CN112813032A - Stem cell evaluation multichannel imaging system and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a stem cell evaluation multichannel imaging system and a preparation method and application thereof. The stem cell evaluation multichannel imaging system comprises: the tumor cell dual-channel imaging unit mainly comprises tumor cells transfected by plasmid genes, wherein the reporter genes of the plasmid genes comprise an Rfluc gene and a Gluc gene; and the contrast agent nano unit mainly comprises an amphiphilic polymer, a contrast agent and a membrane-penetrating peptide, and is provided with a hydrophilic outer shell and a hydrophobic inner core, wherein the contrast agent is wrapped in the hydrophobic inner core, and the membrane-penetrating peptide is combined on the surface of the hydrophilic outer shell through electrostatic adsorption. The multi-channel living body imaging technology for evaluating the stem cell tumorigenicity/tumor suppressibility can realize preclinical evaluation of stem cells of different source systems, realize safety evaluation of different injection modes and dosages of the stem cells, and can be used for development and evaluation research of a new stem cell therapy for tumor treatment.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and biological influence, and particularly relates to a stem cell evaluation multichannel imaging system and a preparation method and application thereof.
Background
Stem cell therapy is taken as a modern biomedical hotspot and widely applied to research in various aspects such as myocardial repair, organ transplantation, tumor treatment and the like. Clinically, ASC/MSC is directly used for regenerative medicine treatment of diseases. Research shows that stem cells have tropism to inflammatory environments such as tumors and the like, and can enter a tumor microenvironment with low oxygen content to interact with the inflammatory environments. The tropism is regulated by cytokines such as TGF beta, VEGF and the like, the chemokines play a decisive role together, the chemotactic factor has a complex signal pathway regulation principle, and stem cells from different sources and strains often show different tumor promotion or tumor inhibition characteristics. In addition, different functionalized MSCs (mesenchymal stem cells) are currently designed for the treatment of tumors. The stem cell therapy has obtained great results in preclinical research, however, when the stem cells are implanted into the body as a therapeutic agent for treatment, the tumorigenicity and the tumorigenicity of the body also exist. The action of stem cells and lesions is mutual, inflammatory reaction, angiogenesis and further proliferation of malignant cells of the lesions can be promoted or inhibited in microenvironment of the lesions, and the safety and the effectiveness of the stem cells are not evaluated by a reasonable and scientific method at present.
Disclosure of Invention
The invention mainly aims to provide a stem cell evaluation multichannel imaging system, a preparation method and application thereof, thereby overcoming the defects in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
the embodiment of the invention provides a stem cell evaluation multichannel imaging system, which comprises:
the tumor cell dual-channel imaging unit mainly comprises tumor cells transfected by plasmid genes, wherein the reporter genes of the plasmid genes comprise an Rfluc gene and a Gluc gene;
and the contrast agent nano unit mainly comprises an amphiphilic polymer, a contrast agent and a membrane-penetrating peptide, and is provided with a hydrophilic outer shell and a hydrophobic inner core, wherein the contrast agent is wrapped in the hydrophobic inner core, and the membrane-penetrating peptide is combined on the surface of the hydrophilic outer shell through electrostatic adsorption.
The embodiment of the invention also provides a preparation method of the multi-channel imaging system for stem cell evaluation, which comprises the following steps:
constructing a double-reporter gene plasmid vector, and then transfecting tumor cells by a liposome method to obtain a tumor cell dual-channel imaging unit;
and mixing the amphiphilic polymer with a contrast agent to form a polymersome, and then mixing the polymersome with the membrane-penetrating peptide to form the contrast agent nano unit.
The embodiment of the invention also provides application of the stem cell evaluation multichannel imaging system in the field of fluorescence imaging.
The embodiment of the invention also provides a multi-channel imaging method for stem cell evaluation, which comprises the following steps:
providing the stem cell evaluation multi-channel imaging system;
establishing a mouse tumor model by using the tumor cell dual-channel imaging unit;
marking the stem cells by using a contrast agent nano unit to obtain marked stem cells;
and inputting the marked stem cells and fluorescein into a target region, thereby realizing simultaneous tracing fluorescence imaging of the stem cells and the tumor cells.
The embodiment of the invention also provides a preclinical evaluation method for promoting tumor/inhibiting tumor of stem cells, which comprises the following steps:
providing the stem cell evaluation multi-channel imaging system;
establishing a mouse tumor model by using the tumor cell dual-channel imaging unit;
marking the stem cells by using a contrast agent nano unit to obtain marked stem cells;
and inputting the marked stem cells and fluorescein into a target area, and respectively monitoring stem cell distribution, tumor growth number and metastasis infiltration state through fluorescence imaging and bioluminescence imaging, thereby evaluating the influence of exogenous stem cells on the tumor occurrence and development of the mouse.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention provides a tumor cell strain for stably expressing Rfluc and Gluc genes, which is an ideal cell for establishing a tumor animal model, combines a plasmid gene and a tumor cell, and carries out gene modification on the tumor cell by using a liposome transfection method to obtain the tumor cell strain capable of simultaneously expressing ZsGreen protein, Rfluc luciferase and Gluc luciferase. Under the condition of normal proliferation and growth of the tumor cells, the Rfluc is stably and continuously expressed in the cells, and ZsGreen is also expressed in cis; the Gluc gene sequence is positioned at the downstream of an NF-kB promoter, the expression quantity is regulated and controlled by NF-kB, and ZsGreen protein is used for carrying out cell monoclonal culture screening, the Rfluc luciferase indicates the number of tumor cells, and the Gluc luciferase indicates the infiltration transfer activity of the tumor cells, so that the problem of in-situ nondestructive detection which is difficult to realize by a traditional method is solved, and an important experimental animal model is provided for predicting the safety of a stem cell therapy;
(2) the stem cell evaluation multichannel imaging technology provided by the invention has the following effects: the excellent fluorescence characteristic of the near-infrared two-region fluorescence quantum dots is utilized, the in-vivo distribution monitoring of the stem cells which are visualized in real time and are lossless can be realized, the imaging time resolution can reach 30ms, the spatial resolution can reach 25 mu m, the stem cell treatment injection mode is guided rationally, and the method is used for tracing and dosage indication of therapeutic cells such as stem cells and the like; meanwhile, the real-time lossless imaging state monitoring of tumor cells is realized, and the survival state, the growth number and the transfer activity of the tumor are detected in real time;
(3) the invention is beneficial to establishing a common working strategy of a plasmid gene, a cell line and a near-infrared two-region contrast agent, and provides a new method for endowing the plasmid gene with a new function of the cell line and marking cells by the near-infrared two-region contrast agent.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic design diagram of a preclinical evaluation method for tumorigenicity/tumorigenicity inhibition of stem cells in an exemplary embodiment of the invention;
FIG. 2 is a schematic diagram of the sequence of the reporter gene of the plasmid gene according to an exemplary embodiment of the present invention;
FIG. 3 is a fluorescent microscope image of ZsGreen expressing transgenic U87MG cells taken in example 1 of the present invention;
FIG. 4 is an Rfluc, Gluc bioluminescence image of transgenic U87MG cells taken in example 1 of the present invention;
FIGS. 5a to 5c are near-infrared two-zone fluorescence imaging images photographed in embodiment 1 of the present invention;
FIG. 6 is a graph of near-infrared two-zone fluorescence imaging taken in example 1 of the present invention, wherein stem cells labeled with a contrast agent nano-unit are injected into a tumor model mouse via tail vein for 1 hour;
FIG. 7 shows the contrast agent nano unit Tat-PEG-Ag in example 1 of the present invention2Schematic diagram of Se synthesis.
Detailed Description
In view of the defects of the prior art, the inventor provides the technical scheme of the invention through long-term research and extensive practice, and mainly combines the Rfluc luciferase and the Gluc luciferase as functional enzymes to indicate the growth state and the metastatic infiltration activity of tumors. The ZsGreen green fluorescent protein is used for monoclonal cell screening, and can also be used for flow cytometry sorting. ZsGreen and Rfluc gene sequences are positioned behind a widely expressed promoter EF1 alpha and are expressed in cis, wherein the content of Rfluc and the number of cells have a good linear relation. After the Gluc gene sequence is positioned on an NF-kB promoter, the content of the Gluc gene sequence is positively correlated with the activity of NF-kB. The continuous and efficient expression of ZsGreen protein, Rfluc luciferase and Gluc luciferase is realized, and the problems that the traditional method is difficult to realize, the distribution condition of stem cells in a living body is monitored, and the influence of the stem cells on host tumor cells is detected in situ without damage are solved.
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
One aspect of an embodiment of the present invention provides a stem cell evaluation multi-channel imaging system, including:
the tumor cell dual-channel imaging unit mainly comprises tumor cells transfected by plasmid genes, wherein the reporter genes of the plasmid genes comprise an Rfluc gene and a Gluc gene; and the number of the first and second groups,
the contrast agent nano unit mainly comprises an amphiphilic polymer, a contrast agent and a membrane-penetrating peptide, and is provided with a hydrophilic outer shell and a hydrophobic inner core, wherein the contrast agent is wrapped in the hydrophobic inner core, and the membrane-penetrating peptide is combined on the surface of the hydrophilic outer shell through electrostatic adsorption.
In some more specific embodiments, the stem cell evaluation multi-channel imaging system comprises:
tumor cell dual channel imaging unit, mainly consisting of tumor cells stably transfected with plasmid genes having the following reporter genes: the gene comprises an EF1 alpha promoter, an Rfluc gene and an NF-kB promoter, wherein the Rfluc gene is expressed by transcription of the EF1 alpha promoter, the Gluc gene is expressed by transcription of the NF-kB promoter, after the plasmid gene successfully and stably transfects human tumor cells, cells express Rfluc luciferase and Gluc luciferase, the transfected tumor cells stably carry and continuously express the Rfluc luciferase, and the Gluc luciferase is expressed according to the activity of NF-kB in the cells;
the contrast agent nano unit mainly comprises an amphiphilic polymer, a contrast agent and a membrane-penetrating peptide, and is provided with a hydrophilic outer shell and a hydrophobic inner core, wherein the contrast agent is wrapped in the hydrophobic inner core, and the membrane-penetrating peptide is combined on the surface of the hydrophilic outer shell through electrostatic adsorption; the contrast agent nanometer unit can efficiently and low-toxicity mark stem cells, so that the stem cells can be imaged in living bodies in a near-infrared two-region window with high sensitivity.
The design sequence of the reporter gene of the plasmid gene of the present invention is schematically shown in FIG. 2.
In some more specific embodiments, the tumor cell dual-channel imaging unit mainly consists of a plasmid gene edited by CRISPR/CAS9 and a tumor cell line; after the plasmid gene stably transfects the tumor cell, the tumor cell can continuously and stably express the Rfluc luciferase and the Gluc luciferase, and the Rfluc luciferase and the Gluc luciferase respectively catalyze luciferin sylvite and coelenterazine to carry out oxidative luminescence; the potassium fluorescein salt and the coelenterazine are enriched at the tumor part of the mouse tumor model respectively by the modes of intraperitoneal injection and tail vein injection; (ii) Rfluc luciferase and Gluc luciferase at the tumor, the redox reaction being carried out by the substrate-enriched catalytic substrate; wherein the reaction emits light, and when sufficient potassium luciferin salt and coelenterazine substrate are given, the quantity of the reaction light has good linear relation with the expression quantity of the Rfluc luciferase and the Gluc luciferase.
In some more specific embodiments, the stem cell evaluation multi-channel imaging system further comprises fluorescein, and is not limited thereto.
Further, the fluorescein includes any one or a combination of two of potassium fluorescein, coelenterazine, and is not limited thereto.
Further, the Rfluc luciferase catalyzes luciferin to generate oxidation reaction to emit bioluminescence, and the light quantity statistics and the cell number are in a linear relation; the Gluc luciferase catalyzes luciferin to generate oxidation reaction to emit bioluminescence, and the light quantity statistics is in direct proportion to the activity of NF-kB in cells.
In some more specific embodiments, the plasmid gene transfected tumor cells are capable of expressing Rfluc luciferase and/or Gluc luciferase.
Further, the tumor cells transfected by the plasmid gene can express ZsGreen protein.
Further, the Rfluc luciferase and/or Gluc luciferase can react with luciferin to display fluorescence.
In some more specific embodiments, the contrast agent nanosystems are capable of labeling stem cells.
In some more specific embodiments, the contrast agent comprises near-infrared two-region quantum dots, and is not limited thereto.
Further, the near-infrared two-region quantum dots comprise Ag2Se、Ag2S、Ag2Te, and is not limited thereto.
In some more specific embodiments, the cell-penetrating peptide comprises a cell-penetrating cationic short peptide, and is not limited thereto.
Further, the cell-penetrating peptide comprises Tat and HR9、hLF、ANTP、Penetration、DPV3、(Ka)4、(RXR)4Any one or a combination of two or more of them, and is not limited thereto.
In some more specific embodiments, the amphiphilic polymer includes any one or a combination of two or more of long chain distearoylphosphatidylethanolamine-polyethylene glycol, long chain distearoylphosphatidylethanolamine-polyethylene glycol derivatives, functionalized long chain distearoylphosphatidylethanolamine-polyethylene glycol, and is not limited thereto.
Furthermore, the molecular weight of the long-chain distearoyl phosphatidyl ethanolamine-polyethylene glycol is 10-100000 Da.
Further, the long-chain distearoylphosphatidylethanolamine-polyethylene glycol comprises distearoylphosphatidylethanolamine-polyethylene glycol 2000.
In another aspect of the embodiments of the present invention, there is provided a method for preparing the above multi-channel imaging system for stem cell evaluation, including:
constructing a double-reporter gene plasmid vector, and then transfecting tumor cells by a liposome method to obtain a tumor cell dual-channel imaging unit;
and mixing the amphiphilic polymer with a contrast agent to form a polymersome, and then mixing the polymersome with the membrane-penetrating peptide to form the contrast agent nano unit.
In some more specific embodiments, the preparation method comprises:
constructing a plasmid vector containing an Rfluc gene and a Gluc gene;
and co-incubating the plasmid vector and the liposome at 4-45 ℃ for 0.1-100h, and screening to obtain the tumor cell dual-channel imaging unit.
In some more specific embodiments, the preparation method comprises:
mixing or ultrasonically treating an amphiphilic polymer, a contrast agent and a solvent to enable a hydrophobic layer of the amphiphilic polymer to wrap the contrast agent to form a polymersome;
and carrying out solvent removal treatment on the polymer vesicle, and mixing the polymer vesicle with the cell-penetrating peptide to form the contrast agent nano unit.
Further, the solvent includes pure water, PBS, or other neutral buffer, and is not limited thereto.
In some more specific embodiments, the method for preparing the tumor cell dual-channel imaging unit specifically comprises:
double reporter gene sequences are inserted into a vector plasmid pROSA26-Puro-DNR, and comprise ZsGreen and Rfluc gene sequences controlled by an EF1 alpha promoter, and Gluc gene sequences controlled by an NF-kB promoter, so that a double reporter gene pROSA26-Puro-DNR plasmid system is constructed; transfecting tumor cells by a liposome method and the plasmid gene pROSA26-Puro-DNR and the auxiliary plasmid gene of the double reporter genes to prepare transgenic tumor cells; and performing fluorescence microscope characterization, flow cytometry screening and single cell clone culture on the transgenic tumor cell to obtain the tumor cell dual-channel imaging unit.
In another aspect of the embodiments of the present invention, an application of the aforementioned multi-channel imaging system for stem cell evaluation in the field of fluorescence imaging is also provided.
Another aspect of the embodiments of the present invention also provides a multi-channel imaging method for stem cell evaluation, including:
providing the stem cell evaluation multi-channel imaging system;
establishing a mouse tumor model by using the tumor cell dual-channel imaging unit;
marking the stem cells by using a contrast agent nano unit to obtain marked stem cells;
and inputting the marked stem cells and fluorescein into a target region, thereby realizing simultaneous tracing fluorescence imaging of the stem cells and the tumor cells.
Further, the labeled stem cells and the fluorescein can be infused in any one of the manners of tail vein injection, in-situ injection and intraperitoneal injection, but not limited thereto.
Yet another aspect of the embodiments of the present invention provides a method for preclinical assessment of tumorigenicity/tumorigenicity of stem cells, comprising:
providing the stem cell evaluation multi-channel imaging system;
establishing a mouse tumor model by using the tumor cell dual-channel imaging unit;
marking the stem cells by using a contrast agent nano unit to obtain marked stem cells;
and inputting the marked stem cells and fluorescein into a target area, and respectively monitoring stem cell distribution, tumor growth number and metastasis infiltration state through fluorescence imaging and bioluminescence imaging, thereby evaluating the influence of exogenous stem cells on the tumor occurrence and development of the mouse.
A schematic design diagram of a preclinical evaluation method for tumorigenicity/tumorigenicity inhibition of stem cells in a specific embodiment of the invention is shown in FIG. 1.
In some more specific embodiments, the method for preclinical evaluation of tumorigenicity/tumorigenicity of stem cells comprises:
the plasmid gene and the tumor cell are prepared into a dual-channel imaging system by adopting the method, and the transgenic tumor cell is utilized to carry out mouse tumor modeling. After stem cells are efficiently marked without damage by using a contrast agent, the stem cells are marked in different modes: tail vein injection, in-situ injection or intraperitoneal injection, and the marked stem cells are delivered into the body. The distribution of stem cells in vivo, the tumor growth number and the metastatic infiltration state are respectively monitored through fluorescence imaging and bioluminescence imaging, and the influence of exogenous stem cells on the tumor occurrence and development of mice is evaluated.
In some embodiments of the invention, a multi-channel imaging system with simultaneous expression of bifluc luciferase and Gluc luciferase dual reporter genes is formed using lipofection in conjunction with a dual reporter plasmid gene and a human brain glioma U87MG cell line. In living cells, Rfluc luciferase catalyzes D-luciferin sylvite redox reaction to generate and emit fluorescence, and Gluc luciferase catalyzes coelenterazine redox reaction to generate and emit fluorescence, so that the catalysis effect with high efficiency and accurate positioning is realized. The D-fluorescein potassium salt and coelenterazine are directly injected into a living body for reaction, the problem of in-situ nondestructive detection which is difficult to realize by the traditional method is solved, and a new method is provided for monitoring the states of attributes such as tumor cell apoptosis, proliferation, metastasis and the like in the presence of stem cells.
The technical solutions of the present invention are further described in detail below with reference to several preferred embodiments and the accompanying drawings, which are implemented on the premise of the technical solutions of the present invention, and a detailed implementation manner and a specific operation process are provided, but the scope of the present invention is not limited to the following embodiments.
The experimental materials used in the examples used below were all available from conventional biochemical reagents companies, unless otherwise specified.
Example 1 tumor promotion/tumor inhibition evaluation of Stem cells Multi-channel in vivo imaging technique
1. Preparation of tumor cell line carrying bioluminescent luciferase gene
Establishing a human brain glioma U87MG cell line carrying a bioluminescent luciferase gene:
1.1 construction of plasmid carrying Rfluc and Gluc gene expression cassette
Designing and synthesizing Rfluc and Gluc gene segments of double reporter gene sequences, inserting the reporter gene into a pCas-Guide-ROSA26 vector, and constructing a plasmid vector pCas-Guide-ROSA26-luc containing the Rfluc and Gluc genes;
1.2 Liposome transfection and selection
One day prior to infection, U87MG cells were digested and counted, the cells plated in 24-well plates, the cells incubated overnight at 37 ℃, the cryopreserved liposomes thawed, and 50 μ Ι _ of 1:2 liposome diluent was prepared and added to the cells; adding low-sugar culture medium into each well to reach final concentration of 6 μ g/mL, shaking the well plate, incubating at 37 deg.C for 2h, removing the culture medium containing liposome and gene plasmid, and adding 500 μ L complete culture medium; carrying out single cell sorting by using a flow cytometer, and carrying out single cell cloning culture; culturing the U87MG cells by using an L-DMEM medium containing 10 wt% FBS and 1 wt% penicillin streptomycin, and continuing culturing;
1.3 Bioshape characterization of transgenic U87MG cells: the growth curve of the transgenic U87MG cell is not significantly different from that of the U87MG cell, and the tumorigenicity capacity is not different from that of the U87MG cell;
2. establishing a human brain glioma U87MG mouse tumor model expressing luciferase gene
Amplifying human brain glioma U87MG cells stably expressing double reporter genes in a culture dish in a large quantity, digesting the tumor cells in logarithmic growth phase by 0.25% of pancreatin and washing the tumor cells for 2 times by PBS, and modulating the tumor cells to the density of 1 × 10 by PBS7Placing the cell suspension in an ice bath for later use; injecting 10% chloral hydrate 50 muL into the abdominal cavity of an Nv/6 mouse with the weight of about 20g for anesthesia, injecting 100 muL of transgenic U87MG tumor cells into the left leg and the right leg subcutaneously, and establishing a human brain glioma U87MG subcutaneous tumor model (a fluorescence microscopic picture of the transgenic U87MG cells expressing ZsGreen is shown in figure 3, the first column is a hochests staining nuclear channel, the third column of the ZsGreen protein channel of the second column is a fusion picture of the two);
3, Nv/6 mouse human brain glioma U87MG subcutaneous tumor model receives different MSC injection mode experiments
3.1 tail vein injection of MSC:
control group: inoculating tumor cells to a mouse human brain glioma dipleg ectopic tumor model, modeling for 5 days, and injecting 200 mu L PBS into a living body through tail vein;
MSC group: inoculation of mouse human brain glioma two-leg ectopic tumor modelOn day 5 of tumor cell modeling, MSC was measured by contrast agent nano-unit (contrast agent nano-unit Tat-PEG-Ag)2Se synthesis scheme as shown in FIG. 7) and labeled with 3X 105A dose of 200 mu L is injected into a living body through tail vein (stem cell tail vein near infrared second zone is labeled for 1 hour as shown in figure 6, and stem cells marked by a contrast agent nano unit are injected into a tumor model mouse through tail vein for 1 hour of fluorescence imaging graph);
3.2 in situ injection of MSC:
control group: inoculating a mouse human brain glioma dipleg ectopic tumor model into tumor cells for modeling on day 5, and injecting 20 mu L PBS into tumor tissues in situ;
MSC group: the mouse human brain glioma dipleg ectopic tumor model is inoculated with tumor cells for modeling on day 5, and MSC is marked by a contrast agent nano unit and then is marked by 3 multiplied by 105A dose of 20. mu.L was injected in situ into tumor tissue (stem cells were labeled in situ on day 1, 4, 7 as shown in FIGS. 5 a-5 c).
4. Dynamic observations of different treatment groups of subcutaneous tumor models:
breeding an Nv mouse of a transgenic human brain glioma U87MG subcutaneous tumor model in an SPF environment; the subcutaneous tumor model starts from the beginning of each group of experimental measures, tumors grow to 4 th, 7 th, 12 th, 18 th and 21 th days, the mice are anesthetized and then subjected to multichannel imaging, bioluminescence imaging of the reaction of Rfluc luciferase and Gluc luciferase with corresponding luciferin is detected, and continuous observation is carried out for 1-3 weeks to obtain continuous images of the tumor growth condition of each mouse (the images of the Rfluc channel imaging and the Gluc channel imaging are shown in figure 4); quantitative data of each imaging time point of each mouse is obtained by using a professional image analysis system of a living body imaging instrument, and the data directly and accurately display the size of the tumor.
The method can also be used for detecting and quantifying distant micrometastasis of tumors; and accurately evaluating the effect of implementing the intervention measures of each group of MSC injected into the living body according to the obtained picture and quantitative data.
Example 2 evaluation of tumor inhibition of genetically modified adipose-derived Stem cells by multichannel in vivo imaging System
1. Establishing a luciferase gene-expressing human brain glioma U87MG in-situ tumor mouse model, establishing a dual-reporter gene-expressing human brain glioma U87MG in-situ tumor mouse model, amplifying human brain glioma U87MG cells stably expressing the dual reporter genes in a culture dish in a large quantity, digesting the tumor cells in logarithmic growth phase by 0.25 percent of pancreatin and washing the cells with PBS for 2 times, and modulating the cells with the density of 2 multiplied by 10 by PBS8Placing the cell suspension in an ice bath for later use; injecting 50 mu L of 10% chloral hydrate into the abdominal cavity of an Nv/6 mouse of about 20g for anesthesia, fixing the mouse by using a brain stereotaxic apparatus, enabling the right striatum of the mouse to pass through a 29-th needle hole (stereotaxic: 1.8mm on the side, 0.6mm in front and 3mm in depth), and injecting 5 mu L of transgenic U87MG cells into the hole to establish a human brain glioma U87MG brain tumor model;
2. establishing fat-derived stem cells (hADSCs-TRAIL) carrying TRAIL gene, which can express and secrete TRAIL. Meanwhile, cell marking is carried out by utilizing a contrast agent nano unit;
hADSCs-TRAIL stem cell in situ delivery to tumor models for experiments;
3.1 Single injection: human brain glioma U87MG brain tumor model was established on day 5 by injecting 3X 10 injections into the right striatum contralateral to the brain tumor57 mu LhADSCs-TRAIL;
3.2 multiple injections: human brain glioma U87MG brain tumor model was established on day 5 by injecting 3X 10 injections into the right striatum contralateral to the brain tumor5One/7 μ LhADSCs-TRAIL, 10 days later, 3X 10 re-injection5(ii) hADSCs-TRAIL;
3.3 negative controls: cell manipulation performed in groups (1) and (2), which were replaced with PBS;
4. dynamic observations of different treatment groups of brain tumor models: breeding a transgenic human brain glioma U87MG in-situ tumor model Nv mouse in an SPF environment; the in situ tumor mouse model starts from the reception of each set of experimental measures, the tumor grows to 4 th, 9 th, 12 th, 18 th and 21 th days, and the mouse is anesthetized and then subjected to multichannel imaging: bioluminescence imaging for detecting the reaction of the Rfluc luciferase and the Gluc luciferase with the corresponding luciferin; detecting near-infrared two-zone fluorescence imaging, and continuously observing for 1-3 weeks to obtain continuous images of tumor growth condition of each mouse and migration path images of stem cells; quantitative data of each imaging time point of each mouse is obtained by using a professional image analysis system of a living body imaging instrument, the growth of the tumor of an experimental group can be directly and accurately displayed to be obviously inhibited through an imaging picture and the quantitative data, and the survival period of the mouse is prolonged; at the same time, stem cells migrate to the contralateral tumor;
the method can also be used for detecting and quantifying distant micrometastasis of tumors; according to the obtained pictures and quantitative data, the effect of implementing the intervention measures of hADSCs-TRAIL injection into the living body of each group is accurately evaluated.
Example 3 evaluation of safety of Induced Pluripotent Stem Cells (iPSCs) for skin tissue repair
1. Preparation of tumor cell line carrying bioluminescent luciferase gene
Establishing a human skin cancer cell SK-MEL-5 cell line carrying a bioluminescent luciferase gene;
2. establishing human skin cancer cell SK-MEL-5 in situ mouse model expressing luciferase gene, digesting tumor cells in logarithmic growth phase with 0.25% pancreatin and washing with PBS for 3 times, and modulating the density with PBS to 5 × 106Placing the cell suspension in an ice bath for later use; injecting 4% chloral hydrate 100 mu L into the abdominal cavity of an Nv/6 mouse of about 20g for anesthesia, and injecting 100 mu L of transgenic SK-MEL-5 tumor cells into the back subcutaneously to establish a human skin cancer cell SK-MEL-5 in-situ model;
3. firstly, marking iPSCs by using a contrast agent nano unit, then carrying out an iPSCs repairing experiment on an in-situ tumor model of skin cancer cells SK-MEL-5 of a mouse in an experimental group, and not carrying out the iPSCs repairing experiment on a control group;
4. dynamic observations of different treatment groups of tumor models: feeding an Nv mouse of an in-situ tumor model of transgenic human skin cancer cells SK-MEL-5 in an SPF environment; the tumor model is anesthetized on days 1, 7, 14 and 21 from the beginning of the operation of a repair experiment, and then multichannel imaging is carried out, bioluminescence imaging for detecting the reaction of Rfluc luciferase and Gluc luciferase with corresponding luciferin and near-infrared two-region fluorescence imaging are carried out, so as to obtain an image of the growth condition of each group of tumors; quantitative data of each imaging time point of each mouse is obtained by using a professional image analysis system of a living body imaging instrument, and the data directly and accurately display the size of the tumor.
The method can also be used for detecting the survival effect of the iPSCs on residual skin tumor cells after skin cancer surgery and during iPSCs transplanted cell differentiation tissue repair, monitoring tumor recurrence and accurately evaluating the prognosis effect of iPSCs applied to tissue repair.
Example 4 assessment of treatment of non-Small cell Lung cancer with mesenchymal Stem cell-carried mRNA (hMSC-miR-150-5p)
1. Establishing a human non-small cell lung cancer H460SM in-situ tumor mouse model expressing double reporter genes, inoculating H460SM cells to the left lung of a SCID mouse by adopting a chest wall puncture method, and establishing a human non-small cell lung cancer H460SM in-situ tumor mouse model;
2. establishing a human mesenchymal stem cell (hMSC-miR-150-5p) carrying miR-150-5p gene segment, wherein the cell can deliver miR-150-5p to the non-small cell lung cancer, directly acts on tumor cells, and simultaneously carries out cell marking by utilizing a contrast agent nano unit;
3. in the experimental group, hMSC-miR-150-5p is injected into tumor-bearing mice through tail vein, and in the control group, PBS with the same volume is injected;
4. dynamic observations of different treatment groups of tumor models: feeding transgenic human non-small cell lung cancer H460SM in situ tumor model SCID mice in SPF environment; the in situ tumor mouse model performs multichannel imaging after anesthetizing the mouse from the experimental operation of each group to days 1, 3, 6, 10, 15 and 21: bioluminescence imaging for detecting the reaction of the Rfluc luciferase and the Gluc luciferase with the corresponding luciferin; detecting near-infrared two-region fluorescence imaging to obtain a mouse tumor growth condition image and a stem cell migration path image; quantitative data of each imaging time point of each mouse is obtained by using a professional image analysis system of a living body imaging instrument, the growth of the tumor of an experimental group can be directly and accurately displayed to be obviously inhibited through an imaging picture and the quantitative data, and the survival period of the mouse is prolonged; meanwhile, stem cells accumulate in the lungs;
the method can also be used for detecting the inhibition effect of the stem cell enzyme-carried/prodrug suicide gene on the tumor, and accurately evaluating the intervention effect of the functional stem cell on the living tumor according to the obtained picture and quantitative data.
In addition, the inventors of the present invention have also made experiments with other materials, process operations, and process conditions described in the present specification with reference to the above examples, and have obtained preferable results.
The aspects, embodiments, features and examples of the present invention should be considered as illustrative in all respects and not intended to be limiting of the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.
The use of headings and chapters in this disclosure is not meant to limit the disclosure; each section may apply to any aspect, embodiment, or feature of the disclosure.
Throughout this specification, where a composition is described as having, containing, or comprising specific components or where a process is described as having, containing, or comprising specific process steps, it is contemplated that the composition of the present teachings also consist essentially of, or consist of, the recited components, and the process of the present teachings also consist essentially of, or consist of, the recited process steps.
Unless specifically stated otherwise, use of the terms "comprising", "including", "having" or "having" is generally to be understood as open-ended and not limiting.
It should be understood that the order of steps or the order in which particular actions are performed is not critical, so long as the teachings of the invention remain operable. Further, two or more steps or actions may be performed simultaneously.
While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated any use of the terms first, second, etc. do not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one element from another.
Claims (10)
1. A stem cell evaluation multi-channel imaging system, comprising:
the tumor cell dual-channel imaging unit mainly comprises tumor cells transfected by plasmid genes, wherein the reporter genes of the plasmid genes comprise an Rfluc gene and a Gluc gene;
and the contrast agent nano unit mainly comprises an amphiphilic polymer, a contrast agent and a membrane-penetrating peptide, and is provided with a hydrophilic outer shell and a hydrophobic inner core, wherein the contrast agent is wrapped in the hydrophobic inner core, and the membrane-penetrating peptide is combined on the surface of the hydrophilic outer shell through electrostatic adsorption.
2. The stem cell evaluation multichannel imaging system according to claim 1, characterized by further comprising fluorescein; preferably, the fluorescein comprises fluorescein potassium salt and/or coelenterazine.
3. The stem cell evaluation multi-channel imaging system of claim 1, wherein: the tumor cells transfected by the plasmid gene can express the Rfluc luciferase and/or the Gluc luciferase; preferably, the Rfluc luciferase and/or Gluc luciferase is capable of exhibiting bioluminescence in reaction with luciferin;
and/or, the contrast agent nanosystems are capable of labeling stem cells.
4. The stem cell evaluation multi-channel imaging system of claim 1, wherein: the above-mentionedThe contrast agent comprises near-infrared two-region quantum dots; preferably, the near-infrared two-region quantum dot comprises Ag2Se、Ag2S、Ag2Any one or a combination of two or more of Te;
and/or, the cell-penetrating peptide comprises a cell-penetrating cationic short peptide; preferably, the cell-penetrating peptide comprises Tat and HR9、hLF、ANTP、Penetration、DPV3、(Ka)4、(RXR)4Any one or a combination of two or more of them;
and/or the amphiphilic polymer comprises any one or the combination of more than two of long-chain distearoyl phosphatidyl ethanolamine-polyethylene glycol, long-chain distearoyl phosphatidyl ethanolamine-polyethylene glycol derivatives and functionalized long-chain distearoyl phosphatidyl ethanolamine-polyethylene glycol;
preferably, the molecular weight of the long-chain distearoyl phosphatidyl ethanolamine-polyethylene glycol is 10-100000 Da; preferably, the long chain distearoylphosphatidylethanolamine-polyethylene glycol comprises distearoylphosphatidylethanolamine-polyethylene glycol 2000.
5. A method for preparing a multi-channel imaging system for stem cell evaluation according to any one of claims 1 to 4, comprising:
constructing a double-reporter gene plasmid vector, and then transfecting tumor cells by a liposome method to obtain a tumor cell dual-channel imaging unit;
and mixing the amphiphilic polymer with a contrast agent to form a polymersome, and then mixing the polymersome with the membrane-penetrating peptide to form the contrast agent nano unit.
6. The production method according to claim 5, characterized by comprising:
constructing a plasmid vector containing an Rfluc gene and a Gluc gene;
and co-incubating the plasmid vector and the liposome at 4-45 ℃ for 0.1-100h, and screening to obtain the tumor cell dual-channel imaging unit.
7. The production method according to claim 5, characterized by comprising:
mixing or ultrasonically treating an amphiphilic polymer, a contrast agent and a solvent to enable a hydrophobic layer of the amphiphilic polymer to wrap the contrast agent to form a polymersome;
and carrying out solvent removal treatment on the polymer vesicle, and mixing the polymer vesicle with the cell-penetrating peptide to form the contrast agent nano unit;
preferably, the solvent comprises pure water and/or PBS.
8. Use of the stem cell evaluation multi-channel imaging system of any one of claims 1-4 in the field of fluorescence imaging.
9. A multi-channel imaging method for stem cell evaluation, comprising:
providing a stem cell evaluation multi-channel imaging system of any one of claims 1-4;
establishing a mouse tumor model by using the tumor cell dual-channel imaging unit;
marking the stem cells by using a contrast agent nano unit to obtain marked stem cells;
and inputting the marked stem cells and fluorescein into a target region, thereby realizing simultaneous tracing fluorescence imaging of the stem cells and the tumor cells.
10. The multi-channel imaging method according to claim 9, characterized in that: the labeled stem cells and the fluorescein can be input in any one of tail vein injection, in-situ injection and intraperitoneal injection.
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