CN101659940A - Human tumor stem cell line and application thereof - Google Patents
Human tumor stem cell line and application thereof Download PDFInfo
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Abstract
The invention provides a human tumor stem cell line T3A-A3, which is obtained by separation of liver cancer tissue excised in an operation of a primary hepatic carcinoma patient. The separated cells can be in long-term passage culture in vitro, can grow rapidly and retain stem cell property after more than 100 times of passage. The cell can express markers of multiple stem cells, has self-updatingcapability of stem cells and directional differentiation potentials for different tumor cells, and also has tumor property, tumorigenicity ability and metastatic ability. The invention is a powerfultool for preparing stem cell drugs for targeting tumor, due to the facts that the cells can be in long-term passage culture in vitro and retain unchanged stem cell property, and the tumorigenicity ability and metastatic ability thereof are strong in immunodeficient mice.
Description
Technical field
The invention belongs to the oncobiology field, be specifically related to a kind of human tumor stem cell line and uses thereof.
Background technology
Malignant tumour is one of principal disease that threatens human health.Present global cancer condition is serious day by day, and its M ﹠ M all rises year by year.Therefore, it is very important developing more efficiently oncotherapy scheme and medicine.
In recent years, more and more studies show that, in same tumor tissues, tumour cell is becoming on knurl ability, differentiation degree and the transfer ability to have heterogeneity, wherein have only quantity a part of cell seldom to have the ability of initial tumour, this class cell be called as tumor stem cell (cancer stem cells, CSCs) or tumorigenesis cancer cells (Tumogeniccancer cells).Tumor stem cell is the cell colony that not only has stem cell character but also have tumour character, and they are the same with stem cell, and unlimited multiplication capacity, self ability, multidirectional differentiation potential, transfer ability are all arranged, and similar adjusting and controlling growth mechanism.They express the sign of stem cell, as CD133, and CD90, CD44 etc.; Express the correlation factor of regulating stem cell self, multidirectional differentiation and propagation path, as Wnt/ β-catenin, Notch, Hedgehog/SMO, Bmi and Oct3/4.Simultaneously, tumor stem cell also has tumour character, and this is the basic place that tumor stem cell is different from normal stem cell.
The proposition of cancer stem-cell hypothesis reaches in recent years in the obtained progress in this field, has been familiar with tumour from a brand-new angle.Though it is found that tumor stem cell quantity is few, can in the immunodeficient mouse body, become knurl, and keep tumor growth, CSCs may be " seed cell " of metastases, plays an important role in metastases and recurrence.Therefore, tumor stem cell may play a key role in generation, development and the transfer of tumour.
The generation development of tumor stem cell and tumour is closely related.When with former generation isolating tumor cell transplantation in the immune deficiency mouse body, only there is a few cell of expressing the stem cell sign can become knurl, thereby start the generation of tumour, keep growth of tumor, and can lose this potential behind most of tumour cell experience atomization.Therefore, Most scholars thinks that the generation of tumour may come from tumor stem cell.Up to the present, except that leukemia,, confirmed the existence of CSCs in the multiple noumenal tumour such as prostate cancer in mammary cancer, glioblastoma.Tumor stem cell also participates in the progress of tumour, though tumor stem cell also can be in dormant state as normal stem cell, but after being subjected to factor such as somatomedin and stimulating, the CSCs of dormancy is activated, and will cause the fast breeding of cell and the rapid progress of disease.
The major cause that present malignant tumour is difficult to cure is to shift and recurrence.Recently have the scholar to propose: CSCs may be the basic reason of metastases, and proposition CSCs is the viewpoint of metastases " seed " cell.CSCs has powerful proliferation potential, and in a single day they arrive remote part just clonal expansion can take place rapidly, even but not CSCs is moved to remote part, also seldom have cell can form colony or clone.Mammary cancer bone study of metastasis is found, in metastasis, expressed breast carcinoma stem cell phenotype CD44
+CD24
-/LowCell account for 72%, and in the primary tumor, the cell count of same cell phenotype has only＜10%, infers in the tumour and has only CSCs could form metastasis.Except having transfer ability, CSCs still is the root of tumor recurrence, because CSCs can be in relative immobilized dormant state, and expresses Teat pipette usually, therefore makes it be not easy to be killed by the chemicotherapy treatment.Hypoxemia tabernacle environment that while CSCs is in and extracellular matrix on every side play a protective role to it, and himself is the DNA repair mechanism efficiently, to the selectively activate of dna damage reaction, also can cause tumor stem cell that chemicotherapy is produced tolerance.
The CSCs major part is in dormant state, have lower division and multiplication capacity, and present chemicotherapy mainly is at the high proliferation cell of division stage, therefore can not efficient targeting CSCs, finally cause tumor recurrence, transfer and treatment failure.Therefore, therapeutic strategy in the future should be that target is removed CSCs, thereby obtains the effective treatment to tumour.The medicine of exploitation target CSCs at first needs the tumor stem cell line of a stable in properties, be the medicine that instrument is just developed special target CSCs easily with this tumor stem cell, but present this clone is not appeared in the newspapers as yet.
Summary of the invention
A technical problem to be solved by this invention provide a kind of can be at the external human tumor stem cell line that goes down to posterity for a long time and cultivate.
The invention provides a kind of human tumor stem cell line, this human tumor stem cell line T3A-A3 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (it abbreviates CGMCC as) on September 21st, 2009, and its deposit number is CGMCC No.3291.The preservation address is the Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.
Human tumor stem cell line T3A-A3 of the present invention is that the contriver obtains in the separation of human liver cancer capillary endothelium process from the tumor tissues of liver cancer patient, this cell is through becoming knurl is separated and external single cell clone purifying filters out the propagation can the strongest cell clone at the immunodeficient mouse secondary, in of the go down to posterity cultivation of external process above 100 times, cell is still grown rapidly, and keeps stem cell character.This clone both expressed multiple stem cell sign, have stem cell self, have potential to different tumour cell directed differentiation; Also have simultaneously tumour character, become knurl ability and transfer ability.
Confirm by the RT-PCR method, human tumor stem cell line of the present invention is expressed the sign of multiple stem cell: CD34, ABCG2, Nestin, C-kit and SCF, also expression gene Notch-1, Bmi-1, β-catenin, SMO and the Oct-4 relevant with stem cells hyperplasia and self, T3A-A3 clone is also expressed iPS cell associated transcription factor simultaneously: Sox2, Nanog, Lin28, C-myc, Oct-4 and KLF4.These iPS cell associated transcription factors are extremely important to keeping stem cell character, it is reported in the human fibroblast of differentiation and maturation wherein 4 gene Oct-4 of transfection, Sox2, C-myc and KLF4 or Oct-4, Sox2, Nanog, and Lin28 will cause the human fibroblasts of end differentiation eventually to be transformed into pluripotent stem cell.
Human tumor stem cell line karyomit(e) of the present invention is the polyploid caryogram, and has very strong one-tenth knurl ability and transfer ability behind the immunoprophylaxis defective mouse.Minimum 1000 cells can be in the subcutaneous one-tenth knurl of NOD/SCID mouse, and is inoculated in the distant metastasis that positions such as lung, colon, stomach can take place behind the liver.
Human tumor stem cell line of the present invention has multidirectional differentiation potential.When this cell is in (conditioned medium of tumor tissues or tumour cell) under the different tumor environments, has ability to the differentiation of respective type tumour.When cultivating, adding melanoma or lymphadenomatous culture supernatant or tissue homogenate, T3A-A3 is broken up to corresponding tumour cell.
Another characteristics of human tumor stem cell line of the present invention are to have ubiquitous possibility in the clinical tumor tissue.Human tumor stem cell line of the present invention as the immunogen preparing monoclonal antibody, is screened by carry out cellular immunofluorescence dyeing on normal cell, tumour cell and T3A-A3 cell then, pick out the special antibody of T3A-A3.Use the special antibody of the anti-T3A-A3 of this strain (liver, brain, esophagus, stomach) on (liver cancer, giant cells glioma, mammary cancer, glioma sarcomatosum, the esophageal carcinoma and stomach signet ring cell cancer) and healthy tissues on different people's tumor tissues to carry out immunohistochemical staining then.Found that: in 6 kinds of people's tumor tissues that detected, all have stained positive cell in various degree, and do not see the stained positive cell in the healthy tissues.May there be the tumor stem cell that has with the T3A-A3 common trait in this results suggest in the different tumor tissues.
Another technical problem to be solved by this invention provides the application of human tumor stem cell line in preparation or screening targeting tumor stem cells medicine.The medicine that human tumor stem cell line of the present invention is used to develop target CSCs.For example, prepare and filter out the special antibody of T3A-A3, pick out to have by experiment in vitro and suppress and the antibody of killer T 3A-A3 cell, can be with the main component of this antibody as the resisting tumour stem cells medicine; Or the antibody of picking out specific recognition T3A-A3 prepares the targeted drug of resisting tumour stem cells as the target instrument.
The main advantage of human tumor stem cell line of the present invention is:
1. human tumor stem cell line T3A-A3 of the present invention both expressed multiple stem cell sign, have stem cell self, have potential to different tumour cell directed differentiation; Also have simultaneously tumour character, become knurl ability and transfer ability.
2. human tumor stem cell line of the present invention can externally go down to posterity cultivation for a long time and keep tumor stem cell character constant, is suitable as the cell material of research and development tumor stem cell related drugs.
3. human tumor stem cell line of the present invention has ubiquitous possibility in the various clinical tumor tissues, therefore the resisting tumour stem cells medicine with its exploitation has potential huge applications value.
Further specify the present invention below in conjunction with the drawings and specific embodiments, but should be pointed out that drawings and Examples example only of the present invention as an illustration, do not limit the scope of the invention.
Description of drawings
Fig. 1. the foundation of human tumor stem cell line T3A-A3; A: the separation of people's tumor stem cell; 1: minicell clone (T3A cell) appears in the liver cancer capillary endothelium of separation and Culture when reaching for the 8th generation; 2: the T3A cell behind the ubcellular clone purification; 3: when T3A becomes the tumor tissue secondary separation cell, still have the minicell clone; 4. the T3A-A3 cell behind the single cell clone purifying; The B:MTT method is screened the strongest single cell clone of multiplication capacity from the T3A cell, wherein the T3A-A3 rate of propagation is the fastest.
Fig. 2 .RT-PCR analyzes T3A-A3 cell stem cell sign and stem cell Expression of Related Genes; The expression of stem cell sign on the A:T3A-A3 cell; On the B:T3A-A3 cell with stem cell self and multiplication capacity Expression of Related Genes; C:iPS cell associated retroviral is in the expression of T3A-A3 cell;
The chromosome karyotype analysis of Fig. 3 .T3A-A3, A: the fetal liver cell of normal control is the diploid caryogram; B: the tumour cell of contrast is the polyploid caryogram; The C:T3A-A3 cell is the polyploid caryogram;
Fig. 4. detect melanoma cell (SK-MEL-1 cell) or organization condition substratum and induce the gp100 of back T3A-A3 cell to express; The detected result of A:RT-PCR; B:Western blot detected result;
1:SK-MEL-1 cell among each figure; The 2:T3A-A3 cell; 3: the T3A-A3 cell after the melanoma cell conditioned medium is induced; 4: the T3A-A3 cell after the melanoma tissue conditioned medium is induced;
Fig. 5. detect lymphoma cell (Daudi cell) or organization condition substratum and induce the CD10 of back T3A-A3 cell to express; The detected result of A:RT-PCR; B:Western blot detected result;
1:Daudi cell among each figure; The 2:T3A-A3 cell; 3: the T3A-A3 cell after the lymphoma cell conditioned medium is induced; 4: the T3A-A3 cell after the lymphoma tissue conditioned medium is induced;
The immunohistochemical staining of Fig. 6 .T3A-A3 monoclonal antibody in different people tumor tissue section; A: liver cancer; B: giant cell type glioblastoma multiforme; C: mammary cancer; D: glioma sarcomatosum; E: esophagus cancer; F: stomach signet ring cell cancer.
Embodiment
The foundation of embodiment 1. human tumor stem cell lines
Former generation separates: human tumor stem cell line of the present invention is to obtain from the process of people's liver cancer tissue separation and Culture capillary endothelium.Tumor tissues is taken from the specimens from pri of the liver cancer patient of being in hospital of Cancer Hospital of Chinese Academy of Medical Sciences, and pathological diagnosis is a hepatocellular carcinoma.At first according to document description method (Wu LQ, et al.Phenotypic andfunctional differences between human liver cancer endothelial cells and liver sinusoidalendothelial cells.J Vasc Res 2008; 45:78-86) separation and Culture capillary endothelium from people's liver cancer tissue that clinical operation downcuts a group occurs and breeds cell clone rapidly when this capillary endothelium reached for the 8th generation.Cell volume is little, and refractivity is strong, and propagation is (Figure 1A-1) rapidly.We come out these cellular segregation by ubcellular clone's method, particularly, nutrient solution in the culturing bottle is outwelled, wash for several times with the DMEM basic medium, remove floating cell, then at microscopically, with the elbow suction pipe minicell clone is scraped and picks up, be inoculated in another culturing bottle, the use stem cell media is cultivated, and concrete composition is to have added among the DMEM of following material: the special-purpose foetal calf serum of 5% stem cell, LIF (10ng/ml), bFGF (100 μ g/ml), heparin (40u/ml).T3A cell proliferation behind the purifying is (Figure 1A-2) rapidly.
Secondary becomes knurl and cellular segregation: with above-mentioned T3A cell inoculation immunodeficient mouse, treat into knurl after, tumor tissue is downcut and shreds, the type i collagen enzyme with 0.01%, 37 ℃ digest tumor tissues.Stop digestion with FCS behind the 20min, basic medium is washed 3 times.Then with cell inoculation in stem cell media.Occur once more in the T3A culturing process of secondary separation propagation rapidly, minicell clone (Figure 1A-3) that refractivity is strong, then as following through the single cell clone purifying with become the knurl ability to screen the fastest clone T3A-A3 the strongest (Figure 1A-4) that obtain growing with becoming the knurl ability.
Single cell clone screening: secondary is become isolated cells counting in the tumor tissue, be inoculated in 96 orifice plates by the density of 0.5 cells/well.Treat to choose the hole that only contains a cell behind the cell attachment, perform mark.Changed a not good liquor in per 3 days, treat that cell grows to 80% when converging, it is reached 24 orifice plates one to one continues to cultivate, 80% reaches it T25 culturing bottle after converging again, 80% reaches the T75 culturing bottle after converging again, totally 20 of the single cell clones that finally obtains, in the T75 culturing bottle, grow to converge after, get the part cell and carry out MTT multiplication capacity mensuration, surplus frozen standby.
MTT measures: all single cell clones that obtained are inoculated in respectively in the 96 porocyte culture plates, and inoculum density is 5 * 10
2Individual cells/well is measured 0 day respectively with mtt assay after the overnight incubation, and 1 day, 3 days, 5 days, 7 days cell proliferation situation, each time point is provided with 3 multiple holes.Take out culture plate at corresponding time point respectively, every hole adds 0.5mg/ml MTT 50ul, continues to cultivate 4h, carefully siphons away then to add dimethyl sulfoxide (DMSO) 150ul in the hole behind the nutrient solution, after vibration is dissolved Viola crystallina fully gently, at microplate reader wavelength 492nm place's photometry density value (OD value).Found that clone's T3A-A3 growth the fastest (Figure 1B).
Become the screening of knurl ability: according to MTT result, select different cell clones, six kinds of different concns inoculation NOD/SCID mouse of 10000,50000 cell/inoculation points respectively by 500,1000,2500,5000.Found that, under identical cell inoculation concentration, it is the strongest that the fastest T3A-A3 of growth that mtt assay is measured clones into the knurl ability, and the latent period that forms tumour when showing equal cell count is the shortest, and become the required minimum cell count of knurl minimum, only 1000 cells can become knurl.
The affirmation of human tumor stem cell line: use the RT-PCR method and detect, find sign of T3A-A3 cell expressing stem cell and the genes involved (as described in embodiment 2) of stem cell.And after going down to posterity for many times, T3A-A3 still expresses these genes.Therefore confirm that T3A-A3 is human tumor stem cell line (called after T3A-A3).This human tumor stem cell line T3A-A3 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (it abbreviates CGMCC as) on September 21st, 2009, and its deposit number is CGMCC No.3291.The preservation address is the Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.
Embodiment 2RT-PCR detects the expression of stem cell genes involved at the T3A-A3 cell
Use stem cell media that T3A-A3 is incubated in the T25 culturing bottle, grow to 90% converge after, digest collecting cell with 0.1% trypsinase-0.1%EDTA, add the aseptic PBS of 10ml, 1200rpm/min, the centrifugal 8min of room temperature, cell precipitation carry out the extraction of total RNA with the total RNA extraction reagent box of Qiagen company.Get the total RNA of 2ug then, use the reverse transcription test kit of Qiagen company to carry out reverse transcription.Detect the expression conditions of the relevant and differentiation of stem cells hyperplasia, self then with PCR method.Goal gene that is increased and primer sequence thereof such as following table, described primer are given birth to worker's biotechnology company limited by Shanghai and are synthesized:
The primer sequence of each gene specific in the table 1.PCR amplified reaction
| Goal gene | Primer sequence | Annealing temperature | Product length (bp) |
| ??β-actin | Upstream: 5 '-TGGCACCACACCTTCTACAATGAGC-3 ' downstream: 5 '-GCACAGCTTCTCCTTAATGTCACGC-3 ' | ??55℃ | ??396 |
| ??Notch-1 | Upstream: 5 '-ATCGGGCACCTGAACGTGGCG-3 ' downstream: 5 '-CACGTCTGCCTGGCTCGGCTC-3 ' | ??60℃ | ??600 |
| ??Bmi-1 | Upstream: 5 '-GGAGACCAGCAAGTATTGTCCTTTTG-3 ' downstream: 5 '-CATTGCTGCTGGGCATCGTAAG-3 ' | ??55℃ | ??370 |
| ??β-catenin | Upstream: 5 '-ACTGGCAGCAACAGTCTTACC-3 ' downstream: 5 '-TTTGAAGGCAGTCTGTCGTAAT-3 ' | ??60℃ | ??836 |
| ??SMO | Upstream: 5 '-ATCTCCACAGGAGAGACTGGTTCGG-3 ' downstream: 5 '-AAAGTGGGGCCTTGGGAACATG-3 ' | ??57℃ | ??242 |
| ??Oct-4 | Upstream: 5 '-CGACCATCTGCCGCTTTGAG-3 ' downstream: 5 '-CCCCCTGTCCCCCATTCCTA-3 ' | ??60℃ | ??573 |
| ??CD34 | Upstream: 5 '-ACAACCTTGAAGCCTAGCCTG-3 ' | ??55℃ | ??348 |
| Downstream: 5 '-CAAGACCAGCAGTAGACACTG-3 ' | |||
| ??ABCG2 | Upstream: 5 '-GGCCTCAGGAAGACTTATGT-3 ' downstream: 5 '-AAGGAGGTGGTGTAGCTGAT-3 ' | ??55℃ | ??342 |
| ??Nestin | Upstream: 5 '-AGAGGGGAATTCCTGGAG-3 ' downstream: 5 '-CTGAGGACCAGGACTCTCTA-3 ' | ??55℃ | ??495 |
| ??C-kit | Upstream: 5 '-GGCTCTTCTCAACCATCTGTG-3 ' downstream: 5 '-ATTTGCCGGTGTTGGTGGCTT-3 ' | ??56℃ | ??217 |
| ??SCF | Upstream: 5 '-CTCCTATTTAATCCTCTCGTC-3 ' downstream: 5 '-TACTACCATCTCGCTTATCCA-3 ' | ??52℃ | ??177 |
| ??Sox2 | Upstream: 5 '-GGCAGCTACAGCATGATGCAGGAGC-3 ' downstream: 5 '-CTGGTCATGGAGTTGTACTGCAGG-3 ' | ??60℃ | ??131 |
| ??Nanog | Upstream: 5 '-GCTTGCCTTGCTTTGAAGCA-3 ' downstream: 5 '-TTCTTGACTGGGACCTTGTC-3 ' | ??55℃ | ??256 |
| ??Lin28 | Upstream: 5 '-GGAGGCCAAGAAAGGGAATA-3 ' downstream: 5 '-CCGCCCCATAAATTCAAGAT-3 ' | ??55℃ | ??178 |
| ??C-myc | Upstream: 5 '-TCTTGACATTCTCCTCGGTG-3 ' downstream: 5 '-TACCCTCTCAACGACAGCAG-3 ' | ??60℃ | ??478 |
| ??Oct-4 | Upstream: 5 '-GAGCAAAACCCGGAGGAGT-3 ' downstream: 5 '-TTCTCTTTCGGGCCTGCAC-3 ' | ??55℃ | ??310 |
| ??KLF4 | Upstream: 5 '-CCAGCCAGAAAGCACTAC-3 ' downstream: 5 '-GACTCACCAAGCACCATC-3 ' | ??55℃ | ??409 |
The PCR reaction conditions is 94 ℃ of 2min of pre-sex change, circulating reaction 40 times, and cycling condition is: 94 ℃ of 15s of sex change, annealing 30s, annealing temperature sees the above table, and extends 72 ℃ of 45s.The end wheel extends to 72 ℃, 8min.At last, the PCR product detects amplified production through 2% agarose electrophoresis, observes on Alphaimager 2200 gel imaging systems.The result as shown in Figure 2, T3A-A3 cell expressing stem cell marker molecule CD34, ABCG2, Nestin, C-kit and SCF (Fig. 2 A); Express gene Notch-1, Bmi-1, β-catenin, SMO, the Oct-4 (Fig. 2 B) relevant with self with the propagation of stem cell; Express iPS cell associated transcription factor Sox2, Nanog, Lin28, C-myc, Oct-4 and KLF4 (Fig. 2 C).
The self ability assessment of embodiment 3.T3A-A3 cell
Colony formation experiment and secondary colony form the poly-HEMA that tests dehydrated alcohol preparation 10g/L, and (P3932 sigma), wraps by 96 orifice plates, to suppress cell adhesion.The T3A-A3 cell is inoculated in 96 orifice plates by 20 cells/well, establishes 9 multiple holes.Substratum is 0.9% methylcellulose gum (H4100, stemcell company) (final concentration of each additive is respectively the special-purpose foetal calf serum of 5% stem cell, 10ng/ml LIF, 100 μ g/ml FGF, 40u/ml heparin in the substratum), and volume of culture is the 0.2ml/ hole.Place 37 ℃, 5%CO
2, saturated humidity is cultivated.Inverted microscope is observed down, surpasses the colony of counting of 50 cells.Be incubated at the T3A-A3 cell of 0.9% methylcellulose gum, can see colony after 14 days and form; Colony is chosen, and basic medium is washed 2 times, is made into single cell suspension, carry out the colony formation experiment second time by above-mentioned steps again, still can see colony after 14 days and form, it is positive to illustrate that the secondary colony forms experiment, also is that experiment in vitro confirms that the T3A-A3 cell has the self ability.
Secondary becomes knurl experiment whether to have secondary and become the knurl ability in order to detect the T3A-A3 cell, get and grow to the 80% T3A-A3 cell that converges, concentration by 50000 cell/inoculation points is inoculated into the NOD/SCID mouse, separating tumor cell the tumor tissues after becoming knurl, by 50000 cells/point, inoculate 5 NOD/SCID mouse once more.Found that, after 1 month, two mouse wherein are at 50000 cells/can become knurl, and 5 mouse all can be in 50000 cells/become knurl in the time of 3 months, and the histological type of tumour becomes the histological type of knurl consistent with the first time, illustrates that the T3A-A3 cell has secondary and becomes the knurl ability.The angle that this experiment is tested in the body has confirmed that the T3A-A3 cell has the self ability.
The analysis of the tumour character of embodiment 4.T3A-A3 cell
In the T25 culturing bottle, change liquid and add the colchicine working fluid the day before yesterday by results with the T3A-A3 cell cultures for chromosome karyotype analysis, and final concentration is 0.06 μ g/ml-0.16 μ g/ml, makes cell stop at metaphase, harvested cell behind the 16h.The hypotonic processing 30min of KCl (0.075M), the methyl alcohol of fresh configuration: Glacial acetic acid (3: 1) fixed cell, Nikon microscope (Nikon 80i type will be adopted after the cell film-making, Japan), PCI IMAQ analyzing card, (CYTOSCAN, U.S. associating biotechnology company) carries out chromosome karyotype analysis by chromosome image analytical work station.Be contrast with fetal liver cell and tumour cell simultaneously.The result shows that the fetal liver cell that contrasts as normal stem cell is diploid cell (Fig. 3 A), and tumour cell in contrast is polyploid caryogram (Fig. 3 B), and the T3A-A3 cell also is polyploid cell (Fig. 3 C).
The tumorigenesis ability detects in order to detect the one-tenth knurl ability of T3A-A3 cell, and with 500,1000, the concentration of 2500,5000,10000,50000 cell/inoculation points is subcutaneous to the NOD/SCID mouse with the T3A-A3 cell inoculation.Observe the tumor growth situation after the inoculation weekly, be recorded as knurl latent period, become required minimum cell count of knurl and tumour size.Observed animal 6 months continuously.Found that T3A-A3 cell (2500-50000 cell/inoculation point) can become knurl in 5-8 week.The inoculation of 1000 cells o'clock forms can seeing tumour 13 weeks.These results show that the T3A-A3 cell has stronger tumorigenicity.
Transfer ability detects and gets 10 NOD/SCID mouse, is divided into two groups at random: experimental group and control group.An experimental group animal per mouse liver inoculation T3A-A3 cell 1 * 10
6, every mouse liver inoculation of control animals human hepatoma cell strain Bel7402 cell 1 * 10
6Inoculate back 2 months and put to death animal, two treated animal abdominal cavities are opened in operation, and the mouse liver of inoculation T3A-A3 all has tumor growth, and finds that metastasis has appearred in 3 mouse a long way off, illustrates that the T3A-A3 cell has high transfer character.And tumor growth is obvious on the NOD/SCID mouse liver of human hepatoma cell strain Bel-7402 cell inoculation, but does not find that all metastasis forms.The primary tumor and the metastasis tumor tissues that form is fixing respectively, carry out finding after the histological examination both histological type unanimity.
The multidirectional differentiation potential analysis of embodiment 5 T3A-A3 cells
In order to analyze the multidirectional differentiation potential of T3A-A3 cell, whether in different tumor microenvironments, observing it can be to corresponding tumour cell directed differentiation with the T3A-A3 cell cultures for we.When the tumour cell of selecting to be used for to carry out directional induction, follow two principles: the one, big with the tumor type difference in T3A-A3 source; The 2nd, this tumour has unique distinctive mark thing.In view of the above, we have selected two kinds of tumor types: melanoma and lymphoma.Used cell is the ATCC collection: melanoma cells SK-MEL-1 and lymphoma cell Daudi, and available from China Concord Medical Science University's Institute of Basic Medical Sciences's cell centre.
For the tumor microenvironment of inducing differentiation is provided to T3A-A3, we are with SK-MEL-1 and Daudi cell the tumor cell culture supernatant behind anoxic-reoxygenation and the tumour cell of ultrasonication repeatedly, as cell conditioned medium.Simultaneously, in order to simulate the microenvironment of tumor growth in vivo better, we downcut the tumor cell inoculation immunodeficient mouse tumor tissue after the one-tenth knurl and prepare homogenate as the organization condition substratum.
The preparation melanoma cell (SK-MEL-1) of tumour cell conditioned medium is the cell of suspension growth, is incubated in the MEM substratum that has added 10%FCS and 2mM glutamine.Lymphoma cell (Daudi) is the cell of suspension growth also, is incubated in the RPMI1640 substratum of 10%FCS and 2mM glutamine.With these two kinds of cells at 37 ℃, normal oxygen, 5%CO
2Environment in be cultured to the logarithmic growth state, place then to be connected with 95%N
2And 5%CO
2Hypoxia device anoxic 24h, put back to the environment reoxygenation 12h of normal cultivation subsequently, so anoxic-reoxygenation is 3 times repeatedly, respectively collecting cell and substratum.Use the Ultrasonic Cell Disruptor smudge cells after counting total cell count, wash cell debris with the nutrient solution of a small amount of anoxic-reoxygenation then, low-temperature and high-speed centrifugal (12000 rev/mins) 20min gets supernatant, 0.22 μ m filter filtration sterilization, and-20 ℃ are frozen standby.
It is subcutaneous that the preparation of tumor tissues conditioned medium is inoculated the NOD/SCID mouse respectively with SK-MEL-1 and Daudi, and 2 * 10
6Cell/point.For obtaining more tumor tissues, inoculation 6 points on every mouse, 5 mouse of every kind of cell inoculation.Treat to perform the operation when diameter of tumor reaches 1cm and downcut tumor tissues, put into the liquid nitrogen quick-frozen after weighing, will organize grinding with mortar then, add liquid nitrogen while grinding and keep low-temperature condition.With after grinding organize powder be suspended from the DMEM basic medium (10mg/ml) carry out ultrasonic, the centrifugal 20min of low-temperature and high-speed (12000 rev/mins) then.Get supernatant, 0.22 μ m filter filtration sterilization ,-20 ℃ are frozen standby.
T3A-A3 induces differentiation phase to melanomatous directed differentiation, the SK-MEL-1 cell conditioned medium of aforementioned preparation and the DMEM substratum that contains 20%FCS mixed at 1: 1 use, and the organization condition substratum is diluted to the 0.1mg/ml use with the DMEM substratum of 20%FCS.Cultivated the T3A-A3 cell respectively 21 days with these two kinds of conditioned mediums.The expression of melanoma cell specific sign (gp100) on the T3A-A3 cell has carried out detecting (Fig. 4) from mRNA and protein level respectively before and after relatively inducing then.
At first utilize the RT-PCR method to detect in the mRNA level.Particularly, after inducing 21 days, collecting cell uses the total RNA extraction reagent box of Qiagen company to carry out the extraction of total RNA.Get the total RNA of 2ug then, use the reverse transcription test kit of Qiagen company to carry out reverse transcription.Detect the expression conditions of gp100 then with PCR method.The primer sequence that uses is: upstream primer: 5 '-AGTTCTAGGGGGCCCAGTGTCT-3 ' and downstream primer: 5 '-GGGCCAGGCTCCAGGTAAGTAT-3 ' (giving birth to worker's biotechnology company limited by Shanghai synthesizes).The PCR reaction conditions is 94 ℃ of 2min of pre-sex change, circulating reaction 40 times, and condition is 94 ℃ of 15s, 64 ℃ of 30s, 72 ℃ of 45s.The end wheel extends to 72 ℃, 8min.With β-actin as internal reference.At last, the PCR product detects amplified production through 2% agarose electrophoresis, observes on Alphaimager 2200 gel imaging systems.The result is as figure, the melanomatous special sign gp100 of the T3A-A3 cell expressing after inducing, and inductive T3A-A3 does not have gp100 expression (Fig. 4 A).
Subsequently, we utilize Western Blot to analyze and induce the proteic expression of back T3A-A3 cell gp100.After inducing 21 days, scrape cell in the culturing bottle (operation on ice) with cell.With cell transfer in the aseptic EP pipe of 1.5ml (about 4 * 10
6Individual cell/pipe), adds the 1 * cell pyrolysis liquid (Cell Signaling company) of 200 μ l precoolings, put and react 15min on ice; 4 ℃, 12000rpm/min, centrifugal 15min gets supernatant, abandons precipitation.Measure protein concentration, packing ,-20 ℃ of storages are standby.To go into XCell SureLockTM Mini-Cell electrophoresis chamber available from the NuPAGE Novex Bis-Tris Mini mucilage binding of Invitrogen company then, inside groove adds 200ml 1 * NuPAGE MES SDS RunningBuffer (NP0002) (adding 500 μ l NuPAGE antioxidants), and water jacket adds 600ml 1 * NuPAGE MESSDS Running Buffer.Ready protein sample is mixed last sample with sample-loading buffer.200V, constant voltage, electrophoresis 35min.Use the dried system that transfers from one department to another of iBlotTM of Invitrogen company to change film then, concrete steps are carried out to specifications.With film with 5% skim-milk/TBST room temperature sealing 2h after, successively with the mouse anti human gp100 (ab15228 that is diluted to 1: 200 with confining liquid, abcam) monoclonal antibody and confining liquid are diluted to 1: 60000 horseradish peroxidase (Horse-radish peroxidase, HRP) hatch 2h on the anti-mouse two anti-room temperature shaking tables of labelled goat, internal reference uses mouse anti human β-actin monoclonal antibody anti-as one, and two is anti-identical.TBS washes that the chemical luminescence reagent kit with millipore company carries out color reaction after 3 times, and with film and X-ray sheet exposure 10-20sec, punching observations.As scheme to show T3A-A3 cell expressing gp100 after inducing, and the gp100 albumen of the T3A-A3 cell expressing of organization condition substratum after inducing is than cell conditioned medium inductive strong (Fig. 4 B).
T3A-A3 uses the Daudi cell and the organization condition substratum of aforementioned preparation to lymphadenomatous directed differentiation, according to induce the identical method preparation of differentiation phase to induce the substratum of differentiation to melanoma, cultivates the T3A-A3 cell respectively 21 days.The expression of the special sign (CD10) of lymphoma cell on the T3A-A3 cell before and after relatively inducing then, detect from mRNA and protein level respectively, concrete grammar is identical when detecting gp100, and wherein RT-PCR uses CD10 special primer upstream: 5 '-CTGGAGTTCATAATGGATCTTGTAAGC-3 ' and downstream: 5 '-CATCCAAGTGAGGTCATCTAAAGTCTG-3 ' (it is synthetic that worker's biotechnology company limited is given birth in Shanghai).Western blot uses mouse anti human CD10 anti-as one.(concrete grammar and test conditions and T3A-A3 to described in the melanoma directed differentiation identical)
The result shows, after cell conditioned medium and organization condition substratum were induced 21 days, the T3A-A3 cell was at the lymphadenomatous special sign CD10 of mRNA horizontal expression, and wherein organization condition substratum inductor is better than cell conditioned medium inductor (Fig. 5 A); On protein level, the T3A-A3 cell after cell conditioned medium is induced is not seen the proteic expression of tangible CD10, but the T3A-A3 cell expressing CD10 albumen (Fig. 5 B) of organization condition substratum after inducing.
The dyeing of embodiment 6T3A-A3 monoclonal antibody in people's healthy tissues and tumor tissue section
Whether the cell that has a human tumor stem cell line sample feature of the present invention for analysis ubiquity in mankind tumor tissue, we have prepared the special monoclonal antibody of T3A-A3, and have carried out the immunohistochemical staining analysis on the various clinical tumor tissues.Particularly, with the T3A-A3 cell with 4% Paraformaldehyde 96 stationary liquid in the fixing 30min of room temperature, basic medium thorough washing 3 times is got precipitation, is suspended among the PBS, with 10
7It is subcutaneous that the concentration of/ml divides multiple spot to be seeded to BALB/c mouse (available from Beijing dimension tonneau China Experimental Animal Center), and every mouse inoculum size is 0.5ml.2 week back supplementary immunizations once, later on week about supplementary immunization once, immunity is 8 times altogether, puts to death animal on the 4th day behind the last booster immunization, gets spleen, separating Morr. cell merges with the SP2/0 cell.Cell after merging is inoculated in 96 orifice plates according to the density of 1 cell in every hole.Mark has only the hole of a clonal growth after 7 days, detects the antibody titer in these holes after 10 days.Select the high hole of tiring and inoculate 96 orifice plates by the density of 1 cells/well once more.Pick out the monoclonal hybridoma strain of tiring high after repeating 4 times.This cell strain is inoculated into the BALB/c mouse abdominal cavity of paraffin oil processing after 7 days, treats that ascites produces very obviously back execution animal, get ascites, utilize affinity column HiTrap
TMRProtein A FF (available from AmershamBiosciences company) is purified into antibody.Utilize these antibody that obtain on T3A-A3 cell, human normal cell line (huve cell, liver cell), human tumor cells (liver cancer cell, breast cancer cell), to carry out cellular immunofluorescence dyeing subsequently, finally pick out the only antibody of stained positive on T3A-A3.
Utilize the special monoclonal antibody of this strain T3A-A3 (liver, brain, esophagus, stomach) on (liver cancer, giant cells glioma, mammary cancer, glioma sarcomatosum, the esophageal carcinoma and stomach signet ring cell cancer) and healthy tissues in the different mankind tumor tissues to carry out immunohistochemical staining at last.Found that: in 6 kinds of clinical tumor tissues that detected, all have stained positive cell (Fig. 6) in various degree, and do not see the stained positive cell in the healthy tissues.May there be the tumor stem cell with common trait in this results suggest in the different tumor tissues.
Claims (2)
1, a kind of human tumor stem cell line is characterized in that: on September 21st, 2009 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number is CGMCC No.3291.
2, the application of the described human tumor stem cell line T3A-A3 of claim 1 in preparation or screening targeting tumor stem cells medicine.
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