CN114672463A - Stem cell line capable of carrying out multi-modal tracing, preparation method and application thereof - Google Patents
Stem cell line capable of carrying out multi-modal tracing, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a stem cell line capable of carrying out multi-modal tracing and a preparation method thereof, wherein the stem cell line comprises: umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, human induced pluripotent stem cells and vascular endothelial progenitor cells all over-expressing three proteins of FTH1, Sr39tk and EGFP. The invention further improves the application of the stem cell line as a tracer for MRI-PET multi-modal imaging. The stem cell line provided by the invention can be used for living cell tracing of multi-modal imaging combining MRI and PET, can effectively detect the movement and differentiation of transplanted cells, explores the action mechanism of the transplanted cells, is favorable for promoting stem cell treatment to be applied to clinic, and can provide a new scheme for preparing a living tracing stem cell reagent for treating human cerebral infarction.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a stem cell line capable of carrying out multi-modal tracing, and a preparation method and application thereof.
Background
The incidence of stroke has been increasing in developing countries over the last 40 years [1], with cerebral infarction accounting for 80% -85%. Cerebral infarction (ischemic stroke) is a clinically common acute cerebrovascular disease and one of three death reasons endangering human health. Acute cerebral infarction can cause rapid reduction of blood supply at ischemic parts, lead to necrosis of neurons, glial cells, vascular endothelial cells and the like, and then produce inflammatory reaction, blood brain barrier destruction, inflammatory medium infiltration and cause irreversible injury. The traditional thinking and methods are mostly followed in the treatment of cerebral apoplexy, such as acute thrombolysis, percutaneous vascular intervention, aspirin anticoagulant, acupuncture rehabilitation physiotherapy in recovery period and the like, and thrombolysis and interventional therapy have respective indications and limitations, and meanwhile, serious dysfunction such as paralysis, slurred speech and the like are left in most survivors after the conventional treatment of cerebral infarction, and further recovery and difficulty are caused.
Intravenous injection of recombinant tissue plasminogen activator (rtPA) is currently the only accepted method for treating ischemic stroke, but it has strict time window limitations and does not stimulate neural repair and regeneration. Animal experiments show that cerebral ischemia and hypoxia can stimulate endogenous neural stem cell proliferation and migrate and differentiate towards ischemic injury to repair nerves [2,3], but the spontaneous repair effect is very limited [4 ]. Therefore, if exogenous stem cell implantation can be applied to treat cerebral infarction, better curative effect can be obtained. In recent years, stem cell transplantation has a good effect in the basic and clinical research of cerebral infarction treatment, and breakthrough progress is brought to the cerebral infarction treatment.
The mechanisms by which stem cells treat cerebral infarction include promotion of nerve regeneration and inhibition of inflammatory responses. Experiments prove that the stem cell transplantation treatment can improve the nerve function of the cerebral infarction mice, and is mainly realized by reducing neurodegeneration, inhibiting the formation of colloid scars, promoting the regeneration of endogenous nerves, stabilizing blood brain barriers and the like [5,6 ]. Hassani et al [7] found that endogenous neurons can regenerate a small amount under normal conditions, that cerebral infarction enhances this effect, and that transplantation of neural stem cells can continuously enhance this proliferative activity. Transplanted stem cells can also adapt to post-infarction microenvironment, secrete various tissue trophic factors, regulate immune response and promote the stability of internal environment [8 ]. The mechanism by which stem cells influence endogenous nerve regeneration is not yet elucidated, but may be related to the interaction of growth factors, inflammatory mediators. Growth factors such as granulocyte colony stimulating factor, vascular endothelial growth factor, transforming growth factor alpha, ciliary neurotrophic factor, etc. have been shown to be involved in nerve regeneration following cerebral infarction [9-12 ]. The stem cell transplantation can improve the concentration of brain-derived neurotrophic factor, fibroblast growth factor, vascular endothelial growth factor and the like [6], and promote tissue remodeling and function recovery.
Meanwhile, research shows that the stem cell treatment can inhibit inflammatory reaction in brain. Tobin et al [13] discovered that the inflammatory response after cerebral infarction damages brain tissue not only in the acute phase but also in the chronic damage of ischemic penumbra. Bacigaluppi et al [5] demonstrate that neural stem cells can reduce delayed neuronal degeneration by inhibiting inflammatory responses. Cytokines secreted by stem cells also modulate post-infarct inflammatory responses [14 ]. The stem cell transplantation is used for treating cerebral infarction, and has the possibility of promoting endogenous nerve regeneration and brain tissue self-repair. Studies show that the application of stem cell transplantation to treat rats 48h after cerebral infarction can promote cell proliferation and migration of sub-ventricular membrane zone (SVZ) and maturation of new neurons, inhibit inflammatory reaction of microglia and macrophages, and improve behavioral functions of mice, and the effects can last for 14 weeks after infarction [15 ]. Peruzzotti-Jametti et al [16] indicated that the effects of inflammatory mediators on post-infarct nerve regeneration were variable, possibly related to differences in the phenotype, cellular molecular pathways responsible for the inflammatory mediators.
Stem cell therapy has shown increasing utility in cerebral infarction. However, at present, in addition to the use of hematopoietic stem cells for the treatment of blood diseases in China, most stem cell therapies are still in the stage of basic research or experimental clinical research, and the application of stem cell therapy to clinical treatment has a long way. The in vivo tracking technology of stem cells observes transplanted stem cells in vivo with high spatial resolution on the premise of not damaging the transplanted cells and target organs, and tracks the functions and survival conditions exerted by the transplanted cells [17-19 ]. The in vivo tracking technology of stem cells is to track the survival, distribution, function and the like of stem cells in a non-invasive manner in a living body, wherein the in vivo tracking technology mainly comprises optical imaging, radionuclide imaging, MRI and the like. The multi-modal imaging technology integrates the advantages of 2 or more imaging technologies, can effectively detect the movement and differentiation of transplanted cells, explores the action mechanism [20], makes the basic research more accurate, is favorable for promoting stem cell treatment to be applied to clinic and finally benefits patients.
The multi-mode reporter gene imaging is to transfer the reporter gene into stem cells, express related enzymes or proteins, and trace the stem cells through an imaging device [21 ]. The signal intensity of the imaging mode is not reduced along with cell division or diffusion, so the imaging mode can be used for tracking stem cells for a long time. The current commonly used reporter genes include Green Fluorescent Protein (GFP), firefly luciferase (Fluc), NIS and the like, and the fluorescent protein can help tissue section to observe fluorescence. The stem cell Magnetic Resonance Imaging (MRI) tracer technique has high spatial and temporal resolution as one of the earliest non-invasive imaging means for monitoring transplanted stem cells in vivo. The MRI reporter genes currently used for stem cell tracking are mainly ferritin reporter genes [22 ]. Ferritin is a 450kD globular protein composed of multiple heavy and light protein subunits, with either elevated heavy chains or associated light chains, which results in increased intracellular iron stores. Huang et al [23] transfects the mesenchymal stem cells with lentivirus containing ferritin heavy chain 1(FTH1) gene shuttle plasmid (pCDH-CMV-MCS-EF1-copGFP) and the bone mesenchymal stem cells marked by superparamagnetic iron oxide nanoparticles, and the two are respectively implanted into SD rats with cerebral ischemia reperfusion injury, and the MRI signal intensity of the bone mesenchymal stem cells marked by superparamagnetic iron oxide nanoparticles is observed to gradually weaken along with time, the cells marked by FTH1 show the same signal intensity within 10-60 days, and the FTH1 marker is more stable than the superparamagnetic iron oxide nanoparticles and is more suitable for long-term tracking. However, MRI sensitivity is relatively low and requires large contrast agent doses, which makes labeling of cells difficult. The medical tracing technology for stem cell nucleus is a cell tracing technology which adopts Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) to display the distribution and state of marked cells in living body by detecting radioactive tracer. The current reporter genes commonly used for nuclide imaging are herpes simplex virus type 1 thymidine kinase (HSV1-TK) or mutant strain Sr39TK [24], dopamine D2 receptor [25], human sodium iodine transporter [26 ]. PET has very high sensitivity but limited spatial resolution and can be partially compensated by combining it with MRI. In contrast to MRI, PET imaging selects suitable nuclides, enables detection of cells for long periods of time and enables assessment of their viability. MRI and PET can be highly complementary, and clinical MRI-PET scanners have recently become a reality. PET-MRI provides a powerful multi-modality approach to combined imaging of future human cell distribution, targeted delivery, viability and therapeutic efficacy.
Multimodal imaging is increasingly used in research related to stem cell allograft because of its unique advantages. Under the background of the current new medical mode, the research focus of stem cell multi-modal imaging lies in the continuous improvement of imaging instruments, the discovery of new stem cell specific molecular targets and reporter genes and the construction of multi-modal probes. Therefore, for modern and even future disease treatments, especially stem cell therapy and immunotherapy, the combination of multimodal techniques and in-vivo cell tracking with integrated smart probes would have great potential.
Reference:
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Disclosure of Invention
The technical problem to be solved by the present invention is to provide a stem cell line capable of performing multi-modal tracking, a preparation method thereof and an application thereof, in view of the above disadvantages in the prior art.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a stem cell line capable of multimodal tracking comprising: umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, human induced pluripotent stem cells and vascular endothelial progenitor cells all over-expressing three proteins of FTH1, Sr39tk and EGFP.
The invention also provides a preparation method of the stem cell line capable of carrying out multi-modal tracing, which comprises the following steps:
1) providing umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, human induced pluripotent stem cells and vascular endothelial progenitor cells;
2) cloning target genes FTH1, Sr39tk and EGFP to a pLVX vector to construct a lentivirus expression vector pLVX-FTH1-Sr39 tk-EGFP;
3) packaging lentiviruses to obtain a recombinant lentivirus containing FTH1, Sr39tk and EGFP genes;
4) The stem cell line capable of performing multi-modal tracing is characterized in that umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, human induced pluripotent stem cells and vascular endothelial progenitor cells are respectively infected by recombinant lentiviruses containing FTH1, Sr39tk and EGFP genes, so that four stem cells which respectively over-express three proteins of FTH1, Sr39tk and EGFP are obtained.
Preferably, the step of providing umbilical cord mesenchymal stem cells in step 1) specifically comprises:
1-1-1) cleaning the umbilical cord, shearing the umbilical cord into tissue blocks, and then placing the tissue blocks into a culture bottle for culture;
1-1-2) after 40% -50% of tissues in the bottle climb out of the cells, carrying out passage on the cells, and washing off tissue blocks and redundant impurities falling from the bottle by using PBS;
1-1-3) adding trypsin to digest the cells, then adding CO2Incubating in an incubator at 37 ℃;
1-1-4) observing the digested cells under an inverted microscope, adding a complete culture medium to neutralize and digest when the cells slightly fall off and the edges of the cells which do not fall off are round, and blowing and beating by using a pipette to enable the cells to fall off completely;
1-1-5) centrifuging the cell suspension, discarding the supernatant, resuspending the cell pellet with complete culture medium, blowing to obtain cell suspension, subculturing according to the required cell amount, adding culture medium, and adding CO at 37 deg.C 2The incubator continues to cultivate.
Preferably, the step of providing adipose-derived mesenchymal stem cells in step 1) specifically comprises:
1-2-1) cleaning adipose tissues, then shearing the adipose tissues into tissue blocks, and removing blood vessels and connective tissues;
1-2-1) transferring the adipose tissues into a centrifugal tube, adding equivalent volume of collagenase type I, and performing shock digestion in a water bath kettle at 37 ℃;
1-2-3) adding PBS with the same volume to stop digestion, blowing and uniformly mixing, and centrifuging the suspension;
1-2-4) the suspension after centrifugation is divided into three layers, wherein the upper layer is a fat layer, the middle layer is a liquid layer, and the lower layer is a cell layer; removing the upper and middle layers and the lower layer of liquid by a suction pipe, and taking the fat layer;
1-2-5) resuspending the fat layer with PBS, sieving with a 300-mesh cell sieve, and centrifuging;
1-2-6) resuspending the cell pellet in a culture medium and culturing;
1-2-7) discarding the old culture medium containing floating cells and replacing with a new culture medium;
1-2-8) carrying out passage when the cell fusion degree reaches more than 80%, discarding the original culture medium, adding PBS to clean the cells, discarding the PBS;
1-2-9) adding trypsin to digest the cells, so that the cells are uniformly confluent with the trypsin, and then placing the cell culture dish into CO2Incubating in an incubator at 37 ℃;
1-2-10) observing the digested cells under an inverted microscope, adding a complete culture medium to neutralize and digest when the cells slightly fall off and the edges of the cells which do not fall off are round, and blowing and beating by using a pipette to enable the cells to fall off completely;
1-2-11) centrifuging the cell suspension, discarding the supernatant, resuspending the cell pellet with complete medium, blowing to obtain cell suspension, passaging according to the required cell amount, adding medium, and adding C at 37 deg.CO2The incubator continues to cultivate.
Preferably, the step of providing the human induced pluripotent stem cells in the step 1) specifically comprises:
1-3-1) adding an E8 culture medium into a centrifuge tube, then adding the human induced pluripotent stem cell liquid into the centrifuge tube, and uniformly mixing;
1-3-2), centrifuging, removing the supernatant, and adding an E8 culture medium into a centrifuge tube for blowing, beating and resuspending;
1-3-3) inoculating the cell suspension into a pore plate coated with matrigel in advance, and culturing in an incubator at 37 ℃;
1-3-4) changing the culture medium every day, and carrying out passage amplification on cells when the fusion degree of the cells reaches more than 80%;
1-3-5) subculturing, absorbing and discarding the old cell culture medium, adding PBS to wash the pore plate, and discarding the PBS;
1-3-6) adding the digestive juice, and incubating in an incubator;
1-3-7) removing the digestive juice, adding E8 culture medium containing Y27632 inhibitor, blowing down the cells, adding into a new centrifugal tube, and centrifuging;
1-3-8) resuspend the cell pellet using E8 medium and transfer to a fresh matrigel coated well plate.
Preferably, the step of providing vascular endothelial progenitor cells in step 1) specifically comprises:
1-4-1) pouring the cord blood into a centrifuge tube 1, pouring the lymphocyte separation liquid into a centrifuge tube 2, adding the cord blood in the centrifuge tube 1 into the centrifuge tube 2, wherein the addition requirement is as follows: inclining the centrifugal tube 2 by 60 degrees, sucking the cord blood in the centrifugal tube 1 by using a Pasteur pipette, and marking an S-shaped drainage band above the tube wall of the centrifugal tube 2 to ensure that the cord blood has a path which is enough to slowly flow into the lymphocyte separation liquid, and finally vertically placing the centrifugal tube 2;
1-4-2) adding the cord blood into the lymphocyte separation liquid, centrifuging, and after the centrifugation is finished, generating four layers: plasma, white blood cells, lymphocyte isolates and red blood cells; taking out the plasma layer and the leucocyte layer, adding the plasma layer and the leucocyte layer into 2 new centrifuge tubes, supplementing PBS to each tube, and centrifuging;
1-4-3) centrifuging, removing PBS and removing red cell masses visible to naked eyes at the bottom of the tube, then supplementing PBS to each tube, and centrifuging;
1-4-4) centrifuging, then removing the supernatant, suspending the cell sediment by using a culture medium, and then spreading the cells into a culture bottle for culture;
1-4-5) adding complete medium into culture flask, adding CO at 37 deg.C2Continuously culturing in an incubator;
1-4-6), washing off the cells which are not attached to the wall, continuously culturing the attached cells, and culturing the vascular endothelial progenitor cells;
1-4-7) carrying out passage expansion of cells when the cell fusion degree reaches more than 80%;
1-4-8), absorbing and discarding the old cell culture medium, adding PBS to wash the pore plate, and discarding the PBS;
1-4-9) adding digestive juice, and incubating in an incubator;
1-4-10) removing the digestive juice, adding E8 culture medium containing Y27632 inhibitor, blowing down the cells, adding into a new centrifugal tube, and centrifuging;
1-4-11) resuspend the cell pellet using E8 medium and transfer to a new matrigel coated well plate.
Preferably, the step 3) specifically includes:
3-1) culturing 293T cells in advance;
3-2) configuration of plasmid transfection systems A and B:
the plasmid transfection system A comprises Opti-MEM and PEI, and the plasmid transfection system B comprises the lentivirus expression vector plasmids pLVX-FTH1-Sr39tk-EGFP, Opti-MEM and three packaging plasmids obtained in the step 2): VSV-G, pMDL, pRSV;
3-3) adding the plasmid transfection system A into the plasmid transfection system B, uniformly mixing, and standing at room temperature;
3-4) adding the mixture obtained in the step 3-3) into Opti-MEM, and uniformly mixing;
3-5) taking the cultured 293T cells, discarding the original culture medium in the 293T cells, and adding the mixture obtained in the step 3-4) into the 293T cells and submerging the 293T cells;
3-6) in CO2Culturing in an incubator;
3-7) taking supernatant fluid of 293T cells, and centrifuging in a refrigerated centrifuge;
3-8) centrifuging, taking the supernatant, and centrifuging again;
3-9), removing the supernatant, leaving the virus precipitate, and resuspending the precipitate with PBS to obtain the recombinant lentivirus.
Preferably, the step 4) specifically includes:
4-1) four cells of interest were previously: culturing umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, human induced pluripotent stem cells and vascular endothelial progenitor cells, and then respectively carrying out lentivirus infection according to the following steps;
4-2) adding the recombinant lentivirus obtained in the step 3) and polybrene into a serum-free culture medium, and uniformly blowing;
4-3) removing the old culture medium in the target cell, adding the recombinant lentivirus mixed solution obtained in the step 4-2) into the target cell, and enabling the recombinant lentivirus mixed solution to be over the target cell;
4-4) carrying out lentivirus infection, and supplementing a fresh complete culture medium in the middle;
4-5) finally obtaining umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, human induced pluripotent stem cells and vascular endothelial progenitor cells which all over express three proteins of FTH1, Sr39tk and EGFP, namely the stem cell line capable of carrying out multi-modal tracing.
Preferably, the gene sequence of FTH1 is shown as SEQ ID No.1, the gene sequence of Sr39tk is shown as SEQ ID No.2, the gene sequence of EGFP is shown as SEQ ID No.3, and the gene sequence of the lentiviral expression vector pLVX-FTH1-Sr39tk-EGFP is shown as SEQ ID No. 4.
The invention also provides the application of the stem cell line capable of carrying out multi-modal tracing or the stem cell line capable of carrying out multi-modal tracing prepared by the method as a tracer for MRI-PET multi-modal imaging.
The beneficial effects of the invention are: the stem cell line can be used for living cell tracing of multi-modal imaging combining MRI and PET, can effectively detect the movement and differentiation of transplanted cells, explores the action mechanism of the transplanted cells, is beneficial to promoting stem cell treatment to be applied to clinic, and can provide a new thought and scheme for preparing a living body tracing stem cell reagent for treating human cerebral infarction.
Drawings
FIG. 1 is a plasmid map of pLVX-FTH1-Sr39tk-EGFP and pLVX-EGFP constructed in an example of the present invention;
FIG. 2 is an agarose gel electrophoresis cleavage map of pLVX-FTH1-Sr39tk-EGFP and pLVX-EGFP recombinant vectors constructed in an example of the present invention;
FIG. 3 shows the results of 293T cells transfected with two recombinant vectors constructed in the examples of the present invention for 24 hours;
FIGS. 4-7 are the results of fluorescence quantitative PCR and Western blot analysis of four stem cells constructed in the examples of the present invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or combinations thereof.
The invention provides a stem cell line capable of carrying out multi-modal tracing, which comprises: umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, human induced pluripotent stem cells and vascular endothelial progenitor cells all over-expressing three proteins of FTH1, Sr39tk and EGFP.
The invention further provides a preparation method of the stem cell line capable of carrying out multi-modal tracing, which comprises the following steps:
1) providing umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, human induced pluripotent stem cells and vascular endothelial progenitor cells;
2) cloning target genes FTH1, Sr39tk and EGFP to a pLVX vector to construct a lentivirus expression vector pLVX-FTH1-Sr39 tk-EGFP;
3) Packaging lentiviruses to obtain a recombinant lentivirus containing FTH1, Sr39tk and EGFP genes;
4) the stem cell line capable of performing multi-modal tracing is characterized in that umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, human induced pluripotent stem cells and vascular endothelial progenitor cells are respectively infected by recombinant lentiviruses containing FTH1, Sr39tk and EGFP genes, so that four stem cells which respectively over-express three proteins of FTH1, Sr39tk and EGFP are obtained.
The invention further provides the application of the stem cell line capable of performing multi-modal tracing or the stem cell line capable of performing multi-modal tracing prepared by the method as a tracer for MRI-PET multi-modal imaging.
More detailed examples are provided herein below.
Example 1 isolated culture of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, human induced pluripotent stem cells and vascular endothelial progenitor cells
1. Umbilical cord mesenchymal stem cells (UC-MSCs) isolated culture
1.1 taking materials
Selecting pregnant women, pregnant women and new-born fetuses with healthy body. After the operation, the umbilical cord with the length not shorter than 25cm is taken, the umbilical cord is placed into the umbilical cord protection solution after being washed by physiological saline, the umbilical cord is transported under the environment of 4 ℃, and the separation of primary cells is completed within 48 hours. It will be appreciated that umbilical cord tissue from postpartum parturients, which is typically discarded as medical waste, is subject to no ethical debate regarding the isolation and extraction of stem cells from umbilical cord tissue, which may also be obtained from scientific or other sources.
1.2 Primary isolation and extraction of umbilical cord mesenchymal Stem cells
1.2.1 pouring 75% alcohol into a 50ml centrifuge tube, clamping the whole umbilical cord from a collection bottle by using long forceps, putting into the alcohol, repeatedly oscillating and cleaning, and cleaning for 2-3 min;
1.2.2 after washing, the umbilical cord was put into a beaker, poured into PBS (1% double antibody), and washed repeatedly 3 times. When the washing liquid is poured, the long forceps are used for propping the umbilical cord without dropping umbilical cord tissues;
1.2.3 clamping the umbilical cord into a renal disc, holding forceps with a left hand and holding scissors with a right hand, and cutting the umbilical cord into long sections of 2-3 cm;
1.2.4 repeatedly scraping two ends of the umbilical cord section by using scissors to scrape blood coagulation as much as possible, and scraping and straightening the umbilical cord;
1.2.5 the cord segments were placed in a new beaker and washed repeatedly 3 times with PBS (1% double antibody). When the washing liquid is poured, the long forceps are used for propping the umbilical cord without dropping umbilical cord tissues;
1.2.6 putting the cleaned umbilical cord section into a new renal disc, and starting a first section to process the umbilical cord;
1.2.7, cutting off the umbilical cord along the umbilical vein (the thickest blood vessel) by scissors, tearing off a layer of blood vessel endothelium by using forceps, naturally preventing the umbilical cord from curling after tearing off, and removing two umbilical arteries;
1.2.8 tearing off the Wharton's jelly slowly to clean the jelly as much as possible; putting the torn glue into the bottom of a 50ml centrifuge tube, and shearing the glue by using large and long tweezers, wherein the broken glue is better;
1.2.9 scraping the crushed materials with a small medicine spoon, taking the crushed glue, paving the crushed glue, and paving the crushed glue into a T75 culture bottle;
1.3 passage expansion of cells
1.3.1 when 40% -50% of tissues in a bottle climb out of cells, carrying out passage of the cells; washing the tissue blocks and the redundant impurities falling from the bottle by PBS for 2 times;
1.3.2 adding 2ml of trypsin to digest cells, lightly shaking the culture dish to ensure that the cells are uniformly covered by the trypsin, and then putting the cell culture dish into a CO2 incubator to incubate for 1min at 37 ℃;
1.3.3 observing the digested cells under an inverted microscope, adding a complete culture medium for neutralization and digestion as soon as possible when the cells slightly fall off and the edges of the cells which do not fall off are round, preventing the cells from being damaged due to over digestion, and slightly blowing the bottom of a culture dish by using a pipette to ensure that the cells all fall off;
1.3.4 adding the cell suspension into a 15ml centrifuge tube, centrifuging at 1000rpm for 3min, discarding the supernatant, resuspending the cell pellet with 1ml of complete culture medium, gently blowing to form a cell suspension, passaging according to the required cell amount, and passing by 10cm2Adding 10ml of culture medium into a cell culture dish, and adding CO at 37 DEG C2The incubator continues to culture.
2. Adipose-derived mesenchymal stem cells (AD-MSCs) isolated culture
2.1 taking materials
Adipose tissue is derived from fat extract of a patient who has undergone liposuction surgery (the source is waste which is medical waste, and ethical disputes do not exist), and after the adipose tissue is collected, the adipose tissue is placed in a preservation solution and transported at 4 ℃, and primary cells are separated within 48 hours.
2.2 Primary isolation and extraction of adipose mesenchymal Stem cells
2.2.1 transfer adipose tissue into 10cm petri dishes in a clean bench and wash three times with PBS plus double antibody. Cutting adipose tissue into small pieces with scissors and forceps, and removing blood vessel and connective tissue under dissecting microscope;
2.2.2 transferring the separated adipose tissue blocks into a 50ml centrifuge tube (PBS is pre-added in the centrifuge tube), weighing, then recording the volume number, adding I-type collagenase with the same volume, and carrying out shock digestion for 30min in a water bath kettle at 37 ℃;
2.2.3 adding PBS with the same volume to stop digestion, blowing and uniformly mixing, and then centrifuging the suspension for 500g for 10 min;
2.2.4 the centrifuged suspension was divided into three layers, the upper layer being the fat layer, the middle layer being the liquid layer and the lower layer being the cell layer. Removing the lower two layers of liquid by using a suction pipe, and sucking off the fat layer as much as possible;
2.2.5 resuspending the cell layer with PBS, sieving with a 300 mesh cell sieve, and centrifuging for 350g 5 min;
2.2.6 cell pellets were resuspended in DMEM + 10% FBS medium and counted at 8X 104/cm2The density inoculation of (3);
2.2.748 hours later, the old medium containing floating cells was discarded and replaced with new medium.
2.3 passage expansion of cells
2.3.1 when the cell growth state is good and the fusion degree reaches more than 80%, passage can be carried out, the original culture medium is discarded, 2ml of 1 XPBS is added to clean the cells, and PBS is discarded;
2.3.2 Add 2ml Trypsin to digest the cells, gently shake the dish to evenly flood the cells with Trypsin, then place the cell culture dish in CO2Incubating in an incubator at 37 ℃ for 1 min;
2.3.3 observing the digested cells under an inverted microscope, adding the cells into a complete culture medium for neutralization and digestion as soon as possible when the cells slightly fall off and the edges of the cells which do not fall off are round, preventing the cells from being damaged due to over digestion, and slightly blowing the bottom of a culture dish by using a pipette to ensure that the cells all fall off;
2.3.4 adding the cell suspension to a 15ml centrifuge tube, centrifuging at 1000rpm for 4min, discarding the supernatant, resuspending the cell pellet with 1ml of complete culture medium, gently blowing to form a cell suspension, passaging according to the desired cell amount, 10cm2Adding 10ml of culture medium into a cell culture dish, and adding CO at 37 DEG C2The incubator continues to cultivate.
3. Subculture of human Induced Pluripotent Stem Cells (iPSCs)
3.1 cell origin
Human induced pluripotent stem cell lines (iPSCs) were purchased from companies and are epidermis-derived human induced pluripotent stem cell lines.
3.2 recovery and passage expansion of cells
3.2.1 firstly, quickly putting the cells in the freezing tube into a water bath kettle at 37 ℃, slightly shaking, taking the freezing tube into a biological safety cabinet when the ice-like objects in the tube are completely melted, and carrying out subsequent operation;
3.2.2 adding 3ml of E8 culture medium into a 15ml centrifuge tube, opening the freezing tube carefully, slowly dripping the melted cell sap into the culture medium, and mixing the cell sap gently and evenly;
3.2.3200 g, centrifuging for 3min, sucking and discarding the supernatant, adding a proper amount of E8 culture medium, and gently blowing and resuspending.
3.2.4 inoculating the cell suspension into a pore plate coated with matrigel in advance, and placing the pore plate in an incubator at 37 ℃ for culture;
3.2.5 changing culture medium every day, and performing passage expansion of cells when the cell fusion degree reaches more than 80%;
3.2.6 passages, the old cell culture medium is aspirated and discarded, 1ml PBS is added into each hole to wash the hole plate, and the PBS is discarded;
3.2.7 Add 0.5ml of digest (PBS containing 0.5mM EDTA and 0.22% NaCl) and incubate for 5min in the incubator;
3.2.8 aspirate off the digest, add 1ml E8 medium containing 10. mu.M Y27632 inhibitor, gently blow the cells down and add to a 15ml centrifuge tube;
3.2.9 centrifuging the cell suspension, centrifuging at 200g for 3 min;
3.2.10 cell pellets were resuspended using E8 medium and transferred to a fresh matrigel-coated well plate.
4. Isolated culture of vascular Endothelial Progenitor Cells (EPCs)
4.1 cell origin: EPCs are derived from mononuclear cells isolated from human cord blood.
4.1.1 after the delivery of the healthy lying-in woman under the aseptic condition, immediately clamping the umbilical cord at the side of the fetus by using a hemostatic clamp, selecting the blood vessel bursting part of the umbilical cord at the end of the mother body as a puncture part, disinfecting and collecting, wherein a blood collection bag with the volume of 200ml contains a sodium citrate anticoagulant, transporting the blood collection bag at 4 ℃, and completing the separation of primary cells within 4 hours; it is to be understood that umbilical cord tissue from a parturient post-partum, which is typically discarded as medical waste, does not present ethical debate to the collection of cord blood from umbilical cord tissue, although cord blood may also be obtained from commercial, scientific, or other sources;
4.1.2 taking the blood collection bag into a biological safety cabinet after disinfection, cutting a sample outlet, and pouring a blood sample into a 50ml centrifuge tube;
4.1.3 the lymphocyte isolates were poured into two 50ml centrifuge tubes, 15ml each. A 25ml blood sample per tube was slowly added to the lymphocyte isolate, adding the following: inclining the centrifuge tube filled with the separation liquid by 60 degrees, drawing an S-shaped drainage band above the tube wall of the separation liquid after sucking a blood sample by a Pasteur pipette, ensuring that the blood sample has a path which is enough to slowly flow into the separation liquid, and slowly erecting the centrifuge tube at last;
4.1.4 adding the whole blood sample into the separating medium, centrifuging at 800g for 25min, and reducing the speed by 1 at 20 ℃;
4.1.5 after centrifugation was complete, four layers appeared: plasma, white blood cells, lymphocyte isolates, and red blood cells. Carefully taking out the plasma layer and the leucocyte layer, adding the plasma layer and the leucocyte layer into 2 new 50ml centrifuge tubes, adding PBS into each tube to 40ml, centrifuging for 10min at 600g, and increasing the speed by 5 and reducing the speed by 5;
4.1.6 after centrifugation, the tube bottom has a macroscopic red cell mass, most PBS is discarded, only 1-2ml of supernatant is left for resuspending cells, each tube is supplemented with PBS to 40ml, and centrifugation is carried out at the speed;
4.1.7 centrifugation followed by discarding the supernatant, resuspending the cell pellet with media, and following a 2X 10 protocol 6The cells were plated in T25 flasks for culture.
4.2 subculture of cells
4.2.1 plating the isolated mononuclear cells into a T25 flask, adding complete medium (FBSS medium containing 10ng/ml VEGF, 20ng/ml bFGF and EGF), and CO at 37 deg.C2The incubator continues to culture.
4.2.2 standing the cells for 3h, washing off the cells which are not attached to the wall, continuously culturing the attached cells for 4 weeks, and changing the whole solution every 3 days to culture the EPCs.
4.3 passage expansion of cells
4.3.1 when the growth state of the cells is determined to be good and the fusion degree reaches more than 80%, passage can be carried out, the original culture medium is discarded, 2ml of 1 XPBS is added to wash the cells, and PBS is discarded;
4.3.2 Add 2ml Trypsin to digest the cells, gently shake the dish to evenly flood the cells with Trypsin, then place the cell culture dish in CO2Incubating in an incubator at 37 ℃ for 1 min;
4.3.3 observing the digested cells under an inverted microscope, adding the cells into a complete culture medium for neutralization and digestion as soon as possible when the cells slightly fall off and the edges of the cells which do not fall off are round, preventing the cells from being damaged due to over digestion, and slightly blowing the bottom of a culture dish by using a pipette to ensure that the cells all fall off;
4.3.4 adding the cell suspension into a 15ml centrifuge tube, centrifuging at 1000rpm for 4min, discarding supernatant, suspending the cell pellet with 1ml of complete culture medium, gently blowing to obtain cell suspension, subculturing according to desired cell amount, and subculturing by 10cm 2Adding 10ml of culture medium into a cell culture dish, and adding CO at 37 DEG C2The incubator continues to cultivate.
Example 2 construction of lentiviral expression vector pLVX-FTH1-Sr39tk-EGFP
In the embodiment, target genes FTH1, Sr39tk and EGFP are cloned to a pLVX vector by a PCR (polymerase chain reaction), enzyme digestion and enzyme ligation method, and meanwhile, an EGFP reporter gene is fused and expressed on the vector in order to conveniently judge the expression efficiency of the virus later, so that the lentiviral expression vector pLVX-FTH1-Sr39tk-EGFP is obtained. Referring to FIG. 1, plasmid maps of pLVX-FTH1-Sr39tk-EGFP and pLVX-EGFP constructed in this example were shown.
And then the pLVX-FTH1-Sr39tk-EGFP vector is subjected to in vitro amplification of the target gene through PCR, and the agarose gel electrophoresis band result is correct. FIG. 2 is an agarose gel electrophoresis cleavage map of pLVX-FTH1-Sr39tk-EGFP (A) and pLVX-EGFP (B) recombinant vectors, wherein M is 10000bp marker, 1: FTH1 fragment, 2: sr39tk fragment, 3: EGFP fragment, 4: an EGFP fragment.
And then, a pLVX-EGFP lentiviral vector is constructed by the same method, and the pLVX vector only contains the EGFP gene and can be used as a control vector in the experiment.
In the embodiment, the gene sequence of FTH1 is shown as SEQ ID No.1, the gene sequence of Sr39tk is shown as SEQ ID No.2, the gene sequence of EGFP is shown as SEQ ID No.3, and the gene sequence of lentiviral expression vector pLVX-FTH1-Sr39tk-EGFP is shown as SEQ ID No. 4.
Example 3 construction of recombinant lentiviruses
1. Lentiviral packages
1.1 Pre-plating 293T cells into 10cm2Culturing cells in a large dish by using a complete culture medium, and performing subsequent experiments when the cell fusion amount is 70-80%;
1.2 2 taking 2 EP tubes of 2ml, adding 400 μ l of Opti-MEM and three packaging plasmids and target plasmids (pLVX-FTH1-Sr39tk-EGFP) into the first tube, and adding 400 μ l of Opti-MEM and 50 μ l of PEI into the second tube, wherein the three packaging plasmids respectively have the following mass: VSV-G2.5. mu.g, pMDL 6.5. mu.g, pRSV3.5. mu.g, the target plasmid mass is: 10 mu g of the mixture; calculating the adding volume according to the concentration of the plasmids, and sequentially adding the four plasmids into a culture medium;
1.3, uniformly mixing the two tubes of culture media, adding the culture medium added with PEI into the culture medium with the plasmid, slightly blowing, uniformly mixing, and standing at room temperature for 10 min;
1.4 adding the mixed 1ml of culture medium into 3ml of Opti-MEM, and uniformly mixing;
1.5 from CO2Taking out the cultured 293T cells from the incubator, discarding the original culture medium in the cells, and adding 4ml of mixed culture medium to ensure that the culture medium can submerge the cells;
1.6 in CO2Supplementing the culture medium to 10ml after the culture in the incubator is carried out for 6-8h, and then culturing for 48 h;
2 Lentiviral concentration
2.1 taking out 10ml of 293T cell supernatant from the large dish and putting the supernatant into a 50ml centrifuge tube;
2.2 placing the mixture into a refrigerated centrifuge for centrifugation, and centrifuging the mixture at 4 ℃ for 10min at 1000 g;
2.3 after centrifugation, putting the supernatant into a sterilized high-speed centrifuge tube, weighing, and then balancing by using the same high-speed centrifuge tube until the weight is accurate to a decimal point and 3 positions are the same;
2.4 centrifuging with a high-speed centrifuge at 4 deg.C for 3 hr at 50000 g;
2.5 after centrifugation, the bottom of the centrifuge tube can see the deposition of grey white particles, the supernatant is discarded and is sufficiently sucked, and the deposition is not sucked away;
2.6 resuspending the pellet gently with ice PBS to obtain virus, subpackaging and storing in-80 deg.C refrigerator for a long time.
The two recombinant vectors are respectively transfected into 293T cells, and the plasmid transfection efficiency is observed after 24 hours; and the virus suspension is recovered after 48 h. Referring to FIG. 3, the results of transfection of 293T cells with two recombinant vectors are shown for 24 hours.
Example 4 construction of Stem cell lines that can undergo multimodal tracking
1. Firstly, four cells (umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, human induced pluripotent stem cells and vascular endothelial progenitor cells) are dissolved from a culture dish, centrifuged and resuspended into cell suspension, and the cell suspension is added into each well at a speed of 1 multiplied by 106The density of individual cells was plated in six well plates, cultured in complete medium, and the plates were placed in 37 ℃ CO 2After culturing for 24 hours in an incubator, performing a lentivirus infection experiment;
2. before infection experiments, preparing a virus solution by using a sterile EP tube; adding a serum-free culture medium into an EP (Eppendorf) tube in a volume of 500 mu l per well, simultaneously adding recombinant virus and polybrene (polybrene) into the tube, adding the virus into the culture medium in a volume of 1 (MOI), calculating a MOI (MOI) according to a virus titer TU/ml x (virus volume/cell number), calculating a virus volume according to the virus titer and the cell number, adding the obtained volume into the serum-free culture medium, simultaneously adding polybrene (2.5 mu g/ml) to improve the infection efficiency of the virus, and gently and uniformly blowing the culture medium;
3. when in infection, the old culture medium in the cells is discarded, the mixed virus solution is added into each hole with the volume of 500 mul, and the hole plate is gently shaken to ensure that the virus solution is fully paved on the cells;
4. each well was supplemented with 1.5ml of fresh complete medium 6-8h after infection;
5. and (3) continuously culturing for 48h, then removing the original culture medium, adding a fresh culture medium, culturing for 24h, and performing fluorescence quantitative PCR (polymerase chain reaction) and Western blot experiment (immunoblot test) to verify whether the recombinant virus starts to express in the four cells.
Example 5 fluorescent quantitative PCR
1. Extraction of cellular RNA
1.1 taking out the cells which are well plated, removing the culture medium, adding PBS for washing, removing the PBS, and placing on ice for 15 min;
1.2 preparation of lysate: 350ul of TRK Lysls buffer +7ul of beta-mercaptoethanol, 350ul of TRK Lysls buffer is added into each hole, and blown cells completely drop;
1.3 adding into 1.5ml EP tube, centrifuging at 14000rpm for 5min, taking the supernatant into a new 1.5ml EP tube, adding equal volume of 350ul 70% alcohol, mixing uniformly, and adding into a purification column;
1.4 centrifuging 14000r for 1min, and discarding the liquid in the collecting tube;
1.5 adding 300ul RNA wash buffer I, centrifuging for 30s at 14000r, and discarding the liquid in the collection tube;
1.6 adding 500ul RNA wash buffer I, centrifuging for 30s at 14000r, and discarding the liquid in the collection tube;
1.7 adding 500ul RNA wash buffer II, centrifuging for 30s at 14000r, and discarding the liquid in the collection tube;
1.8 adding 500ul RNA wash buffer II, centrifuging for 30s at 14000r, and discarding the liquid in the collection tube;
idling at 1.9, centrifuging at 14000r for 1min, abandoning the collecting pipe, and placing in a new EP pipe;
1.10 add 30ul DEPC water, 14000r centrifuge for 1min, abandon the purification column;
1.11 the liquid in the tube is purified RNA, and the concentration of the extracted RNA is detected.
2. Reverse transcription
2.1 the reverse transcription system is:
2.2 after the system is mixed evenly, the instantaneous centrifugation is carried out. And (3) loading a PCR instrument, and performing the following procedures:
2.3 reverse transcription of RNA into cDNA, and storing at-80 ℃.
3PCR
3.1 the PCR reaction system is: (primers are shown in Table 1 below)
3.2 after mixing the system, carrying out instantaneous centrifugation. The PCR instrument is loaded, and the program is as follows:
TABLE 1 PCR primer sequences
Example 6 immunoblot assay
1. Preparation of protein samples
1.1 each group of samples at least needs 2 holes of a six-hole plate, the culture plate is taken out, a culture medium is not discarded, and a cell layer at the bottom of each hole is scraped by a cell scraper;
1.2 put into EP tube after scraping, insert on ice in time. Centrifuging at 3000rpm for 5min at 4 deg.C;
1.3 cleavage protein: sample + protein lysate (RIPA, 1:1) + protease inhibitor (100: 1). Repeatedly beating cell precipitate with lysis solution, and standing on ice or 4 deg.C for 15 min;
1.4, ultrasonic: performing ultrasonic treatment for 5-6 times. Before ultrasonic treatment, the probe is cleaned by ice water, the temperature is reduced, and each sample is cleaned by the ice water and wiped by non-woven fabrics after ultrasonic treatment;
1.5 SDS loading buffer was added, sample: SDS loading buffer 4: 1;
decocting in water at 1.6100 deg.C for 10min, and storing at-20 deg.C for one month. Taking out again, and decocting with water for 5 min.
2 glue making
2.1 using 1.0mm glass plate and comb, clamping on the groove, checking whether water leakage exists, adding sterile water, and making the water flush with the plate surface;
2.2 preparation of separation gel: preparing with a 50ml centrifuge tube, adding separation gel, adding 1ml isopropanol, flattening the gel surface, removing air bubbles, and solidifying for 40-50 min;
2.3, preparing concentrated glue: the mixture was dispensed into a 50ml centrifuge tube, the isopropanol was decanted off, and the tube was wiped clean with a nonwoven cloth. Adding concentrated gel, immediately inserting 1.0mm comb, and coagulating for 20 min.
3 sample loading
3.1, taking down the rubber plate, pulling out the comb, and paving the obliquely pulled rubber by using the syringe needle;
3.2 putting the rubber plate into an electrophoresis apparatus, adding the recovered 1 × Running buffer into the outer layer, and adding a new 1 × Running buffer into the middle layer;
3.3 mixing the samples uniformly before loading, loading 10 mul of samples on one hole, loading 3 multiple holes on each sample, and adding protein markers on two sides of the samples;
3.4 after the sample loading is finished, connecting the red line with the positive electrode and connecting the black line with the negative electrode, running for 15min at 120V, fully running the concentrated gel on the sample, and then regulating the voltage to better separate the sample in the separation gel, wherein the voltage is regulated to 150V50 min.
4-turn film
4.1 shearing a PVDF membrane with the same size as the glue, and putting the membrane into methanol for activation;
4.2 taking out the rubber clamping plate, prying the glass plate by using a green plate, removing redundant rubber, and not concentrating the rubber and rubber except strips;
4.3 pouring a 1 × transfer buffer on a white disc, putting glue and a black and red plate in the buffer, and fully soaking, wherein the sandwich structure is blackboard + sponge + filter paper + glue + membrane + filter paper + sponge + red plate, and when the front side and the back side are noticed, no air bubbles can exist between every two layers;
4.4 put into an electrophoresis apparatus, the positive electrode is aligned to the positive electrode, the negative electrode is aligned to the negative electrode, and 1 × transfer buffer is poured until the membrane plate is submerged. The film is rotated in ice at 150V 240mA for 2 h.
5 blocking and antibody incubation
5.1 preparation of sealing liquid: skimmed milk powder (5%) + TBST, membrane sealing on shaking table for 30 min;
5.2 washing with TBST for 3 times, 5min each time;
5.3 applying primary antibody, diluting according to the antibody instruction, and incubating at 4 ℃ overnight or at room temperature for 3 h;
5.4 washing with TBST for 3 times, 5min each time;
5.5 applying a second antibody, diluting according to the antibody instruction, and keeping the temperature at room temperature for 1 h;
5.6 Wash 3 times with TBST for 5min each.
6 development
After the developing working solution is prepared, the PVDF film is clamped by tweezers and dipped on the non-woven fabric, the developing solution is added into a Western Blot box, the front side and the back side of the film are dipped with the developing solution, the film is placed in the middle of a plate of an instrument, and the machine is used for detecting.
After UC-MSCs, AD-MSCs, iPSCs and EPCs are respectively infected by the two recombinant lentiviral vectors, in order to prove that exogenous genes on the recombinant vectors are normally expressed, the invention uses a fluorescent quantitative PCR technology and Western blot to carry out gene detection and quantitative analysis according to embodiments 5 and 6. As can be seen from the Western blot results of all the panels A in FIGS. 4-7, the protein levels of different genes were normally expressed after infection with the pLVX-FTH1-Sr39tk-EGFP recombinant vector. From all the graphs B, the relative expression amounts of the foreign gene mRNA of the two recombinant vectors were significantly different. Fig. 4 is an analysis result of the umbilical cord mesenchymal stem cell expression recombinant vector, fig. 5 is an analysis result of the adipose mesenchymal stem cell expression recombinant vector, fig. 6 is an analysis result of the human induced pluripotent stem cell expression recombinant vector, and fig. 7 is an analysis result of the vascular endothelial progenitor cell expression recombinant vector.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Suzhou Institute of Biomedical Engineering Technology, Chinese Academy of Sciences
The invention name is as follows: stem cell line capable of carrying out multi-modal tracing, preparation method and application thereof
Serial number (5 'to 3')
SEQ ID No. 1 ：
ATGACCACCGCGTCTCCCTCGCAAGTGCGCCAGAACTACCACCAGGACTCGGAGGCTGCCATCAACCGCCAGATCAACCTGGAGTTGTATGCCTCCTACGTCTATCTGTCCATGTCTTGTTATTTTGACCGGGATGATGTGGCCCTGAAGAACTTTGCCAAATACTTTCTCCATCAATCTCATGAAGAGAGGGAACATGCTGAGAAACTGATGAAGCTGCAGAACCAGCGAGGTGGACGAATCTTCCTGCAGGATATAAAGAAACCTGACCGTGATGACTGGGAGAGCGGGCTGAATGCAATGGAGTGTGCACTGCACTTGGAAAAGAGTGTGAATCAGTCACTACTGGAACTTCACAAACTGGCTACTGACAAGAATGATCCCCACTTATGTGACTTCATTGAGACGCATTACCTGAATGAGCAGGTGAAATCCATTAAAGAACTGGGTGACCACGTGACCAACTTACGCAAGATGGGAGCCCCTGAATCTGGCATGGCAGAATATCTCTTTGACAAGCACACCCTGGGACACGGTGATGAGAGC
SEQ ID No. 2：
ATGGCTTCGTACCCCTGCCATCAACACGCGTCTGCGTTCGACCAGGCTGCGCGTTCTCGCGGCCATAGCAACCGACGTACGGCGTTGCGCCCTCGCCGGCAGCAAGAAGCCACGGAAGTCCGCCTGGAGCAGAAAATGCCCACGCTACTGCGGGTTTATATAGACGGTCCTCACGGGATGGGGAAAACCACCACCACGCAACTGCTGGTGGCCCTGGGTTCGCGCGACGATATCGTCTACGTACCCGAGCCGATGACTTACTGGCAGGTGCTGGGGGCTTCCGAGACAATCGCGAACATCTACACCACACAACACCGCCTCGACCAGGGTGAGATATCGGCCGGGGACGCGGCGGTGGTAATGACAAGCGCCCAGATAACAATGGGCATGCCTTATGCCGTGACCGACGCCGTTCTGGCTCCTCATATCGGGGGGGAGGCTGGGAGCTCACATGCCCCGCCCCCGGCCCTCACCATCTTCCTCGACCGCCATCCCATCGCCTTCATGCTGTGCTACCCGGCCGCGCGATACCTTATGGGCAGCATGACCCCCCAGGCCGTGCTGGCGTTCGTGGCCCTCATCCCGCCGACCTTGCCCGGCACAAACATCGTGTTGGGGGCCCTTCCGGAGGACAGACACATCGACCGCCTGGCCAAACGCCAGCGCCCCGGCGAGCGGCTTGACCTGGCTATGCTGGCCGCGATTCGCCGCGTTTACGGGCTGCTTGCCAATACGGTGCGGTATCTGCAGGGCGGCGGGTCGTGGCGGGAGGATTGGGGACAGCTTTCGGGGACGGCCGTGCCGCCCCAGGGTGCCGAGCCCCAGAGCAACGCGGGCCCACGACCCCATATCGGGGACACGTTATTTACCCTGTTTCGGGCCCCCGAGTTGCTGGCCCCCAACGGCGACCTGTACAACGTGTTTGCCTGGGCCTTGGACGTCTTGGCCAAACGCCTCCGTCCCATGCACGTCTTTATCCTGGATTACGACCAATCGCCCGCCGGCTGCCGGGACGCCCTGCTGCAACTTACCTCCGGGATGGTCCAGACCCACGTCACCACCCCCGGCTCCATACCGACGATCTGCGACCTGGCGCGCACGTTTGCCCGGGAGATGGGGGAGGCTAAC
SEQ ID No. 3：
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA
SEQ ID No. 4：
TGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGCCTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTATCGATAAGCTTGGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGACTCTACTAGAGGATCTATTTCCGGTGAATTCCTCGAGACTAGTTCTAGAGCCACCATGACCACCGCGTCTCCCTCGCAAGTGCGCCAGAACTACCACCAGGACTCGGAGGCTGCCATCAACCGCCAGATCAACCTGGAGTTGTATGCCTCCTACGTCTATCTGTCCATGTCTTGTTATTTTGACCGGGATGATGTGGCCCTGAAGAACTTTGCCAAATACTTTCTCCATCAATCTCATGAAGAGAGGGAACATGCTGAGAAACTGATGAAGCTGCAGAACCAGCGAGGTGGACGAATCTTCCTGCAGGATATAAAGAAACCTGACCGTGATGACTGGGAGAGCGGGCTGAATGCAATGGAGTGTGCACTGCACTTGGAAAAGAGTGTGAATCAGTCACTACTGGAACTTCACAAACTGGCTACTGACAAGAATGATCCCCACTTATGTGACTTCATTGAGACGCATTACCTGAATGAGCAGGTGAAATCCATTAAAGAACTGGGTGACCACGTGACCAACTTACGCAAGATGGGAGCCCCTGAATCTGGCATGGCAGAATATCTCTTTGACAAGCACACCCTGGGACACGGTGATGAGAGCGGATCCGCCACAAACTTTTCCCTATTGAAGCAAGCAGGAGATGTTGAGGAAAATCCAGGACCCCCTAGGATGGCTTCGTACCCCTGCCATCAACACGCGTCTGCGTTCGACCAGGCTGCGCGTTCTCGCGGCCATAGCAACCGACGTACGGCGTTGCGCCCTCGCCGGCAGCAAGAAGCCACGGAAGTCCGCCTGGAGCAGAAAATGCCCACGCTACTGCGGGTTTATATAGACGGTCCTCACGGGATGGGGAAAACCACCACCACGCAACTGCTGGTGGCCCTGGGTTCGCGCGACGATATCGTCTACGTACCCGAGCCGATGACTTACTGGCAGGTGCTGGGGGCTTCCGAGACAATCGCGAACATCTACACCACACAACACCGCCTCGACCAGGGTGAGATATCGGCCGGGGACGCGGCGGTGGTAATGACAAGCGCCCAGATAACAATGGGCATGCCTTATGCCGTGACCGACGCCGTTCTGGCTCCTCATATCGGGGGGGAGGCTGGGAGCTCACATGCCCCGCCCCCGGCCCTCACCATCTTCCTCGACCGCCATCCCATCGCCTTCATGCTGTGCTACCCGGCCGCGCGATACCTTATGGGCAGCATGACCCCCCAGGCCGTGCTGGCGTTCGTGGCCCTCATCCCGCCGACCTTGCCCGGCACAAACATCGTGTTGGGGGCCCTTCCGGAGGACAGACACATCGACCGCCTGGCCAAACGCCAGCGCCCCGGCGAGCGGCTTGACCTGGCTATGCTGGCCGCGATTCGCCGCGTTTACGGGCTGCTTGCCAATACGGTGCGGTATCTGCAGGGCGGCGGGTCGTGGCGGGAGGATTGGGGACAGCTTTCGGGGACGGCCGTGCCGCCCCAGGGTGCCGAGCCCCAGAGCAACGCGGGCCCACGACCCCATATCGGGGACACGTTATTTACCCTGTTTCGGGCCCCCGAGTTGCTGGCCCCCAACGGCGACCTGTACAACGTGTTTGCCTGGGCCTTGGACGTCTTGGCCAAACGCCTCCGTCCCATGCACGTCTTTATCCTGGATTACGACCAATCGCCCGCCGGCTGCCGGGACGCCCTGCTGCAACTTACCTCCGGGATGGTCCAGACCCACGTCACCACCCCCGGCTCCATACCGACGATCTGCGACCTGGCGCGCACGTTTGCCCGGGAGATGGGGGAGGCTAACCCTAGGGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTACCGGTATCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAACGCGTCTGGAACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTAATTCTGCAGTCGAGACCTAGAAAAACATGGAGCAATCACAAGTAGCAATACAGCAGCTACCAATGCTGATTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAGGTGGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGAGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGCCTTGACATTGCTAGCGTTTACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAACTGGATAACTCAAGCTAACCAAAATCATCCCAAACTTCCCACCCCATACCCTATTACCACTGCCAATTACCTGTGGTTTCATTTACTCTAAACCTGTGATTCCTCTGAATTATTTTCATTTTAAAGAAATTGTATTTGTTAAATATGTACTACAAACTTAGTAGTTTTTAAAGAAATTGTATTTGTTAAATATGTACTACAAACTTAGTAGT
Claims (10)
1. A stem cell line capable of multimodal tracking, comprising: umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, human induced pluripotent stem cells and vascular endothelial progenitor cells all over-expressing three proteins of FTH1, Sr39tk and EGFP.
2. A method of preparing a stem cell line capable of undergoing multimodal tracking according to claim 1, comprising the steps of:
1) providing umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, human induced pluripotent stem cells and vascular endothelial progenitor cells;
2) cloning target genes FTH1, Sr39tk and EGFP to a pLVX vector to construct a lentivirus expression vector pLVX-FTH1-Sr39 tk-EGFP;
3) Packaging lentiviruses to obtain a recombinant lentivirus containing FTH1, Sr39tk and EGFP genes;
4) the stem cell line capable of performing multi-modal tracing is characterized in that umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, human induced pluripotent stem cells and vascular endothelial progenitor cells are respectively infected by recombinant lentiviruses containing FTH1, Sr39tk and EGFP genes, so that four stem cells which respectively over-express three proteins of FTH1, Sr39tk and EGFP are obtained.
3. The method for preparing the stem cell line capable of performing multimodal tracking according to claim 2, wherein the step of providing the umbilical cord mesenchymal stem cells in the step 1) specifically comprises the following steps:
1-1-1) cleaning an umbilical cord, shearing the umbilical cord into tissue blocks, and then placing the tissue blocks into a culture bottle for culture;
1-1-2) after 40% -50% of tissues in the bottle climb out of the cells, carrying out passage on the cells, and washing off tissue blocks and redundant impurities falling from the bottle by using PBS;
1-1-3) adding trypsin to digest the cells, and then adding CO2Incubating in an incubator at 37 ℃;
1-1-4) observing the digested cells under an inverted microscope, adding a complete culture medium to neutralize and digest when the cells slightly fall off and the edges of the cells which do not fall off are round, and blowing and beating by using a pipette to enable the cells to fall off completely;
1-1-5) centrifuging the cell suspension, discarding the supernatant, resuspending the cell pellet with complete culture medium, blowing to obtain cell suspension, subculturing according to the required cell amount, adding culture medium, and adding CO at 37 deg.C2The incubator continues to culture.
4. The method for preparing the stem cell line capable of performing multimodal tracking according to claim 3, wherein the step of providing the adipose-derived mesenchymal stem cells in the step 1) specifically comprises the following steps:
1-2-1) cleaning adipose tissues, then shearing the adipose tissues into tissue blocks, and removing blood vessels and connective tissues;
1-2-1) transferring the adipose tissues into a centrifugal tube, adding equivalent volume of collagenase type I, and performing shock digestion in a water bath kettle at 37 ℃;
1-2-3) adding PBS with the same volume to stop digestion, blowing and uniformly mixing, and centrifuging the suspension;
1-2-4) the suspension after centrifugation is divided into three layers, wherein the upper layer is a fat layer, the middle layer is a liquid layer, and the lower layer is a cell layer; removing the upper and middle layers and the lower layer of liquid by a suction pipe, and taking the fat layer;
1-2-5) resuspending the fat layer with PBS, sieving with a 300-mesh cell sieve, and centrifuging;
1-2-6) resuspending the cell pellet in a culture medium and culturing;
1-2-7) discarding the old culture medium containing floating cells and replacing with a new culture medium;
1-2-8) carrying out passage when the cell fusion degree reaches more than 80%, discarding the original culture medium, adding PBS to clean the cells, discarding the PBS;
1-2-9) adding trypsin to digest the cells, so that the cells are uniformly covered by the trypsin, and then putting the cell culture dish into CO2Incubating in an incubator at 37 ℃;
1-2-10) observing the digested cells under an inverted microscope, adding a complete culture medium to neutralize and digest when the cells slightly fall off and the edges of the cells which do not fall off are round, and blowing and beating by using a pipette to enable the cells to fall off completely;
1-2-11) centrifuging the cell suspension, discarding the supernatant, resuspending the cell pellet with complete medium, blowing to obtain cell suspension, subculturing according to the required cell amount, adding medium, and adding CO at 37 deg.C2The incubator continues to cultivate.
5. The method for preparing a stem cell line capable of performing multimodal tracking according to claim 3, wherein the step of providing the human induced pluripotent stem cells in the step 1) specifically comprises:
1-3-1) adding an E8 culture medium into a centrifuge tube, then adding the human induced pluripotent stem cell liquid into the centrifuge tube, and uniformly mixing;
1-3-2), removing the supernatant, adding an E8 culture medium into a centrifuge tube, and blowing, beating and resuspending;
1-3-3) inoculating the cell suspension into a pore plate coated with matrigel in advance, and culturing in an incubator at 37 ℃;
1-3-4) replacing the culture medium every day, and carrying out passage expansion on cells when the cell fusion degree reaches more than 80%;
1-3-5) subculturing, absorbing and discarding the old cell culture medium, adding PBS to wash the pore plate, and discarding the PBS;
1-3-6) adding the digestive juice, and incubating in an incubator;
1-3-7) absorbing and removing the digestive juice, adding an E8 culture medium containing a Y27632 inhibitor, blowing and beating cells, adding the cells into a new centrifugal tube, and centrifuging;
1-3-8) resuspend the cell pellet using E8 medium and transfer to a fresh matrigel coated well plate.
6. The method for preparing a stem cell line capable of performing multimodal tracking according to claim 3, wherein the step of providing vascular endothelial progenitor cells in the step 1) specifically comprises:
1-4-1) pouring the cord blood into a centrifuge tube 1, pouring the lymphocyte separation liquid into a centrifuge tube 2, adding the cord blood in the centrifuge tube 1 into the centrifuge tube 2, wherein the addition requirement is as follows: inclining the centrifugal tube 2 by 60 degrees, sucking the cord blood in the centrifugal tube 1 by using a Pasteur pipette, and marking an S-shaped drainage band above the tube wall of the centrifugal tube 2 to ensure that the cord blood has a path which is enough to slowly flow into the lymphocyte separation liquid, and finally vertically placing the centrifugal tube 2;
1-4-2) adding the cord blood into the lymphocyte separation liquid, centrifuging, and after the centrifugation is finished, generating four layers: plasma, white blood cells, lymphocyte isolates and red blood cells; taking out the plasma layer and the leucocyte layer, adding the plasma layer and the leucocyte layer into 2 new centrifuge tubes, supplementing PBS to each tube, and centrifuging;
1-4-3) centrifuging, discarding PBS and discarding, then supplementing PBS to each tube, and centrifuging;
1-4-4) centrifuging, then removing the supernatant, suspending cell sediment by using a culture medium, and then paving cells into a culture bottle for culture;
1-4-5) adding complete medium into culture flask, adding CO at 37 deg.C2Continuously culturing in an incubator;
1-4-6), washing off the cells which are not attached to the wall, continuously culturing the attached cells, and culturing the vascular endothelial progenitor cells;
1-4-7) carrying out passage expansion of cells when the cell fusion degree reaches more than 80%;
1-4-8), absorbing and discarding the old cell culture medium, adding PBS to wash the pore plate, and discarding the PBS;
1-4-9) adding digestive juice, and incubating in an incubator;
1-4-10) removing the digestive juice, adding E8 culture medium containing Y27632 inhibitor, blowing down the cells, adding into a new centrifugal tube, and centrifuging;
1-4-11) resuspend the cell pellet using E8 medium and transfer to a new matrigel coated well plate.
7. The method for preparing a stem cell line capable of undergoing multimodal tracing according to claim 2, wherein the step 3) specifically comprises:
3-1) pre-culturing 293T cells;
3-2) configuration of plasmid transfection systems A and B:
The plasmid transfection system A comprises Opti-MEM and PEI, and the plasmid transfection system B comprises the lentivirus expression vector plasmids pLVX-FTH1-Sr39tk-EGFP, Opti-MEM and three packaging plasmids obtained in the step 2): VSV-G, pMDL, pRSV;
3-3) adding the plasmid transfection system A into the plasmid transfection system B, uniformly mixing, and standing at room temperature;
3-4) adding the mixture obtained in the step 3-3) into Opti-MEM, and uniformly mixing;
3-5) taking the cultured 293T cells, discarding the original culture medium in the 293T cells, and adding the mixture obtained in the step 3-4) into the 293T cells and submerging the 293T cells;
3-6) in CO2Culturing in an incubator;
3-7) taking supernatant fluid of 293T cells, and centrifuging in a refrigerated centrifuge;
3-8) centrifuging, taking the supernatant, and centrifuging again;
3-9), removing the supernatant, leaving the virus precipitate, and resuspending the precipitate with PBS to obtain the recombinant lentivirus.
8. The method for preparing a stem cell line capable of undergoing multimodal tracing according to claim 2, wherein the step 4) specifically comprises:
4-1) four cells of interest were previously: culturing umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, human induced pluripotent stem cells and vascular endothelial progenitor cells, and then respectively carrying out lentivirus infection according to the following steps;
4-2) adding the recombinant lentivirus obtained in the step 3) and polybrene into a serum-free culture medium, and uniformly blowing;
4-3) removing the old culture medium in the target cell, adding the recombinant lentivirus mixed solution obtained in the step 4-2) into the target cell, and enabling the recombinant lentivirus mixed solution to be over the target cell;
4-4) carrying out lentivirus infection, and supplementing a fresh complete culture medium in the middle;
4-5) finally obtaining umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, human induced pluripotent stem cells and vascular endothelial progenitor cells which all over express three proteins of FTH1, Sr39tk and EGFP, namely the stem cell line capable of carrying out multi-modal tracing.
9. The method for preparing the stem cell line capable of performing multimodal tracking according to claim 2, wherein the gene sequence of FTH1 is shown as SEQ ID No.1, the gene sequence of Sr39tk is shown as SEQ ID No.2, the gene sequence of EGFP is shown as SEQ ID No.3, and the gene sequence of the lentiviral expression vector pLVX-FTH1-Sr39tk-EGFP is shown as SEQ ID No. 4.
10. Use of a multi-modal traceable stem cell line according to claim 1 or produced by the method of any one of claims 2 to 9 as a tracer for MRI-PET multi-modal imaging.
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