CN102250828A - CD24 (glycosyl phosphatidyl inositol-linked surface mucin) and new use of CD24 antibody - Google Patents
CD24 (glycosyl phosphatidyl inositol-linked surface mucin) and new use of CD24 antibody Download PDFInfo
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Abstract
The invention discloses CD24 and a new use of a CD24 antibody. The new use refers to the use of the CD24 antibody in enrichment of precursor cells of pancreas. In the invention, the CD24 antibody can be used to enrich the precursor cells of pancreas to a certain degree, so that the effect of various signals from endothelium or interstitial substance on further differentiation maturity can be screened out accurately; and thus, the study on the whole pancreas differentiation process is promoted greatly.
Description
Technical field
The present invention relates to the new purposes of CD24 and CD24 antibody.
Background technology
Human embryo stem cell not only can be for the treatment of from now on diabetes especially type i diabetes provides competent donor islet cell source, and can become the ideal in vitro study model of exploring human pancreas's growth course.Up to the present, at inducing human embryo stem cell to having obtained some progress aspect pancreas precursor and the insulin secretory cell directional induction differentiation.According to the substep revulsion, the atomization of human embryo stem cell at first induces it to be divided into entoderm in the embryo growth course of pancreas in the analogue body, further is induced to differentiate into pancreas precursor cell and insulin secretory cell on this basis.Main at present coexpression such as PDX1, HNF1b, SOX9 and the HNF6 with some pancreas development related genes waits mark pancreas precursor cell, and human embryo stem cell can obtain this class pancreas precursor cell by external evoked differentiation, but they are lower to the efficient of sophisticated functional beta cell directional differentiation, and are transplanted in the diabetic mice model body reverting diabetes symptom effectively.The pancreas precursor cell in Kroon study group report human embryo stem cell source is transplanted in the immunodeficient mouse body and can be formed sophisticated islet cells after 3 months, but the tumorigenicity of precursor cell can not promoted it from now on to clinical application.Therefore, compared to the pancreas precursor cell, sophisticated islet cells is better donorcells source, remains one of bottleneck of diabetes stem cell therapy but how to induce the pancreas precursor cell to produce islet cells effectively.
What pancreas allelotaxis process had comprised a series of complexity organizes interphase interaction and signal transmission.In the growth course of tire pancreas, interaction between epithelium-mesenchyme, epithelium-endothelium and endothelium-mesenchymal cell and plays a significant role in the keeping of pancreas precursor, pars exocrina pancreatis and the specialization of internal secretion portion, the differentiation and the maturation of islet cells simultaneously and collaborative the generation.Yet these functions are found on as the zebra fish animal model lower animal mostly, for human pancreas's growth course, still lack relevant research report.In the atomization of embryonic stem cell, there are the dissimilar cell masses of blended; And become very complicated and difficult of the process that the molecular signal that the inducing function insulin secretory cell produces make is explored in cell and intercellular interaction.Can the specialization endoderm cell and the chemical molecular and the coherent signal path thereof of pancreas precursor cell although can search out some with chemical method for screening, it is incomplete being based on the information that impure mixed system screening obtains.Purifying pancreas precursor cell can help further further investigation and regulate islet cells differentiation and sophisticated key signal path.Therefore, will be all significant if can search out the surface markers gene of pancreas precursor cell for separation and further investigation pancreas precursor cell.But in view of the limitation that researching human body is grown, the pancreas precursor cell of originating with human embryo stem cell is a research model, and the surface markers gene of seeking its purifying is one of best at present research system.
The relevant report that the gene studies of pancreas precursor cell surface markers has been arranged in the past few years, successively.Suzuki etc. utilize c-Met, and the acceptor of pHGF (hepatocyte growth factor) comes to have in the sorting adult mice pancreatic precursor cell of height multiplication capacity and multidirectional differentiation potential.CD133 has been used to separate the embryonic pancreas precursor cell in newborn and adult mouse body, and this class isolated cells can produce external secretion, internal secretion and the vessel cell of pancreas.In other report, in 15.5 days embryonic pancreas of mouse, the low presentation markup of the CD133 positive/CD49f a group internal secretion precursor cell, and this presentation markup proof is also guarded in people's tire pancreas.Except this example report, because the restriction of the aspect of drawing materials yet there are no other relevant reports about human pancreas's precursor cell surface markers.Because people and rodent pancreas development have very big-difference, also be not very clear that body tag before the pancreas of whether reporting at present is applicable to people's pancreas precursor cell at present on mouse model.Therefore, the vitro differentiation model of human embryo stem cell will be grown the research platform that provides to imitate very much for the research human pancreas, help exploring the specific marker of human pancreas's precursor cell.Up to the present, yet there are no the relevant report of the surface markers of the pancreatic cell of originating in the research of now delivering about human embryo stem cell.
The aminoacid sequence NCBI Reference Sequence:NP_037362.1 of CD24.Specifically shown in SEQ ID NO:1.The aminoacid sequence NCBI Reference Sequence:NP_000200.1 of PDX1 (pancreatic and duodenal homeobox 1).Specifically shown in SEQ ID NO:2.The aminoacid sequence NCBI Reference Sequence:NP_000337.1 of SOX9.Specifically shown in SEQ ID NO:3.The aminoacid sequence NCBI Reference Sequence:NP_004489.1 of HNF6 (hepatocyte nuclear factor 6).Specifically shown in SEQ ID NO:4.The aminoacid sequence NCBI Reference Sequence:NP_000449.1 of HNF1b (hepatocyte nuclear factor 1-beta).Specifically shown in SEQ ID NO:5.The aminoacid sequence NCBI Reference Sequence:NP_005506.3 of HB9 (Hlxb9).Specifically shown in SEQ ID NO:6.The aminoacid sequence NCBI Reference Sequence:NP_068556.2 of FOXA2 (forkhead box A2).Specifically shown in SEQ ID NO:7.NKX6.1 (NK6 homeobox 1) aminoacid sequence NCBI Reference Sequence:NP_006159.2.Specifically shown in SEQ ID NO:8.
Summary of the invention
An object of the present invention is to provide the application of antibody in enrichment pancreas precursor cell of CD24; Or, the application of the antibody of CD24 in the test kit of preparation enrichment pancreas precursor cell; Or, the application of CD24 in enrichment pancreas precursor cell; Or CD24 is as the application of target in enrichment pancreas precursor cell.
In the above-mentioned application, described pancreas precursor cell is to be differentiated by the human embryo stem cell that exsomatizes.
In above-mentioned arbitrary application, described stripped human embryo stem cell is clone H1 or clone H9.
In above-mentioned arbitrary application, the described vitro differentiation that is divided into.
In above-mentioned arbitrary application, the described method that is differentiated by the human embryo stem cell that exsomatizes comprises the steps:
1) uses described stripped human embryo stem cell instead cell culture fluid I, cultivated 4 days;
2) cell that step 1) is obtained is directly used cell culture fluid II instead, cultivates 6-8 days;
3) with step 2) cell that obtains directly uses cell culture fluid III instead, cultivated 3-5 days;
Described cell culture fluid I prepares as follows and obtains: with DMEM/F12 substratum, B27, activin A and wortmannin mix, obtain cell culture fluid I, wherein DMEM/F12 substratum, B27, the proportioning of activin A and wortmannin is 1ml: 5 μ l: 100ng: 0.5nmol;
Described cell culture fluid II prepares as follows and obtains: with the IMDM substratum, the F12 substratum, B27, RA, FGF7 and NOGGIN mix, obtain cell culture fluid II, IMDM substratum wherein, F12 substratum, B27, RA, the proportioning of FGF7 and NOGGIN is 0.5ml: 0.5ml: 10 μ l: 2nmol: 20ng: 100ng;
Described cell culture fluid III prepares as follows and obtains: with the DMEM substratum, N2 and EGF mix, and obtain cell culture fluid III, DMEM substratum wherein, and the proportioning of N2 and EGF is 1ml: 10 μ l: 50ng;
In the described step 1), culture condition is: temperature is 37 ℃, 5% carbonic acid gas;
Described step 2) in, culture condition is: temperature is 37 ℃, 5% carbonic acid gas;
In the described step 3), culture condition is: temperature is 37 ℃, 5% carbonic acid gas;
In the described step 1), the inoculum density of cell is 50%-80%.
In above-mentioned arbitrary application, described pancreas precursor cell is to possess following 1) and/or 2) shown in the cell of condition:
1) is expressed as follows at least a in the albumen: PDX1, SOX9, HNF6, HNF1b, HB9 and FOXA2;
2) can be divided into the Regular Insulin positive cell.
In above-mentioned arbitrary application, described Regular Insulin positive cell is at least a cell that possesses in following I, II and the III condition:
I, excreting insulin; II, expressing protein PDX1; III, expressing protein NKX6.1.
In above-mentioned arbitrary application, described enrichment is to carry out in the cell that described step 3) obtains; The antibody of described CD24 is the monoclonal antibody of polyclonal antibody or the anti-CD24 of anti-CD24.
In above-mentioned arbitrary application, the polyclonal antibody of described anti-CD24 is the how anti-FL80 in rabbit source, and the monoclonal antibody of described anti-CD24 is the monoclonal antibody ML5 in mouse source or the monoclonal antibody SN3 in mouse source.
Another object of the present invention provide a kind of from cell mass the method for enrichment purpose pancreas precursor cell.
Provided by the present invention from cell mass the method for enrichment purpose pancreas precursor cell, comprise the steps: to utilize the method pair cell group of airflow classification to separate with CD24 antibody, collect the CD24 positive cell, promptly obtain purpose pancreas precursor cell.
Described cell mass is to be differentiated by the human embryo stem cell that exsomatizes; Specifically differentiate as follows:
1) uses described stripped human embryo stem cell instead cell culture fluid I, cultivated 4 days;
2) cell that step 1) is obtained is directly used cell culture fluid II instead, cultivates 6-8 days;
3) with step 2) cell that obtains directly uses cell culture fluid III instead, cultivates 3-5 days, obtains described cell mass;
Described cell culture fluid I prepares as follows and obtains: with DMEM/F12 substratum, B27, activin A and wortmannin mix, obtain cell culture fluid I, wherein DMEM/F12 substratum, B27, the proportioning of activin A and wortmannin is 1ml: 5 μ l: 100ng: 0.5nmol;
Described cell culture fluid II prepares as follows and obtains: with the IMDM substratum, the F12 substratum, B27, RA, FGF7 and NOGGIN mix, obtain cell culture fluid II, IMDM substratum wherein, F12 substratum, B27, RA, the proportioning of FGF7 and NOGGIN is 0.5ml: 0.5ml: 10 μ l: 2nmol: 20ng: 100ng;
Described cell culture fluid III prepares as follows and obtains: with the DMEM substratum, N2 and EGF mix, and obtain cell culture fluid III, DMEM substratum wherein, and the proportioning of N2 and EGF is 1ml: 10 μ l: 50ng;
In the described step 1), culture condition is: temperature is 37 ℃, 5% carbonic acid gas;
Described step 2) in, culture condition is: temperature is 37 ℃, 5% carbonic acid gas;
In the described step 3), culture condition is: temperature is 37 ℃, 5% carbonic acid gas;
In the described step 1), the inoculum density of cell is 50%-80%.
In the described method, described pancreas precursor cell is to possess following 1) and/or 2) shown in the cell of condition:
1) is expressed as follows at least a in the albumen: PDX1, SOX9, HNF6, HNF1b, HB9 and FOXA2;
2) can be divided into the Regular Insulin positive cell.
Described Regular Insulin positive cell is at least a cell that possesses in following I, II and the III condition:
I, excreting insulin;
II, expressing protein PDX1;
III, expressing protein NKX6.1.
In the described method, the antibody of described CD24 is the monoclonal antibody of polyclonal antibody or the anti-CD24 of anti-CD24.
In the described method, the polyclonal antibody of described anti-CD24 is the how anti-FL80 in rabbit source, and the monoclonal antibody of described anti-CD24 is the monoclonal antibody ML5 in mouse source or the monoclonal antibody SN3 in mouse source.
In above-mentioned arbitrary application or the separation method, the aminoacid sequence of described CD24 is shown in SEQ ID NO:1; The aminoacid sequence of described PDX1 is shown in SEQ ID NO:2; The aminoacid sequence of described SOX9 is shown in SEQ ID NO:3; The aminoacid sequence of described HNF6 is shown in SEQ ID NO:4; The aminoacid sequence of described HNF1b is shown in SEQ ID NO:5; The aminoacid sequence of described HB9 is shown in SEQ ID NO:6; The aminoacid sequence of described FOXA2 is shown in SEQ ID NO:7; The aminoacid sequence of described NKX6.1 is shown in SEQ ID NO:8.
The present invention has found that a brand-new specific surfaces tagged molecule of pancreas precursor cell in human embryo stem cell source is CD24.The CD24 coexpression is in PDX1 and the distinctive crucial transcription factor of other pancreas precursor cell, and has only the CD24 positive cell to break up to become the Regular Insulin positive cell.These digital proofs CD24 can be used for the pancreas precursor cell in enrichment human embryo stem cell source.Only need to use surface molecular mark of CD24, just the very big enrichment human embryo stem cell of the degree pancreas precursor cell of originating.The present invention will make purpose pancreas precursor cell obtain the enrichment of certain degree, to help accurately screening of the effect of the signal in various endotheliums or a matter source like this, thereby the research of whole pancreas differentiation will be played considerable pushing effect further differentiation and maturation.
Description of drawings
Fig. 1 is the existence of pancreas precursor cell in the human embryo stem cell atomization.
Fig. 2 is for determining the coexpression of CD24 and PDX1.
Fig. 3 is for determining CD24 and the PDX1 coexpression under different clones and different differentiation condition.
Fig. 4 expresses the transcription factor of pancreas precursor cell key for immunofluorescence proof CD24 positive cell.
Fig. 5 expresses the transcription factor of pancreas precursor cell key for quantitative PCR proof CD24 positive cell.
Fig. 6 can form the clone for the CD24 positive cell and keep the expression of CD24 and PDX1, and the CD24 negative cells can not.
Fig. 7 can be divided into the Regular Insulin positive cell for the CD24 positive cell, and the CD24 negative cells can not.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Specific proteins coexpression in embodiment 1, CD24 and the pancreas precursor cell
One, determines CD24 and the PDX1 coexpression in the pancreatic cell in human embryo stem cell source
Pancreas differentiation culture concrete steps are as follows:
1): use human embryonic stem cell H1 instead cell culture fluid I (inoculum density is 80%), under temperature is 37 ℃, 5% carbon dioxide conditions, cultivated 4 days;
2) cell that step 1) is obtained is directly used cell culture fluid II instead, under temperature is 37 ℃, 5% carbon dioxide conditions, cultivates 6 days;
3) with step 2) cell that obtains directly uses cell culture fluid III instead, under temperature is 37 ℃, 5% carbon dioxide conditions, cultivates 5 days, obtains the pancreas precursor cell;
Described cell culture fluid I prepares as follows and obtains: with DMEM/F12 substratum, B27, activin A and wortmannin mix, obtain cell culture fluid I, wherein DMEM/F12 substratum, B27, the proportioning of activin A and wortmannin is 1ml: 5 μ l: 100ng: 0.5nmol;
Described cell culture fluid II prepares as follows and obtains: with the IMDM substratum, the F12 substratum, B27, RA, FGF7 and NOGGIN mix, obtain cell culture fluid II, IMDM substratum wherein, F12 substratum, B27, RA, the proportioning of FGF7 and NOGGIN is 0.5ml: 0.5ml: 10 μ l: 2nmol: 20ng: 100ng;
Described cell culture fluid III prepares as follows and obtains: with the DMEM substratum, N2 and EGF mix, and obtain cell culture fluid III, DMEM substratum wherein, and the proportioning of N2 and EGF is 1ml: 10 μ l: 50ng.
Detect whether coexpression of a plurality of transcription factors and CD24 with immuno-fluorescent antibody technique.Wherein, the antibody of using is as follows:
Detect the antibody of CD24: the how anti-FL80 of the anti-CD24 in rabbit source is available from Santa Cruz, and catalog number is sc-11406; The monoclonal antibody SN3 of the anti-CD24 in mouse source is available from Santa Cruz, and catalog number is sc-19585; And the monoclonal antibody ML5 in mouse source, be to use the PE mark, available from BD Bioscience, catalog number is 555428.
The antibody that detects PDX1 is the how anti-of rabbit source, and this antibody is available from Abcam, and catalog number is ab47267; And goat source is how anti-, and this antibody is available from Abcam, and catalog number is ab47383.
The antibody that detects SOX9 is the how anti-of rabbit source; This antibody is available from Santa Cruz, and catalog number is sc-20095.The antibody that detects HNF6 is the how anti-of rabbit source; This antibody is available from Santa Cruz, and catalog number is sc-13050.The antibody that detects HNF1b is the how anti-of sheep source; This antibody is available from Santa Cruz, and catalog number is sc-7411.The antibody that detects HB9 is the monoclonal antibody in mouse source; This antibody is available from Developmental Studies Hybridoma Bank, and catalog number is 81.5C10.The antibody that detects FOXA2 is the how anti-of sheep source; This antibody is available from R﹠amp; D Systems, catalog number is AF2400.The antibody that detects CD133 is the how anti-of rabbit source; This antibody is available from Santa Cruz, and catalog number is SC-30220.The antibody that detects E-cadherin is the monoclonal antibody in mouse source; This antibody is available from Abcam, and catalog number is ab1416.The antibody that detects N-cadherin is the monoclonal antibody in mouse source; This antibody is available from BD Bioscience, and catalog number is 610920.The antibody that detects NCAM is the monoclonal antibody in mouse source; This antibody is available from Santa Cruz, and catalog number is SC-7326.The antibody that detects VE-cadherin is the how anti-of sheep source; This antibody is available from Santa Cruz, and catalog number is SC-6458.The antibody that detects HCAM is the monoclonal antibody in rat source; This antibody is available from Santa Cruz, and catalog number is SC-18849.The antibody that detects PDGFR α is the how anti-of rabbit source; This antibody is available from Abcam, and catalog number is ab5460.
The monoclonal antibody ML5 that more than removes CD24 is the PE mark, all the other all antibody are non-straight labeling antibody, purchasing the antibody that comes with Jackson ImmunoResearch company respectively in the detection redyes: many anti-(catalog number is 705-026-147) of the anti-goat in the donkey source of Rhodamine mark, many anti-(catalog number is 705-096-147) of the anti-goat in the donkey source of FITC mark, many anti-(catalog number is 715-026-150) of the anti-mouse in the donkey source of Rhodamine mark, many anti-(catalog number is 711-096-152) of the anti-rabbit in the donkey source of FITC mark, many anti-(catalog number is 705-496-147) of the anti-goat in the donkey source of DyLight-649 mark.Many anti-(catalog number is 712-096-153) of the Chinese People's Anti-Japanese Military and Political College mouse in the donkey source of FITC mark.
Result: in as above pancreas differentiated system, have the stage of a plurality of transcription factor co expression in the atomization, comprise PDX1, HNF1b, SOX9, FOXA2, HNF6 and PROX1 (Figure 1A), and these PDX1 or HNF6 positive cell are not expressed SOX17 (Figure 1B).The immunofluorescence of being carried out PDX1 and many surface moleculars this stage dyes altogether, finds that tentatively the dyeing of CD24 and PDX1 is quite mated, and, in the CD24 negative cells, do not observe PDX1 painted (Fig. 1 C).
Two, several method is confirmed the coexpression of CD24 and PDX1
Differentiated system is with identical described in the experiment one.
1, dyes altogether with three kinds of CD24 antibody and PDX1 respectively
At first, there were some differences in different commercially available antibodies because CD24 is in the news, so selected 3 kinds of commercially available antibodies altogether, comprised the how anti-FL80 in a rabbit source, the monoclonal antibody ML5 and the SN3 in two mouse sources.
Detect whether coexpression of PDX1 and CD24 with immuno-fluorescent antibody technique.
The antibody that detects CD24 is as follows respectively:
Detect the antibody of CD24: the how anti-FL80 of the anti-CD24 in rabbit source is available from Santa Cruz, and catalog number is sc-11406; And the monoclonal antibody SN3 of the anti-CD24 in mouse source is available from Santa Cruz, and catalog number is sc-19585; And the monoclonal antibody ML5 in mouse source, be to use the PE mark, this antibody is available from BD Bioscience, and catalog number is 555428.The antibody that detects PDX1 is the how anti-of goat source, and this antibody is available from Abcam, and catalog number is ab47383.
More than all antibody all purchase the antibody that comes and redye with Jackson ImmunoResearch company: the anti-rabbit in many anti-(catalog number is 715-026-150) of the anti-mouse in many anti-(catalog number is 705-096-147) of the anti-goat in the donkey source of FITC mark and the donkey source of Rhodamine mark or the donkey source of Rhodamine mark resist (catalog number is 711-026-152) more.
The result shows that three antibody all demonstrate and good the dying altogether of PDX1 (Fig. 2 A).
2, flow cytometer showed
Method: purchase the cell fixation/penetrating test kit (catalog number is 554714) that comes according to BD Biosciences and carry out.(resisting of goat source available from R﹠amp more with using PDX1 antibody after the cell fixation; D Systems, catalog number is AF2419) and CD24 antibody (the monoclonal antibody SN3 in mouse source, available from Santa Cruz, catalog number is sc-19585) dyeing, afterwards again with fluorescence two anti-redying.Analyze with flow sorter.Two resist many anti-(catalog number is 715-116-150) of the anti-mouse of originating available from the donkey of Jackson ImmunoResearch:PE mark.
From the result of PDX1 and CD24, stream data is presented in the embryonic stem cell of differentiation and mainly only has two groups of cells, two positives and jack to jack adapter (Fig. 2 B), and this has also illustrated coexpression relation of PDX1 and CD24.
3, airflow classification
Obtained the negative and CD24 positive cell of CD24 with airflow classification.This two groups of gene expression of cells situations have been analyzed with quantitative PCR technique.The PDX1 expression amount of CD24 positive cell on average is 26 times of negative cells group, is 2.5 times (Fig. 2 C) of mixed cellularity group before the sorting.
The primer of quantitative PCR detection PDX1 is 5 '-GGTGGAGCTGGCTGTCATGT-3 ', 5 '-CGCGCTTCTTGTCCTCCTC-3 '.
Three, the coexpression of PDX1 and CD24 detects in the different differentiated systems
Two clones of generally acknowledging have at present been selected, H1 and H9; And two kinds of differentiation schemes that application is maximum, based on Matrigel and based on feeder layer cells (Feeder).Respectively each clone is all used two kinds of differentiated systems, differentiated system is with experiment one.
Experimental result shows, in two kinds of clones and two kinds of differentiation schemes, there are the good relation of dying altogether (Fig. 3) all the time in CD24 and PDX1.
Four, the CD24 positive cell is expressed the transcription factor of pancreas precursor cell key
Really represented the pancreas precursor cell, the expression conditions of analysis CD24 positive cell group in order to prove the CD24 positive cell group.Pancreas precursor cell group has special transcription factor expression network, and these crucial transcription factors and CD24 are carried out immunofluorescence analysis.Human embryo stem cell is that H1 and human embryo stem cell are that H9 all detects.Differentiated system is with consistent described in the experiment one.
As a result, the CD24 positive cell not only with the PDX1 coexpression, and and other crucial transcription factor, comprise SOX9, HNF6, HNF1B and HB9 have good coexpression relation (Fig. 4, picture is from H9, the result of H1 is similar) herein.
Five, further prove multiple transcription factor coexpression in the CD24 positive cell
Human embryo stem cell and differentiated system are with consistent described in the experiment one.
Collect CD24 positive cell and CD24 negative cells.And utilize quantitative PCR detection to test the expression of crucial transcription factor described in four.
The primer that detects Pdx1 is 5 '-GGTGGAGCTGGCTGTCATGT-3 ', 5 '-CGCGCTTCTTGTCCTCCTC-3 '; The primer that detects Foxa2 is 5 '-CTGAGCGAGATCTACCAGTGGA-3 ', 5 '-CAGTCGTTGAAGGAGAGCGAGT-3 '; The primer that detects Hnf6 is 5 '-TGTGGAAGTGGCTGCAGGA-3 ', 5 '-TGTGAAGACCAACCTGGGCT-3 '; The primer that detects Hes1 is 5 ' AGCACACTTGGGTCTGTGC-3 ', 5 '-TGAAGAAAGATAGCTCGCGG-3 '; The primer that detects Prox1 is 5 '-CAATTTCCACACCGCCAAC-3 ', 5 '-TCAGTGGAACTGGCCATCTG-3 ';
The result: with the experiment four in the immunofluorescence data consistent, comprise Pdx1, Foxa2, Hnf6, Hes1 and Prox1, the expression amount of the mRNA of these transcription factors in the CD24 positive cell group apparently higher than negative cells group (Fig. 5).
Above result proves that the CD24 positive cell is expressed the transcription factor of pancreas precursor cell key.
Embodiment 2, with the antibody enrichment pancreas precursor cell of anti-CD24
Human embryonic stem cell H1 is available from WiCell Research Institute, and NIH is numbered WA01; Human embryonic stem cell H9 is available from WiCell Research Institute, and NIH is numbered WA09.
The DMEM/F12 substratum is available from Invitrogen, and catalog number is 11330-032; KnockOut SR substratum is available from Invitrogen, and catalog number is 10828-028; BFGF is available from PeproTech, and catalog number is AF-100-18B;
B27 is available from Invitrogen, and catalog number is 17504044; Activin A is available from PeproTech, and catalog number is 120-14; Wortmannin is available from Sigma, and catalog number is W1628;
The IMDM substratum is available from Invitrogen, and catalog number is 12440-061; The F12 substratum is available from Invitrogen, and catalog number is 11765-054; RA is available from Sigma, and catalog number is R2625; FGF7 is available from PeproTech, and catalog number is AF-100-19; NOGGIN is available from PeproTech, and catalog number is 120-10C;
The DMEM substratum is available from Invitroge, and catalog number is C11965500BT; N2 is available from Invitrogen, and catalog number is 17502-048; EGF is available from PeproTech, and catalog number is AF-100-15;
The monoclonal antibody ML5 of the anti-CD24 in the straight target mouse of PE source is available from BD Bioscience, and catalog number is 555428.
One, the pre-cultivation: cultivate and on the mouse embryo fibroblasts that mitomycin was handled, carry out.Substratum is as follows: every 0.8ml DMEM/F12 substratum, 0.2ml KnockOut SR substratum and 10ng bFGF are mixed obtaining.
Two, differentiation culture
1): use human embryonic stem cell H1 instead cell culture fluid I (inoculum density is 80%), under temperature is 37 ℃, 5% carbon dioxide conditions, cultivated 4 days;
2) cell that step 1) is obtained is directly used cell culture fluid II instead, under temperature is 37 ℃, 5% carbon dioxide conditions, cultivates 6 days;
3) with step 2) cell that obtains directly uses cell culture fluid III instead, under temperature is 37 ℃, 5% carbon dioxide conditions, cultivates 5 days, obtains the pancreas precursor cell;
Described cell culture fluid I prepares as follows and obtains: with DMEM/F12 substratum, B27, activin A and wortmannin mix, obtain cell culture fluid I, wherein DMEM/F12 substratum, B27, the proportioning of activin A and wortmannin is 1ml: 5 μ l: 100ng: 0.5nmol;
Described cell culture fluid II prepares as follows and obtains: with the IMDM substratum, the F12 substratum, B27, RA, FGF7 and NOGGIN mix, obtain cell culture fluid II, IMDM substratum wherein, F12 substratum, B27, RA, the proportioning of FGF7 and NOGGIN is 0.5ml: 0.5ml: 10 μ l: 2nmol: 20ng: 100ng;
Described cell culture fluid III prepares as follows and obtains: with the DMEM substratum, N2 and EGF mix, and obtain cell culture fluid III, DMEM substratum wherein, and the proportioning of N2 and EGF is 1ml: 10 μ l: 50ng.
Three, with CD24 antibody enrichment purpose pancreas precursor cell
Monoclonal antibody ML5 with the anti-CD24 in the straight target mouse of PE source carries out airflow classification with the cell that above-mentioned steps two obtains, and the cell that above-mentioned steps two is obtained is divided into two groups: the positive group of CD24 and CD24 is negative organizes.
The positive group of CD24 cell is purpose pancreas precursor cell (can be divided into the pancreas precursor cell of pancreas).
Four, the CD24 positive that detects enrichment organizes whether cell is real positive pancreas precursor cell
1, verifies by cell surface marker protein expression situation
Whether detect each marker protein with immuno-fluorescent antibody technique expresses in the CD24 positive cell.During detection used antibody all with embodiment 1 in consistent described in the experiment one.
The result: the CD24 positive cell is expressed as follows albumen: PDX1, SOX9, HNF6, HNF1b, HB9 and FOXA2.These albumen all are the distinctive transcription factor expression networks of pancreas precursor cell group, therefore, illustrate that the CD24 positive cell promptly is the pancreas precursor cell.
2, verify by ability of cell proliferation
The positive group of CD24 and the negative group of CD24 cell be inoculated in respectively test two cell culture fluid III, under temperature is 37 ℃, 5% carbon dioxide conditions, cultivate a week.Detect the growthhabit of cell.
The result: with CD24 positive cell and negative cells group's unicellular heavy shop, the CD24 positive cell can form a large amount of clones, and these clones have kept the expression of CD24 and PDX1; By contrast, the CD24 negative cells almost can not form the clone, does not also express CD24 or PDX1 (Fig. 6).
Repetition, unanimity are as a result established in experiment 3 times.Show that the CD24 positive cell demonstrates certain multiplication capacity.The CD24 negative cells almost can not form the clone.By contrast, the CD24 positive cell can form, and embodying the CD24 positive cell like this has certain multiplication capacity.
3, obtain the Regular Insulin positive cell and verify by whether breaking up
Differentiation method: the CD24 positive cell and the CD24 negative cells of sorting is described respectively after medium ii I cultivates a week by experiment four-2, use following cell culture fluid instead: with DMEM/F12 substratum, B27, bFGF and nicotinamide mix, wherein DMEM/F12 substratum, B27, the proportioning of bFGF and nicotinamide is 1ml: 5 μ l: 10ng: 10 μ mol.Under temperature is 37 ℃, 5% carbon dioxide conditions, cultivate a week, can detection obtain the Regular Insulin positive cell.
The cell that differentiates of proof is the method for Regular Insulin positive cell: detect the cell that differentiates and whether express PDX1, NKX6.1 and expression of insulin (INS) whether.Specific as follows:
The antibody that detects NKX6.1 is the monoclonal antibody in mouse source, and available from Developmental Studies Hybridoma Bank, catalog number is F55A12;
The antibody that detects INS is the how anti-of cavy (Guinea Pig) source, and available from Dako, catalog number is A0564;
The antibody that detects PDX1 is the how anti-of goat source, and this antibody is available from Abcam, and catalog number is ab47383.
More than all antibody all purchase the antibody that comes and redye with Jackson ImmunoResearch company: the anti-cavy in many anti-(catalog number is 715-026-150) of the anti-mouse in many anti-(catalog number is 705-096-147) of the anti-goat in the donkey source of FITC mark and the donkey source of Rhodamine mark or the donkey source of Rhodamine mark resist (catalog number is 706-026-148) more.
Experimental data shows that under identical differentiation condition, the CD24 positive cell can further differentiate the endocrine cell of expressing PDX1, NKX6.1 and Regular Insulin, but the CD24 negative cells does not have this differentiation capability (Fig. 7).
Comprehensive above-mentioned experiment shows that the CD24 positive cell is purpose pancreas precursor cell (being the Regular Insulin positive cell) in the cell that is obtained by human embryonic stem cell H1 differentiation.
The enrichment of embodiment 3, checking CD24 obtains differentiation on human embryonic stem cell H9 pancreas precursor cell
Method: basic identical described in one to four with experiment, different that be to use human embryonic stem cell is H9.
Result: do not have significant difference with result described in the experiment four.
Claims (10)
1.CD24 the application of antibody in enrichment pancreas precursor cell; Or, the application of the antibody of CD24 in the test kit of preparation enrichment pancreas precursor cell; Or, the application of CD24 in enrichment pancreas precursor cell; Or CD24 is as the application of target in enrichment pancreas precursor cell.
2. application according to claim 1 is characterized in that: described pancreas precursor cell is to be differentiated by the human embryo stem cell that exsomatizes.
3. application according to claim 1 and 2 is characterized in that: described stripped human embryo stem cell is clone H1 or clone H9.
4. according to claim 1 or 2 or 3 described application, it is characterized in that: the described vitro differentiation that is divided into.
5. according to arbitrary described application among the claim 1-4, it is characterized in that: the described method that is differentiated by the human embryo stem cell that exsomatizes comprises the steps:
1) uses described stripped human embryo stem cell instead cell culture fluid I, cultivated 4 days;
2) cell that step 1) is obtained is directly used cell culture fluid II instead, cultivates 6-8 days;
3) with step 2) cell that obtains directly uses cell culture fluid III instead, cultivated 3-5 days;
Described cell culture fluid I prepares as follows and obtains: with DMEM/F12 substratum, B27, activin A and wortmannin mix, obtain cell culture fluid I, wherein DMEM/F12 substratum, B27, the proportioning of activin A and wortmannin is 1ml: 5 μ l: 100ng: 0.5nmol;
Described cell culture fluid II prepares as follows and obtains: with the IMDM substratum, the F12 substratum, B27, RA, FGF7 and NOGGIN mix, obtain cell culture fluid II, IMDM substratum wherein, F12 substratum, B27, RA, the proportioning of FGF7 and NOGGIN is 0.5ml: 0.5ml: 10 μ l: 2nmol: 20ng: 100ng;
Described cell culture fluid III prepares as follows and obtains: with the DMEM substratum, N2 and EGF mix, and obtain cell culture fluid III, DMEM substratum wherein, and the proportioning of N2 and EGF is 1ml: 10 μ l: 50ng;
In the described step 1), culture condition is: temperature is 37 ℃, 5% carbonic acid gas;
Described step 2) in, culture condition is: temperature is 37 ℃, 5% carbonic acid gas;
In the described step 3), culture condition is: temperature is 37 ℃, 5% carbonic acid gas;
In the described step 1), the inoculum density of cell is 50%-80%.
6. according to arbitrary described application among the claim 1-5, it is characterized in that: described pancreas precursor cell is to possess following 1) and/or 2) shown in the cell of condition:
1) is expressed as follows at least a in the albumen: PDX1, SOX9, HNF6, HNF1b, HB9 and FOXA2;
2) can be divided into the Regular Insulin positive cell.
7. according to arbitrary described application among the claim 1-6, it is characterized in that: described Regular Insulin positive cell is at least a cell that possesses in following I, II and the III condition:
I, excreting insulin;
II, expressing protein PDX1;
III, expressing protein NKX6.1.
8. according to arbitrary described application among the claim 1-7, it is characterized in that: described enrichment is to carry out in the cell that described step 3) obtains; The antibody of described CD24 is the monoclonal antibody of polyclonal antibody or the anti-CD24 of anti-CD24.
9. according to arbitrary described method among the claim 1-8, it is characterized in that: the polyclonal antibody of described anti-CD24 is the how anti-FL80 in rabbit source, and the monoclonal antibody of described anti-CD24 is the monoclonal antibody ML5 in mouse source or the monoclonal antibody SN3 in mouse source.
10. the method for an enrichment purpose pancreas precursor cell from cell mass comprises the steps: to utilize the method pair cell group of airflow classification to separate with CD24 antibody, collects the CD24 positive cell, promptly obtains purpose pancreas precursor cell.
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| CN116554330A (en) * | 2023-07-04 | 2023-08-08 | 天津旷博同生生物技术有限公司 | Anti-human CD24 engineering antibody and application thereof |
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| CN105646713A (en) * | 2014-01-22 | 2016-06-08 | 中国药科大学 | Monoclonal antibody and application thereof |
| CN105646712A (en) * | 2014-01-22 | 2016-06-08 | 中国药科大学 | Monoclonal antibody and application thereof |
| CN105646713B (en) * | 2014-01-22 | 2019-07-12 | 中国药科大学 | A kind of monoclonal antibody and its application |
| CN104725511A (en) * | 2015-02-16 | 2015-06-24 | 华南农业大学 | Separation culture method for pig intestinal stem cells |
| CN104725511B (en) * | 2015-02-16 | 2018-01-12 | 华南农业大学 | A kind of isolated culture method of pig intestinal stem cell |
| CN106699879A (en) * | 2016-12-19 | 2017-05-24 | 西安斯凯达生物制品有限公司 | Establishment method and application of type-A avian influenza specific monoclonal antibody |
| CN112512640A (en) * | 2018-06-04 | 2021-03-16 | 肿瘤免疫股份有限公司 | Method of use of CD24 for the prevention and treatment of graft versus host disease and mucositis |
| CN116554330A (en) * | 2023-07-04 | 2023-08-08 | 天津旷博同生生物技术有限公司 | Anti-human CD24 engineering antibody and application thereof |
| CN116554330B (en) * | 2023-07-04 | 2023-09-01 | 天津旷博同生生物技术有限公司 | Anti-human CD24 engineering antibody and application thereof |
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