CN1940062B - Liver-cancer stem cell, its separation and use - Google Patents
Liver-cancer stem cell, its separation and use Download PDFInfo
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- CN1940062B CN1940062B CN2005100300810A CN200510030081A CN1940062B CN 1940062 B CN1940062 B CN 1940062B CN 2005100300810 A CN2005100300810 A CN 2005100300810A CN 200510030081 A CN200510030081 A CN 200510030081A CN 1940062 B CN1940062 B CN 1940062B
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Abstract
A liver cancer stem cell, its separation and use are disclosed. The process is carried out by sorting by specific AC133/CD133/1 single-clone antibody and magnetic ball, collecting liver-cancer stem cell and separating to obtain the CD133+ liver cancer stem cell. It can be used to screen effective anti-cancer medicine or medicinal target, diagnose and treat bi-functional specific single-clone antibody of liver cancer.
Description
Technical field
The present invention relates to stem cell, relate in particular to a kind of liver-cancer stem cell.
Background technology
Along with progress of science and technology, the research of diagnosing tumor, treatment has also obtained remarkable progress, and especially the introducing of advanced persons' such as CT, NMR, PET modernized diagnositc equipment is clinical makes the diagnosis of tumour stride forward major step to " accurately ", " in early days ".Same aspect treatment because the introduction of various in recent years new bios treatment, chemotherapeutics also makes the medication effect of tumour be significantly improved, but the genesis mechanism of kinds of tumors (comprising liver cancer) is not illustrated as yet so far.In recent years because the proposition [Reya etc. of " cancer stem cell (cancer stem cell; be called for short CSC) " notion, Nature (2001) 414:105-111], people have had obvious transformation to the idea of oncotherapy recruitment evaluation, even think that the good again conventional chemotherapy medicine of curative effect accounts for quantitative proportion bigger " common tumour cell " in also can only the kill tumor tissue, tumor mass is obviously dwindled even " disappearance ", and for " the very tumour cell " of (less thaies 100,000/) few in number wherein--be feel simply helpless [Bissell etc., Cancer Cell (2005) 7:17-23 of cancer stem cell; Patrawala etc., Cancer Research (2005) 65:6207-6219], and there are indications after the conventional chemotherapy pharmacological agent tumour because the balance of cancer stem cell and common tumour cell is broken, understand the asymmetric division of irritation cancer stem cell or be divided into common tumour cell, finally cause the recurrence even the transfer of tumour.
Therefore this area presses for the method that a kind of separation obtains liver-cancer stem cell, also needs resulting liver-cancer stem cell is used for the screening and the preparation of antitumor drug.
Summary of the invention
Purpose of the present invention just provides the method that a kind of separation obtains liver-cancer stem cell, and with separate the liver-cancer stem cell that obtains be used for the screening of important effective antitumor medicine or medicine target, be used for the preparation of the difunctional monoclonal antibody specific of diagnosing cancer of liver/treatment, and obtain a kind ofly to screen inhibitor that specificity suppresses the liver-cancer stem cell growth, promote the agent of liver-cancer stem cell apoptosis induced, kill the method for the cytotoxic drug of liver-cancer stem cell.
In one aspect of the invention, provide a kind of separation method that obtains liver-cancer stem cell from hepatoma cell line, it comprises step:
(a) CD133/1 monoclonal antibody (being also referred to as the AC133 monoclonal antibody) is mixed with the hepatoma cell line that contains liver-cancer stem cell, form " CD133/1 monoclonal antibody-liver-cancer stem cell " binary complex;
(b) the unconjugated free CD133/1 of centrifugal removal monoclonal antibody forms the throw out that contains " CD133/1 monoclonal antibody-liver-cancer stem cell " binary complex;
(c) throw out of step (b) is suspended, and mix, form " second antibody-CD133/1 monoclonal antibody-liver-cancer stem cell " ternary complex with the anti-mouse IgG1 second antibody of marked by magnetic bead;
(d), thereby obtain liver-cancer stem cell with the ternary complex that forms in the magnetic field separation step (c).
This separation method that obtains liver-cancer stem cell from hepatoma cell line in another preference comprises step:
(a) will hatch certain hour on AC133/CD133/1 monoclonal antibody (IgG1, Miltenyi Biotec) and hepatoma cell line thorough mixing, the ice bath, with the damping fluid repetitive scrubbing that contains BSA, centrifugally remove unconjugated free IgG1 molecule;
(b) with step (a) thus hepatoma cell line and the second antibody thorough mixing of the anti-mouse IgG1 of marked by magnetic bead of proper volume and on ice bath, hatch and formed liver-cancer stem cell-magnetic bead mixture in 30 minutes;
(c) allow the liver-cancer stem cell-magnetic bead mixture of step (b) acquisition and the hepatoma cell line suspension that is not labeled add the separator column that is installed in the stationary magnetic field together, with the damping fluid repetitive scrubbing that contains BSA for several times, do not make and flowed out separator column, and be attracted on the separator column by the liver cancer cell of marked by magnetic bead (being liver-cancer stem cell) by the liver cancer cell of marked by magnetic bead;
(d) separator column that will be adsorbed with liver-cancer stem cell shifts out magnetic field, gets separator column with the separator column core, obtains isolating liver-cancer stem cell.
In another preference, a kind of separation method that obtains liver-cancer stem cell from hepatoma cell line also is provided, it comprises step:
(a) magnetic bead is mixed with hepatoma cell line, be fixed with the AC133/CD133/1 monoclonal antibody on the described magnetic bead, thereby form liver-cancer stem cell-magnetic bead mixture;
(b) liver-cancer stem cell-magnetic bead mixture in the separating step (a);
(c) liver-cancer stem cell is disintegrated down from mixture, obtain isolating liver-cancer stem cell.
In described separation method, described hepatoma cell line and liver-cancer stem cell are from the people.
More preferably, preferred liver cancer cell is (a) SMMC7721, (b) BEL7402, (c) Huh-7, (d) Hep3B, (e) MHCC-LM3, (f) MHCC-97L.
In another aspect of the present invention, a kind of liver-cancer stem cell that is obtained by above-mentioned separation method is provided, it is people's liver-cancer stem cell; In vivo or externally can be divided into liver cell; Can form tumour; It has resistant function to apoptosis and the cell-cycle arrest that tumour necrosis factor brings out.
In another aspect of the present invention, provide the application of liver-cancer stem cell provided by the invention in screening antineoplastic drugs.
It has anti-medicine effect to tumor chemotherapeutic drug commonly used, wherein preferred (a) endoxan (b) Platinol (c) methotrexate (d) vincristine sulphate (e) 5 FU 5 fluorouracil (f) Platinol (g) Zorubicin.
In another aspect of the present invention, provide the application of liver-cancer stem cell provided by the invention in preparation diagnosis and treatment liver-cancer stem cell monoclonal antibody specific.
In another aspect of the present invention, a kind of method of screening the inhibitor that suppresses the liver-cancer stem cell growth is provided, comprise step:
(a) in test group, in the liver-cancer stem cell culture, add material standed for to be screened, and detect the propagation situation of liver-cancer stem cell;
(b) the propagation situation with liver-cancer stem cell in the propagation situation of liver-cancer stem cell in step (a) test group and the control group that does not add material standed for compares, if the propagation of the liver-cancer stem cell in the test group is lower than (preferably being starkly lower than) control group statistically, just show that this material standed for is the compound that suppresses the liver-cancer stem cell growth.
Separation provided by the invention obtains the method for liver-cancer stem cell can the enrichment liver-cancer stem cell; And separate the CD133 that obtains
+Liver-cancer stem cell can be used for screening antineoplastic drugs, can be used to prepare diagnosis and treatment liver-cancer stem cell monoclonal antibody specific, the method for the inhibitor that screens the growth of inhibition liver-cancer stem cell etc. can be provided.
Description of drawings
The expression of stem cell markers CD133 in Fig. 1 hepatoma cell line
Cancer stem cell in Fig. 2 people's liver cancer related tissue
Enlarge multiple: (d, g, j, m) * 100, (a-c, e, h, k, n) * 400, (f, i, l) * 630
Fig. 3 CD133
+The body outer clone of liver-cancer stem cell forms
Enlarge multiple: * 200
Fig. 4 CD133
+Differentiation characteristic in the body of liver-cancer stem cell
B enlarges multiple: * 400; C enlarges multiple: * 1000
Fig. 5 CD133
+Become knurl in the body of liver-cancer stem cell
Fig. 6 CD133
+Liver-cancer stem cell is to the resistant function of cancer therapy drug
Fig. 7 CD133
+And CD133
-The SMMC-7721 cell is to the resistance curve of Vincristine
Fig. 8 CD133
+And CD133
-The SMMC-7721 cell is to the resistance curve of 5 '-Fluorouracil
Fig. 9 CD133
+And CD133
-The SMMC-7721 cell is to the resistance curve of Cisplatin
Figure 10 CD133
+And CD133
-The SMMC-7721 cell is to the resistance curve of Adriamycin
Figure 11 CD133+ liver-cancer stem cell brings out the resistant function of apoptosis and cell-cycle arrest to tumour necrosis factor (TNF α)
Embodiment
CD133 is an embryonic stem cell, the specific marker thing of the adult stem cell of bone marrow stem cell and portion of tissue, we are by stem cell markers such as CD117/c-kit to relatively generally acknowledging at present, AC133/CD133/1, CD34, Dlk/Pref-1, CD45, CD90, CD28 etc. are by the strict screening of a series of experiments, finally choose CD133 as the liver-cancer stem cell marker, other antigenic defective shows that mainly (a) efficiency of separation is low (as CD117/c-kit, the separation results that the contriver did not once obtain with the antibody of 6 different manufacturers) providing a kind of method (b) of screening the inhibitor that suppresses the liver-cancer stem cell growth, representative poor (as Dlk/Pref-1 is the embryonic stem cell sign, express all very low and difference is not obvious the adult stem cell that comprises tumor stem cell and non-stem cell), (c) the cytodifferentiation ability of sorting is low (as CD34, CD45, the liver cancer cell differentiation capability of CD28 antibody sorting is lower).With specific AC133/CD133/1 monoclonal antibody with magnetic bead sorting system successful enrichment from hepatoma cell line, separate liver-cancer stem cell.
The alleged hepatoma cell line of the present invention is the Bel7402, wherein preferred (a) SMMC7721, (b) BEL7402, (c) Huh-7, (d) Hep3B, (e) MHCC-LM3, (f) MHCC-97L.Except that normal liver tissue, has CD133 positive cell few in number in liver cancer, the other liver of cancer, the cirrhotic tissue.
The alleged AC133/CD133/1 monoclonal antibody of the present invention (monoclonal anti-CD133/1 IgG1, pure), available from German Miltenyi Biotec company.The alleged magnetic bead sorting system of the present invention is well-known to those skilled in the art, comprises step:
(a) AC133/CD133/1 monoclonal antibody (mouse IgG1) is mixed with hepatoma cell line hatch, make it and express the antigenic cell of CD133 (liver-cancer stem cell) and combine;
(b) magnetic bead is mixed with the hepatoma cell line of step (a), be fixed with the specific antibody of anti-mouse IgG1 on the described magnetic bead, thereby form liver-cancer stem cell-magnetic bead mixture;
(c) with the liver-cancer stem cell-magnetic bead mixture in the magnetic-type sorter separating step (b);
(d) separator column of adsorption step (c) liver-cancer stem cell-magnetic bead mixture is removed magnetic field, the cell of step (c) is got, obtain isolating liver-cancer stem cell with magnetic-type separator column core.
The present invention separates the CD133 that obtains
+Liver-cancer stem cell can be cloned under the condition of no feeder cell in (in soft agar medium) external formation, the 0.6% low melting point agar that the contains 10% foetal calf serum-stem cell media shop 6-well Tissue Culture Plate (bottom) of its method for preparing down with 42 ℃, treat that it is after solidifying under 37 ℃, respectively with containing 5 * 10
3Individual CD133
+Or CD133
-SMMC7721-GFP is unicellular-and the soft agar suspension covers bottom glue, makes its curing under 37 ℃, adds the 2ml stem cell media at last in each hole, changes a fresh culture in per 5 days, promptly.
The present invention separates the CD133 that obtains
+The existing stem cell characteristic of liver-cancer stem cell has liver cancer cell and liver source characteristic again; In vivo with external all have be divided into ripe hepatocellular carcinoma cell and have the characteristic of the cell of normal stem cell phenotype.The present invention separates the CD133 that obtains on the other hand
+Liver-cancer stem cell has the ability that forms tumour in vivo, and 500 cells can form obvious tumour in 6 weeks in the immunodeficient mouse body.The present invention separates the CD133 that obtains
+Liver-cancer stem cell has anti-medicine effect to tens kinds of tumor chemotherapeutic drugs commonly used, wherein for cancer therapy drug (a) endoxan (b) Platinol (c) methotrexate (d) vincristine sulphate (e) 5 FU 5 fluorouracil (f) Platinol (g) Zorubicin is had tangible resistant function.In addition, the present invention separates the CD133 that obtains
+Liver-cancer stem cell has resistant function, CD133 to apoptosis and the cell-cycle arrest that tumour necrosis factor (TNF α) brings out
+The cell cycle of liver-cancer stem cell is subjected to the influence of TNF α hardly, and CD133
-Cell mainly blocks the phase in G1.
Show that according to the above the present invention separates the CD133 that obtains
+Liver-cancer stem cell can be used for screening antineoplastic drugs; Can be used to prepare the liver-cancer stem cell monoclonal antibody specific with diagnosis and treatment tumour; Can comprise step in order to obtain a kind of method of screening the inhibitor that suppresses the liver-cancer stem cell growth: (a) in test group, in the liver-cancer stem cell culture, add material standed for to be screened, and detect the propagation situation of liver-cancer stem cell; (b) the propagation situation with liver-cancer stem cell in the propagation situation of liver-cancer stem cell in step (a) test group and the control group that does not add material standed for compares, if the propagation of the liver-cancer stem cell in the test group is lower than (preferably being starkly lower than) control group statistically, just show that this material standed for is the compound that suppresses the liver-cancer stem cell growth.
Major advantage of the present invention is:
1, can the enrichment liver-cancer stem cell;
2, separate the CD133 that obtains
+Liver-cancer stem cell is of many uses.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1
The separation of cancer stem cell, evaluation in the hepatoma cell line
The enrichment from hepatoma cell line of specific AC133/CD133/1 monoclonal antibody and magnetic bead sorting system, separate stem cells:
(1) expression of stem cell markers CD133 in the hepatoma cell line: Fig. 1 a-f is the Bel7402, (a) SMMC7721 wherein, (b) BEL7402, (c) Huh-7, (d) Hep3B, (e) MHCC-LM3, (f) MHCC-97L, the immunocytochemistry detected result shows all a small amount of CD133+ cell (arrow) in the above-mentioned clone;
(2) CD133 of magnetic bead sorting institute efficiently concentrating
+Cell, wherein cell CD133 before (g) sorting
+The positive cell ratio is low, (h) CD133 after the sorting
+Positive ratio improves＜yellowish brown greatly 〉.
Western blot (i) result has also confirmed this-point, wherein CD133 after "+" expression sorting
+Positive cell, CD133 after "-" expression sorting
-Negative cell, β-actin are confidential reference items.
Embodiment 2
Identification, the evaluation of cancer stem cell in people's liver cancer related tissue
The expression (immunohistochemical methods detection) of stem cell markers CD133 in paraffin embedding liver cancer, the other liver of cancer, liver cirrhosis, the normal liver tissue section: Fig. 2 a-c is a liver cancer tissue, and d-i is the other hepatic tissue of cancer, and j-1 is a cirrhotic tissue, and m, n are normal liver tissue.
As can be seen from Figure 2, except that normal liver tissue, have CD133 positive cell (yellowish brown) few in number in liver cancer, the other liver of cancer, the cirrhotic tissue, white arrow indicates class Hering ' s canal structure.
CD133
+The body outer clone formation ability of liver-cancer stem cell detects
Form experiment (Colony formation assay) with soft-agar cloning and detect CD133
+The clonality of liver-cancer stem cell in soft agar, the concrete operations step is:
(1) with 42 ℃ of 0.6% low melting point agar that contain 10% foetal calf serum of preparing down-stem cell media shop 6-well Tissue Culture Plate (bottom);
After solidifying under (2) 37 ℃, respectively with containing 5 * 10
3Individual CD133
+Or CD133
-SMMC7721-GFP is unicellular-and the soft agar suspension covers bottom glue;
(3) 37 ℃ solidify down, add the 2ml stem cell media in each hole, fresh culture of replacing in per 5 days;
After (4) 3 weeks 15 being cloned under the inverted fluorescence microscope more than the cell are counted, taken pictures, data statistic analysis.
The results are shown in Figure 3: bar graph (a) shows that the clone forms number, is expressed as mean number ± s.d. (n=12 low-power field); Bar graph (b) shows clone's diameter, is expressed as mean number ± s.d. (μ m); (c) be representative picture.
The result shows CD133
+No matter liver cancer cell is from forming clone's quantity, still from the size that forms the clone all than CD133
-The cell height all has significant difference: * *, p＜0.001[wherein clone number: p=5.43416E-11; Clone diameter: p=5.1314E-07]
CD133
+Differentiation characteristic detects in the body of liver-cancer stem cell
1, the immunity of GFP and human albumin is located altogether
This experiment has detected CD133
+Liver-cancer stem cell is at 2-AAF/PHx that duplicates with immune deficiency NOD/SCID mouse and the intravital differentiation capability of DMSO/PHx model mouse, and the concrete operations step is:
(1) the female NOD/SCID of SPF in age in 6-7 week 2-AAF/PHx and each group of DMSO/PHx model of duplicating is 9, and every mouse is through tail intravenous inoculation 1 * 10
4CD133
+-SMMC7721-GFP cell;
(2) inoculation 1,2,3 week back every group of difference no pain is put to death 3 mouse, gets its liver and makes frozen section;
(3) fluorescent microscope is observed down the go forward side by side fluorescence immunoassay cytochemistry of pedestrian's albumin (Albumin) of green fluorescent protein (GFP) positive cell (green) and is detected (redness) and locate altogether, DAPI redyes nuclear (blueness), gather same field-of-view image respectively, at last stack (three looks).
The result: Fig. 4 a shows the AAF/PHx group, stack look (yellow fluorescence) in the middle of the overlapping appearance of some cell GFP green fluorescence and people Albumin red fluorescence, " liver cell " that the secretion human albumin is described differentiated by GFP male liver-cancer stem cell, and the DMSO/PHx group is for observing similar phenomenon, illustrate that the AAF shortage makes the regeneration of mouse liver cell itself and splitting function not be suppressed, the division differentiation function of exogenous liver-cancer stem cell fails to be mobilized.
2, the original position PCR of Alu sequence (in situ PCR) detects
The original position PCR that we have also carried out above-mentioned hepatic tissue serial section for the reliability of verifying above-mentioned liver-cancer stem cell detects the CD133 of the AAF/PHx group of result shown in Fig. 4 b
+Find that the Alu sequence male is dispersed in or bunch shape distribution liver cell in-the SMMC-7721-GFP inoculation mouse liver, and in the DMSO/PHx group mouse of inoculation, do not detect the Alu positive cell with like cell.
Illustrate that these positive cells are exactly the noble cells that derives from people's liver-cancer stem cell.
3, Y chromosome fluorescence in situ hybridization (FISH) detects
In order further to confirm people's liver-cancer stem cell in the intravital differentiation of mouse, we have carried out Y chromosome FISH with AAF/PHx and the DMSO/PHx model that female immunodeficient mouse duplicates, and the concrete operations step is:
(1) the female NOD/SCID of SPF in age in 6-7 week 2-AAF/PHx and each group of DMSO/PHx model of duplicating is 9, and every mouse is through tail intravenous inoculation 1 * 10
4CD133
+-SMMC-7721 cell;
(2) inoculation 1,2,3 week back every group of difference no pain is put to death 3 mouse, gets its liver and makes frozen section;
(3) revise a little by the schedule of operation that its probe prepares manufacturer's recommended with the direct fluorescent probe of Y-chromosome, hybridize (green), DAPI redyes nuclear (blueness), gathers same field-of-view image respectively, at last stack (double-colored).
The result is at the CD133 of the group of the AAF/PHx shown in Fig. 4 c
+Find that the Y chromosome male is dispersed in the distribution liver cell in the-SMMC-7721 inoculation mouse liver, and in the DMSO/PHx group mouse of inoculation, do not detect the Y chromosome positive cell with like cell.
Illustrate that these positive liver cells are differentiated by people's liver-cancer stem cell exactly.
4, Western blot (Fig. 4 d)
Verified tail intravenous inoculation CD133
+In the AAF/PHx murine liver tissue of-SMMC7721-GFP cell very strong specificity GFP is arranged, CK19, the CK8/18 band and in the DMSO/PHx murine liver tissue its corresponding protein almost detect less than, Beta-actin is confidential reference items.
CD133
+Become knurl in the body of liver-cancer stem cell
Become knurl experiment (in vivo tumor formation assay) to detect CD133 in the body
+Liver-cancer stem cell is in the subcutaneous formation tumour of immune deficiency NBX mouse ability, and the concrete operations step is:
(1) every test BNX mouse is subcutaneous inoculates 500 CD133 respectively
+-SMMC7721-GFP cell and 1 * 10
5CD133
-The SMMC-7721 feeder cell;
(2) every contrast BNX mouse is subcutaneous inoculates 500 CD133 respectively
--SMMC7721-GFP cell and 1 * 10
5CD133
-The SMMC-7721 feeder cell;
No pain is put to death all animals after (3) 4 weeks, takes pictures, gets its tumour and weigh.
Result such as Fig. 5.
A shows that the test mouse becomes knurl obviously greater than the contrast mouse
The b bar graph shows the heavy mean number ± s.d. (n=6) of knurl, p=7.91912E-05
c?Western?blot
The result shows inoculation CD133
+The one-tenth tumor tissue CD133 of cell expresses will be higher than CD133 far away
-The one-tenth tumor tissue of cell.
CD133
+Liver-cancer stem cell is to the resistant function of cancer therapy drug
(1) every hole inoculates 2 * 10 respectively in the 96-well Tissue Culture Plate
3CD133
+And CD133
-The SMMC-7721 cell;
(2) respectively with cancer therapy drug 125 μ g/ml endoxan, 25 μ g/ml Platinol Platinols, 5 μ g/ml methotrexates, (d) 0.25ng/ml vincristine sulphate (e) 5 μ g/ml 5 FU 5 fluorouracils, (f) 5 μ g/ml Platinols, (g) the 2.5ng/ml Zorubicin is handled cell;
(3) conventional MTT tests (490nm) at 0h, 12h, and 24h, 36h and 48h time point detect its absorbancy respectively, establish 3 repeating holes, do not add the anticarcinogen hole as blank.
Result such as Fig. 6-10 shows CD133
+Cell is to cancer therapy drug (a) endoxan, (b) Platinol (DDP), (c) methotrexate, (d) vincristine sulphate, (e) 5 FU 5 fluorouracil, (f) Platinol, (g) Zorubicin and CD133
-Cell all has tangible resistant function (at 48 hours treatment site: a, p=1.42E-07; B, p=4.39E-05; C, p=7.70E-07) (*, p＜0.01; *, p＜0.001). at CD133
+Cell pastille and do not have difference on the statistics between the pastille group (at 48 hours treatment site: a, p=0.788; B, p=0.489; C, p=0.745).
Embodiment 7
CD133
+Liver-cancer stem cell brings out the resistant function of apoptosis and cell-cycle arrest to tumour necrosis factor (TNF α)
(1) 10ng/ml TNF α handles the CD133 of sorting respectively
+-SMMC7721-GFP and CD133
--SMMC7721-GFP cell;
After (2) 24 hours, press flow cytometer and detect apoptosis and collecting cell, preservation, the censorship respectively of the ordinary method of cell cycle.
Result such as Fig. 7 show, the CD133 of sorting
-(on a) cell and CD133
+(under a) cell compares the cell-cycle arrest effect sensitivity of tumour necrosis factor (TNF α), CD133
+The cell cycle of cell is subjected to TNF α to influence CD133 hardly
-Cell mainly blocks the phase in G1.
The cell of the same processing dyes with the nuclear DNA of PI, and by dna content analyzing and testing apoptosis rate, the result shows the CD133 of sorting
-(on the b) cell and CD133
+It is responsive that (under the b) cell compares the apoptosis effect that tumour necrosis factor (TNF α) causes, and CD133
+Cell is to apoptosis-induced 11% the resistant function of having an appointment of TNF α.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. a separation method that obtains liver-cancer stem cell from hepatoma cell line is characterized in that, comprises step:
(a) CD133/1 monoclonal antibody (being also referred to as the AC133 monoclonal antibody) is mixed with the hepatoma cell line that contains liver-cancer stem cell, form " CD133/1 monoclonal antibody-liver-cancer stem cell " binary complex;
(b) the unconjugated free CD133/1 of centrifugal removal monoclonal antibody forms the throw out that contains " CD133/1 monoclonal antibody-liver-cancer stem cell " binary complex;
(c) throw out of step (b) is suspended, and mix, form " second antibody-CD133/1 monoclonal antibody-liver-cancer stem cell " ternary complex with the anti-mouse IgG1 second antibody of marked by magnetic bead;
(d), thereby obtain liver-cancer stem cell with the ternary complex that forms in the magnetic field separation step (c).
2. separation method as claimed in claim 1 is characterized in that described hepatoma cell line and liver-cancer stem cell are from the people.
3. separation method as claimed in claim 1 is characterized in that, the described hepatoma cell line that contains liver-cancer stem cell is selected from down group: (a) SMMC7721, (b) BEL7402, (c) Huh-7, (d) Hep3B, (e) MHCC-LM3, (f) MHCC-97L.
4. the liver-cancer stem cell that separation method according to claim 1 obtains is characterized in that, is people's liver-cancer stem cell.
5. liver-cancer stem cell as claimed in claim 4 is characterized in that, in vivo or externally can be divided into liver cell.
6. liver-cancer stem cell as claimed in claim 4 is characterized in that, can form tumour.
7. liver-cancer stem cell as claimed in claim 4 is characterized in that, apoptosis and cell-cycle arrest that tumour necrosis factor is brought out have resistant function.
8. the application of liver-cancer stem cell as claimed in claim 4 in screening antineoplastic drugs.
9. the application of liver-cancer stem cell as claimed in claim 4 in preparation diagnosis and treatment liver-cancer stem cell monoclonal antibody specific.
10. a method of screening the inhibitor that suppresses the liver-cancer stem cell growth is characterized in that, comprises step:
(a) in test group, in liver-cancer stem cell as claimed in claim 4, add material standed for to be screened, and detect the propagation situation of liver-cancer stem cell;
(b) the propagation situation with liver-cancer stem cell in the propagation situation of liver-cancer stem cell in step (a) test group and the control group that does not add material standed for compares, if the propagation of the liver-cancer stem cell in the test group is lower than control group statistically, just show that this material standed for is the compound that suppresses the liver-cancer stem cell growth.
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| US9945842B2 (en) | 2010-09-03 | 2018-04-17 | Abbvie Stemcentrx Llc | Identification and enrichment of cell subpopulations |
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| EP2022848A1 (en) * | 2007-08-10 | 2009-02-11 | Hubrecht Institut | A method for identifying, expanding, and removing adult stem cells and cancer stem cells |
| CN102199574B (en) * | 2011-03-31 | 2013-05-29 | 浙江大学 | Separation method for cell cluster having tumorigenic potential in liver cancer tissue |
| CN103923214B (en) * | 2014-04-24 | 2016-01-20 | 中国人民解放军第三军医大学第一附属医院 | Anti-liver-cancer stem cell monoclonal antibody and application thereof |
| CN111733136B (en) * | 2020-06-29 | 2021-11-30 | 中山大学孙逸仙纪念医院 | Method for improving separation efficiency of CD90posi cells |
| CN112986559A (en) * | 2021-03-09 | 2021-06-18 | 北京市创伤骨科研究所 | Application of H gene and expression product thereof in identification, screening or sorting of liver cancer stem cells |
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2005
- 2005-09-28 CN CN2005100300810A patent/CN1940062B/en not_active Expired - Fee Related
Non-Patent Citations (8)
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| 余爽等.人胎儿脑室区神经前体细胞的分离纯化、培养及鉴定.解剖学报36 3.2005,36(3),第260-261，263页. |
| 余爽等.人胎儿脑室区神经前体细胞的分离纯化、培养及鉴定.解剖学报36 3.2005,36(3),第260-261,263页. * |
| 余爽等.免疫磁珠体外纯化人胎脑中CD133阳性干细胞的实验研究.神经解剖学杂志20 3.2004,9(3),全文. |
| 余爽等.免疫磁珠体外纯化人胎脑中CD133阳性干细胞的实验研究.神经解剖学杂志20 3.2004,9(3),全文. * |
| 杜英等.免疫磁珠法分离和纯化人胚胎神经干细胞.郑州大学学报（医学版）38 1.2003,9(3),全文. |
| 杜英等.免疫磁珠法分离和纯化人胚胎神经干细胞.郑州大学学报(医学版)38 1.2003,9(3),全文. * |
| 柴文祥等.人脐血CD133+和CD133-细胞分化未内皮细胞的实验研究.中国临床康复9 11.2005,9(3),全文. |
| 柴文祥等.人脐血CD133+和CD133-细胞分化未内皮细胞的实验研究.中国临床康复9 11.2005,9(3),全文. * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9778264B2 (en) | 2010-09-03 | 2017-10-03 | Abbvie Stemcentrx Llc | Identification and enrichment of cell subpopulations |
| US9945842B2 (en) | 2010-09-03 | 2018-04-17 | Abbvie Stemcentrx Llc | Identification and enrichment of cell subpopulations |
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| Publication number | Publication date |
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| CN1940062A (en) | 2007-04-04 |
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