells, and to use the isolated liver cancer stem cells for important effective anticancer drugs or drug target screening, and for the preparation of bifunctional specific monoclonal antibodies for diagnosis/treatment of liver cancer . The separation method for obtaining liver cancer stem cells from liver cancer stem cell lines is to use specific AC133/CD133/1 monoclonal antibody and magnetic beads to sort and enrich liver cancer stem cells, and the isolated CD133 ⁺ liver cancer stem cells have various Features, wide range of uses.

Description

A kind of liver cancer stem cell and its separation method and application

technical field

The present invention relates to stem cells, in particular to a liver cancer stem cell.

Background technique

With the advancement of science and technology, the research on tumor diagnosis and treatment has also made great progress, especially the introduction of advanced modern diagnostic equipment such as CT, NMR, PET, etc. has made the diagnosis of tumors move towards "accurate" and "early stage". A big step. Also in terms of treatment, due to the introduction of various new biological treatments and chemotherapy drugs in recent years, the drug treatment effect of tumors has been significantly improved, but the mechanism of occurrence of various tumors (including liver cancer) has not yet been elucidated. In recent years, due to the introduction of the concept of "cancer stem cell (CSC)" [Reya et al., Nature (2001) 414: 105-111], people's concept of evaluating the effect of tumor treatment has changed significantly. Good conventional chemotherapy drugs can only kill the "common tumor cells" that account for a large proportion of the tumor tissue, and the tumor mass can be significantly reduced or even "disappeared" during treatment. One) of the "very tumor cells" - that is, cancer stem cells are helpless [Bissell et al., Cancer Cell (2005) 7: 17-23; Patrawala et al., Cancer Research (2005) 65: 6207-6219], and there are indications that After conventional chemotherapy drugs treat tumors, because the balance between cancer stem cells and normal tumor cells is broken, it will stimulate cancer stem cells to asymmetrically divide or differentiate into normal tumor cells, eventually leading to tumor recurrence or even metastasis.

Therefore, there is an urgent need in the field for a method for isolating liver cancer stem cells, and it is also necessary to use the obtained liver cancer stem cells for screening and preparation of antitumor drugs.

Contents of the invention

The purpose of the present invention is to provide a method for isolating liver cancer stem cells, and to use the isolated liver cancer stem cells for important effective anticancer drugs or drug target screening, and for the preparation of bifunctional specific monoclonal antibodies for diagnosis/treatment of liver cancer , and obtain a method for screening inhibitors that specifically inhibit the growth of liver cancer stem cells, inducers that promote apoptosis of liver cancer stem cells, and cytotoxic drugs that kill liver cancer stem cells.

In one aspect of the present invention, there is provided a method for separating liver cancer stem cells from liver cancer cell lines, which comprises the steps of:

(a) CD133/1 monoclonal antibody (also known as AC133 monoclonal antibody) is mixed with a liver cancer cell line containing liver cancer stem cells to form a binary complex of "CD133/1 monoclonal antibody-liver cancer stem cells";

(b) centrifuging to remove unbound free CD133/1 monoclonal antibody, forming a precipitate containing binary complexes of "CD133/1 monoclonal antibody-liver cancer stem cells";

(c) suspending the precipitate of step (b), and mixing it with the anti-mouse IgG1 secondary antibody labeled with magnetic beads to form a "secondary antibody-CD133/1 monoclonal antibody-liver cancer stem cell" ternary complex;

(d) using a magnetic field to separate the ternary complex formed in step (c), thereby obtaining liver cancer stem cells.

In another preferred example, the separation method for obtaining liver cancer stem cells from liver cancer cell lines includes the steps of:

(a) AC133/CD133/1 monoclonal antibody (IgG1, Miltenyi Biotec) was fully mixed with the liver cancer cell line, incubated on ice for a certain period of time, washed repeatedly with BSA-containing buffer, and centrifuged to remove unbound free IgG1 molecules;

(b) fully mixing the liver cancer cell line in step (a) with an appropriate volume of magnetic bead-labeled anti-mouse IgG1 secondary antibody and incubating on an ice bath for 30 minutes to form a liver cancer stem cell-magnetic bead complex;

(c) Let the liver cancer stem cell-magnetic bead complex obtained in step (b) and the unlabeled liver cancer cell line suspension be added together to a separation column installed in a constant magnetic field, and washed several times with a buffer solution containing BSA, so that no The liver cancer cells labeled with magnetic beads flow out of the separation column, while the liver cancer cells labeled with magnetic beads (ie liver cancer stem cells) are adsorbed on the separation column;

(d) removing the separation column with adsorbed liver cancer stem cells out of the magnetic field, and punching out the separation column with a separation column core to obtain separated liver cancer stem cells.

In another preferred example, a method for separating liver cancer stem cells from liver cancer cell lines is also provided, which includes the steps of:

(a) mixing the magnetic beads with the liver cancer cell line, the magnetic beads are immobilized with AC133/CD133/1 monoclonal antibody, thereby forming a liver cancer stem cell-magnetic bead complex;

(b) separating the liver cancer stem cell-magnetic bead complex in step (a);

(c) dissociating the liver cancer stem cells from the complex to obtain isolated liver cancer stem cells.

In said separation method, said liver cancer cell line and liver cancer stem cells are from human.

More preferably, the preferred liver cancer cell lines are (a) SMMC7721, (b) BEL7402, (c) Huh-7, (d) Hep3B, (e) MHCC-LM3, (f) MHCC-97L.

In another aspect of the present invention, there is provided a liver cancer stem cell obtained by the above-mentioned separation method, which is a human liver cancer stem cell; can differentiate into liver cells in vivo or in vitro; can form tumors; it can induce tumor necrosis factor Resistance to apoptosis and cell cycle arrest.

In another aspect of the present invention, application of the liver cancer stem cells provided in the present invention in screening antitumor drugs is provided.

It is resistant to commonly used tumor chemotherapy drugs, among which (a) cyclophosphamide (b) cisplatin (c) methotrexate (d) vincristine sulfate (e) 5-fluorouracil (f) cisplatin (g) Adriamycin.

In another aspect of the present invention, the application of the liver cancer stem cells provided in the present invention in the preparation of specific monoclonal antibodies for diagnosis and treatment of liver cancer stem cells is provided.

In another aspect of the present invention, a method of screening inhibitors for inhibiting the growth of liver cancer stem cells is provided, comprising the steps of:

(a) in the test group, adding the candidate to be screened to the liver cancer stem cell culture, and detecting the proliferation of the liver cancer stem cell;

(b) compare the proliferation of liver cancer stem cells in the test group in step (a) with the proliferation of liver cancer stem cells in the control group without adding the candidate, if the proliferation of liver cancer stem cells in the test group is statistically lower than (preferably Significantly lower than) the control group, it shows that the candidate is a compound that inhibits the growth of liver cancer stem cells.

The method for isolating liver cancer stem cells provided by the present invention can enrich liver cancer stem cells; and the isolated CD133 ⁺ liver cancer stem cells can be used for screening anti-tumor drugs, can be used for the preparation of specific monoclonal antibodies for diagnosis and treatment of liver cancer stem cells, can provide A method for screening inhibitors that inhibit the growth of liver cancer stem cells, etc.

Description of drawings

Figure 1 Expression of stem cell marker CD133 in liver cancer cell lines

Figure 2 Cancer stem cells in human liver cancer-related tissues

Magnification factor: (d, g, j, m) × 100, (a-c, e, h, k, n) × 400, (f, i, l) × 630

Figure 3 In vitro clone formation of CD133 ⁺ liver cancer stem cells

Magnification factor: ×200

Figure 4 In vivo differentiation characteristics of CD133 ⁺ liver cancer stem cells

b magnification factor: ×400; c magnification factor: ×1000

Figure 5 In vivo tumorigenicity of CD133 ⁺ liver cancer stem cells

Fig. 6 Resistance of CD133 ⁺ liver cancer stem cells to anticancer drugs

Figure 7 The resistance curves of CD133 ⁺ and CD133 ^- SMMC-7721 cells to Vincristine

Figure 8 Resistance curves of CD133 ⁺ and CD133 ^- SMMC-7721 cells to 5'-Fluorouracil

Figure 9 The drug resistance curves of CD133 ⁺ and CD133 ^- SMMC-7721 cells to Cisplatin

Figure 10 The drug resistance curve of CD133 ⁺ and CD133 ^- SMMC-7721 cells to Adriamycin

Figure 11 Resistance of CD133+ liver cancer stem cells to apoptosis and cell cycle arrest induced by tumor necrosis factor (TNFα)

Detailed ways

CD133 is a specific marker for embryonic stem cells, bone marrow stem cells and adult stem cells in some tissues. We compared the currently recognized stem cell markers such as CD117/c-kit, AC133/CD133/1, CD34, Dlk/Pref-1, CD45, CD90, CD28, etc. were strictly screened through a series of experiments, and finally CD133 was selected as a liver cancer stem cell marker. The defects of other antigens are mainly manifested in (a) low sorting efficiency (such as CD117/c-kit, the inventors used 6 Antibodies from different manufacturers have not obtained good sorting results) provide a method for screening inhibitors that inhibit the growth of liver cancer stem cells (b) poor representation (such as Dlk/Pref-1 is a marker of embryonic stem cells, The expression of adult stem cells and non-stem cells is very low and the difference is not obvious), (c) the sorted cells have low differentiation ability (such as CD34, CD45, CD28 antibody sorted liver cancer cells have low differentiation ability). Liver cancer stem cells were successfully enriched and isolated from liver cancer cell lines with specific AC133/CD133/1 monoclonal antibody and magnetic bead sorting system.

The liver cancer cell lines referred to in the present invention are human liver cancer cell lines, wherein preferred (a) SMMC7721, (b) BEL7402, (c) Huh-7, (d) Hep3B, (e) MHCC-LM3, (f) MHCC- 97L. In addition to normal liver tissue, there are a small number of CD133 positive cells in liver cancer, paracancerous liver, and liver cirrhosis.

The AC133/CD133/1 monoclonal antibody (monoclonal anti-CD133/1 IgG1, pure) referred to in the present invention was purchased from Miltenyi Biotec, Germany. The so-called magnetic bead sorting system of the present invention is well known to those skilled in the art, comprises steps:

(a) Incubate the AC133/CD133/1 monoclonal antibody (mouse IgG1) with the liver cancer cell line to bind to the cells (liver cancer stem cells) expressing the CD133 antigen;

(b) mixing the magnetic beads with the liver cancer cell line in step (a), on which the specific antibody against mouse IgG1 is immobilized, thereby forming a liver cancer stem cell-magnetic bead complex;

(c) using a magnetic sorter to separate the liver cancer stem cell-magnetic bead complex in step (b);

(d) removing the separation column of the liver cancer stem cell-magnetic bead complex adsorbed in step (c) from the magnetic field, and using a magnetic separation column core to beat out the cells in step (c) to obtain isolated liver cancer stem cells.

The CD133 ⁺ liver cancer stem cells isolated by the present invention can form clones in vitro (in soft agar medium) without feeder cells, and the method is to use 0.6% low-melting point agar containing 10% fetal bovine serum prepared at 42°C -Spread 6-well cell culture plate (bottom) with stem cell medium, after it solidifies at 37°C, cover the bottom layer with soft agar suspension containing 5× ¹⁰³ CD133 ⁺ or CD133 ^- SMMC7721-GFP single cells , solidified at 37°C, and finally added 2ml of stem cell culture medium to each well, and replaced with fresh medium every 5 days.

The CD133 ⁺ liver cancer stem cells isolated by the present invention have both stem cell characteristics and characteristics of liver cancer cells and liver origin; both in vivo and in vitro have the characteristics of differentiation into mature hepatic liver cancer cells and cells with normal stem cell phenotype. On the other hand, the CD133 ⁺ liver cancer stem cells isolated by the present invention have the ability to form tumors in vivo, and 500 cells can form obvious tumors in immunodeficiency mice within 6 weeks. The CD133 ⁺ liver cancer stem cells separated by the present invention have drug resistance to dozens of commonly used tumor chemotherapeutic drugs, among which anticancer drugs (a) cyclophosphamide (b) cisplatin (c) methotrexate (d) vinca sulfate Neobase (e) 5-fluorouracil (f) cisplatin (g) doxorubicin has obvious resistance. In addition, the CD133 ⁺ liver cancer stem cells isolated by the present invention are resistant to apoptosis and cell cycle arrest induced by tumor necrosis factor (TNFα), and the cell cycle of CD133 ⁺ liver cancer stem cells is hardly affected by TNFα, while CD133 ^- cells Mainly arrested in the G1 phase.

According to the above, it shows that the CD133 ⁺ liver cancer stem cells isolated by the present invention can be used for screening anti-tumor drugs; can be used for the preparation of liver cancer stem cell-specific monoclonal antibodies to diagnose and treat tumors; can be used to obtain a screening method for inhibiting the growth of liver cancer stem cells A method for an inhibitor comprising the steps of: (a) adding a candidate to be screened to liver cancer stem cell cultures in a test group, and detecting the proliferation of liver cancer stem cells; The proliferation of the stem cells is compared with the proliferation of the liver cancer stem cells in the control group to which no candidate was added, and if the proliferation of the liver cancer stem cells in the test group is statistically lower (preferably significantly lower) than the control group, it indicates that the candidate It is a compound that inhibits the growth of liver cancer stem cells.

The main advantages of the present invention are:

1. Can enrich liver cancer stem cells;

2. The isolated CD133 ⁺ liver cancer stem cells are widely used.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed.

Example 1

Isolation and Identification of Cancer Stem Cells from Liver Cancer Cell Lines

Specific AC133/CD133/1 monoclonal antibody and magnetic bead sorting system enrich and isolate stem cells from liver cancer cell lines:

(1) Expression of stem cell marker CD133 in liver cancer cell lines: Figure 1a-f are human liver cancer cell lines, in which (a) SMMC7721, (b) BEL7402, (c) Huh-7, (d) Hep3B, (e) MHCC-LM3, (f) MHCC-97L, immunocytochemistry results showed that there were a small number of CD133+ cells (arrows) in the above cell lines;

(2) CD133 ⁺ cells efficiently enriched by magnetic bead sorting, in which (g) the proportion of CD133 ^{+ positive cells is low before sorting, and (h) the proportion of CD133 + positive} cells is greatly increased after sorting <yellow brown>.

The results of Western blot (i) also confirmed this point, where "+" indicates that the sorted CD133 ⁺ positive cells, "-" indicates that the sorted CD133 ^- negative cells, β-actin is an internal reference.

Example 2

Identification and Characterization of Cancer Stem Cells in Human Liver Cancer-Related Tissues

Expression of stem cell marker CD133 in paraffin-embedded liver cancer, paracancerous liver, liver cirrhosis, and normal liver tissue sections (immunohistochemical detection): Figure 2a-c is liver cancer tissue, d-i is paracancerous liver tissue, j-1 is liver Hardened tissue, m, n are normal liver tissues.

It can be seen from Figure 2 that, in addition to normal liver tissue, there are a small number of CD133-positive cells (yellow brown) in liver cancer, paracancerous liver, and cirrhosis tissue, and the white arrow marks the Hering’s canal-like structure.

Example 3

In vitro clonogenicity detection of CD133 ⁺ liver cancer stem cells

The colony formation ability of CD133 ⁺ liver cancer stem cells in soft agar was detected by soft agar colony formation assay, and the specific operation steps were as follows:

(1) Spread 6-well cell culture plates (bottom) with 0.6% low melting point agar-stem cell culture medium containing 10% fetal bovine serum prepared at 42°C;

(2) After curing at 37°C, cover the bottom layer with soft agar suspension containing 5× ¹⁰³ CD133 ⁺ or CD133 ^- SMMC7721-GFP single cells;

(3) solidify at 37°C, add 2ml of stem cell medium to each well, and replace with fresh medium every 5 days;

(4) After 3 weeks, the clones with more than 15 cells were counted and photographed under an inverted fluorescence microscope, and the data were statistically analyzed.

The results are shown in Figure 3: the stick graph (a) shows the number of clones formed, expressed as the mean ± s.d. (n=12 low power fields); the stick graph (b) shows the diameter of the clone, expressed as the mean ± s.d. (μm); ( c) is a representative picture.

The results showed that CD133 ⁺ liver cancer cells were significantly higher than CD133 ^- cells in terms of the number of clones and the size of clones: **, p<0.001 [the number of clones: p=5.43416E-11; Diameter: p = 5.1314E-07]

Example 4

Detection of in vivo differentiation characteristics of CD133 ⁺ liver cancer stem cells

1. Immunolocalization of GFP and human albumin

This experiment detects the differentiation ability of CD133 ⁺ liver cancer stem cells in 2-AAF/PHx and DMSO/PHx model mice replicated with immunodeficiency NOD/SCID mice. The specific steps are as follows:

(1) The 2-AAF/PHx and DMSO/PHx models replicated by SPF female NOD/SCID at the age of 6-7 weeks were 9 mice in each group, and each mouse was inoculated with 1×10 ⁴ CD133 ⁺ -SMMC7721-GFP via the tail vein cell;

(2) 1, 2, and 3 weeks after inoculation, 3 mice in each group were sacrificed painlessly, and their livers were taken to make frozen sections;

(3) Observe green fluorescent protein (GFP) positive cells (green) under a fluorescent microscope and perform fluorescent immunocytochemical detection (red) and co-localization of human albumin (Albumin), DAPI counterstained nuclei (blue), collected separately Images of the same field of view, finally superimposed (three colors).

Results: Figure 4a shows the AAF/PHx group, some cells overlapped with GFP green fluorescence and human Albumin red fluorescence to appear an intermediate superimposed color (yellow fluorescence), indicating that the "hepatocytes" that secrete human albumin were differentiated from GFP-positive liver cancer stem cells. However, similar phenomena were not observed in the DMSO/PHx group, indicating that the lack of AAF prevented the regeneration and division of mouse liver cells from being inhibited, and the division and differentiation of exogenous liver cancer stem cells could not be mobilized.

2. In situ PCR (in situ PCR) detection of Alu sequence

In order to verify the reliability of the above-mentioned liver cancer stem cells, we also carried out the in situ PCR detection of the serial sections of the above-mentioned liver tissues. As shown in Figure 4b, the Alu sequence was found in the liver of the mice inoculated with CD133 ⁺ -SMMC-7721-GFP in the AAF/PHx group Positive hepatocytes were scattered or clustered, but no Alu-positive cells were detected in the DMSO/PHx group mice inoculated with the same cells.

It shows that these positive cells are differentiated cells derived from human liver cancer stem cells.

3. Y chromosome fluorescence in situ hybridization (FISH) detection

In order to further confirm the differentiation of human liver cancer stem cells in mice, we performed Y chromosome FISH with the AAF/PHx and DMSO/PHx models replicated in female immunodeficient mice. The specific steps are as follows:

(1) The 2-AAF/PHx and DMSO/PHx models replicated by SPF female NOD/SCID at the age of 6-7 weeks were 9 mice in each group, and each mouse was inoculated with 1×10 ⁴ CD133 ⁺ -SMMC-7721 via the tail vein cell;

(2) 1, 2, and 3 weeks after inoculation, 3 mice in each group were sacrificed painlessly, and their livers were taken to make frozen sections;

(3) Use the Y-chromosome direct fluorescent probe to slightly modify the operation procedure recommended by the probe preparation manufacturer, carry out hybridization (green), counterstain the nucleus with DAPI (blue), collect images of the same field of view, and finally superimpose (two-color ).

Results As shown in Figure 4c, Y chromosome-positive scattered hepatocytes were found in the livers of mice inoculated with CD133 ⁺ -SMMC-7721 in the AAF/PHx group, while Y was not detected in the mice inoculated with the same cells in the DMSO/PHx group Chromosomally positive cells.

It shows that these positive liver cells are differentiated from human liver cancer stem cells.

4. Western blot (Figure 4d)

It was verified that there were strong specific GFP, CK19, CK8/18 bands in the liver tissues of AAF/PHx mice inoculated with CD133 ⁺ -SMMC7721-GFP cells in the tail vein, and their corresponding proteins were almost identical in the liver tissues of DMSO/PHx mice Undetectable, Beta-actin is used as internal reference.

Example 5

In vivo tumorigenesis of CD133 ⁺ liver cancer stem cells

In vivo tumor formation assay (in vivo tumor formation assay) was used to detect the ability of CD133 ⁺ liver cancer stem cells to form tumors subcutaneously in immunodeficient NBX mice. The specific steps are as follows:

(1) Each experimental BNX mouse was subcutaneously inoculated with 500 CD133 ⁺ -SMMC7721-GFP cells and 1×10 ⁵ CD133 ^- SMMC-7721 feeder cells;

(2) Each control BNX mouse was subcutaneously inoculated with 500 CD133 ^- -SMMC7721-GFP cells and 1×10 ⁵ CD133 ^- SMMC-7721 feeder cells;

(3) After 4 weeks, all animals were sacrificed painlessly, photographed, and their tumors were weighed.

The result is shown in Figure 5.

a shows that the tumor formation in the experimental mice was significantly larger than that in the control mice

b Bar graph shows mean tumor weight±s.d.(n=6), p=7.91912E-05

c Western blot

The results showed that the expression of CD133 in the tumorigenic tissue inoculated with CD133 ⁺ cells was much higher than that in the tumorigenic tissue of CD133 ^- cells.

Example 6

Anticancer drug resistance of CD133 ⁺ liver cancer stem cells

(1) Inoculate 2×10 ³ CD133 ⁺ and CD133 ^- SMMC-7721 cells in each well of a 96-well cell culture plate;

(2) Anticancer drugs 125 μg/ml cyclophosphamide, 25 μg/ml cisplatin, 5 μg/ml methotrexate, (d) 0.25ng/ml vincristine sulfate (e) 5 μg/ml 5 - Fluorouracil, (f) 5 μg/ml cisplatin, (g) 2.5ng/ml doxorubicin to treat the cells;

(3) Conventional MTT assay (490nm) was used to detect the absorbance at 0h, 12h, 24h, 36h and 48h time points respectively. Three replicate wells were set up, and the wells without anticancer drugs were used as blank control.

The results shown in Figure 6-10 show CD133 ⁺ cells anticancer drugs (a) cyclophosphamide, (b) cisplatin (DDP), (c) methotrexate, (d) vincristine sulfate, (e) 5- Fluorouracil, (f) cisplatin, (g) doxorubicin and ^CD133- cells all have obvious resistance effects (at the 48-hour treatment point: a, p=1.42E-07; b, p=4.39E-05 ; c, p=7.70E-07) (*, p<0.01; **, p<0.001). There was no statistically significant difference between drug-containing and non-medicated groups in CD133 ⁺ cells (at the 48-hour treatment point : a, p=0.788; b, p=0.489; c, p=0.745).

Example 7

Resistance of CD133 ⁺ liver cancer stem cells to tumor necrosis factor (TNFα)-induced apoptosis and cell cycle arrest

(1) Treat sorted CD133 ⁺ -SMMC7721-GFP and CD133 ^- -SMMC7721-GFP cells with 10ng/ml TNFα respectively;

(2) After 24 hours, the cells were collected, preserved, and sent for inspection according to the conventional methods for detecting cell apoptosis and cell cycle by flow cytometry.

The results are shown in Figure 7. Compared with CD133 ⁺ (a lower) cells, the sorted CD133 ^- (a) cells are sensitive to the cell cycle arrest effect of tumor necrosis factor (TNFα), and the cell cycle of CD133 ⁺ cells is hardly affected. Under the influence of TNFα, ^CD133- cells were mainly arrested in G1 phase.

The cells treated as above were stained with nuclear DNA of PI, and the apoptosis rate was detected by DNA content analysis. The results showed that the sorted CD133 ^- (b upper) cells were more resistant to tumor necrosis factor (TNFα) than CD133 ⁺ (b lower) cells The induced apoptosis is more sensitive, while CD133 ⁺ cells have about 11% resistance to TNFα-induced apoptosis.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. A separation method for obtaining liver cancer stem cells from liver cancer cell lines, characterized in that, comprising steps: (a) CD133/1 monoclonal antibody (also known as AC133 monoclonal antibody) is mixed with a liver cancer cell line containing liver cancer stem cells to form a binary complex of "CD133/1 monoclonal antibody-liver cancer stem cells"; (b) centrifuging to remove unbound free CD133/1 monoclonal antibody, forming a precipitate containing binary complexes of "CD133/1 monoclonal antibody-liver cancer stem cells"; (c) suspending the precipitate of step (b), and mixing it with the anti-mouse IgG1 secondary antibody labeled with magnetic beads to form a "secondary antibody-CD133/1 monoclonal antibody-liver cancer stem cell" ternary complex; (d) using a magnetic field to separate the ternary complex formed in step (c), thereby obtaining liver cancer stem cells. 2. The separation method according to claim 1, wherein said liver cancer cell line and liver cancer stem cells are from human. 3. separation method as claimed in claim 1, is characterized in that, described liver cancer cell line that contains liver cancer stem cell is selected from the group: (a) SMMC7721, (b) BEL7402, (c) Huh-7, (d) ) Hep3B, (e) MHCC-LM3, (f) MHCC-97L. 4. A liver cancer stem cell obtained by the separation method according to claim 1, characterized in that it is a human liver cancer stem cell. 5. The liver cancer stem cells according to claim 4, characterized in that they can differentiate into liver cells in vivo or in vitro. 6. The liver cancer stem cells according to claim 4, capable of forming tumors. 7. The liver cancer stem cells according to claim 4, characterized in that they are resistant to tumor necrosis factor-induced apoptosis and cell cycle arrest. 8. An application of the liver cancer stem cells as claimed in claim 4 in screening antitumor drugs. 9. A use of the liver cancer stem cells as claimed in claim 4 in the preparation of specific monoclonal antibodies for diagnosis and treatment of liver cancer stem cells. 10. A method of screening inhibitors for suppressing the growth of liver cancer stem cells, characterized in that it comprises the steps of: (a) in the test group, adding the candidate to be screened to the liver cancer stem cells as claimed in claim 4, and detecting the proliferation of the liver cancer stem cells; (b) comparing the proliferation of the liver cancer stem cells in the test group in step (a) with the proliferation of the liver cancer stem cells in the control group to which no candidate was added, if the proliferation of the liver cancer stem cells in the test group is statistically lower than that in the control group , indicating that the candidate is a compound that inhibits the growth of liver cancer stem cells.

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