CN114196764B - Detection method for distribution of organ tissues of human cell preparation intravenous infusion animal body - Google Patents

Detection method for distribution of organ tissues of human cell preparation intravenous infusion animal body Download PDF

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CN114196764B
CN114196764B CN202111657687.2A CN202111657687A CN114196764B CN 114196764 B CN114196764 B CN 114196764B CN 202111657687 A CN202111657687 A CN 202111657687A CN 114196764 B CN114196764 B CN 114196764B
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胡芹
王张凡
向娥
饶巍
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Guangzhou Hamilton Biotechnology Co ltd
Wuhan Hamilton Cells Technology Co ltd
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Abstract

The invention discloses a method for detecting distribution of viscera tissues of a human cell preparation intravenous infusion animal body, which relies on a primer with specificity to mitochondrial DNA in the human cell, and adopts a real-time quantitative PCR method to detect the concentration of the human cell preparation DNA of experimental animal peripheral blood and viscera tissue samples collected in the test. The cell distribution detection method is used for tracing the directional distribution of the human cells implanted in the animal body, and has the advantages of safety, sensitivity, specificity, longevity and the like during animal experiments.

Description

Detection method for distribution of organ tissues of human cell preparation intravenous infusion animal body
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a detection method for distribution of viscera and tissues of a human cell preparation which is input into an animal body by veins.
Background
In recent years, due to the regenerative and repair capabilities of tissue cells, various stem cells (such as human umbilical cord mesenchymal stem cells, UC-MSCs) have attracted attention in the treatment of various diseases, such as myocardial injury, lung injury, liver diseases, osteoarthritis, diabetes and complications thereof. In the process of drug development, research on metabolism and movement of drugs in vivo is a necessary item, and in order to study metabolism and movement of cellular drugs in vivo, human cells must be transplanted into experimental animals and tracked through animal experiments. After transplantation, human cells may migrate from the site of transplantation with blood flow or by other means to other organs or tissues, so the detection method must be able to detect human cells in different organs and tissues of the experimental animal, including blood. However, stem cell labeling tracer studies evaluating cell engraftment, distribution, survival, migration, differentiation and function are significantly delayed from the observation of overall functional improvement, becoming a bottleneck factor and a new research hotspot for the discussion of stem cell therapeutic mechanisms.
Current detection means include both non-invasive and invasive types. Non-invasive detection relies mainly on in vivo imaging techniques (in vivo imaging system, IVIS) (e.g. using fluorescent in vitro labelling of cells) or other markers (such as position markers, transgenes and magnetic ions etc.) and then tracking the cell preparation in vivo to determine the viability and fate of the cell preparation, thus qualitatively reflecting the distribution of the cell preparation in vivo at the overall level. By using the method, firstly, the cell preparation entering the body needs to be marked or modified, the operation is complex, the process is complicated, and some marking means can influence the activity of the cell preparation and the in-vivo clearance rate. The invasive detection mainly comprises collecting body fluid or tissue sample, extracting total DNA of tissue or body fluid sample, detecting the content of housekeeping gene (such as beta-actin, globin, GAPDH gene) on human cell chromosome DNA in DNA sample by quantitative PCR (Q-PCR) or semi-quantitative PCR method, thereby calculating the content of human cell medicine in tissue or body fluid sample (Zhou Jinming, zhongmin. Cultured mouse bone marrow mesenchymal stem cells and their positioning distribution in vivo after transplantation [ J ]. J. 2002, 22 (3): 167-169; deng Weimin, li Changhong, liao Lianming, etc.. Adult bone marrow derived multipotent mesenchymal stem cells differentiate skin stem cells and skin tissue [ J ]. J. Cell biology, 2003, 25 (2): 98-104;Deng W,Han Q,Liao L,et al.Engrafted bone marrow-derived flk- (1+) mesenchymal stem cells regenerate skin tissue Eng,2005,11 (1-2): 110-119). The method can be used for researching the distribution of human cell medicines in animals with low homology with human, such as mice, rats and the like, but the human and monkey gene sequences are basically consistent due to the high homology, so that the method can not distinguish the chromosomal DNA of the human and the monkey, and can not detect the content of the human cells in the monkey. Therefore, the method is only used for researching the distribution condition of the human cell medicine in the rodent body at present, and the application range is narrow.
Therefore, there is an urgent need for a method capable of effectively researching the distribution characteristics of human cell medicines in various animal bodies to research the safety of human cell medicines as therapeutic medicines so as to solve the problems existing in the prior art.
Disclosure of Invention
The invention aims to provide a detection method for distribution of human cell preparation internal organs and tissues which are input into an animal body by veins. Mitochondrial DNA (mtDNA) is a further genome that is independent of the nuclear chromosome, is simple in structure, has a small molecular weight, and is easy to compare between eukaryotic (especially mammalian) species, so that human-specific gene fragments can be found by alignment of the intergeneric mitochondrial genome sequences. The expression of housekeeping genes in various tissues and cells of organisms is relatively constant, the conservation is high, the expression level is less influenced by environmental factors, and the housekeeping genes are often used as reference substances when detecting the change of the expression level of the genes. Commonly used internal genes such as beta-actin, GAPDH,18s-rRNA, etc. The invention designs a primer with specificity relative to human source intracellular mitochondrial DNA of animals and a primer of an animal internal reference gene relative to human. The qPCR is adopted to detect the target gene fragment, and the product specificity of the target gene fragment is found to be good, and the correlation coefficient of the Ct value and the template content is high. In order to compare the target gene content between different samples and make up for the difference of sample purity and concentration in the preparation process, the invention uses the reference gene to correct the DNA dosage, thereby establishing a set of simple, universal and high-sensitivity qPCR analysis method. The method can be used for improving the current limiting conditions, quickly and effectively researching the distribution characteristics of organs and tissues after the human cell preparation is infused into various animal bodies, and determining the treatment safety and effectiveness of the cell medicine to be detected.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the invention provides a method for detecting human cells, which is characterized in that a fragment shown in SEQ ID NO.1 is used as a template, a specific primer is designed, and an RT-qPCR method is adopted for detection.
According to the invention, the method detects the distribution of cells of human origin in the animal body, animal tissue or animal cell culture. Preferably, the human cell preparation is administered intravenously to the visceral organ tissue or blood distribution in the experimental animal.
According to the invention, the detection method comprises the following steps:
(1) According to human and rat mitochondrial genomes provided in NCBI-Genome, obtaining fragments with the largest difference through comparison, wherein the nucleotide sequences of the fragments are shown as SEQ ID NO.1, and designing a human cell mitochondrial DNA specific primer A and a rat DNA reference gene specific primer by taking the obtained fragments as templates;
(2) Extracting cells of a human cell preparation and organ tissues or blood DNA of a rat, respectively carrying out gradient dilution, designating the relative percentage concentration of the initial concentration of the DNA subjected to gradient dilution as 100%, and respectively establishing standard curves of RT-qPCR amplification of a human cell mitochondrial DNA specific primer A and a rat internal reference gene specific primer by utilizing real-time quantitative RT-qPCR, wherein the X axis of the standard curves is the relative percentage concentration of various DNA, and the Y axis is the Ct value obtained by amplifying the DNA of each relative percentage concentration by the primer A or the rat internal reference gene specific primer;
(3) Collecting organ tissues and blood of a rat according to a time point to be observed after the humanized cell preparation is administered to the rat, extracting total DNA of a sample, and carrying out RT-qPCR reaction to obtain Ct values amplified by the primer A and the rat internal reference gene primer;
(4) And (3) calculating the Ct value obtained in the step (3) through the standard curve in the step (2), respectively obtaining the relative percentage concentration of the primer A or the DNA template amplified by the rat internal reference gene primer in the total DNA sample of each organ tissue or blood, dividing the relative percentage concentration of the DNA template amplified by the human cell mitochondrial DNA specific primer A by the relative percentage concentration of the DNA template amplified by the rat internal reference gene specific primer, and characterizing the content of the human DNA in the organ tissue or blood of the rat by the obtained ratio.
Preferably, the step (2) includes: respectively diluting a certain amount of human cell DNA and rat DNA according to a 2-20-fold gradient, and respectively carrying out RT-qPCR; preferably 5-15 fold gradient dilution; more preferably 10-fold gradient dilution; and drawing a standard curve of the relative percent concentration-Ct value of the DNA according to the cycle number Ct value generated by the reaction system, namely respectively establishing a standard curve of RT-qPCR amplification of the primer A and the reference gene primer in the rat.
Preferably, the human cell mitochondrial DNA specific primer A is homo1, and the homo1 sequence is as follows:
forward：5′-CCATCCTCCGTGAAATCAAT-3′(SEQ ID NO.2)
reverse：5′-GGGAACGTGTGGGCTATTTA-3′(SEQ ID NO.3)；
preferably, the rat reference gene is GAPDH, and the GAPDH primer sequence is as follows:
forward：5′-GCAAGTTCAACGGCACAG-3′(SEQ ID NO.4)
reverse：5′-GCCAGTAGACTCCACGACA-3′(SEQ ID NO.5)。
still preferably, in the step (2), preparing a DNA of a human cell or a rat with a concentration of 25 ng/. Mu.l, and taking 25 ng/. Mu.l as a starting concentration of the DNA of the gradient dilution, wherein the relative percentage concentration of the DNA is 100%, and the DNA of the gradient dilution is diluted 2 times, 20 times, 200 times, 2000 times, 20000 times and 200000 times in sequence to obtain DNA gradient dilution samples with relative percentage concentrations of 50%, 5%, 0.5%, 0.05%, 0.005% and 0.0005%, respectively, taking 1. Mu.l of each sample for RT-qPCR, setting 3 multiple wells for each concentration, taking Ct mean value as Y axis, and taking the relative percentage concentration of the DNA of the human cell as X axis, and establishing a standard curve of the mitochondrial DNA specific primer homo1 of the human cell; a standard curve of the rat reference gene GAPDH was established in the same manner.
Further preferably, in the step (2), the target gene fragment is amplified according to the following RT-qPCR reaction conditions:
more preferably:
preferably, the RT-qPCR reaction system in the step (2): 1. Mu.L of DNA template, 0.5. Mu.M of primer Homo 1/upstream and downstream of rat GAPDH, 2 Xgold SYBR Green 5. Mu.L each, using ddH 2 O was made up to a total reaction volume of 10. Mu.L.
Preferably, in the step (3), after the humanized cell preparation is administered to the rat, the total DNA of each organ tissue and whole blood is extracted by using a DNA extraction kit, and the extracted total DNA of the sample is subjected to RT-qPCR reaction; each organ tissue comprises heart, liver, spleen, lung and kidney; the human cell preparation comprises somatic cells (such as epithelial cells), multipotent stem cells formed by in vitro induction, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, hepatic stem cells, epidermal stem cells, retina stem cells and the like, preferably human mesenchymal stem cells, more preferably human umbilical cord mesenchymal stem cells; the RT-qPCR reaction conditions are the same as those used in standard curve establishment.
Preferably, in the step (4), the relative percentage concentration of the primer Homo1 in the total DNA of the sample and the DNA template amplified by the rat GAPDH primer is calculated according to the Ct value generated by the RT-qPCR reaction of the primer Homo1 and the rat GAPDH primer on the total DNA of each organ tissue sample or blood by using the standard curve of the step (2), and the ratio of the two is used to characterize the content of the human DNA in the organ tissue or blood of the rat.
In a second aspect, the invention provides a kit for detecting human cells, which comprises a designed human cell mitochondrial DNA specific primer A and a rat internal reference gene specific primer by taking the sequence with the nucleotide sequence shown as SEQ ID NO.1 as a template.
According to the invention, the kit is for determining the distribution of cells of human origin in an animal body, animal tissue or animal cell culture. Preferably, the kit is used for determining the distribution of human cell preparations into the viscera tissue or blood of an experimental animal. Preferably, the human cell mitochondrial DNA specific primer A is homo1, and the homo1 sequence is as follows:
forward：5′-CCATCCTCCGTGAAATCAAT-3′(SEQ ID NO.2)
reverse：5′-GGGAACGTGTGGGCTATTTA-3′(SEQ ID NO.3)。
preferably, the rat reference gene is GAPDH, and the GAPDH primer sequence is as follows:
forward：5′-GCAAGTTCAACGGCACAG-3′(SEQ ID NO.4)
reverse：5′-GCCAGTAGACTCCACGACA-3′(SEQ ID NO.5)。
in a third aspect, the present invention provides a primer, wherein the primer sequence is:
forward：5′-CCATCCTCCGTGAAATCAAT-3′(SEQ ID NO.2)
reverse：5′-GGGAACGTGTGGGCTATTTA-3′(SEQ ID NO.3)。
in a fourth aspect, the present invention provides the use of the above primers and/or kits,
according to the invention, the primers and/or the kit are used for determining the distribution of human cells in an animal body, an animal tissue or an animal cell culture. Preferably, the primers and/or kits are used for determining the intravenous infusion of a human cell preparation into the internal organ tissue or blood distribution of an experimental animal.
The primers and/or kits may also be used in pharmacokinetic studies according to the invention.
The beneficial effects are that:
by adopting the prior art method, only the enrichment of human cells in a certain part of an animal can be qualitatively detected, but the distribution of other tissues cannot be detected, and the content in the enriched part cannot be accurately quantified. Compared with the prior art, the detection method provided by the invention is used for researching the distribution of the humanized cell preparation in the animal body, and has the following specific advantages:
(1) The somatic cell preparation is directly researched, the modification of the somatic cell preparation is not needed, the test method is simple, and the result is reliable; (2) The experimental process is simple, only the conventional DNA extraction and RT-qPCR technology is needed, large and complex experimental equipment is not needed, and the experimental process can be carried out in a common laboratory; (3) The specific fragments of human mitochondrial DNA relative to other species are used for amplification, so that the specificity is high, the amplification is easy, the detection sensitivity is greatly improved, the detection limit can be as low as 0.125 pg/mu L, and the requirement of pharmacokinetic study on the detection limit of cell drug residues in animals is met; (4) Compared with the traditional PCR method and flow cytometry (FACS) method, the RT-qPCR method has the advantages that the repeatability in and among experiments is greatly improved, the reliability of experimental results is ensured, and the accuracy requirement of pharmacokinetic study on the measurement of the cell drug metabolism curve in animals is met.
Drawings
FIG. 1 is a diagram of the alignment of a DNA sequence with the largest difference between human mitochondrial genome and rat mitochondrial genome;
FIG. 2 amplification curve obtained by mixing UC-MSCs and rat tubular epithelial cell NRK52E DNA according to a certain ratio and performing RT-qPCR amplification with primer homo1 in example 1;
wherein, "1" to "7" represent the percentage concentrations of UC-MSCs DNA in the above mixed DNA as 100%, 50%, 5%, 0.5%, 0.05%, 0.005%, 0.0005%, respectively, and the concentration of UC-MSCs DNA at 25 ng/. Mu.L is set as 100%;
FIG. 3 melting curves obtained by RT-qPCR amplification with primer homo1 at% concentration of UC-MSCs DNA of 100%, 50%, 5%, 0.5%, 0.05%, 0.005%, 0.0005% and 0%, respectively (i.e., NRK52E DNA) in example 1;
FIG. 4A standard curve (expressed as mean.+ -. SEM) established between Ct values obtained by RT-qPCR amplification of UC-MSCs DNA with primer Homo1 at relative percent concentration of 0.0005% -50% in example 1;
FIG. 5A standard curve (expressed as mean.+ -. SEM) established between Ct values obtained by RT-qPCR amplification of rat GAPDH primer at relative percent concentration of 0.0001% -100% of NRK52E DNA;
FIG. 6 is a graph of the reproducibility of the RT-qPCR reaction system of the human specific primer homo1 of example 2;
A-F are standard curves after independent RT-qPCR operation;
FIG. 7 shows the relative percentage concentration ratio of UC-MSCs primer Homo1 RT-qPCR amplified template DNA to the relative percentage concentration of rat GAPDH primer RT-qPCR amplified template DNA in rat blood DNA over time (expressed as mean.+ -. SEM) at various time points after tail vein injection of UC-MSCs;
FIG. 8 is a graph comparing the relative percentage concentration of UC-MSCs primer Homo1 RT-qPCR amplified template DNA to the relative percentage concentration ratio (expressed as mean.+ -. SEM) of rat GAPDH primer RT-qPCR amplified template DNA in rat organ tissue DNA at various time points after tail vein injection of UC-MSCs in example 3;
FIG. 9. Distribution of UC-MSCs primer Homo1 RT-qPCR amplified template (i.e., human DNA) in kidney of normal and AKI rats (expressed as mean.+ -. SEM) at 24 and 48 hours, respectively, after tail intravenous injection of UC-MSCs in example 4, with significant differences denoted by P < 0.05;
fig. 10. Distribution of UC-MSCs primer Homo1 RT-qPCR amplified template (i.e. human DNA) in organ tissues of normal and DN rats (expressed as mean ± SEM) 2 weeks after tail vein injection of UC-MSCs in example 5, P <0.01 represents a very significant difference.
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.
Unless defined otherwise or clearly indicated by context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Example 1: human specific gene fragment and primer verification and establishment of standard curve
Searching Homo sapiens mitochondrion and Rat sapiens mitochondrion in NCBI-Genome to find the human mitochondrial Genome (RefSeq: NC_ 012920.1), rat mitochondrial Genome (RefSeq: NC_ 001665.2); the two pairs of sequences which are found on https:// www.justbio.com/hoted-tools.html are aligned, a DNA sequence with the largest difference and the largest difference is found, the alignment result of the two pairs of sequences is shown in figure 1, and the nucleotide sequence of the DNA is shown in SEQ ID NO. 1. Designing primers by using the obtained DNA segment as a template and using Primer 3 software, and simultaneously designing animal internal reference gene primers by using animal internal reference genes (such as rat GAPDH) as a template; the primer length is 15 to 50 bases, preferably 17 to 30 bases, more preferably 20 to 27 bases; the designed primer is compared with Homo sapiens and Rat sapiens in NCBI-Blast, a pair of sequences with the best comparison result (namely the least obtained non-specific amplified product) is found as a final primer, and the sequences of the human specific primer Homo1 and the Rat internal reference gene GAPDH primer are as follows:
Human Homo1:
5′-CCATCCTCCGTGAAATCAAT-3′(forward)
5′-GGGAACGTGTGGGCTATTTA-3′(reverse)
Rat GAPDH:
5′-GCAAGTTCAACGGCACAG-3′(forward)
5′-GCCAGTAGACTCCACGACA-3′(reverse)
and delivering the designed primer sequence to Tianyi Yuanhui biotechnology Co.
1) Designing an RT-qPCR reaction system according to the specific primer Homo1 of the mitochondrial DNA of the humanized cell:
extracting cell DNA by using a DNA extraction kit, and amplifying target gene fragments by RT-qPCR according to the following reaction conditions:
the reaction system: 1. Mu.L of DNA template, 0.5. Mu.M each of the upstream and downstream primers of Human Homo1/Rat GAPDH, 2 Xgold SYBR Green 5. Mu.L, and ddH 2 O was made up to a total reaction volume of 10. Mu.L.
2) Verifying the applicability of the qPCR reaction system in extracting human mesenchymal stem cells (UC-MSCs) and rat renal tubular epithelial cells NRK52E DNA:
UC-MSCs DNA samples were prepared at a concentration of 25 ng/. Mu.L, and the UC-MSCs DNA was subjected to gradient dilution with 25 ng/. Mu.L of NRK52E DNA, and in FIG. 2, "1" to "7" represent that the UC-MSCs DNA concentrations were 100%, 50%, 5%, 0.5%, 0.05%, 0.005%, 0.0005%, respectively, and the UC-MSCs DNA concentration was set at 25 ng/. Mu.L was 100%.
The human homo1 primer amplified a single specific peak in the above samples (FIGS. 2, 3) and no product was amplified in NRK52E DNA. Therefore, the human homo1 primer designed by the invention has better specificity. Similarly, rat GAPDH primer amplified a single specific peak in NRK52E DNA and no product in UC-MSCs DNA (results not shown).
3) Making standard curve of RT-qPCR amplification of human cell mitochondrial DNA specific primer Homo1 and rat internal reference gene GAPDH primer
The relative percentage concentration of 25 ng/. Mu.L UC-MSCs DNA was designated as 100% with ddH 2 O is diluted 2 times, 20 times, 200 times, 2000 times, 20000 times and 200000 times respectively, so that DNA relative percentage concentration is 50%, 5%, 0.5%, 0.05%, 0.005% and 0.0005% respectively, 1 mu L is taken for RT-qPCR, 3 compound holes are arranged for each concentration, ct average value is taken as Y axis, UC-MSCs DNA relative percentage concentration value is taken as X axis, and a standard curve of the human cell mitochondrial DNA specific primer homo1 is established; the organ tissues or blood DNA of the rat in the control group are extracted, and a standard curve of the reference gene GAPDH of the rat is established according to the same method, wherein the control group is a rat which is not treated by modeling and MSC under the same feeding condition. As can be seen from FIG. 4, the standard curve R of the human cell mitochondrial DNA specific primer homo1 2 0.996, amplification efficiency 107.11%, limit of detection: concentration was 0.125 pg/. Mu.L, limit of quantitation: the concentration was 0.125 pg/. Mu.L. Standard curve R of rat reference gene GAPDH 2 The amplification efficiency was 102.54% at 0.994, as shown in FIG. 5.
Every time the amount of RT-qPCR amplified template of human Homo1 primer in rat organ tissue or blood DNA sample after administration is detected, standard curves of RT-qPCR amplification of Homo1 primer of human UC-MSCs DNA and GAPDH primer of control rat organ tissue or blood DNA are respectively established according to the method.
Example 2 detection of the reproducibility of the RT-qPCR reaction System of the human specific primer Homo1
Sample preparation: with ddH 2 O5 parts of human UC-MSCs cell DNA sample with the concentration of 25 ng/. Mu.L is prepared and used as a repeatability verification test sample to be placed on an ice box for standby. Sample DNA was diluted as in 3) of example 1 and data was derived from 5 independent RT-qPCR runs to prepare a standard curve for RT-qPCR amplification of the human cell mitochondrial DNA specific primer Homo 1. As shown in FIG. 6, the slope of the 5 th order standard curve is-3.0303 to-3.198, the intercept is 34.043 to 35.121, R 2 All are larger than 0.99, and the amplification efficiency is between 90 and 110 percent. Therefore, the repetitive test of the human cell mitochondrial DNA specific primer Homo1 meets the regulations and has better repeatability.
TABLE 1 repeatability detection of qPCR reaction System of human specific primer Homo1
EXAMPLE 3 distribution of UC-MSCs in visceral tissue and blood following intravenous injection into the rat tail
1) Male SD rats (6-8 weeks; 180-200 g) was purchased from food and drug safety evaluation center (Wuhan, china) in Hubei province. Rats were fed in light/dark cycles for 12 hours under standard sterile conditions and maintained on standard laboratory feed. All animal experiments of the study were carried out according to the guidelines for laboratory animal care and use of the animal welfare committee of China and were approved by the animal research center (hereinafter referred to as the safety evaluation center) of the food and drug safety evaluation center of Hubei province.
2) Rat tail intravenous injection 2.0X10 6 UC-MSCs (suspended in 500. Mu.L PBS) were collected from the canthus vein plexus within 0, 5, 10, 15, 30, 45 minutes after injection, and rats were anesthetized with 2% sodium pentobarbital 1, 2, 4, 24, 48 hours and 1, 2 weeks after injection, and the hearts were collected, and then euthanized, and the heart, liver, spleen, lung, kidney of the rats were collected, flash frozen at-80℃to extract DNA of organ tissues and blood samples and were processedAnd (5) one-step analysis.
3) Biological distribution of UC-MSCs in visceral tissues and blood was assessed by detection of human Homo1 and rat GAPDH. UC-MSCs (Homo 1) peaked 30 minutes after rat tail intravenous injection, and then declined rapidly, disappearing for 2 hours (FIG. 7). In the lung, UC-MSCs reached a peak within 4 hours after injection. After 24-48 hours, the UC-MSCs ratio in the lung is rapidly reduced and gradually reduced to the lowest. There was no significant difference in the UC-MSCs ratio in the tissues after 1 week of injection (fig. 8). Previous follow-up studies showed that most of the fluorescent or isotopically labeled MSCs accumulated in the lungs after intravenous injection, which is consistent with the results of the present invention, and that the primary cause of the blockage of MSCs in the lungs may be cell entrapment in the pulmonary capillaries and vascular endothelial adhesion.
EXAMPLE 4 distribution of UC-MSCs in kidneys of normal and acute kidney injured rats
1) Male SD rats (6-8 weeks; 180-200 g) was purchased from food and drug safety evaluation center (Wuhan, china) in Hubei province. Rats were fed in light/dark cycles for 12 hours under standard sterile conditions and maintained on standard laboratory feed. After 1 week of adaptive feeding, animals were randomly divided into control and Acute Kidney Injury (AKI) model groups, each with n=4. All animal experiments of the study were carried out according to the guidelines for laboratory animal care and use of the animal welfare committee of China and were approved by the animal research center (hereinafter referred to as the safety evaluation center) of the food and drug safety evaluation center of Hubei province.
2) Ischemia reperfusion injury (Ischemia-reperfusion injury, IRI) is the most common cause of AKI (acute kidney injury), so a rat AKI model can be established by IRI surgery. Rats were anesthetized with pentobarbital sodium intraperitoneal injection, then an abdominal incision was made by midline ureterectomy and right nephrectomy, left renal ischemia was induced by closing the micro-saw tooth jaws on the renal arteriovenous for 45 minutes, and then left kidneys were removed to begin reperfusion. When the kidney turns from pale to red, the kidney IRI surgery is judged to be successful. Rats in the control group were anesthetized, the abdomen was cut and sutured, and all rats were recovered and cared for after surgery. After AKI model was established, the control group and AKI group rats were injected into tail vein by 2.0X10 respectively 6 UC-MSCs (suspension)In 500 μlpbs), kidneys were harvested 24 and 48 hours after injection, respectively, -quick frozen at-80 ℃, DNA was extracted and further analyzed.
The proportion of UC-MSCs (Homo 1) in AKI rat kidneys was significantly higher than normal rats 24 and 48 hours after tail vein injection, and furthermore, the proportion of UC-MSCs (Homo 1) in AKI rat kidneys was significantly increased 48 hours compared to 24 hours after injection (fig. 9), showing that UC-MSCs was enriched at the injured site of model animals.
EXAMPLE 5 distribution of UC-MSCs in the kidneys of normal and chronic kidney injured (diabetic nephropathy) rats
(1) Male SD rats (6-8 weeks; 180-200 g) were purchased from food and drug safety evaluation center (Wuhan, china) of Hubei province. Rats were fed in light/dark cycles for 12 hours under standard sterile conditions and maintained on standard laboratory feed. After 1 week of adaptive feeding, animals were randomly divided into control and Diabetic Nephropathy (DN) groups, each group n=4. All animal experiments of the study were carried out according to the guidelines for laboratory animal care and use of the animal welfare committee of China and were approved by the animal research center (hereinafter referred to as the safety evaluation center) of the food and drug safety evaluation center of Hubei province.
(2) A single intraperitoneal injection of 60mg/kg streptozotocin (Sigma, USA) was used to induce the rat DN model, and the control group was given an equal volume of saline treatment. After 24 hours, the blood glucose of the rats was measured, and rats having blood glucose concentrations exceeding 16.7mM for three consecutive days were confirmed as diabetic rats. After 6 weeks, urine of diabetic rats was collected with a metabolism cage, and the urine volume and urine protein concentration of the rats were measured, and the proteinuria of 30mg/24h or more was confirmed as diabetic nephropathy. Rats in control and DN groups were injected tail vein 2.0X10, respectively 6 UC-MSCs (suspended in 500. Mu.L PBS) were collected after 2 weeks from heart, liver, spleen, lung, kidney tissues of rats in the control group and DN group, respectively, and rapidly frozen at-80℃and then DNA was extracted for further analysis.
(3) Except for the heart, the proportion of UC-MSCs (Homo 1) in the remaining organ tissues of DN rats was higher than that of the rats in the control group, especially in the spleen and kidneys (P < 0.01) (FIG. 10). In the examples of the present invention, the migration of UC-MSCs to the kidney of the DN model rats was more than that of the control rats, consistent with a great deal of evidence that the injected UC-MSCs migrated more readily to the site of inflammation or injury.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
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Guangzhou Han Mitonian biotechnology Co.Ltd
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Claims (10)

1. A method of detecting cells of human origin, the method comprising the steps of:
1) According to human and rat genomes provided in NCBI-Genome, obtaining a part of sequences with the largest difference through comparison, wherein the nucleotide sequence of the sequences is shown as SEQ ID NO.1, and a primer homo1 with specificity of human cell mitochondrial DNA and a primer with specificity of rat DNA internal reference gene GAPDH are designed by taking the obtained sequence as a template; the specific primer homo1 is as follows:
forward：5′-CCATCCTCCGTGAAATCAAT-3′(SEQ ID NO.2)
reverse：5′-GGGAACGTGTGGGCTATTTA-3′(SEQ ID NO.3)；
the GAPDH primer sequences were as follows:
forward：5′-GCAAGTTCAACGGCACAG-3′(SEQ ID NO.4)
reverse：5′-GCCAGTAGACTCCACGACA-3′(SEQ ID NO.5)；
2) Extracting human cell preparation cells and rat organ tissues or blood DNA, preparing human cell DNA or rat DNA with the concentration of 25 ng/mu L, taking 25 ng/mu L as the initial concentration of DNA subjected to gradient dilution, and sequentially diluting the DNA with the relative percentage concentration of 100%, namely, obtaining DNA gradient dilution samples with the relative percentage concentrations of 50%, 5%, 0.5%, 0.05%, 0.005% and 0.0005% respectively by 2 times, 20 times, 200 times, 2000 times, 20000 times and 200000 times, respectively, taking 1 mu L of each sample to perform RT-qPCR, setting 3 compound holes at each concentration, taking Ct average value as Y axis, and taking the relative percentage concentration of human cell DNA as X axis, and establishing a standard curve of a human cell mitochondrial DNA specific primer homo1; establishing a standard curve of a rat reference gene GAPDH according to the same method;
3) After the humanized cell preparation is administered to a rat, collecting organ tissues and blood of the rat according to a time point to be observed, extracting total DNA of a sample, and carrying out RT-qPCR reaction, wherein the RT-qPCR reaction conditions are the same as those established by a standard curve to obtain Ct values of primer Homo1 and amplification of a rat internal reference gene GAPDH primer;
4) And 2) respectively obtaining the relative percentage concentration of the primer Homo1 and the DNA template amplified by the rat internal reference gene GAPDH primer in the total DNA sample of each organ tissue or blood by utilizing the standard curve of the step 2), dividing the relative percentage concentration of the DNA template amplified by the human cell mitochondrial DNA specific primer Homo1 by the relative percentage concentration of the DNA template amplified by the rat internal reference gene GAPDH specific primer, and characterizing the content of the human DNA in the organ tissue or blood of the rat by the obtained ratio.
2. The method according to claim 1, wherein in the step 3), after the humanized cell preparation is administered to the rat, the total DNA of each organ tissue and whole blood is extracted using a DNA extraction kit, and the extracted total DNA of the sample is subjected to RT-qPCR reaction; each organ tissue comprises heart, liver, spleen, lung and kidney; the human cell preparation comprises somatic cells, multipotent stem cells formed by in vitro induction, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, hepatic stem cells, epidermal stem cells and retina stem cells.
3. The method of claim 2, wherein the human cell preparation is human umbilical mesenchymal stem cells.
4. A method according to any one of claims 1 to 3, wherein in step 2) the gene fragment of interest is amplified according to the following RT-qPCR reaction conditions:
5. the method according to claim 4, wherein in the step 2), the target gene fragment is amplified according to the following RT-qPCR reaction conditions:
the RT-qPCR reaction system in the step 2) is as follows: 1. Mu.L of DNA template, 0.5. Mu.M of primer Homo 1/upstream and downstream of rat GAPDH, 2 Xgold SYBR Green 5. Mu.L each, using ddH 2 O was made up to a total reaction volume of 10. Mu.L.
6. A primer, characterized in that the primer sequence is:
forward：5′-CCATCCTCCGTGAAATCAAT-3′(SEQ ID NO.2)
reverse:5'-GGGAACGTGTGGGCTATTTA-3' (SEQ ID NO. 3); the primers are used to determine the distribution of human cells in an animal body, animal tissue or animal cell culture.
7. The kit is characterized by comprising a designed human cell mitochondrial DNA specific primer Homo1 and a rat reference gene GAPDH specific primer by taking a sequence with a nucleotide sequence shown as SEQ ID NO.1 as a template; the homo1 sequence is as follows:
forward：5′-CCATCCTCCGTGAAATCAAT-3′(SEQ ID NO.2)
reverse：5′-GGGAACGTGTGGGCTATTTA-3′(SEQ ID NO.3)；
the GAPDH primer sequences were as follows:
forward：5′-GCAAGTTCAACGGCACAG-3′(SEQ ID NO.4)
reverse：5′-GCCAGTAGACTCCACGACA-3′(SEQ ID NO.5)；
the kit is used for determining the distribution of human cells in an animal body, an animal tissue or an animal cell culture.
8. Use of the primer of claim 6 and/or the kit of claim 7 for determining the distribution of human cells in an animal body, an animal tissue or an animal cell culture.
9. The use according to claim 8, wherein the primers and/or the kit are used for determining the distribution of human cell preparations into the internal organ tissue or blood of experimental animals intravenously.
10. The use according to claim 8, wherein the primers and/or kits are used in pharmacokinetic studies.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109498616A (en) * 2018-12-29 2019-03-22 中国医科大学附属第医院 EGCG improves the application in liver inflammation and insulin resistance drug in preparation
CN112080569A (en) * 2020-09-30 2020-12-15 北京银丰鼎诚生物工程技术有限公司 Specific primer, kit and method for detecting in-vivo transplanted human cells
CN112501263A (en) * 2020-12-04 2021-03-16 云南舜喜再生医学工程有限公司 Method for detecting directional distribution of human-derived mesenchymal stem cells in animal body
CN113462757A (en) * 2021-07-15 2021-10-01 南京艾尔普再生医学科技有限公司 Quantitative detection method of human cells in pharmacokinetic research

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109498616A (en) * 2018-12-29 2019-03-22 中国医科大学附属第医院 EGCG improves the application in liver inflammation and insulin resistance drug in preparation
CN112080569A (en) * 2020-09-30 2020-12-15 北京银丰鼎诚生物工程技术有限公司 Specific primer, kit and method for detecting in-vivo transplanted human cells
CN112501263A (en) * 2020-12-04 2021-03-16 云南舜喜再生医学工程有限公司 Method for detecting directional distribution of human-derived mesenchymal stem cells in animal body
CN113462757A (en) * 2021-07-15 2021-10-01 南京艾尔普再生医学科技有限公司 Quantitative detection method of human cells in pharmacokinetic research

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