CN112877294A - Preparation and application of gene-modified mesenchymal stem cell exosome - Google Patents
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Abstract
The invention discloses a gene modified mesenchymal stem cell exosome, a preparation method thereof and application thereof in allergic rhinitis treatment. The invention has obvious treatment effect on allergic rhinitis, has great economic and social benefits and good market prospect.
Description
Technical Field
The invention belongs to the technical field of biological pharmacy, relates to preparation and application of a genetically modified mesenchymal stem cell exosome, and particularly relates to preparation and application of the genetically modified mesenchymal stem cell exosome in treatment of allergic rhinitis.
Background
Allergic rhinitis, also known as allergic rhinitis, is a allergic disease of the nasal mucosa type I induced by IgE-mediated release of histamine, kinins, leukotriene, etc. The onset symptoms are mainly manifested as nasal itching, nasal obstruction, sneezing, thin nasal discharge, dysosmia and the like, and along with the progress of the disease process, the onset symptoms can be accompanied by various diseases such as low memory, pharyngitis and the like, and the work and life quality of patients are seriously affected. The disease is one of the chronic diseases which are always common in human beings, the incidence rate of the disease is on the trend of increasing year by year due to the influence of global climate change, environmental pollution, social and psychological stress and the like in recent years, the disease is regarded as epidemic disease of the 21 st century, and about 25 percent of people in the world are deeply disturbed by the disease.
The current treatment of allergic rhinitis mainly consists of three main aspects, allergen avoidance, pharmacotherapy and immunotherapy. Known allergens should be protected from contact with the allergens, but contact with allergens such as pollen, dust mites and certain foods cannot be avoided in life. Immunotherapy mainly refers to allergen-specific immunotherapy, but the treatment process is complicated, the treatment period is long (usually about 3 years), the effect is slow, the cost is high, most patients are difficult to persist, and the treatment safety is uncertain. Medication remains the first treatment option for allergic rhinitis, and clinical medications are mainly glucocorticoid, antihistamine, decongestant, anticholinergic, and mast cell membrane stabilizer class 5. In treatment, glucocorticoid and antihistamine are mainly used, and the other 3 types of drugs are used as auxiliary drugs. Antihistamines have good effects on rhinocnesmus, sneezing and thin nasal discharge, but have no obvious effect on nasal obstruction; decongestants are effective in relieving nasal mucosal congestion, but are ineffective against other symptoms; anticholinergic agents have good effects in relieving watery nasal discharge, but are ineffective in treating nasal obstruction and sneezing symptoms, and also have side effects of causing nasal cavity dryness and bleeding; the mast cell membrane stabilizer has good effect of preventing allergic rhinitis, has no antagonism on generated allergic medium, and has poor curative effect on acute attack of allergic rhinitis; glucocorticoid can block allergic inflammation reaction of nasal mucosa from multiple links, can effectively prevent and treat symptoms of allergic rhinitis, is the most effective first-line medicament for treating allergic rhinitis at present, but has slow onset of action, takes effect after being taken for days or weeks, has side effect of local stimulation, and the side effect and long-term curative effect of long-term local application of glucocorticoid still need to be evaluated by evidence-based medicine.
In view of the above, the present invention aims to provide a genetically modified mesenchymal stem cell exosome, a preparation method and an application thereof, so as to solve one or more technical problems.
Disclosure of Invention
In order to solve one or more technical problems in the prior art, the applicant researches and discovers that the mesenchymal stem cell exosome modified by the IL-27 gene has a good effect of treating allergic rhinitis, the immunoregulation and antiallergic functions of the mesenchymal stem cell exosome are enhanced by secreting and expressing the IL-27 factor, the nasal microenvironment can be well repaired, the symptoms of the allergic rhinitis can be improved, and the problems of side effects and poor treatment effect of the existing allergic rhinitis treatment medicines can be solved.
In order to achieve the purpose, the invention provides the following technical scheme:
according to an aspect of the present invention, there is provided a genetically modified mesenchymal stem cell exosome for allergic rhinitis, characterized by being obtained by:
designing and obtaining a nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27, wherein the nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27 comprises a tPA signal peptide coding sequence, an IL-27B chain coding sequence, a linker sequence and an IL-27A chain coding sequence, and is shown as SEQ ID NO. 1;
transferring the nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27 into mesenchymal stem cells to obtain the genetically modified mesenchymal stem cells;
culturing the gene modified mesenchymal stem cell to obtain the gene modified mesenchymal stem cell exosome.
According to still another aspect of the present invention, there is also provided a method for preparing a genetically modified mesenchymal stem cell exosome for allergic rhinitis, characterized by the steps of:
a. designing and obtaining a nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27, wherein the nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27 comprises a tPA signal peptide coding sequence, an IL-27B chain coding sequence, a linker sequence and an IL-27A chain coding sequence, and is shown as SEQ ID NO. 1;
b. transferring the nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27 into mesenchymal stem cells to obtain the genetically modified mesenchymal stem cells;
c. culturing the gene modified mesenchymal stem cell to obtain the gene modified mesenchymal stem cell exosome.
According to another aspect of the present invention, in step a, the nucleic acid sequence for efficiently expressing IL-27 in the mammalian cells is synthesized by a human, wherein the IL-27B chain and the IL-27A chain are linked by a linker consisting of GGGS GGGS GGGS, and the amino acid sequence shown in SEQ ID No.2 is synthesized by a human cell-preferred codon, wherein the synthesis is carried out by introducing a tPA signal peptide, a Kozak sequence and a BamHI site at the upstream of the gene and introducing a stop codon and an EcoRI site at the downstream of the gene.
According to another aspect of the present invention, step b is specifically: the artificially synthesized nucleic acid sequence and pHBAd adenovirus expression vector are respectively cut by BamHI and EcoRI to construct pHBAd-IL27 recombinant plasmid, pHBAd-IL27 recombinant plasmid and pHBAd-BHG plasmid are transfected into a packaging virus 293A cell by using Lipofilter transfection reagent, the cell is diseased and falls off from the bottom, and a mature adenovirus containing the nucleic acid sequence of the mammalian cell for efficiently expressing IL-27 is obtained; infecting the mesenchymal stem cells with the mature adenovirus to obtain genetically modified mesenchymal stem cells.
According to another aspect of the present invention, step c is specifically: culturing the gene modified mesenchymal stem cell, collecting supernatant and extracting the gene modified mesenchymal stem cell exosome.
According to another aspect of the present invention, there is also provided a use of the aforementioned genetically modified mesenchymal stem cell exosome in treatment of allergic rhinitis.
According to still another aspect of the present invention, there is also provided a method for preparing a nasal spray for allergic rhinitis, characterized by comprising the steps of:
preparing a gene modified mesenchymal stem cell exosome by adopting the preparation method;
taking deionized water, sequentially adding glycerol, eucalyptol and EDTA-Na2Benzalkonium chloride and the gene modified mesenchymal stem cell exosome are added and stirred to be fully dissolved, and finally, microcrystalline cellulose-sodium carboxymethyl cellulose is added, after the mixture is fully dissolved by stirring, HCl is used for regulating the pH value to be 5.2-6.6, and the nasal spray is obtained;
wherein, each 1000ml of the nasal spray comprises 5.0-15.0mg of the gene modified mesenchymal stem cell exosome, 3.0-7.0g of eucalyptol, 23.0-30.0g of glycerol, 12.0-18.0g of microcrystalline cellulose-sodium carboxymethyl cellulose, EDTA-Na20.5-1.0g of benzalkonium chloride, 0.2g of benzalkonium chloride and deionized water as a solvent.
According to still another aspect of the present invention, there is also provided a nasal spray for allergic rhinitis, characterized in that it comprises per 1000 ml:
the genetically modified mesenchymal stem cell exosome of claim 1, 5.0-15.0 mg;
3.0-7.0g of cineole;
23.0-30.0g of glycerol;
microcrystalline cellulose-sodium carboxymethylcellulose 12.0-18.0 g;
EDTA-Na2 0.5-1.0g；
benzalkonium chloride 0.2 g;
the balance being deionized water.
Compared with the prior art, the invention has the beneficial effects that at least:
1. the mesenchymal stem cell exosome modified by the IL-27 gene is used as an active ingredient for treating the allergic rhinitis, and the IL-27 factor is secreted and expressed, so that the immunoregulation and antiallergic functions of the mesenchymal stem cell exosome are enhanced, the microenvironment of a nasal cavity can be well repaired, the symptom of the allergic rhinitis is improved, the aim of treating the allergic rhinitis is fulfilled, the treatment effect is remarkable, the safety is good, and no side effects such as the dependence of hormone products exist.
2. The exosome obtained by the mesenchymal stem cell modified by the IL-27 gene forms an exosome and simultaneously fills active IL-27 protein into the exosome, so that the mesenchymal stem cell is endowed with a stronger immunoregulation function, and the treatment effect of the secreted exosome on allergic rhinitis is enhanced.
3. The exosome obtained from the mesenchymal stem cell modified by the IL-27 gene forms an exosome and simultaneously fills active IL-27 protein into the exosome, thereby improving the stability of the IL-27 active protein.
4. The exosome obtained by the mesenchymal stem cell modified by the IL-27 gene forms an exosome and simultaneously loads active IL-27 protein into the exosome, and the exosome is used for transmitting biological information such as nucleic acid, protein and the like contained in the exosome, so that the bioavailability of the active factor IL-27 protein is further improved, and the comprehensive effect of the factors can obtain better allergic rhinitis treatment effect;
5. in each 1000ml of nasal spray, eucalyptol (3.0-7.0g) and gene-modified mesenchymal stem cell exosome (5.0-15.0mg) have synergistic effect, so that the treatment effect of allergic rhinitis is further enhanced.
Detailed Description
The present invention is further specifically illustrated below with reference to specific examples, which are intended to be purely exemplary of the invention and are not intended to limit its scope.
According to a preferred embodiment of the present invention, there is provided a genetically modified mesenchymal stem cell exosome characterized by being obtained by the following steps:
designing and obtaining a nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27, wherein the nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27 comprises a tPA signal peptide coding sequence, an IL-27B chain coding sequence, a linker sequence and an IL-27A chain coding sequence, and is shown as SEQ ID NO. 1;
transferring the nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27 into mesenchymal stem cells to obtain the genetically modified mesenchymal stem cells;
culturing the gene modified mesenchymal stem cell to obtain the gene modified mesenchymal stem cell exosome.
Advantageously, the invention constructs a gene sequence for expressing IL-27 by a molecular biological means, constructs a mammalian cell expression vector, further transfects mesenchymal stem cells, and obtains a gene modified mesenchymal stem cell exosome.
According to still another preferred embodiment of the present invention, there is also provided a method for preparing a genetically modified mesenchymal stem cell exosome, characterized by the steps of:
a. designing and obtaining a nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27, wherein the nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27 comprises a tPA signal peptide coding sequence, an IL-27B chain coding sequence, a linker sequence and an IL-27A chain coding sequence, and is shown as SEQ ID NO. 1;
b. transferring the nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27 into mesenchymal stem cells to obtain the genetically modified mesenchymal stem cells;
c. culturing the gene modified mesenchymal stem cell to obtain the gene modified mesenchymal stem cell exosome.
According to still another preferred embodiment of the present invention, in step a, the nucleic acid sequence for efficiently expressing IL-27 in the mammalian cell is synthesized by a human, wherein the IL-27B chain and the IL-27A chain are linked by a linker consisting of GGGS GGGS GGGS and the amino acid sequence shown in SEQ ID No.2 is synthesized by a human cell's preferred codon, and the synthesis is carried out by introducing a tPA signal peptide, a Kozak sequence and a BamHI site at the upstream of the gene and a stop codon and an EcoRI site at the downstream of the gene.
According to another preferred embodiment of the present invention, step b is specifically: the artificially synthesized nucleic acid sequence and pHBAd adenovirus expression vector are respectively cut by BamHI and EcoRI to construct pHBAd-IL27 recombinant plasmid, pHBAd-IL27 recombinant plasmid and pHBAd-BHG plasmid are transfected into a packaging virus 293A cell by using Lipofilter transfection reagent, the cell is diseased and falls off from the bottom, and a mature adenovirus containing the nucleic acid sequence of the mammalian cell for efficiently expressing IL-27 is obtained; infecting the mesenchymal stem cells with the mature adenovirus to obtain genetically modified mesenchymal stem cells.
According to another preferred embodiment of the present invention, step c is specifically: culturing the gene modified mesenchymal stem cell, collecting supernatant and extracting the gene modified mesenchymal stem cell exosome.
According to still another preferred embodiment of the present invention, there is also provided a use of the aforementioned genetically modified mesenchymal stem cell exosome in the treatment of allergic rhinitis.
According to still another preferred embodiment of the present invention, there is also provided a method for preparing a nasal spray for allergic rhinitis, characterized by comprising the steps of:
preparing a gene modified mesenchymal stem cell exosome by adopting the preparation method;
taking deionized water, sequentially adding glycerol, eucalyptol and EDTA-Na2Benzalkonium chloride and the gene modified mesenchymal stem cell exosome are added and stirred to be fully dissolved, and finally, microcrystalline cellulose-sodium carboxymethyl cellulose is added, after the mixture is fully dissolved by stirring, HCl is used for regulating the pH value to be 5.2-6.6, and the nasal spray is obtained;
wherein, each 1000ml of the nasal spray comprises 5.0-15.0mg of the gene modified mesenchymal stem cell exosome, 3.0-7.0g of eucalyptol, 23.0-30.0g of glycerol, 12.0-18.0g of microcrystalline cellulose-sodium carboxymethyl cellulose, EDTA-Na20.5-1.0g of benzalkonium chloride, 0.2g of benzalkonium chloride and deionized water as a solvent.
According to still another preferred embodiment of the present invention, there is also provided a nasal spray for allergic rhinitis, characterized in that the nasal spray comprises per 1000 ml:
the genetically modified mesenchymal stem cell exosome of claim 1, 5.0-15.0 mg;
3.0-7.0g of cineole;
23.0-30.0g of glycerol;
microcrystalline cellulose-sodium carboxymethylcellulose 12.0-18.0 g;
EDTA-Na2 0.5-1.0g；
benzalkonium chloride 0.2 g;
the balance being deionized water.
Advantageously, the cineole is colorless to yellowish oily liquid, is a main component of eucalyptus oil, has camphor and cool herbal odor, has the effects of dispelling wind, clearing heat, eliminating dampness, detoxifying, resisting bacteria and viruses, and has synergistic effect with the gene-modified mesenchymal stem cell exosome, thereby further enhancing the treatment effect of allergic rhinitis.
According to still another preferred embodiment of the present invention, there is also provided a spray for treating allergic rhinitis comprising mesenchymal stem cell exosomes, having a pH of 5.2-6.6, comprising 5.0-15.0mg of mesenchymal stem cell exosomes, 3.0-7.0g of eucalyptol, 23.0-30.0g of glycerol, 12.0-18.0g of microcrystalline cellulose-sodium carboxymethyl cellulose, EDTA-Na per 1000ml20.5-1.0g of benzalkonium chloride, 0.2g of benzalkonium chloride and deionized water as a solvent.
In a preferred embodiment of the present invention, each 1000ml of the composition comprises 10.0mg of the mesenchymal stem cell exosome, 5.0g of eucalyptol, 26.0g of glycerol, 15.0g of microcrystalline cellulose-sodium carboxymethyl cellulose, EDTA-Na20.5g of benzalkonium chloride, 0.2g of benzalkonium chloride and deionized water as a solvent.
Preferably, the genetically modified mesenchymal stem cell is a mesenchymal stem cell transfected with an IL-27 gene and capable of secreting and expressing a functional IL-27.
Preferably, the vector for transfecting the IL-27 gene is a viral vector such as an adenovirus vector, an adeno-associated viral vector, a retrovirus vector, a lentivirus vector and the like, or a non-viral vector such as pcDNA3.1 and the like.
Further preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells and placental mesenchymal stem cells.
According to another preferred embodiment of the present invention, there is also provided a method for preparing a mesenchymal stem cell exosome nasal spray, comprising the steps of:
(1) selecting a mammalian cell expression vector, and designing and constructing an IL-27 secretion expression vector;
(2) separating, culturing, purifying and identifying mesenchymal stem cells;
(3) transferring the IL-27 secretion expression vector into mesenchymal stem cells, continuously culturing for at least 3 days, collecting culture supernatant, and extracting to obtain stem cell exosome sediment.
Preferably, the preparation method further comprises step (4): proportionally mixing eucalyptol, glycerin, microcrystalline cellulose-sodium carboxymethyl cellulose and EDTA-Na2Adding benzalkonium chloride and the mesenchymal stem cell exosome into deionized water, adjusting the pH value, and filtering and sterilizing by using a 0.45-micrometer sterile filter membrane to obtain the allergic rhinitis treatment spray containing the mesenchymal stem cell exosome.
Preferably, the construction of IL-27 secretion expression vector is through molecular biology and genetic engineering means to IL-27 protein expression cassette subcloning to mammalian cell expression vector.
In a preferred embodiment of the present invention, the expression vector is an adenoviral vector.
Further preferably, the step (1) is: artificially synthesizing a nucleic acid sequence (shown as SEQ ID NO. 1) which can be used for efficiently expressing IL-27 in mammalian cells, and comprises a Kozak sequence, a tPA signal peptide sequence, an IL-27B chain (EBI3) sequence, a linker sequence and an IL-27A chain (p28) sequence. The nucleic acid sequence capable of being used for efficiently expressing IL-27 in mammalian cells is connected into a pHBAd adenovirus expression vector through BamHI and EcoRI endonucleases. The nucleic acid sequence capable of being used for efficiently expressing IL-27 in mammalian cells can encode an amino acid sequence shown as SEQ ID NO. 2.
In a preferred embodiment of the present invention, the mesenchymal stem cell is an umbilical cord mesenchymal stem cell.
Further preferably, the step (2) is: cleaning fresh umbilical cord with sterile normal saline to remove blood, removing veins, envelopes and arteries, cutting Wharton's jelly, inoculating the cut Wharton's jelly into a culture dish, adding a mesenchymal stem cell culture medium, culturing in a 5% CO2 cell culture box at 37 ℃ for 6-8 days, and allowing a large amount of spindle-shaped cells to climb out of tissue blocks to obtain the mesenchymal stem cells.
The respective parts of the present invention will be described in detail with reference to examples. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 Artificial Synthesis of a DNA sequence encoding IL-27 (shown in SEQ ID NO. 1)
The mature protein sequence of the B chain of IL-27 comprises 209 amino acid residues, the mature protein sequence of the A chain of IL-27 comprises 215 amino acid residues, and the amino acid sequence is artificially synthesized by a linker consisting of GGGS GGGS GGGS between the B chain and the A chain according to the codon preferred by human cells. For the synthesis, a tPA signal peptide (MDAMKRGLCCVLLLCGAVFVSP), Kozak sequence and BamHI site were introduced upstream of the gene, and a stop codon and EcoRI site were introduced downstream of the gene.
Example 2 construction of expression vector
The synthesized gene fragment and pHBAd adenovirus expression vector were digested with BamHI and EcoRI, respectively, and incubated at 37 ℃ overnight. And (4) cutting the enzyme digestion product by agarose gel electrophoresis to obtain a target fragment, and then recovering by using an agarose gel DNA recovery kit. And (3) mixing the recovered target gene subjected to enzyme digestion with a vector according to the weight ratio of 3: 1, and connecting the mixture at constant temperature overnight by using a T4 DNA Ligase 16 ℃ PCR instrument to construct a pHBAd-IL27 recombinant plasmid. The ligation product was transformed into competent Escherichia coli DH 5. alpha. and the transformed competent cells were spread on LB plate medium containing ampicillin (100. mu.g/mL) uniformly, and after the bacterial solution was completely absorbed, it was cultured by inversion at 37 ℃ for 12-16 h. 4-8 single colonies with good growth status were picked from the transformation plate with a sterile tip, inoculated in 5mL LB medium containing ampicillin (100. mu.g/mL), and cultured overnight at 37 ℃ with vigorous shaking (225 rpm). Plasmid DNA was extracted using a rapid plasmid miniprep kit, the recombinant plasmid pHBAd-IL27 was identified by double-cutting with BamHI and EcoRI, digested at 37 ℃ for 3h, and correct clones were identified by endonuclease mapping. And selecting the clone with the correct enzyme cutting map, and sending the clone to a sequencing company for sequencing verification. Taking clone with correct sequencing verification, extracting by using a plasmid large-extraction kit to obtain a large amount of recombinant plasmid pHBAd-IL27, then transfecting a pHBAd-IL27 plasmid and pHBAd-BHG plasmid by using a lipoiter transfection reagent to package virus 293A cells, collecting virus when most cells are diseased and fall off from the bottom, repeatedly freezing and thawing for 3 times, centrifuging for 5min at 3000r/min, collecting supernatant to obtain a large amount of mature adenovirus containing the IL-27 sequence, detecting and calculating virus titer, and storing in a refrigerator at-80 ℃ for subsequent experiments.
Example 3 obtaining umbilical cord mesenchymal Stem cells
Cleaning fresh umbilical cord with sterile normal saline to remove blood, shearing into 4cm small sections, removing veins, envelopes and 2 arteries, shearing Wharton's jelly, inoculating the minced Wharton's jelly into a 10cm culture dish, adding a mesenchymal stem cell culture medium, culturing for 6-8 days in a 5% CO2 cell culture box at 37 ℃, allowing a large amount of fusiform cells to climb out of a tissue block, namely the mesenchymal stem cells, and performing passage amplification and purification identification to obtain the umbilical cord mesenchymal stem cells.
Example 4 transfection of umbilical cord mesenchymal Stem cells
Inoculating umbilical cord mesenchymal stem cells into a T175 culture bottle, when the cell density reaches about 80%, infecting the umbilical cord mesenchymal stem cells by using secretion expression IL-27 adenovirus with the multiplicity of infection (MOI) of 200, changing the solution after 6h, continuously culturing for 72h, collecting supernatant, extracting exosome, and taking 2ml of supernatant for detecting the expression condition of IL-27 by an ELISA method.
Example 5 preparation of umbilical cord mesenchymal Stem cell exosomes
The method adopts a differential centrifugation method to extract exosomes, and comprises the following specific steps: centrifugation was carried out at 2000 Xg for 10 minutes at 4 ℃ to obtain the supernatant. Centrifugation was carried out at 4 ℃ at 10000 Xg for 30 minutes to collect the supernatant. Centrifugation was continued at 100000 Xg for 120 min at 4 ℃ to remove the supernatant and the pellet was resuspended in PBS and centrifuged again at 100000 Xg for 120 min to obtain an exosome pellet. The BCA method quantitated after resuspension using saline.
Example 6 preparation of spray for allergic rhinitis
The spray for treating allergic rhinitis comprises mesenchymal stem cell exosome, eucalyptol, glycerol, microcrystalline cellulose-sodium carboxymethyl cellulose, EDTA-Na2And benzalkonium chloride in water.
The microcrystalline cellulose-sodium carboxymethylcellulose mainly plays the roles of a suspending agent and a thickening agent in the nasal spray, is beneficial to the distribution and the retention time of active ingredients in the nasal cavity, improves the utilization rate of the medicine, and has the concentration of 0.91-2.1 percent, and the preferred concentration in the scheme is 1.5 percent.
In this example, the formulation was prepared according to the following formulation:
formula composition of epi-I mesenchymal stem cell exosome spray for allergic rhinitis
Taking the deionized water according to the prescription amount, and sequentially adding glycerol, eucalyptol and EDTA-Na2Benzalkonium chloride and the mesenchymal stem cell exosome are fully dissolved by adding and stirring, finally, microcrystalline cellulose-sodium carboxymethylcellulose is added, after the mixture is fully dissolved by stirring, HCl is used for regulating the pH value to be about 6.0, then a sterile filter membrane of 0.45 mu m is used for filtering and sterilizing, and the mixture is filled into a sterile nasal spray bottle, thus obtaining the allergic rhinitis treatment spray containing the mesenchymal stem cell exosome.
Example 7 study of the therapeutic action of the spray for allergic rhinitis on allergic rhinitis in rats
1) Establishment of rat allergic rhinitis model
60 male Wistar rats with the body mass of 180-200g are randomly extracted 10 rats to be used as a blank group of normal rats, 5 mu l of olive oil solution of 10 percent Toluene Diisocyanate (TDI) is dripped into each nostril at each side of the rest rats by using a microsyringe, the time of each nostril is 1 time per day, the model is stimulated and molded continuously for 7 days, and the blank group of the normal rats is given olive oil with the same dosage in the same way. 30min after the last stimulation, the rats are observed for nasal discharge, sneezing and rhinorrhea, and the scores are used for judging whether the model building is successful. The scoring criteria were: 1-2 times of nasal obstruction, 1-3 sneezes, and 1 minute of nasal discharge to each nostril; the nasal discharge is caused by multiple times of scratching, sneezing is caused by 4-10, and the nasal discharge exceeds the anterior nares by 2 minutes respectively; violently scratching the nasal surface, rubbing everywhere, sneezing more than 11, and counting 3 minutes each. The scores of all symptoms are superposed, and if the total score exceeds 5 scores, the molding is successful.
2) Experimental study on treatment of rat allergic rhinitis model
2.1 grouping and administration of rats
The rats after successful modeling are divided into a model blank group, a positive control group and low, medium and high dose groups of exosome spray, 10 rats in each group are divided into groups, then a dosing experiment is carried out, the blank group uses a micro-sampler to give 15 mu l of physiological saline to each nostril, the positive control group uses the micro-sampler to give 15 mu l of mometasone furoate nasal spray to each nostril, and the low, medium and high dose groups of exosome spray use the micro-sampler to give 5 mu l, 10 mu l and 15 mu l of exosome spray to each nostril. Meanwhile, 5 mul of olive oil solution with 10 percent of Toluene Diisocyanate (TDI) per nostril is given to the rats in the model group every other day to maintain sensitization, and the olive oil is given to the blank rats in the same way and in the same dose until the experiment is finished.
2.2 Observation of indices and results
2.2.1 rhinitis behavior index and score:
the behavior indexes of rhinitis symptoms such as nasal pruritus, sneezing and rhinorrhea were evaluated on the successful grouping of model building, the 7 th day of administration and the 14 th day of administration, and counted and calculated according to the above scoring criteria, and the results are shown in the following table:
TABLE II behavioral integration of allergic rhinitis symptoms in rats
Indicates that p < 0.01, with significant differences compared to the model group.
2.2.2 pathological results of HE staining of nasal mucosal tissue
And (3) taking the nasal mucosa respiratory area of each group of rats, placing the nasal mucosa respiratory area in tissue fixing liquid for fixation, preparing wax block slices, carrying out HE staining, and observing the nasal mucositis condition of each group of rats under an optical microscope. Pathological results were graded as follows: "-" neither nasal mucosal epithelium nor submucosal tissue had inflammatory responses and exudates; "+" submucosal congestion with little infiltration of inflammatory cells; "+ +" beneath the mucosa there was a large infiltration of inflammatory cells and inflammatory exudates in the nasal cavity; the mucosa has a large amount of inflammatory cells infiltrated, the tissue under the mucosa has hyperplasia, the mucosa can be seen to fall off, and inflammatory exudates and bleeding can be seen in the nasal cavity.
Pathological result of HE staining of mucosal tissue of rats
These results show that the nasal spray prepared by the gene modified mesenchymal stem cell exosome obtained according to the scheme of the invention has a remarkable treatment effect on allergic rhinitis.
Compared with the prior art, the invention has the beneficial effects that at least:
1. the mesenchymal stem cell exosome modified by the IL-27 gene is used as an active ingredient for treating the allergic rhinitis, and the IL-27 factor is secreted and expressed, so that the immunoregulation and antiallergic functions of the mesenchymal stem cell exosome are enhanced, the microenvironment of a nasal cavity can be well repaired, the symptom of the allergic rhinitis is improved, the aim of treating the allergic rhinitis is fulfilled, the treatment effect is remarkable, the safety is good, and no side effects such as the dependence of hormone products exist.
2. The exosome obtained by the mesenchymal stem cell modified by the IL-27 gene forms an exosome and simultaneously fills active IL-27 protein into the exosome, so that the mesenchymal stem cell is endowed with a stronger immunoregulation function, and the treatment effect of the secreted exosome on allergic rhinitis is enhanced.
3. The exosome obtained from the mesenchymal stem cell modified by the IL-27 gene forms an exosome and simultaneously fills active IL-27 protein into the exosome, thereby improving the stability of the IL-27 active protein.
4. The exosome obtained by the mesenchymal stem cell modified by the IL-27 gene forms an exosome and simultaneously loads active IL-27 protein into the exosome, and the exosome is used for transmitting biological information such as nucleic acid, protein and the like contained in the exosome, so that the bioavailability of the active factor IL-27 protein is further improved, and the comprehensive effect of the factors can obtain better allergic rhinitis treatment effect;
5. in each 1000ml of nasal spray, eucalyptol (3.0-7.0g) and gene-modified mesenchymal stem cell exosome (5.0-15.0mg) have synergistic effect, so that the treatment effect of allergic rhinitis is further enhanced.
The above description is related to embodiments of the present invention, and the description is specific and detailed, but not construed as limiting the scope of the present invention. For example, the genetically modified mesenchymal stem cell exosomes of the present invention may also be used in the treatment of asthma, pulmonary fibrosis or rheumatoid arthritis. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Sequence listing
<110> Sepu Biotechnology (Changchun) Ltd
Preparation and application of <120> gene modified mesenchymal stem cell exosome
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<170> SIPOSequenceListing 1.0
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cccaacagca catctcccgt gagcttcatc gccacctaca gactgggaat ggctgccaga 240
ggccacagct ggccctgcct gcagcagaca cccacttcta caagctgcac catcaccgac 300
gtgcagctgt tcagcatggc tccctacgtg ctgaacgtga ccgccgtgca cccctggggc 360
tctagctcta gcttcgtgcc cttcatcacc gagcacatca tcaagcccga ccctcccgag 420
ggcgtgaggc tgtctcctct ggccgagaga cagcttcaag tacagtggga acctcccggc 480
agctggccct tccccgagat cttcagcctg aagtactgga tcagatacaa gagacaaggc 540
gctgccaggt tccacagagt gggccctatc gaggccacaa gcttcatcct gagagccgtc 600
agacctagag ctagatacta cgtgcaagtg gctgcccaag acctcaccga ctacggcgag 660
ttaagcgatt ggtctctgcc tgccaccgca accatgtcat taggcaaagg aggcgggagc 720
ggcggtgggt ctggcggcgg aagctttcct agacctcccg gcagacctca gctgagcctg 780
caagagctga ggagagagtt caccgtgtct ctgcacctgg ctagaaagct gctgagcgag 840
gtgagaggcc aagcccacag gttcgccgag agccacctgc ctggcgtgaa cctgtacctg 900
ctgcctctgg gcgagcagct gcccgacgtg agcctgacct tccaagcctg gaggagactg 960
agcgaccccg agagactgtg cttcatctct accactctgc agcccttcca cgccctgctg 1020
ggcggcttag gtacccaagg cagatggacc aacatggaga gaatgcagct gtgggccatg 1080
aggctggacc tgagagacct gcagagacac ctgagattcc aagtgctggc tgctggcttc 1140
aacctgcccg aggaggagga ggaggaggag gaggaggaag aggaggaaag aaagggactg 1200
ctccctggtg ccttaggctc tgcactacaa ggccctgccc aagtgagctg gcctcagctg 1260
ctgtctacct acaggttgct gcacagcctg gagctggtgc tgagcagagc agtgagggaa 1320
ctgctcctcc tgagcaaggc tgggcacagc gtgtggcctc tgggcttccc cactctgagc 1380
cctcagccct gataagaatt c 1401
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Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Arg Lys Gly Pro Pro Ala Ala Leu Thr Leu
20 25 30
Pro Arg Val Gln Cys Arg Ala Ser Arg Tyr Pro Ile Ala Val Asp Cys
35 40 45
Ser Trp Thr Leu Pro Pro Ala Pro Asn Ser Thr Ser Pro Val Ser Phe
50 55 60
Ile Ala Thr Tyr Arg Leu Gly Met Ala Ala Arg Gly His Ser Trp Pro
65 70 75 80
Cys Leu Gln Gln Thr Pro Thr Ser Thr Ser Cys Thr Ile Thr Asp Val
85 90 95
Gln Leu Phe Ser Met Ala Pro Tyr Val Leu Asn Val Thr Ala Val His
100 105 110
Pro Trp Gly Ser Ser Ser Ser Phe Val Pro Phe Ile Thr Glu His Ile
115 120 125
Ile Lys Pro Asp Pro Pro Glu Gly Val Arg Leu Ser Pro Leu Ala Glu
130 135 140
Arg Gln Leu Gln Val Gln Trp Glu Pro Pro Gly Ser Trp Pro Phe Pro
145 150 155 160
Glu Ile Phe Ser Leu Lys Tyr Trp Ile Arg Tyr Lys Arg Gln Gly Ala
165 170 175
Ala Arg Phe His Arg Val Gly Pro Ile Glu Ala Thr Ser Phe Ile Leu
180 185 190
Arg Ala Val Arg Pro Arg Ala Arg Tyr Tyr Val Gln Val Ala Ala Gln
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Asp Leu Thr Asp Tyr Gly Glu Leu Ser Asp Trp Ser Leu Pro Ala Thr
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Ala Thr Met Ser Leu Gly Lys Gly Gly Gly Ser Gly Gly Gly Ser Gly
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Gly Gly Ser Phe Pro Arg Pro Pro Gly Arg Pro Gln Leu Ser Leu Gln
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Glu Leu Arg Arg Glu Phe Thr Val Ser Leu His Leu Ala Arg Lys Leu
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Leu Ser Glu Val Arg Gly Gln Ala His Arg Phe Ala Glu Ser His Leu
275 280 285
Pro Gly Val Asn Leu Tyr Leu Leu Pro Leu Gly Glu Gln Leu Pro Asp
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305 310 315 320
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325 330 335
Gly Leu Gly Thr Gln Gly Arg Trp Thr Asn Met Glu Arg Met Gln Leu
340 345 350
Trp Ala Met Arg Leu Asp Leu Arg Asp Leu Gln Arg His Leu Arg Phe
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Gln Val Leu Ala Ala Gly Phe Asn Leu Pro Glu Glu Glu Glu Glu Glu
370 375 380
Glu Glu Glu Glu Glu Glu Glu Arg Lys Gly Leu Leu Pro Gly Ala Leu
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Gly Ser Ala Leu Gln Gly Pro Ala Gln Val Ser Trp Pro Gln Leu Leu
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Ser Thr Tyr Arg Leu Leu His Ser Leu Glu Leu Val Leu Ser Arg Ala
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Claims (8)
1. A genetically modified mesenchymal stem cell exosome for allergic rhinitis, obtained by:
designing and obtaining a nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27, wherein the nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27 comprises a tPA signal peptide coding sequence, an IL-27B chain coding sequence, a linker sequence and an IL-27A chain coding sequence, and is shown as SEQ ID NO. 1;
transferring the nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27 into mesenchymal stem cells to obtain the genetically modified mesenchymal stem cells;
culturing the gene modified mesenchymal stem cell to obtain the gene modified mesenchymal stem cell exosome.
2. A preparation method of a gene modified mesenchymal stem cell exosome for allergic rhinitis is characterized by comprising the following steps:
a. designing and obtaining a nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27, wherein the nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27 comprises a tPA signal peptide coding sequence, an IL-27B chain coding sequence, a linker sequence and an IL-27A chain coding sequence, and is shown as SEQ ID NO. 1;
b. transferring the nucleic acid sequence of the mammalian cell for efficiently expressing the IL-27 into mesenchymal stem cells to obtain the genetically modified mesenchymal stem cells;
c. culturing the gene modified mesenchymal stem cell to obtain the gene modified mesenchymal stem cell exosome.
3. The method according to claim 2, wherein in the step a, the nucleic acid sequence for efficiently expressing IL-27 in the mammalian cell is synthesized by a human, wherein the IL-27B chain and the IL-27A chain are linked by a linker consisting of GGGS GGGS GGGS, and the amino acid sequence shown in SEQ ID NO.2 is synthesized by a human cell-preferred codon, and the synthesis is carried out by introducing a tPA signal peptide, a Kozak sequence and a BamHI site at the upstream of the gene and introducing a stop codon and an EcoRI site at the downstream of the gene.
4. The method according to claim 3, wherein step b is specifically: the artificially synthesized nucleic acid sequence and pHBAd adenovirus expression vector are respectively cut by BamHI and EcoRI to construct pHBAd-IL27 recombinant plasmid, pHBAd-IL27 recombinant plasmid and pHBAd-BHG plasmid are transfected into a packaging virus 293A cell by using Lipofilter transfection reagent, the cell is diseased and falls off from the bottom, and a mature adenovirus containing the nucleic acid sequence of the mammalian cell for efficiently expressing IL-27 is obtained; infecting the mesenchymal stem cells with the mature adenovirus to obtain genetically modified mesenchymal stem cells.
5. The method according to claim 4, wherein step c specifically comprises: culturing the gene modified mesenchymal stem cell, collecting supernatant and extracting the gene modified mesenchymal stem cell exosome.
6. Use of the genetically modified mesenchymal stem cell exosome of claim 1 in the treatment of allergic rhinitis.
7. A method for preparing a nasal spray for allergic rhinitis, characterized by comprising the steps of:
preparing a genetically modified mesenchymal stem cell exosome using the preparation method of claims 2-6;
taking deionized water, sequentially adding glycerol, eucalyptol and EDTA-Na2Benzalkonium chloride and the gene modified mesenchymal stem cell exosome are added and stirred to be fully dissolved, and finally, microcrystalline cellulose-sodium carboxymethyl cellulose is added, after the mixture is fully dissolved by stirring, HCl is used for regulating the pH value to be 5.2-6.6, and the nasal spray is obtained;
wherein said nasal spray comprises said base per 1000ml5.0-15.0mg of modified mesenchymal stem cell exosome, 3.0-7.0g of eucalyptol, 23.0-30.0g of glycerol, 12.0-18.0g of microcrystalline cellulose-sodium carboxymethylcellulose, EDTA-Na20.5-1.0g of benzalkonium chloride, 0.2g of benzalkonium chloride and deionized water as a solvent.
8. A nasal spray for allergic rhinitis characterized in that it comprises per 1000 ml:
the genetically modified mesenchymal stem cell exosome of claim 1, 5.0-15.0 mg;
3.0-7.0g of cineole;
23.0-30.0g of glycerol;
microcrystalline cellulose-sodium carboxymethylcellulose 12.0-18.0 g;
EDTA-Na2 0.5-1.0g；
benzalkonium chloride 0.2 g;
the balance being deionized water.
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