CN113502271A - Mesenchymal stem cells for treating inflammatory bowel disease and application thereof - Google Patents

Abstract

The application discloses mesenchymal stem cells for treating inflammatory bowel disease and application thereof. The mesenchymal stem cells for treating inflammatory bowel disease are formed by transfecting nucleic acid of a chemokine receptor CCR9 into the mesenchymal stem cells. The mesenchymal stem cells for treating the inflammatory bowel disease can specifically target lesion parts of the inflammatory bowel disease, have strong homing capacity, greatly improve the number of the mesenchymal stem cells homing to the lesion parts, and improve the treatment effect of the inflammatory bowel disease; provides a new individual and accurate treatment scheme and idea for the treatment of inflammatory bowel diseases.

Description

Mesenchymal stem cells for treating inflammatory bowel disease and application thereof

Technical Field

The application relates to the field of medicines for treating inflammatory bowel diseases, in particular to mesenchymal stem cells for treating inflammatory bowel diseases and application thereof.

Background

Inflammatory Bowel Disease (IBD) is a chronic and incurable global disease, and the incidence rate of the IBD is higher in areas with more developed economy, and more than 150 thousands of IBD patients are expected within 10 years in China. The patients mainly have the symptoms of easy relapse of abdominal pain and diarrhea, even fistula formation, high curing difficulty and serious influence on the work and life of the patients. To date, the etiology and pathogenesis of inflammatory bowel disease have not been fully elucidated, and immune response abnormalities are an important feature of this type of disease. Studies have shown that among the four major classes of chemokines, the CC Chemokine Receptor (CCR) plays an important role in IBD; of the 19 CCR types currently found, 10 are associated with IBD, including CCR1 throughCCR 10.

Mesenchymal stem cells are the most clinically used pluripotent stem cells at present, and have all the commonalities of stem cells, and are capable of self-renewal and multipotent differentiation. The mesenchymal stem cells have various biological effects such as immunoregulation, loss repair and the like, show good application prospects in various diseases, but have poor treatment effects on partial diseases due to the fact that the homing capacity is reduced due to the change of characters in the in-vitro culture process, and are limited in clinical application. The targeting homing of stem cells to lesion tissues is less, and the poor treatment targeting is a worldwide problem restricting the clinical transformation of the stem cells. Mesenchymal stem cell homing is a complex cascade of processes, from mesenchymal stem cell activation to specific chemotactic adhesion to the site of tissue injury and migration to the injury, involving interactions between various chemokines and receptors. In order to realize efficient and targeted enrichment of damaged tissues by stem cells, the key point is to search high-sensitivity and high-specificity disease individual chemokines and corresponding chemokine receptors.

At present, no relevant research and report exists on how to realize efficient and targeted treatment of inflammatory bowel diseases by using mesenchymal stem cells.

Disclosure of Invention

The purpose of the application is to provide a novel mesenchymal stem cell for treating inflammatory bowel disease and application thereof.

The following technical scheme is adopted in the application:

one aspect of the present application discloses a mesenchymal stem cell for treating inflammatory bowel disease, which is obtained by transfecting a nucleic acid of a chemokine receptor CCR9 into the mesenchymal stem cell.

The preparation method has the advantages that the prepared targeted mesenchymal stem cells have good targeting specificity and better targeting homing capability of the lesion tissues by transfecting the corresponding nucleic acid of inflammatory bowel disease chemokine receptor CCR9 in the mesenchymal stem cells, the mesenchymal stem cells can be efficiently and targeted and enriched in the lesion tissues, and the treatment effect of inflammatory bowel diseases is greatly improved. In one implementation of the present application, the number of targeted mesenchymal stem cells homing to the lesion is increased by about 3-fold.

Preferably, the nucleic acid of the chemokine receptor CCR9 is the sequence shown in Seq ID No. 1.

Preferably, the mesenchymal stem cells are derived from bone marrow, adipose tissue, dental pulp, umbilical cord blood, amniotic fluid or placenta. More preferably, the mesenchymal stem cells are derived from bone marrow.

Preferably, the transfection is electroporation.

The application also discloses a preparation method of the targeting mesenchymal stem cell, which comprises the steps of obtaining CCR9 nucleic acid, and transfecting the CCR9 nucleic acid into the mesenchymal stem cell by an electroporation transfection method, wherein the CCR9 nucleic acid is a sequence shown in Seq ID No. 1.

Still another aspect of the application discloses the use of the targeted mesenchymal stem cells of the application in the preparation of a medicament for the treatment of inflammatory bowel disease.

In another aspect, the present application discloses a medicament for treating inflammatory bowel disease, comprising the mesenchymal stem cells of the present application.

Preferably, the medicine of the application also contains T cells or immune cells which do not influence the function of the targeted mesenchymal stem cells or can be cooperated with the targeted mesenchymal stem cells.

It should be noted that, the existing research shows that some specially modified T cells or immune cells also have the therapeutic effect of inflammatory bowel disease; it is understood that as long as these T cells or immune cells do not adversely affect the mesenchymal stem cells of the present application; under some special use conditions, the medicine can be compatible with the mesenchymal stem cells to prepare inflammatory bowel disease medicines or other multifunctional medicines.

Preferably, the medicament of the present application is in a pharmaceutically acceptable dosage form, including but not limited to a powder, a solvent, a tablet or a capsule.

It can be understood that the key of the drug of the present application lies in the specific targeting of the mesenchymal stem cells, and as for the specific dosage form, the drug dosage form can be determined according to the use condition by referring to the existing drug dosage form.

Preferably, the medicine also contains pharmaceutically acceptable auxiliary materials.

It can be understood that the medicine can contain some auxiliary materials besides the main active ingredients so as to prepare corresponding dosage forms or assist the active ingredients; the type of the auxiliary materials is determined according to the production or use requirements, and is not particularly limited herein.

The beneficial effect of this application lies in:

the mesenchymal stem cells for treating the inflammatory bowel disease can specifically target lesion parts of the inflammatory bowel disease, have strong homing capacity, greatly improve the number of the mesenchymal stem cells homing to the lesion parts, and improve the treatment effect of the inflammatory bowel disease; provides a new individual and accurate treatment scheme and idea for the treatment of inflammatory bowel diseases.

Drawings

FIG. 1 is a graph showing the results of MSC-CCR9 and MSC-EGFP expression of CCR9 in the examples of the present application;

FIG. 2 is a graph showing the change with time of mRNA in the right ear after sensitization in the examples of the present application;

FIG. 3 is a graph of the degree of edema in the right ear after sensitization over time in an example of the present application;

FIG. 4 is a graph of modeling and treatment patterns of CHS mice in the examples of the present application;

FIG. 5 is a line graph showing the degree of ear edema in mice after treatment in the examples of the present application;

FIG. 6 shows the results of the section staining of the CHS + MSC-EGFP group in the examples of the present application;

FIG. 7 shows the results of staining the section of the CHS + MSC-CCR9 group in the examples of the present application;

FIG. 8 is a diagram of an MPO active column in the example of the present application;

FIG. 9 shows the results of infiltration of the inflammatory factor TNF α by ELISA assay in the examples of the present application;

FIG. 10 shows the results of infiltration of the inflammatory factor IFN γ by ELISA analysis in the examples of the present application.

Detailed Description

The present application will be described in detail below with reference to specific embodiments and drawings. The following examples are intended to be illustrative of the present application and should not be construed as limiting the present application.

Example one

First, research on chemotactic factor

In the example, the inflammatory bowel disease is taken as a research object, a blood sample is collected, DNA is extracted, and the chemokine receptor CCR9 of the inflammatory bowel disease patient is sequenced; meanwhile, a tissue sample of a normal person is used as a contrast; changes in CCR9 in inflammatory bowel disease were analyzed by comparison. Based on the effect of CCR9 on inflammatory bowel disease, corresponding transfected nucleic acids were constructed. The details are as follows:

1. sample collection

20mL of fresh peripheral blood of the volunteers is taken, diluted by 1:1 with 1 XPBS, separated by density gradient centrifugation by using Ficoll-Paque lymph separation liquid, and all white membrane layer mononuclear cells are collected and diluted by 1:4 with sterile PBS. Centrifuge at 2000rpm for 10min and discard the supernatant. Sufficient PBS was added and washed twice. The cells were suspended in RPMI-1640 complete medium, i.e.human Peripheral Blood Mononuclear Cells (PBMC) were obtained.

2. Nucleic acid extraction

In the embodiment, the RNA of the peripheral blood mononuclear cell is extracted by adopting a Trizol method, specifically, the obtained human Peripheral Blood Mononuclear Cell (PBMC) is added into 1mL of Trizol solution, evenly blown and mixed to ensure that the cell is fully cracked, and then is kept stand for 5 min; adding 200 μ L chloroform, shaking vigorously and mixing for 20s to make the water phase and organic phase contact sufficiently, standing at room temperature for 15 min; centrifuging at 12000g for 15min at 4 deg.C to obtain three layers, wherein RNA is in the upper water phase, and carefully transferring to another new RNase free EP tube; adding 0.5mL of isopropanol, gently and fully mixing, standing at room temperature for 10min, and precipitating RNA; centrifuging at 12000g for 10min at 4 deg.C, collecting RNA precipitate, and removing supernatant; washing the tube wall twice with 75% ethanol, and air-drying on an ultra-clean bench; the precipitate was dissolved by adding 50. mu.L of DEPC water and the concentration was measured by a NanoDrop ultramicro spectrophotometer.

3. Nucleic acid sequencing

(1) Reverse transcription

Firstly removing genome DNA of RNA extracted by a Trizol method, and then carrying out reverse transcription to obtain first strand cDNA, wherein the specific steps are as follows:

extractedRNA 1. mu.g, DNase I1. mu.L, Buffer DNase I withMgCl₂1 μ L, supplemented with DEPC water to 10 μ L, incubated at 37 deg.C for 30 min; then addingEDTA 1 μ L, and incubating at 65 deg.C for 10 min; then adding oligo (dT) 1. mu.L, and incubating for 10min at 65 ℃; finally, 5XReaction Buffer 5. mu. L, RNase-ribonuclear Inhibitor 1. mu.L,10mM dNTP Mix 2. mu. L, M-MLV RT 1. mu.L, RNase Free Water to 25. mu.L, and incubation at 42 ℃ for 60min were added to obtain cDNA.

(2) PCR reaction and recovery of CCR9cDNA fragment

And (3) PCR reaction: 2XStar mix 10. mu. L, DEPC water 7. mu.L,forward primer 1. mu.L,reverse primer 1. mu. L,cDNA 1. mu.L, total 20. mu.L. Wherein, 2 XStar mix is a conventional PCR amplification reaction buffer solution system and comprises Taq DNA Polymerase, dNTPs, MgCl and the like.

The PCR conditions were 95 ℃ for 10min, then 40 cycles: 15s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃; after the circulation is finished, the temperature is 72 ℃ for 10min, and the standby temperature is 4 ℃.

Wherein, the upstream primer is a sequence shown in Seq ID No.2, and the downstream primer is a sequence shown in Seq ID No. 3.

Seq ID No.2：5’-atggctgatgactatggctctg-3’

Seq ID No.3：5’-tcagagggagagtgctcctga-3’

After the PCR amplification is finished, detecting the PCR amplification product by agarose gel electrophoresis, and performing gel cutting recovery on the PCR amplification product by using an agarose gel DNA recovery kit. The result of agarose gel electrophoresis shows that the PCR amplification product contains an amplification product with the size of about 1074bp, which is consistent with the size of the expected CCR9 fragment.

Cutting and recycling the rubber: cutting the target fragment band by a sharp scalpel, and cutting the gel by using an agarose gel DNA recovery kit to recover a PCR amplification product, namely a CCR9 fragment.

(3) TA cloning and transformation:

the TA cloning system included: 4 mu L of PCR amplification product of 1 mu L, CCR9 of pMD19-T vector, Solution I5 mu L, 10 mu L of reaction system in total, and reacting for 2h at 16 ℃ to obtain the clone plasmid.

And (3) transformation: competent cells DH5 α were removed at-80 ℃ and lysed on ice for 5 min; add 5. mu.L of the cloned plasmid to 30. mu.L ofcompetent cell DH 5. alpha. and flick, place on ice for 30 min; thermally shocking at 42 deg.C for 45s-1min, immediately placing on ice for 2 min; adding 600 microliter LB incubated at 37 deg.C, and incubating for 1h on a shaker at 37 deg.C and 1500 rpm; centrifuging at 3500rpm for 4-5min, discarding supernatant, resuspending 100 μ L of culture solution, or obtaining transformant, spreading 100 μ L of transformant on preheated LB plate containing 50 μ g/mL Amp, and incubating overnight at 37 deg.C; 3-5 colonies were picked for PCR detection and sequencing.

The primers, reaction system and reaction conditions adopted by PCR detection are the same as those of the PCR reaction and CCR9cDNA fragment recovery of the step (2). In this example, 4 colonies were picked for PCR detection. The results showed that the amplification of all 4 colonies gave a CCR9 fragment of about 1074bp, which was in agreement with the expectations.

The 4 colonies were inoculated into 500. mu.L of LB medium, incubated overnight on a shaker at 37 ℃ and 1500rpm, and the resulting bacterial suspension was sequenced by Shanghai Bioengineering Co., Ltd.

Sequencing results show that the PCR amplification of the example obtains a CCR9 fragment of 1074bp, and the specific sequence is shown as Seq ID No. 1.

Seq ID No.1：

atggctgatgactatggctctgaatccacatcttccatggaagactacgttaacttcaacttcactgacttctactgtgagaaaaacaatgtcaggcagtttgcgagccatttcctcccacccttgtactggctcgtgttcatcgtgggtgccttgggcaacagtcttgttatccttgtctactggtactgcacaagagtgaagaccatgaccgacatgttccttttgaatttggcaattgctgacctcctctttcttgtcactcttcccttctgggccattgctgctgctgaccagtggaagttccagaccttcatgtgcaaggtggtcaacagcatgtacaagatgaacttctacagctgtgtgttgctgatcatgtgcatcagcgtggacaggtacattgccattgcccaggccatgagagcacatacttggagggagaaaaggcttttgtacagcaaaatggtttgctttaccatctgggtattggcagctgctctctgcatcccagaaatcttatacagccaaatcaaggaggaatccggcattgctatctgcaccatggtttaccctagcgatgagagcaccaaactgaagtcagctgtcttgaccctgaaggtcattctggggttcttccttcccttcgtggtcatggcttgctgctataccatcatcattcacaccctgatacaagccaagaagtcttccaagcacaaagccctaaaagtgaccatcactgtcctgaccgtctttgtcttgtctcagtttccctacaactgcattttgttggtgcagaccattgacgcctatgccatgttcatctccaactgtgccgtttccaccaacattgacatctgcttccaggtcacccagaccatcgccttcttccacagttgcctgaaccctgttctctatgtttttgtgggtgagagattccgccgggatctcgtgaaaaccctgaagaacttgggttgcatcagccaggcccagtgggtttcatttacaaggagagagggaagcttgaagctgtcgtctatgttgctggagacaacctcaggagcactctccctctga。

Preparation of mesenchymal stem cells

1. Sample collection

Selecting healthy adults, checking up an informed consent according to the national blood donation standard after the adult is qualified, then extracting bone marrow by a clinician, and transporting the bone marrow to a laboratory for isolated culture by a cold chain.

In this example, 20mL of bone marrow of healthy volunteers was diluted 1:1 with 1 XPBS, and mononuclear cells were separated from the bone marrow by density gradient centrifugation using Ficoll-Paque lymph separation medium at 2000rpm for 30min, and the collected mononuclear cells were diluted1X 10⁵Per cm²Inoculating at a density of 75cm²The culture flask is used for culturing. Culturing in L-DMEM medium at 37 deg.C and 5% CO₂After culturing for 3 days under the condition, removing the suspension cells, and continuously culturing by changing the culture solution. After the cells grew to 80% density, the medium was aspirated, washed 2 times with PBS, and digested with 0.125% pancreatin for 1-2min with a passage ratio of 1: 3. Mesenchymal stem cells are isolated from bone marrow donated by healthy donors, and clinical MSC separation, amplification, cryopreservation, resuscitation and the like are performed under the condition of meeting GMP (good manufacturing practice) standards. The growth and morphological characteristics of the primary and passaged cells were observed daily under an inverted microscope and photographed for recording.

2. Mesenchymal stem cell biological evaluation

Digesting in vitro cultured mesenchymal stem cells into single cell suspension, adding the suspension containing 0.1% BSA and 0.05% NaN₃PBS, pH7.4, washing once, discarding supernatant, adjusting cell density to 10⁶Labeling mesenchymal stem cells with flow antibodies CD29, CD34, CD44, CD45, CD73, CD90, CD105 and CD166 in a flow tube, shaking and mixing uniformly, incubating at 4 ℃ in the dark for 30min, and then using a solution containing 0.1% BSA and 0.05% NaN₃PBS, pH7.4, washed twice to remove excess antibody; supernatant was discarded, cells were resuspended in 200. mu.L of 1% PFA, and the mesenchymal stem cell phenotype CD29 was flow-assayed⁺、CD34^-、CD44⁺、CD45^-、CD73⁺、CD90⁺、CD105⁺、CD166⁺And the in vitro culture is proved to have no influence on the phenotype of the mesenchymal stem cells.

And transferring the P2 generation cells to a six-well plate until the length reaches about 60% for standby.

Preparation of specific targeting mesenchymal stem cells

The prepared modified mRNA was introduced into mesenchymal stem cells by electroporation to obtain specific targeting mesenchymal stem cells of the present example, and the prepared specific targeting mesenchymal stem cells were subjected to functional evaluation as follows:

DNA transfection

Inoculating the artificially cultured mesenchymal stem cells to 75cm²In a culture flask, 5% CO at 37 deg.C₂Culturing for 3 days under the condition; the mixture was centrifuged at 2000rpm at 4 ℃ for 30min, and the supernatant was discarded. With a solution containing 0.1% BSA and 0.05% NaN₃PBS, pH7.4, washed once and the supernatant discarded. The cells were washed once with 5mL of electroporation buffer, and the mesenchymal stem cells were made to a density of1X 10 with electroporation buffer⁶Each 0.8mL of cell suspension. 0.8mL of cells were all transferred to a pre-cooled electroporation cuvette, 5. mu.g of PCR amplification product was added, and a water blank was set. DNA and cell suspension were mixed well and incubated on ice for 10 min. The cell suspension was then bombarded at 400V/500. mu.F and the time was recorded. After completion, the electroporation cuvette was removed and left on ice for 10 min. Transfer of bombarded cells toA new centrifuge tube containing the culture medium is filled, a small amount of culture medium is used for washing the electroporation cup, and the electroporation cup is collected into the centrifuge tube; the mixture was centrifuged at 2000rpm at 4 ℃ for 30min, and the supernatant was discarded. Cells were suspended in 20mL of culture medium and transferred to 75cm²In a culture flask, 5% CO at 37 deg.C₂Cultured for 3 days under the condition, and centrifuged to collect the cells.

2. Transfection assay

The cultured mesenchymal stem cells are sorted and purified by a flow cytometer, and RNA extraction, reverse transcription and PCR amplification detection are the same as the nucleic acid extraction of the example 2 and the nucleic acid sequencing of the example 3.

RNA was also extracted by Trizol method and primers for "(2) PCR reaction and recovery of CCR9cDNA fragment" were used.

The PCR amplification product is detected by agarose gel electrophoresis, and the result shows that all the cultured transgenic mesenchymal stem cells can be amplified to obtain a CCR9 gene fragment of about 1074bp, which is consistent with the expectation.

Western Blot detection of protein levels of CCR9 expressed by transfected MSCs

(1) Protein extraction

The MSCs that overexpress CCR9 in culture, labeled as MSC-CCR9, and untransfected control MSCs, labeled as MSC-EGFP, were placed on ice, the culture medium was removed, washed twice with pre-cooled PBS, 1 x SDS loading buffer containing 5% DTT was added, quickly flushed back and forth with a 1mL pipette gun, and the cells were lysed thoroughly. Sucking liquid after blowing, putting the liquid into a 1.5mL Eppendorf centrifugal tube, and ultrasonically crushing the liquid for 3 times at 4 ℃ for 1 second each time; boiling at 100 deg.C for 5min, cooling at 4 deg.C, centrifuging at 15000g at 4 deg.C for 5min, and preparing for electrophoresis or storing at-80 deg.C for use.

Wherein the loading buffer comprises 62.5mM Tris-HCl pH6.8, 2% (w/v) SDS, 10% glycerol, 50mM DTT and 0.1% (w/v) bromophenol blue.

(2) Electrophoretic separation

In this example, a modified polyacrylamide gel (abbreviated as SDS-PAGE) was used for electrophoretic separation, specifically, 4.1mL of separation gel was added, the gel was sealed with deionized water, after an obvious interface appeared, the water seal was removed, and the prepared gel was added to the top of the short glass block. And inserting the comb, wherein when the glue surface has an irregular shape, the glue is well polymerized, and the comb can be pulled out and loaded.

In this order, 15. mu.L of a sample boiled at 100 ℃ for 5 minutes was added to each lane, and electrophoresed at constant pressure of 120V for about 45 minutes in SDS electrophoresis buffer.

(3) Rotary film

During electrophoresis, materials required by the transfer membrane, such as sponge, filter paper, PVDF membrane and the like, in this case, the PVDF membrane is soaked in the transfer membrane buffer solution. After electrophoresis, the gel was removed and the concentrated gel portion was removed. And (3) placing the glue in the membrane transferring solution for balancing for 15-30 minutes to remove SDS attached on the surface of the glue. Then, a membrane sandwich box is arranged from the negative electrode to the positive electrode, the sandwich box is arranged according to the sequence of sponge, a layer of filter paper, gel, a PVDF membrane, a layer of filter paper and sponge, the sandwich box is placed in a transfer tank after being fixed, and the PVDF membrane faces to the direction of the positive electrode. Placing the sandwich box and the ice box in a transfer tank, injecting 600mL of transfer buffer solution, and keeping constant current at 200mA²And (4) hours.

Wherein, the transmembrane buffer comprises 25mM Tris base, 0.2M glycine and 20% methanol pH8.5.

(4) Antigen antibody reaction

After the membrane transfer was complete, the PVDF membrane was removed and washed in 25mL TBS containing 50mM Tris-HCl pH7.4 and 150mM NaCl for 10 minutes. Then transferred to 20mL of blocking solution, blocked at room temperature with shaking for 1 hour, and the corresponding primary antibody diluent diluted with 5% (w/v) skim milk was added. Gently shake overnight at 4 ℃. Wherein the blocking solution is 1 XTSST containing 5% skimmed milk, and TBST is TBS containing 0.05% Tween-20.

The next day, the membrane was washed 3 times with 1 × TBST for 5 minutes each, and then 15mL of a secondary antibody (1:2000) labeled with horseradish peroxidase (HRP) diluted with a blocking solution was added, followed by shaking at room temperature for 1 hour. The film was then washed 3 times with 1 × TBST for 10 minutes each, developed in the dark room, fixed: preparing ECL kit A, B liquid into working liquid, uniformly coating the working liquid on the surface of a PVDF membrane, incubating for 1 minute, removing residual reaction liquid on the surface of the membrane as much as possible, fixing the membrane in an X-ray sensitive box by using a plastic preservative film, placing an X-ray film for proper exposure, taking out the X-ray film, reacting for 1 minute in a developing solution, rinsing the developed film in clear water for several times, reacting in a fixing solution for 1 minute, then rinsing with clear water, and airing the film.

The results are shown in FIG. 1, where MSC-CCR9 expresses the protein, MSC-EGFP does not, and both express the green fluorescent protein EGFP; the transgenic mesenchymal stem cells constructed in the example are shown to be capable of effectively expressing CCR 9.

Example two

In this example, DNFB-induced mice were subjected to contact hypersensitivity to verify the therapeutic effect of the transgenic mesenchymal stem cells constructed in example one on inflammatory bowel disease.

In this example, male BALB/c mice of 6-8 weeks were bred in 16-18g SPF environment and used for model building of contact hypersensitivity model.

DNFB presensitizing solution: preparing a mixed solution, namely acetone: the volume ratio of the olive oil is 4:1, and the olive oil is violently shaken and uniformly mixed;

preparing a pre-sensitization mixed solution: and preparing 0.5% DNFB mixed solution, namely pre-sensitization mixed solution, by using the mixed solution.

DNFB sensitization solution: and preparing a DNFB mixed solution with the concentration of 0.2 percent by using the mixed solution, namely a sensitization mixed solution.

Pre-sensitization: onday 1, a region of 1.5X 1.5cm was scraped off from the skin of the back of the mouse near the head with an electric razor, and 20. mu.L of 0.5% DNFB-presensitized mixture was applied to the scraped skin, and the treated skin was kept as usual.

Sensitization: onday 5, the mouse ears were smeared with 0.2% DNFB solution, 10. mu.L each on both sides.

The animals were divided into 4 groups of 8 animals each, 4 groups including a blank Control (Control) group, a model group (CHS), an MSC EGFP treatment group (CHS + MSC-EGFP) and an MSC CCR9 treatment group (CHS + MSC-CCR 9). Among them, CHS + MSC-CCR9 is the test group injected with the transgenic mesenchymal stem cells prepared in example one.

Onday 2 after molding, the CHS + MSC EGFP group was injected with MSC-EGFP intravenously at an injection rate of1X 10⁶Individual cells/mouse; the CHS + MSC-CCR9 group was injected intravenously with MSC-CCR9, the injection amount was also1X 10⁶One cell/mouse.

The ear thickness of mice was recorded ondays 1, 2, 3, 4, 5 after treatment and measured with a micrometer. The results showed that after sensitization, the right ear of the sensitized mice was increasing in chemokine mRNA after inflammation occurred, as shown in fig. 2; at the same time, the ear thickness gradually increased and the edema deepened, as shown in fig. 3, without a significant change in the corresponding left ear.

OnDay 4, as shown in fig. 4, the mice were treated by applying 20 μ L of 0.5% DNFB pre-sensitization mixture to the back,Day 0, sensitizing the right ear of the mice, and injecting MSC into the tail vein of the mice onDay 2 after sensitization,Day 2, Day 2:

CHS + MSC-EGFP group intravenous MSC EGFP, 1 × 10⁶Cells/stick;

CHS + MSC-CCR9 group intravenous MSC CCR9, 1 × 10⁶Cells/stick;

control group injected PBS.

The thickness of the ears of mice ondays 1, 2, 3, 4 and 5 after treatment was recorded, the thickness of the outer edges of the ears was measured by a micrometer, and the average value was taken by the same operator three times at the same time each day, and the results showed that the thickness of the ears of the mice in the CHS + MSC-CCR9 group decreased most rapidly and the treatment effect was the best, as shown in FIG. 5, the treatment effect of the CHS + MSC-EGFP group was between that of the CHS group and that of the CHS + MSC-CCR9 group.

Slicing, dyeing and observing the number of mouse ear MSCs, specifically, killing the mouse with broken neck, taking the ears of the mice of each experimental group, fixing the ears by using paraformaldehyde with 10 times of the volume of the ears for 6 hours, transferring the ears to 30% sucrose for dehydration, standing overnight at 4 ℃, fixing by OCT (optical coherence tomography), slicing at-20 ℃ by using a freezing microtome, and measuring 7 microns; and (3) immunofluorescence staining: baking the slices, performing baking at 60 ℃ for 30 minutes, tearing off OCT, eluting with 0.01M PBS for 5 minutes × 3 times, drawing a line with an immunohistochemical pen to circle the organized part, dripping goat serum, sealing for 30 minutes, adding primary antibody, incubating overnight at 4 ℃, standing at room temperature for 30 minutes, eluting with 0.01M PBS for 5 minutes, eluting for 3 times, adding secondary antibody, eluting for 30 minutes, eluting with 0.01M PBS for 5 minutes × 3 times, adding DAPI, eluting for 10 minutes, eluting with 0.01M PBS for 5 minutes, eluting for 3 times, and sealing the slices.

The observation results of the sections are shown in fig. 6 and fig. 7, and the results show that the thickness of the ear of the MSC-CCR9 group is obviously lower than that of the ear of the MSC-EGFP group after treatment, and more importantly, the quantity of the MSCs in the ear of the MSC-CCR9 group is obviously higher than that of the ear of the MSC-EGFP group, thereby proving that the MSC-CCR9 can migrate to the inflammation site in a targeted manner.

Infiltration of local inflammatory cells-the detection of Myeloperoxidase (MPO), a enzyme known as peroxidase, is a heme protease of the heme prosthetic group, a member of the heme peroxidase superfamily. Myeloperoxidase is characteristic of neutrophils and is rarely or not at all present in macrophages with strong phagocytosis. Cytochemically, myeloperoxidase is generally used as a marker for neutrophils, and each cell contains a certain amount of enzyme, about 5% of the dry weight of the cell, which has the ability to reduce hydrogen peroxide, and this feature can be used to analyze the activity of the enzyme and quantitatively determine the number of neutrophils.

MPO detection: preparing samples to be detected, namely, ears of each group of mice, weighing, taking corresponding reagents in the kit as homogenate media, adding the homogenate media according to the weight-volume ratio of 1:19 to prepare 5% tissue homogenate, and then detecting MPO activity according to the kit steps. The results are shown in FIG. 8 and show that the MPO activity of the CHS + MSC-CCR9 group is significantly lower than that of the CHS + MSC-EGFP and CHS groups. The infiltration of neutrophils in the MSC-CCR9 group is obviously lower than that of the MSC-EGFP group, and the MSC-CCR9 is proved to be capable of migrating to an inflammatory part in a targeted manner, playing an immune function and reducing inflammatory infiltration.

In this example, the secretion of local inflammatory factors was examined as follows:

and (3) ELISA detection: washing a 96-well ELISA plate for 2 times by using a 1 xwashing buffer, adding a standard substance and a sample to be detected, 100 mu L/well, incubating for 2h at room temperature in a dark place, discarding liquid, washing for 5 times by using the 1 xwashing buffer, adding a detection antibody 100 mu L/well, incubating for 1h at room temperature in the dark place, discarding liquid, washing for 5 times by using the 1 xwashing buffer, removing redundant antibody, adding an enzyme conjugate working solution 100 mu L/well, incubating for 30min at room temperature in the dark place, discarding liquid, washing for 5 times by using the 1 xwashing buffer, adding adisplay substrate 50 mu L/well, incubating for 20min in the dark place, adding atermination solution 50 mu L/well, and detecting a 450nm value by using an enzyme labeling instrument after uniform mixing.

The results are shown in fig. 9 and fig. 10, and show that the infiltration of local inflammatory factors TNF α and IFN γ is significantly lower in the CHS + MSC-CCR9 group than in the CHS + MSC-EGFP and CHS groups, demonstrating that MSC-CCR9 can migrate to the site of inflammation in a targeted manner, exert immune function, and reduce inflammatory response.

The foregoing is a detailed description of the present application in connection with specific embodiments thereof, and implementations of the present application are not to be considered limited to those descriptions. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the basic inventive concepts herein.

SEQUENCE LISTING

<110> Guangzhou meaning Biotechnology Co., Ltd

<120> mesenchymal stem cells for treating inflammatory bowel disease and application thereof

<130> 2021.07.20

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 1074

<212> DNA

<213> CCR9 gene

<400> 1

atggctgatg actatggctc tgaatccaca tcttccatgg aagactacgt taacttcaac 60

ttcactgact tctactgtga gaaaaacaat gtcaggcagt ttgcgagcca tttcctccca 120

cccttgtact ggctcgtgtt catcgtgggt gccttgggca acagtcttgt tatccttgtc 180

tactggtact gcacaagagt gaagaccatg accgacatgt tccttttgaa tttggcaatt 240

gctgacctcc tctttcttgt cactcttccc ttctgggcca ttgctgctgc tgaccagtgg 300

aagttccaga ccttcatgtg caaggtggtc aacagcatgt acaagatgaa cttctacagc 360

tgtgtgttgc tgatcatgtg catcagcgtg gacaggtaca ttgccattgc ccaggccatg 420

agagcacata cttggaggga gaaaaggctt ttgtacagca aaatggtttg ctttaccatc 480

tgggtattgg cagctgctct ctgcatccca gaaatcttat acagccaaat caaggaggaa 540

tccggcattg ctatctgcac catggtttac cctagcgatg agagcaccaa actgaagtca 600

gctgtcttga ccctgaaggt cattctgggg ttcttccttc ccttcgtggt catggcttgc 660

tgctatacca tcatcattca caccctgata caagccaaga agtcttccaa gcacaaagcc 720

ctaaaagtga ccatcactgt cctgaccgtc tttgtcttgt ctcagtttcc ctacaactgc 780

attttgttgg tgcagaccat tgacgcctat gccatgttca tctccaactg tgccgtttcc 840

accaacattg acatctgctt ccaggtcacc cagaccatcg ccttcttcca cagttgcctg 900

aaccctgttc tctatgtttt tgtgggtgag agattccgcc gggatctcgt gaaaaccctg 960

aagaacttgg gttgcatcag ccaggcccag tgggtttcat ttacaaggag agagggaagc 1020

ttgaagctgt cgtctatgtt gctggagaca acctcaggag cactctccct ctga 1074

<210> 2

<211> 22

<212> DNA

<213> Artificial sequence

<400> 2

atggctgatg actatggctc tg 22

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence

<400> 3

tcagagggag agtgctcctg a 21

Claims (10)

1. A mesenchymal stem cell for use in the treatment of inflammatory bowel disease, characterized by: the mesenchymal stem cell is formed by transfecting a nucleic acid of a chemokine receptor CCR9 into the mesenchymal stem cell.

2. The mesenchymal stem cell of claim 1, wherein: the nucleic acid of the chemokine receptor CCR9 is a sequence shown in Seq ID No. 1.

3. Mesenchymal stem cell according to claim 1 or 2, characterized in that: the mesenchymal stem cells are derived from bone marrow, adipose tissue, dental pulp, umbilical cord blood, amniotic fluid or placenta; preferably, the mesenchymal stem cells are derived from bone marrow.

4. Mesenchymal stem cell according to claim 1 or 2, characterized in that: the transfection is electroporation transfection.

5. Method for the preparation of mesenchymal stem cells according to any one of claims 1-4, characterized in that: comprises the steps of obtaining CCR9 nucleic acid and transfecting the CCR9 nucleic acid into mesenchymal stem cells by an electroporation transfection method, wherein the CCR9 nucleic acid is a sequence shown in Seq ID No. 1.

6. Use of mesenchymal stem cells according to any one of claims 1-4 in the manufacture of a medicament for the treatment of inflammatory bowel disease.

7. A medicament for the treatment of inflammatory bowel disease, characterized by: the medicament contains the mesenchymal stem cell of any one of claims 1-4.

8. The medicament of claim 7, wherein: the medicine also contains T cells or immune cells which do not influence the function of the mesenchymal stem cells or can act synergistically with the mesenchymal stem cells.

9. The medicament according to claim 7 or 8, characterized in that: the medicament is a pharmaceutically acceptable dosage form, including but not limited to powder, solvent, tablet or capsule.

10. The medicament according to claim 7 or 8, characterized in that: the medicine also contains pharmaceutically acceptable auxiliary materials.

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