CN102965393B - The preparation method and its usage of people's induced multi-potent stem cells - Google Patents
The preparation method and its usage of people's induced multi-potent stem cells Download PDFInfo
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- CN102965393B CN102965393B CN201210440090.7A CN201210440090A CN102965393B CN 102965393 B CN102965393 B CN 102965393B CN 201210440090 A CN201210440090 A CN 201210440090A CN 102965393 B CN102965393 B CN 102965393B
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Abstract
The present invention relates to a kind of preparation method and its usage of people's induced multi-potent stem cells of cancer target imaging technique field, described preparation method comprises the steps:, by slow-virus transfection human fibroblasts, to obtain people's induced multi-potent stem cells.The invention still further relates to described people's induced multi-potent stem cells and prepare the purposes in target in-vivo tumour focus preparation.People's induced multi-potent stem cells of the present invention can target in-vivo tumour focus, people's induced multi-potent stem cells is by after mark fluorescent magnetic Nano probe, can by specific enrichment after intravenous injection in tumor focus position, also there is molecular image function, tracking cells distribution in vivo under condition of living organism simultaneously.
Description
Technical field
The invention belongs to cancer target imaging technique field, be specifically related to a kind of preparation method and its usage of people's induced multi-potent stem cells.
Background technology
Tumour is considered to one of disease that at present most refractory heals, and gene therapy is the promising means of one for the treatment of tumour.Studies have reported that mescenchymal stem cell (MSCs) can realize oncotherapy as genophore, MSCs is separated to obtain from the tissues such as marrow, fat, corium, and from these tissues, be separated MSCs needs select suitable donor and carry out store period.And to obtain MSCs from single donor be limited, and can not carry out long-term propagation, these all have impact on MSCs carries out gene therapy operability as carrier.Embryonic stem cell is a kind of myeloid-lymphoid stem cell coming from the inner cell mass of blastaea, infinitely can rise in value in vitro and not lose its potential broken up, and embryonic stem cell (ESCs) is relative to other cells, be easy to carry out genetic modification, be more suitable for as genophore, but it is limited that embryonic stem cell has source, the restriction of the factor such as ethics and operative technique, develop comparatively slow, and inducibility pluripotent stem cell (induced pluripotent stem cells, iPSCs) be the multipotential stem cell with embryonic stem cell sample characteristic that reprogramming of somatic cells is obtained, present iPSCs can replace ESCs, thus it is limited to overcome ESCs source, the problems such as ethics restriction, therefore apply iPSCs to wish for tumor research brings treatment as " carrier " cell.
Up to the present, through the literature search to prior art, find that Hannon etc. the 431st volume the 7006th phase 371-378 page was published an article in 2004 " Unlocking the potential of thehuman genome with RNA interference " at " Nature " (nature) magazine, this article reports and passes through transgenic technology, a gene is with the addition of in hESC, this gene can control the anti-cancer molecules that stem cell expresses a kind of TRAIL by name, when this transgenosis stem cell is injected in the long mouse body having brain tumor, transgenosis stem cell is just attached on Tumor cells, and discharge TRAIL molecule.After experiment terminates, the volume averaging of these mouse brain tumors reduces 50%, and maximum even reduces 70%.Within 2012, the large auspicious people such as grade of Cui has delivered article " DiR-labeled Embryonic Stem Cells for Targeted Imaging of in vivoGastric Cancer Cells " at " Theranostics " (diagnosis and treatment) magazine the 2nd volume the 6th phase 618-628 page again, this article reports the effect that mouse embryo stem cell has stomach cancer cell in active targeting identity, and this transporting action may be because Chemokine CXCL12 in body and CXCR4 pairing effect caused.More than study and all found that ESCs has targets identification and the effect for the treatment of tumour, but, up to now, the report also not in iPSCs targets identification interior tumor cell.In fact iPSCs has convenient sources, realizes individualized treatment, anti-immunological rejection, improves the advantages such as patient's autoimmunity, in the early warning and treatment of tumour, have good application prospect, be a kind of instrument having the stem cell base treatment tumour of application prospect.
Prior art is compared, the present invention has following beneficial effect: the invention provides a kind of people's induced multi-potent stem cells and preparing the purposes in target in-vivo tumour focus preparation, people's induced multi-potent stem cells of the present invention can target in-vivo tumour focus, people's induced multi-potent stem cells is by after mark fluorescent magnetic Nano probe, can by specific enrichment after intravenous injection in tumor focus position, also there is molecular image function, tracking cells distribution in vivo under condition of living organism simultaneously.
Summary of the invention
The object of the invention is to make up the deficiencies in the prior art, a kind of preparation method and its usage of people's induced multi-potent stem cells is provided.People's induced multi-potent stem cells of the present invention can target in-vivo tumour focus, people's induced multi-potent stem cells is by after mark fluorescent magnetic Nano probe, can by specific enrichment after intravenous injection in tumor focus position, also there is molecular image function, tracking cells distribution in vivo under condition of living organism simultaneously.
The object of the invention is to be achieved through the following technical solutions:
First object of the present invention is the preparation method providing a kind of people's induced multi-potent stem cells, comprises the steps:, by slow-virus transfection human fibroblasts, to obtain people's induced multi-potent stem cells.
Preferably, described human fibroblasts is fibroblasts of adult human dermis or human foreskin fibroblast.
Preferably, described slow virus is the slow virus carrying Oct4, Sox2, Nanog and Lin28 transcription factor.
Preferably, described human fibroblasts is the human fibroblasts obtained in neoplastic disease human body by the method for original cuiture.
Second object of the present invention is to provide aforementioned people's induced multi-potent stem cells preparing the purposes in target in-vivo tumour focus preparation.
Preferably, described purposes comprises the steps: the preparation of people's induced multi-potent stem cells to be injected into human body, and people's induced multi-potent stem cells can initiative recognition in-vivo tumour focus.
Preferably, described tumour is cancer of the stomach, mammary cancer, prostate cancer, lung cancer, liver cancer, bladder cancer or cerebral glioma.
Preferably, described purposes comprises the steps:, by fluorescent mark people induced multi-potent stem cells, to be injected into human body, tracing in vivo, display people's induced multi-potent stem cells distribution in vivo, and then the position of tumor focus in display body.
Preferably, the material that described fluorescent mark uses is inorganic fluorescent nano material.
Preferably, described inorganic fluorescent nano material is quantum dot, fluorescence magnetic particle, the fluorescent nano particles of coated with silica or up-conversion.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is the alkaline phosphatase staining figure of the people iPSCs in embodiment 1;
Fig. 2 is the immunofluorescence dyeing figure of the people iPSCs in embodiment 1;
Fig. 3 is that in embodiment 1, suspension culture forms the expression (B) of each germinal layer specific gene in the aspect graph (A) of embryoid (EB) and RT-PCR quantitative analysis person iPSCs and EB;
Fig. 4 is visible ray and the fluorescence imaging composite diagram of the people iPSCs cell (B) of unlabelled people iPSCs cell (A) and fluorescence magnetic particle mark in embodiment 2;
Fig. 5 be the people iPSCs cell (B) of unlabelled people iPSCs cell (A) and fluorescence magnetic particle mark in embodiment 2 Prussian blue-core fast red redyes photo figure;
Fig. 6 is FMNPs(A in embodiment 2) and the iPSCs(B of FMNPs mark) 1h and 6h time point mouse living body fluorescent image after tail vein injection enters in tumor-bearing mice body.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.These embodiments are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, such as Sambrook equimolecular clone: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.
The preparation of embodiment 1, people iPSCs
The present embodiment relates to the preparation of a kind of people iPSCs, comprises the steps:
Step one, prepared by cell
Mouse embryo fibroblasts (mouse embryo fibroblast, MEF) purchased from Shanghai Si Dansai Bioisystech Co., Ltd, Secondary Culture to the MEF cell in three ~ five generations as trophocyte;
Original cuiture fibroblasts of adult human dermis (Human Dermal Fibroblasts, HDF): get patient's belly 2 ~ 3cm under aseptic condition
2skin, remove subcutis and epidermal area, use collagenase digesting skin corium, keep temperature to be 37 DEG C, the time is 2 hours, then the skin digested is extruded, and filter with mono layer gauze, nutrient solution DMEM substratum adds 10% foetal calf serum and is placed in incubator and cultivates, primary cell foundation after one week, adopt the method for original cuiture to prepare HDF cell, original cuiture to the HDF cell in three ~ five generations is used for virus transfection and prepares iPSCs;
Step 2, the foundation of people iPSCs
It is in the culture dish of 10cm that 4th generation HDF cell is seeded in diameter, cultivates after 24 hours when cell reaches the degree of converging of about 50%, changes antibiotic-free substratum;
By the slow virus liquid (four kinds of each 5ml of transcription factor virus liquid) carrying Oct4, Sox2, Nanog and Lin28 transcription factor fresh for the 20mL of packaging after 48 hours, this slow virus liquid can be packaged to be by virus, or buy from disclosed commercially available channel, add in substratum, add the polybrene that final concentration is 8 μ g/ml, transduction HDF cell simultaneously;
Discard nutrient solution after 24 hours, again to transduce HDF cell according to the fresh viral mixed solution of same method collecting packing after 72 hours.Be replaced by normal nutrient solution after 24 hours, continue cultivation 72 hours.Screen by the human embryonic stem cell medium containing selective agent Puromycin (10 μ g/mL) after 72 hours, change a nutrient solution later every day, until occur that initialized iPSCs clones;
Step 3, the amplification cultivation of people iPSCs
Reject the cell clone of edge disperse by mechanical process under microscope, the embryonic stem cell sample clone excision that centre is fine and close is that fritter is seeded on the MEF cell of mitomycin C process, with interpolation Y-27632 (10 μm of ol/L, Rho kinase inhibitor) human embryonic stem cell medium cultivate, change human embryonic stem cell medium every day to cultivate, its metamorphosis of close observation, can carry out second pass generation through the cultivations of 5 ~ 7 days;
Step 4, the biological characteristics of people iPSCs and multipotency qualification
Alkaline phosphatase expression identification: by third generation iPSCs cells rinsed with PBS 2 times, 20min is fixed with 4% paraformaldehyde, PBS washs 2 times again, 25mM Tris-Cl (pH9.0) rinsing 1 time, add freshly prepared fast red AP staining fluid, after room temperature places about 15-30 minute, namely positive cell presents redness, and PBS rinsing is with termination reaction, basis of microscopic observation, as shown in Figure 1.
The expression of immunofluorescence technique qualification iPSCs surface marker: iPSCs PBS washes 3 times, then fixes 20 minutes with 4% paraformaldehyde, and PBS washs 2 times, each 3 ~ 5 minutes; 0.2%Triton X-100 changes 5 minutes thoroughly, and PBS washes three times, and 5%BSA room temperature closes 30 minutes, four kinds of primary antibodies (SSEA-3, SSEA4, Tra-1-60, Tra-1-81) are diluted with 1%BSA, the primary antibodie of having diluted is added drop-wise on cell respectively, do not add primary antibodie as experiment contrast, be placed on 4 DEG C and spend the night, PBS washes three times, add two anti-(diluting with 1%BSA) lucifuges and hatch 2 hours, PBS washes 3 times, and DAPI redyes core, be placed in fluorescence microscopy Microscopic observation, as shown in Figure 2.
The formation qualification of embryoid: cell IV collagenase digesting, hatch more than 40min for 37 DEG C, after cell clone comes off, collecting cell group, natural subsidence, removes supernatant.Change human embryos body (EB) substratum into, suspension culture 7 days in without the low attaching plate of feeder layer.Cultivate after 7 days, the EB of formation is proceeded to 0.1% gelatin process culture dish in adherent culture 2 weeks, the significant gene that EB expresses each germinal layer is detected: from EB, extract total serum IgE by RT-PCR, TIANGEN public TIANScript RT Kit is adopted to synthesize cDNA, then be that template utilizes RT-PCR as shown in table 1 below to detect the primer sequence of the significant genetic expression of each germinal layer with cDNA, increase the significant gene of each germinal layer, and the expression in people iPSCs of the significant gene of each germinal layer is as negative control.Result as shown in Figure 3.
Table 1
The preparation of the people iPSCs cell that embodiment 2, fluorescence magnetic particle (FMNPs) mark
The present embodiment relates to the preparation of the people iPSCs cell that fluorescence magnetic particle (FMNPs) marks, and comprises the steps:
Step one, the iPSCs cell in vegetative period of taking the logarithm, adds human embryonic stem cell medium by FMNPs with the concentration of 50 μ g/mL, and the iPSCs not adding FMNPs is negative control group, the CO2 of 5%, and cultivate 2 hours for 37 DEG C, inhale and abandon substratum, PBS washes 3 times;
Step 2, fixes the people iPSCs cell 30 minutes of FMNPs mark, cell is divided into two groups with 2.5% glutaraldehyde solution, the fluorescence radiation situation of one group of people iPSCs marked by FMNPs at fluorescence microscopy Microscopic observation, and result as shown in Figure 4; Other one group of cell does Prussian blue-core fast red and redyes, first use potassium ferrocyanide solution 37 DEG C of incubated cells 30 minutes of 200g/L, use deionized water wash cell afterwards 3 times, cell is redyed 10 ~ 15 minutes again with 2g/L core fast red alum liquor, deionized water wash cell 3 times, examine under a microscope the situation of the Prussian blue NiHCF thin films of the people iPSCs cell marked by FMNPs, result as shown in Figure 5.
Step 3, the people iPSCs cell marked with the IV Collagenase Type digestion FMNPs of 1mg/mL 30 ~ 40 minutes, collecting cell clone afterwards, and it is resuspended with PBS damping fluid, repeatedly blowing and beating cell mass to cell with the rifle head of 1mL is single cell suspension, leaves standstill after 2 minutes, the cell mass do not disperseed, collect upper strata single cell suspension, place stand-by.
The living body fluorescent imaging of cancer of the stomach in the people iPSCs target that compliance test result: FMNPs marks, spike body, first the subcutaneous bearing mouse model of cancer of the stomach is set up, select male nude mouse 6, the body weight 20-22g in 4-5 age in week, wherein nude mice is all purchased from Shanghai Slac Experimental Animal Co., Ltd., every mouse bare subcutaneous injection 1 × 10
6gastric carcinoma cells (MGC803), SPF level environment raises 4-5 week, controls diameter of tumor at about 0.5cm.The subcutaneous tumor-bearing mice of cancer of the stomach is divided into two groups, A group tail vein injection fluorescence dye FMNPs, B group tail vein injection 5 × 10
6the people iPSCs of FMNPs mark, A group and B component do not inject 3 tumor-bearing mices, the ability that all nude mices all after injection 1 hour and 2 hours accept small animal living body fluorescence imaging scanning analysis iPSCs target stomach cancer with lateral position and situation about distributing in vivo, the condition of fluorescence imaging is excitation wavelength 460nm, emission wavelength 630nm, result as shown in Figure 6, A group mouse tail vein injection fluorescence dye after FMNPs2 hour tumor locus there is no fluorescent signal, the people iPSCs that B group mouse marks at tail vein injection FMNPs after 1 hour tumor locus have fluorescent signal, and along with the increase of inject time, the fluorescent signal of tumor locus increases gradually, after injection, the fluorescent signal at 2 hours Tumor positions is obviously better than the injection fluorescent signal of latter 1 hour, this illustrates that iPSCs has the effect of target in-vivo tumour focus.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (3)
1. people's induced multi-potent stem cells is preparing the purposes in target in-vivo tumour focus preparation; The preparation method of described people's induced multi-potent stem cells comprises the steps:, by slow-virus transfection human fibroblasts, to obtain people's induced multi-potent stem cells;
Described human fibroblasts is the human fibroblasts obtained in neoplastic disease human body by the method for original cuiture; Described human fibroblasts is fibroblasts of adult human dermis;
Described slow virus is the slow virus carrying Oct4, Sox2, Nanog and Lin28 transcription factor;
Described tumour is cancer of the stomach.
2. purposes as claimed in claim 1, it is characterized in that, described purposes comprises the steps: by fluorescent mark people induced multi-potent stem cells, be injected into human body, tracing in vivo, display people's induced multi-potent stem cells distribution in vivo, and then the position of tumor focus in display body;
The material that described fluorescent mark uses is inorganic fluorescent nano material.
3. purposes as claimed in claim 2, it is characterized in that, described inorganic fluorescent nano material is fluorescence magnetic particle.
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| CN105929001B (en) * | 2016-04-19 | 2018-04-10 | 南京大学 | The gold electrode and preparation method and application of specific DNA pseudoknot structure modification |
| CN106544315A (en) * | 2016-10-12 | 2017-03-29 | 广东艾时代生物科技有限责任公司 | A kind of method that fat mesenchymal stem cell induces into pluripotent stem cell |
| CN107198779A (en) * | 2016-11-29 | 2017-09-26 | 南京东纳生物科技有限公司 | A kind of nanometer coupled complex of stem cell labeling spike and targeting navigation and its preparation method and application |
| CN111265673B (en) * | 2020-03-02 | 2022-07-26 | 云南中科灵长类生物医学重点实验室 | Method for tracing stem cells of cynomolgus monkey by fluorescence |
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