阅读说明：本技术 以放射性核素89Zr标记的心肌细胞在药动学中的示踪方法 (With radionuclides89Tracing method of Zr marked cardiac muscle cells in pharmacokinetics ) 是由 于九洋 张月辉 陈涛涛 王嘉显 于 2021-09-16 设计创作，主要内容包括：本发明提供通过以放射性核素～(89)Zr标记的心肌细胞在药动学中的示踪方法,具体包括向生物体提供包括放射性标记物的受试物,并进行示踪。监测iPSC来源心肌细胞在体内分布规律,通过标记物在细胞进入机体之后的动态分布和细胞疗效之间建立关系,从而有效提高细胞药物的临床疗效,降低风险,减少药物的相互作用。(The invention provides a method for producing a compound by using a radionuclide 89 The tracing method of Zr marked cardiac muscle cell in pharmacokinetics includes providing organism with test matter including radioactive marker and tracing. The distribution rule of iPSC-derived myocardial cells in vivo is monitored, and the relationship between the dynamic distribution of markers after the cells enter an organism and the cell curative effect is established, so that the clinical curative effect of cell medicines is effectively improved, the risk is reduced, and the interaction of medicines is reduced.)

1. With radionuclides⁸⁹A method for tracing Zr-labeled cardiomyocytes in pharmacokinetics, comprising providing a test substance to a living body;

the test substance comprises a radioactive marker⁸⁹Zr-oxine；

By ingestion⁸⁹Zr-oxine and⁸⁹the Zr marked cardiac muscle cells are detected and traced by a positron emission tomography technology.

2. The radionuclide as claimed in claim 1⁸⁹A method for tracing Zr-labeled cardiomyocytes in pharmacokinetics, wherein the cardiomyocytes are derived from induced pluripotent stem cells.

3. The method of claim 2 with a radionuclide⁸⁹The tracing method of the Zr marked myocardial cells in pharmacokinetics is characterized by comprising the following specific steps:

s1, cell recovery: preparing a cell solvent from 0.9% sodium chloride injection and 20% human serum albumin according to a specific proportion, flushing a cell cryopreservation tube by using the cell solvent, adding a cell resuspension solution, centrifuging, and then adding the cell solvent to dilute a cell stock solution for later use;

s2, cytonuclide labeling: get⁸⁹Reaction of Zr with oxine to give⁸⁹Zr-oxine; and take out⁸⁹Respectively placing Zr-oxine in the recovered myocardial cells (iPSC-CM), incubating for 15min, centrifuging, then re-suspending with 5% human serum albumin, centrifuging and washing for 3 times;

s3, injection and detection: injecting the tested substances into organisms respectively; and monitoring and sketching PET imaging and standard uptake values in vivo at different time points.

4. The method of claim 3 with a radionuclide⁸⁹A method for tracing Zr-labeled cardiomyocytes in pharmacokinetics, which comprises the step of preparing a solution containing Zr in the form of a solution containing Zr in a solvent, wherein the solution is prepared in the step S2⁸⁹The Zr-oxine also comprises the steps of adjusting the PH,get⁸⁹Zr adding HEPES and Na₂CO₃The pH was adjusted to 7.

5. The method of claim 3 with a radionuclide⁸⁹A method for tracing Zr-labeled cardiomyocytes in pharmacokinetics, wherein the organism is a non-human mammal; the non-human mammal may be a mouse, monkey, cow, sheep or dog.

6. The method of claim 3 with a radionuclide⁸⁹The tracing method of Zr marked cardiac muscle cells in pharmacokinetics is characterized in that organisms are respectively G1 monkey, G2 monkey, KM mouse and NCG mouse, PET imaging in the S3 step comprises four distribution images, wherein G1 monkey, G2 monkey, KM mouse and NCG mouse respectively correspond to a first image, a second image, a third image and a fourth image; and the time points of the first image and the second image are consistent, and the time points of the third image and the fourth image are consistent.

7. The method of claim 3 with a radionuclide⁸⁹The tracing method of Zr marked cardiac muscle cells in pharmacokinetics is characterized by further comprising the step S4 of carrying out KM mouse organ in vitro detection, carrying out organ in vitro detection after 192h of test object injection and carrying out data comparison with PET radioactive substance uptake.

8. The method of claim 3 with a radionuclide⁸⁹The tracing method of Zr marked myocardial cells in pharmacokinetics is characterized in that the cell stock solution in S1 refers to the recovered myocardial cells.

9. The method of claim 3 with a radionuclide⁸⁹The tracing method of Zr marked myocardial cells in pharmacokinetics is characterized in that in the step S1, the specific ratio of sodium chloride injection to human serum albumin is 3: 1.

10. The method of claim 9 with a radionuclide⁸⁹Core marked by ZrThe tracing method of muscle cells in pharmacokinetics is characterized in that the Standard Uptake Value (SUV) is determined by a method comprising the following steps:

determining r, wherein r is image reconstruction after PET scanning is completed in a region of interest (ROI), and processing images and data by PMOD software to obtain radioactivity activity concentration (mu Ci/g); definition, radioactivity concentration (i.e. radioactivity value per unit volume);

determining s, wherein s is the administered dose of the injected subject (μ Ci);

determining w, the body weight (g) of the organism; that is to say that the first and second electrodes,

11. the radionuclide as claimed in claim 10⁸⁹A method for tracing Zr-labeled cardiomyocytes in pharmacokinetics, wherein the radiochemical purity (RCP) of the test substance is not less than 90%.

12. The radionuclide as claimed in claim 11⁸⁹A method for tracing Zr-labeled cardiomyocytes in pharmacokinetics, wherein a test substance diluent is a physiological saline solution containing 5% HSA at the time of administration.

Technical Field

The invention relates to the technical field of biological medicine, in particular to a radioactive nuclide⁸⁹A tracing method of Zr marked myocardial cells in pharmacokinetics.

Background

The induced pluripotent stem cells have the potential of differentiating into various functional cells, and similarly, the induced pluripotent stem cells can generate the myocardial cells with the contraction function by regulating corresponding signal paths, so that the myocardial cells with stable electrophysiological properties can be used for researching the pathogenesis of heart diseases and screening drugs for treating the heart diseases, even can be used for carrying out clinical research on treating the heart diseases by the myocardial cells, and further expands the contribution of the myocardial cells in treating diseases such as myocardial infarction, heart failure and the like. The mechanism of the treatment of heart diseases by cardiomyocytes as a drug is not clear, and the survival, retention, proliferation, migration and distribution of cardiomyocytes in vivo are still under further study, so that the need for tracer labeling of cardiomyocytes is necessary.

The human myocardial cells have different biological characteristics from traditional medicines such as small molecules, macromolecules and the like. The system for pharmacokinetic assessment of traditional drugs for drug absorption, distribution, metabolism is not suitable for the assessment of cellular drugs. Therefore, the distribution, metabolism and excretion rules of human iPSC-derived cardiomyocytes in vivo cannot be monitored, and further, the clinical curative effect of cell medicines cannot be improved, the risk is reduced, and the interaction of medicines is reduced.

The existing marking method mainly aims at the traditional medicine, aims at the problem that human-derived myocardial cells cannot be effectively marked, and is difficult to effectively establish the relationship between the dynamic distribution of cell medicine entering an organism and the cell curative effect. In order to solve the above problems, the present solution provides a new technical solution for overcoming the above problems.

Disclosure of Invention

The invention aims to provide⁸⁹The Zr mark can trace the myocardial cell in vivo, and the change or distribution of the myocardial cell in vivo can be accurately reflected by detecting the Zr mark when the myocardial cell is applied to the organism. To achieve the above object, the present invention provides the following technical solutions,

providing a test substance to a biological body;

the test substance comprises a radioactive marker⁸⁹Zr-oxine；

By ingestion⁸⁹Zr-oxine and⁸⁹the Zr marked cardiac muscle cells are detected and traced by a positron emission tomography technology.

In a further improvement, the cardiomyocytes are derived from induced pluripotent stem cells.

A labeling and tracing method for myocardial cells comprises the following specific steps:

s1, cell recovery: preparing a cell solvent from 0.9% sodium chloride injection and 20% human serum albumin according to a specific proportion, flushing a cell cryopreservation tube by using the cell solvent, adding a cell resuspension solution, centrifuging, and then adding the cell solvent to dilute a cell stock solution for later use;

s2, cytonuclide labeling: get⁸⁹Reaction of Zr with oxine to give⁸⁹Zr-oxine; and take out⁸⁹Respectively placing Zr-oxine in the recovered myocardial cells, incubating for 15min, centrifuging, then resuspending with 5% human serum albumin, centrifuging and washing for 3 times;

s3, subject injection and generating results: subjecting the test object⁸⁹Zr-myocardial cells,⁸⁹The Zr-oxine is respectively injected into the bodies of a cynomolgus monkey G1 and a cynomolgus monkey G2,⁸⁹zr-myocardial cells are injected into KM mice and NCG mice; and monitoring and sketching PET imaging and standard uptake values in the tested object at different time points.

In a further improvement, the preparation in the step S2⁸⁹Before Zr-oxine, the pH is adjusted, and the obtained product is taken⁸⁹Zr adding HEPES and Na₂CO₃The pH was adjusted to 7.

In a further improvement, the organism is a non-human mammal; the non-human mammal may be a mouse, monkey, cow, sheep or dog.

In a further improvement, the PET imaging in the S3 step includes four distribution images, wherein G1 monkey, G2 monkey, KM mouse and NCG mouse correspond to the first image, the second image, the third image and the fourth image, respectively; and the time points of the first image and the second image are consistent, and the time points of the third image and the fourth image are consistent.

The further improvement is that the method also comprises the step S4 of testing the organs of KM mice in vitro, testing the organs in vitro 192h after the injection of the test object and comparing the data with the PET radioactive substance uptake.

In a further improvement, the cell stock solution in S1 refers to the recovered myocardial cells.

In a further improvement, in the step S1, the specific ratio of the sodium chloride injection to the human serum albumin is 3: 1.

In a further improvement, the Standard Uptake Value (SUV), the method for determining the SUV includes:

determining r, wherein r is image reconstruction after PET scanning is completed in a region of interest (ROI), and processing images and data by PMOD software to obtain radioactivity activity concentration (mu Ci/g); definition, radioactivity concentration (i.e. radioactivity value per unit volume);

determining s, wherein s is the administered dose of the injected subject (μ Ci);

determining w, the body weight (g) of the organism; that is to say that the first and second electrodes,

in a further improvement, the Radioactive Chemical Purity (RCP) of the subject is not less than 90%.

In a further improvement, the subject is diluted with a saline solution containing 5% HSA when administered.

An organism refers to a subject, which is a non-human mammal.

The cardiomyocytes according to any one of the embodiments of the present invention and the labeling method thereof are intended for non-disease diagnosis or treatment.

According to the technical scheme, the technical scheme of the invention provides the following beneficial effects:

on one hand, the method can realize the stability of the in vivo tracing method and can be combined with living body PET imaging to monitor the distribution of cells in vivo. On the other hand, the distribution rule of human iPSC-derived cardiomyocytes in vivo is monitored, and then the relationship is established between the dynamic distribution of the markers after the cells enter the organism and the cell curative effect, so that the clinical curative effect of the cell medicine is effectively improved, the risk is reduced, and the interaction of the medicine is reduced.

It should be understood that all combinations of the foregoing concepts and additional concepts described in greater detail below can be considered as part of the inventive subject matter of this disclosure unless such concepts are mutually inconsistent.

The foregoing and other aspects, embodiments and features of the present teachings can be more fully understood from the following description taken in conjunction with the accompanying drawings. Additional aspects of the present invention, such as features and/or advantages of exemplary embodiments, will be apparent from the description which follows, or may be learned by practice of specific embodiments in accordance with the teachings of the present invention.

Drawings

The drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures may be represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. Embodiments of various aspects of the present invention will now be described, by way of example, with reference to the accompanying drawings, in which:

FIG. 1 shows G1-M-01 (hereinafter referred to as G1) administered by injection to cynomolgus monkey⁸⁹Zr-cardiomyocytes; G2-M-01 (hereinafter referred to as G2) administered by injection to cynomolgus monkey⁸⁹PET scanning images at different time points after Zr-oxide;

FIG. 2 shows the respective injections of G1 and G2 for cynomolgus monkey⁸⁹Zr-myocardial cells,⁸⁹Histograms of the change in radioactivity uptake of each tissue over time after Zr-oxide;

FIG. 3 shows KM mice injection⁸⁹After Zr-myocardial cells, the radioactive uptake value of each tissue changes along with time;

FIG. 4 shows KM mice injection⁸⁹PET scans at different time points after Zr-cardiomyocytes;

FIG. 5 shows NCG mice injected⁸⁹The change of the radioactivity uptake value of each tissue along with time after the Zr-myocardial cells;

FIG. 6 shows NCG mice injected⁸⁹PET scans at different time points after Zr-cardiomyocytes;

FIG. 7 is a graph comparing biodistribution and histogram data of KM mice after 192h imaging technique; (wherein, biodistribution: ex vivo detection of organs; Chart data: PET biopsy)

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings of the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention. Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.

The use of "first," "second," and similar terms in the description and claims of the present application do not denote any order, quantity, or importance, but rather the terms are used to distinguish one element from another. Similarly, the singular forms "a," "an," or "the" do not denote a limitation of quantity, but rather denote the presence of at least one, unless the context clearly dictates otherwise. The terms "comprises," "comprising," or the like, mean that the elements or items listed before "comprises" or "comprising" encompass the features, integers, steps, operations, elements, and/or components listed after "comprising" or "comprising," and do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. "upper", "lower", "left", "right", and the like are used only to indicate relative positional relationships, and when the absolute position of the object to be described is changed, the relative positional relationships may also be changed accordingly.

Defining:

PET positron emission tomography scanner

PMOD medical analysis software

% ID/g percent injection dose rate per gram of tissue

SUV Standard uptake value

Instrument for measuring activity of radioactive nuclide by activity meter

Mu Ci micro Curie, Primary Unit of radioactivity

ROI refers to the term "region of interest" in image processing "

Radiochemical purity of RCP

SUV Standard uptake value

⁸⁹Zr zirconium-89

Main materials and reagents:

iPSC-CM: supplied by Nanjing El & lt & gt, regenerative medicine science and technology Co., Ltd;

oxine: purchased from Sigma Aldrich;

⁸⁹zr-oxalate: purchased from Nanjing Andi Ke;

⁸⁹zr-cardiomyocytes:⁸⁹zr marks cardiomyocytes.

2 cynomolgus monkeys, normally bred by the Mild (Nanjing) Biotechnology Ltd; license for use of experimental animal: SYXK (threo) 2017-0065.

1 KM mouse and 1 NCG mouse, which are provided by Changzhou Kavens laboratory animals Co., Ltd, and are normally bred by the clean-grade environment IVC system of the laboratory animal center of atomic medical research institute of Jiangsu province. All animal studies strictly comply with national laws and regulations, and all animal experiments are approved by the institutional animal care and ethical committee of atomic medical research institute of Jiangsu province.

Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, which are within the capabilities of persons skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

Example 1 biomarker differentiation

1) Tissue distribution experiments in cynomolgus monkeys (non-human primates)

The normal cynomolgus monkey has 2 normal cynomolgus monkeys, male cynomolgus monkeys and each cynomolgus monkey has a unique animal identification number which is respectively marked as G1-M-01 and G2-M-01. The cynomolgus monkey grouping and dosing schedule in this experiment is detailed in table 1 below:

TABLE 1

2) Kunming (KM) mouse tissue distribution experiment

Normal Kunming (KM) mice 1, male, were scored as KM mice, respectively. The KM mice dosing regimen in this experiment is detailed in Table 2 below:

TABLE 2

3) Tissue distribution experiments in immunodeficient mice (NCG mice)

Normal NCG mice 1, male, NCG mouse dosing regimen in this trial, detailed in table 3 below:

TABLE 3

Example 2 cell recovery

(1) Taking out cell stock solution to be resuscitated from liquid nitrogen tank, including 3 groups of cardiomyocytes (iPSC-CM), each group is 8 × 10⁸Respectively placing the mixture in dry ice for temporary storage; taking out the 20% human serum albumin solution from a refrigerator at 4 ℃, and keeping the solution in the dark for 10 minutes at 15-25 ℃;

(2) adding a cell solvent into a centrifugal tube, wherein the cell solvent is prepared according to the proportion of 0.9% sodium chloride injection and 20% human serum albumin of 3: 1, and fully and uniformly mixing for later use;

(3) taking out cell stock solutions from the dry ice, respectively putting the cell stock solutions into a freezing tube, quickly immersing the lower 1/2 part of the freezing tube into a water bath at 37 +/-1 ℃, quickly shaking for 1-2 minutes until only one small piece of ice remains in the tube, and taking out the freezing tube;

(4) wiping the surfaces of the freezing tubes with 75% alcohol, gently blowing and beating the freezing tubes for about 5 times by using a pipette, and respectively transferring all liquid in each freezing tube into a 50mL centrifugal tube;

(5) a pipettor takes 1ml of cell solvent to wash the cell freezing tube, and the washing liquid is slowly added into the 50ml centrifugal tubes respectively;

(6) slowly adding 19mL of cell resuspension by using a pipettor, and centrifuging for 5min at 15-25 ℃ and 300 g;

(7) and (4) after centrifugation is finished, sucking and removing supernatant, counting after cell solvent is resuspended, adding cell solvent to dilute the cell stock solution to the required concentration, and finishing the administration preparation.

Example 3 radionuclide labeling

Take 50ul⁸⁹Zr, found 633 uCi;

adjusting pH to 7, adding 200ul HEPES and 32.5ul Na₂CO₃，

The concentration of the HEPES is 0.1mol/L, Na₂CO₃The concentration is 1 mol/L;

then adding 5uL Oxine, wherein the concentration of the Oxine is 5ug/uL to obtain⁸⁹Zr-OXINE, 240uCi was measured.

The 3 groups of cardiomyocyte resuscitating fluids of example 1 were removed separately and each group was labeled as follows:

respectively take⁸⁹Zr-Oxine to 3 groups 8X 10 in example 1⁸Incubating in individual cardiomyocytes for 15min at room temperature; centrifuge at 300g for 1 min.

Then re-suspending with 5% human serum albumin, centrifuging for 1min at 300g, and centrifugally washing for 3 times.

Are respectively prepared into⁸⁹Zr-oxine; and group 3⁸⁹Zr-cardiomyocytes.

The test substance is administered in a diluted solution of a physiological saline solution containing 5% HSA.

⁸⁹Zr-oxine is stored in dimethyl sulfoxide (DMSO), and the DMSO content is less than or equal to 10% when the Zr-oxine is administrated.

Example 4 injection and PET imaging thereof

⁸⁹Zr-myocardial cells are injected into the myocardium of a cynomolgus monkey (G1-M-01) with the administration dosage of 1 × 10⁸One cell/one, 1.25mL was injected, with a radioactive dose of 1.5 uCi. A first image is formed by PET scanning.

⁸⁹Zr-oxine is injected into the myocardium of a cynomolgus monkey (G2-M-01) at an administration dose of 1 × 10⁸One cell/one, 1.25mL was injected, with a radioactive dose of 1.5 uCi. A second image is formed by PET scanning.

⁸⁹Zr-myocardial thinnessMyocardial injection of cells in KM mice with administration dose of 2X 10⁶Per cell, a 50uL radioactive dose of 1uCi was injected. A third image is formed by PET scanning.

⁸⁹Zr-myocardial cells are injected into the myocardium of NCG mice, and the administration dose is 5 multiplied by 10⁵One cell/one, 200uL injection, radioactive dose 1 uCi. A fourth image is formed by PET scanning.

The first and second images were subjected to PET whole body static scans at 1.5h, 2h, 4h, 8h, 24h, 2d, 3d, 8d, 14d, 21d post-injection, respectively.

The third image and the fourth image are subjected to PET imaging detection at 1h, 4h, 6h, 16h, 24h, 40h, 48h, 72h, 96h, 120h, 144h, 168h and 192h after injection.

All the scanning time points are kept consistent with the theoretical time in principle, if deviation exists, the allowable deviation range is as follows, the deviation is controlled within +/-5 min at the time point of 0-4h (including 0h and 4 h); controlling the deviation within +/-10 min at the time point of 4-24 h; the deviation is controlled to be +/-5% at the time point of more than or equal to 24h (including 24 h). The observation can be carried out in real time, and the error is reduced. Detecting the amount of reflection by gamma counter, and detecting the distribution of cell tissue

Example 4 delineation of major tissues and organs and acquisition of Radioactive uptake values

Carrying out image reconstruction after PET scanning is finished, processing images and data by adopting PMOD software, and delineating a region of interest (ROI), wherein the ROI of the cynomolgus monkey comprises organs such as heart, liver, spleen, kidney, whole brain, muscle, knee joint, bone marrow, gonad and the like; KM mouse region of interest (ROI) including heart, lung, liver, spleen and bone marrow; NCG mouse region of interest (ROI) includes lung, liver, bone marrow and muscle. The radioactivity concentration (i.e. radioactivity value per volume) of a region of interest (ROI) is obtained. And calculating the percent injection dosage rate (abbreviated as a percent ID/g value) of each organ per gram of tissues according to the dosage of the medicament, or calculating the standard uptake value (abbreviated as a SUV value) of each organ according to the weight of the animal. The formula is as follows:

the Standard Uptake Value (SUV), the method for determining SUV is detailed as follows:

determining r, wherein r is image reconstruction after PET scanning is completed in a region of interest (ROI), and processing images and data by PMOD software to obtain radioactivity activity concentration (mu Ci/g);

determining s, wherein s is the administered dose of the injected subject (μ Ci);

determining w, the body weight (g) of the organism; that is to say that the first and second electrodes,

FIG. 2 depicts Standard Uptake Values (SUVs); figures 3, 5 and 7 illustrate the percentage injection dose rate per gram of tissue (abbreviated as% ID/g value), which is consistent with the theoretical meaning.

Example 5 PET imaging results and Radioactive uptake values

⁸⁹The marked myocardial cells and Zr-oxine are verified to be injected into the bodies of the cynomolgus monkeys to trace the distribution of the cells in the bodies

G1 injection for cynomolgus monkey⁸⁹After Zr-cardiomyocytes, radioactive substances are mainly distributed in the heart, liver and spleen, secondly in the lung, kidney, bone joints, bone marrow-femur, bone marrow-spine and bone marrow-tibia, thirdly in the testis and tibia cortical bone, and the brain and muscle are less distributed.

G2 administration by injection to cynomolgus monkeys⁸⁹After Zr-oxide, radioactive substances are mainly distributed in the heart, liver, spleen and kidney, then lung and marrow-spine, then bone joint and testis, and bone marrow-femur, bone marrow-tibia, tibia cortical bone, brain and muscle are low in distribution. See attached figure 1

From the perspective of the radioactive uptake value and the trend of change, cynomolgus monkeys G1 and G2 showed thatInjection of drugs⁸⁹Zr-myocardial cells,⁸⁹After Zr-oxine, the radioactive uptake values of the liver, spleen, lung, kidney and bone joints are different: the method comprises the following specific steps:

liver and spleen SUV-mean (mean) values G1 cynomolgus monkeys were higher than G2 cynomolgus monkeys, and G1 cynomolgus monkeys liver and spleen SUV-mean values generally showed a first-rising-then-falling trend, whereas G2 cynomolgus monkeys did not change significantly;

the SUV-mean value of the lung of the G1 cynomolgus monkey is higher than that of the G2 cynomolgus monkey, and the SUV-mean value of the lung of the G1 cynomolgus monkey shows a gradually descending trend, while the change of the lung of the G2 cynomolgus monkey is not obvious;

the SUV-mean of the kidney of the G1 cynomolgus monkey is lower than that of the G2 cynomolgus monkey, and the SUV-mean value of the kidney of the G1 cynomolgus monkey generally shows a tendency of ascending before descending, while the SUV-mean value of the kidney of the G2 cynomolgus monkey shows a tendency of gradually descending;

the SUV-mean of the pulmonary bone joint of the cynomolgus monkey G1 is higher than that of the cynomolgus monkey G2, and both show the tendency of ascending before descending. See figure 2 for details.

The method comprises the steps of labeling the myocardial cells inducing differentiation of the pluripotent stem cells, injecting the labeled myocardial cells into a cynomolgus monkey animal body, detecting distribution of radioactive labeling signals in the animal body after cell injection by means of an animal living body imager, and tracing the in-vivo process of the cells based on a PET scanning image in tissues. With single injection⁸⁹The comparison of Zr-oxine shows that the change trend of the radioactive uptake value is more obvious when the marked myocardial cells are injected into the animal body.

⁸⁹Further verifying the distribution of Zr-labeled cardiomyocytes injected into KM mice

As shown in FIG. 4, KM mice were injected with the injection⁸⁹Zr-cardiomyocytes: over time⁸⁹The Zr-myocardial cells are transferred from the heart to the lung, the liver, the bone and the like; the heart intake gradually decreased and began to decrease slowly after 40 h. The liver decreases first, and the decrease amount after 24h begins to level off. The lung slowly decreases with time, and the decrease after 48h is smaller and tends to be stable. The bone intake gradually increased with time, and the amount of change was small after 144 h.

As shown in FIG. 3, KM mice were injected with the injection⁸⁹Zr-cardiomyocytes in the animalThe highest amount of material taken by heart over time⁸⁹The Zr-myocardial cells gradually transfer from the heart to the lung, liver, bone and other parts. The heart intake gradually decreased and began to stabilize after 48 h. The liver is reduced firstly, and the reduction amount is smaller after 24 hours and begins to tend to be stable; the lung slowly decreases with time, and the decrease after 48h is smaller and tends to be stable. The liver intake is about 5.1 times of the lung at 192 h. The bone intake gradually increased with time, and the amount of change was small after 144h, reaching a peak at 192 h. The bone intake at 192h was about 15.4 times that of the lung and 3.0 times that of the liver.

The results show that the change trend of the radioactive uptake value of the marked myocardial cells is also obvious when the marked myocardial cells are injected into animals. Can trace in organism, has normal bioactivity, and accurately reflects the change process or distribution condition of the cell.

⁹Further verify the distribution of Zr-labeled cardiomyocytes injected into NCG mice

As shown in FIG. 6, the NCG mice were injected into myocardium⁸⁹Zr-cardiomyocytes: the myocardial cells are gradually transferred from the lung to the liver, bones and other parts along with the time; the intake of the lung gradually decreases and becomes stable after 48 h. The liver intake increases slowly with time and tends to be stable after 48 h. Bone uptake increased first and began to decrease after 72 h.

As shown in FIG. 5, the NCG mice were injected into myocardium⁸⁹After 1h of Zr-myocardial cells, the intake of the Zr-myocardial cells in the lung of the animal is the highest, and the Zr-myocardial cells are taken in the lung of the animal over time⁸⁹The Zr-myocardial cells are gradually transferred from the lung to the liver and bone parts. The intake of the lung gradually decreases and becomes stable after 48 h. The liver intake increases slowly with time, reaches a peak value at 40h, slightly decreases after 72h, and starts to keep stable after 96 h; the liver intake is about 1.7 times that of lung at 192 hr. The bone intake reaches a peak value in 72h, and then gradually decreases to 192 h; the bone intake at 192h was about 3.1 times that of the lung and 1.8 times that of the liver. Therefore, the marked myocardial cells can be shown to be injected into animal bodies, the variation trend of the radioactive uptake value is also obvious, and the in-vivo channels of the marked cells can be accurately tracedTime distribution.

Example 6 cell tissue distribution (KM mice)

Further verifying the accuracy of tissue radiation uptake value in the living body state

The mice were sacrificed immediately after 192h imaging, and heart, liver, spleen, lung, and joints of the KM mice were taken, and the reflectance and cell tissue distribution were measured using a gamma counter. Biodistribution: detecting each organ in vitro; drawing data: PET biopsy. Reference is made in detail to fig. 7.

KM mouse injection⁸⁹The Zr-myocardial cells are sacrificed after 192h imaging is finished, the heart, the liver, the spleen, the lung and the joints are immediately taken, and the tissue intake is detected. During the detection of the living body, the joint ingestion value is the largest, and then the heart and the liver are the second; during the detection of each organ in vitro, the uptake value of the joint is the largest, and then the uptake values of the heart and the liver are the second. Therefore, the ex vivo detection of each organ is compatible with the biopsy.

The PET delineation uptake value is about 2 times of the in vitro heart uptake value, the PET delineation uptake value is about no obvious difference from the in vitro liver uptake value, the in vitro spleen uptake value is about 5 times of the PET delineation uptake value, the in vitro lung uptake value is about 2 times of the PET delineation uptake value, and the PET delineation uptake value is not obviously different from the in vitro joint uptake value. See fig. 7.

In conclusion, the invention realizes the researches on early distribution, curative effect prediction, visual detection and the like of cells. The distribution rule of human iPSC-derived myocardial cells in vivo is monitored, and a relationship is established between the dynamic distribution and the cell curative effect of the cell medicament after entering an organism, so that the clinical curative effect of the cell medicament is effectively improved, the risk is reduced, and the interaction of the medicaments is reduced. The radioactive isotope labeling is a stable in-vivo tracing method, and provides an effective effect for monitoring the distribution of cells in vivo by combining with living body PET imaging analysis.

Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, the protection scope of the present invention should be determined by the appended claims.