阅读说明：本技术 一种治疗过敏性鼻炎的细胞制剂 (cell preparation for treating allergic rhinitis ) 是由 常智杰 付艳霞 王银银 任芳丽 于 2018-07-19 设计创作，主要内容包括：本发明公开了一种治疗过敏性鼻炎的细胞制剂。本发明首先保护间充质干细胞在制备用于治疗过敏性鼻炎的药物中的应用。本发明还保护一种细胞制剂的制备方法,包括如下步骤：以脐带沃顿区组织块为原料,制备脐带间充质干细胞,得到细胞制剂,制备过程中采用低血清培养基。本发明还保护所述细胞制剂在制备用于治疗过敏性鼻炎的药物中的应用。本发明提供的细胞制剂,适用于过敏性鼻炎的细胞治疗。本发明对于过敏性鼻炎的治疗具有重大的应用价值。(The invention discloses cell preparations for treating allergic rhinitis, which firstly protect the application of mesenchymal stem cells in preparing a medicament for treating the allergic rhinitis and also protect a preparation method of cell preparations.)

1. Application of mesenchymal stem cells in preparing a medicament for treating allergic rhinitis.

2. The use of claim 1, wherein: the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

3. Use according to claim 2, characterized in that: the umbilical cord mesenchymal stem cells are prepared by taking umbilical cord Wolton area tissue blocks as raw materials.

The preparation method of kinds of cell preparation includes the following steps, umbilical cord mesenchymal stem cells are prepared by using umbilical cord Wolton area tissue blocks as raw materials, and a low serum culture medium is adopted in the preparation process.

The preparation method of the cell preparation of kinds comprises the following steps:

(1) culturing isolated umbilical cord Wolton zone tissue blocks by adopting a low-serum culture medium for 7-10 days;

(2) after the step (1) is completed, removing tissue blocks, and culturing cells by adopting a low serum culture medium to reach 80% confluency;

(3) after the step (2) is finished, collecting cells, digesting the cells by pancreatin, and collecting the cells, namely the P0 generation umbilical cord mesenchymal cells;

(4) generation of P0 umbilical cord mesenchymal cell 1: 2, passage, and culturing by adopting a low serum culture medium to reach 80% confluence;

(5) after the step (4) is finished, collecting cells, digesting the cells by pancreatin, and collecting the cells, namely the P1 generation umbilical cord mesenchymal cells;

(6) generation of P1 umbilical cord mesenchymal cell 1: 2, passage, and culturing by adopting a low serum culture medium to reach 80% confluence;

(7) after the step (6) is finished, collecting cells, digesting the cells by pancreatin, and collecting the cells, namely the P2 generation umbilical cord mesenchymal cells;

(8) generation of P2 umbilical cord mesenchymal cell 1: 2, passage, and culturing by adopting a low serum culture medium to reach 80% confluence;

(9) and (5) after the step (8) is finished, collecting cells, digesting by pancreatin, and collecting the cells to obtain the cell preparation.

6. A cell preparation produced by the method of claim 4 or 5.

7. Use of a cell preparation according to claim 6 for the manufacture of a medicament for the treatment of allergic rhinitis.

8, A medicine for treating allergic rhinitis, wherein the active ingredient is mesenchymal stem cells.

9. The medicament of claim 8, wherein: the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

10, A medicament for treating allergic rhinitis, which comprises the cell preparation according to claim 6 as an active ingredient.

Technical Field

The invention relates to cell preparations for treating allergic rhinitis, which are human umbilical cord Volton mesenchymal stem cell preparations, and a preparation method and application thereof.

Background

Allergic Rhinitis (AR), also known as allergic rhinitis, is a common disease of the ear, nose, throat and respiratory tract, and is an allergic disease of the nasal mucosa, which is mainly characterized by itching, sneezing, overflowing and clear nasal discharge and swelling of the nasal mucosa. the prevalence rate of allergic rhinitis is 10% -40%, wherein pollen allergy is commonly seen in Europe and North America, and perennial allergic rhinitis is commonly seen in Asia. although allergic rhinitis is not fatal, the learning and work of patients are affected due to significant discomfort of the nose and the whole body of patients, and if not treated correctly, about 30% of patients develop bronchial asthma, even pulmonary heart disease and other diseases seriously affecting the physical health and the quality of life of patients.

①, AR is an IgE-mediated allergic inflammatory reaction occurring in the nasal cavity, and is a biochemical and cellular cascade reaction caused by the allergen in allergic constitutional individuals, wherein the reaction is caused by the interaction between infiltrated inflammatory cells and normal cells ② family inheritance, AR patients are reported to be mostly specific allergic constitutions, allergic rhinitis patients with family other allergic disease history account for about 60%, the probability of the disease occurring in children of parents and children if both parents and children have AR offspring is 75% -100%, ③ is related to asthma, AR and asthma belong to respiratory tract chronic allergic diseases occurring in different parts, and 40% of allergic rhinitis patients can develop into asthma if effective treatment is not achieved.

Allergic rhinitis is an allergic inflammatory reaction mainly caused by immune reaction of nasal mucosa, which is mediated by IgE under the action of in vitro environmental factors.

Disclosure of Invention

The invention aims to provide cell preparations for treating allergic rhinitis.

The invention firstly protects the application of the mesenchymal stem cells in preparing the medicament for treating allergic rhinitis.

The mesenchymal stem cells are umbilical cord mesenchymal stem cells. The umbilical cord mesenchymal stem cells are prepared by taking umbilical cord Wolton area tissue blocks as raw materials. The umbilical cord Wolton tissue block is used as a raw material to prepare the umbilical cord mesenchymal stem cells, and a low serum culture medium is adopted in the process. The umbilical cord is an in vitro umbilical cord. The umbilical cord mesenchymal stem cells can be primary umbilical cord mesenchymal stem cells and also can be umbilical cord mesenchymal stem cells after passage. The umbilical cord mesenchymal stem cells after passage can be umbilical cord mesenchymal stem cells within 20 passages, specifically umbilical cord mesenchymal stem cells within 10 passages, and more specifically umbilical cord mesenchymal stem cells within 3-5 passages.

The invention also provides a preparation method of the cell preparations, which comprises the following steps of preparing umbilical cord mesenchymal stem cells by taking the umbilical cord Wolton area tissue blocks as raw materials to obtain the cell preparation, wherein a low-serum culture medium is adopted in the preparation process.

The umbilical cord mesenchymal stem cells can be primary umbilical cord mesenchymal stem cells and also can be umbilical cord mesenchymal stem cells after passage.

The preparation method of the primary umbilical cord mesenchymal stem cells sequentially comprises the following steps:

(1) culturing the umbilical cord Wolton area tissue block by adopting a low-serum culture medium until cells climb out;

(2) culturing the cells by adopting a low serum culture medium;

(3) collecting cells, digesting with pancreatin, and collecting the cells, namely the primary umbilical cord mesenchymal cells.

The umbilical cord mesenchymal stem cells after passage can be umbilical cord mesenchymal stem cells within 20 passages, specifically umbilical cord mesenchymal stem cells within 10 passages, and more specifically umbilical cord mesenchymal stem cells within 3-5 passages.

The passage method specifically comprises the following steps: umbilical cord mesenchymal cells 1: and 2, passage, adopting a low serum culture medium to culture cells, collecting the cells, digesting the cells by pancreatin, and collecting the cells.

The invention also provides a preparation method of the cell preparations, which comprises the following steps:

(1) culturing isolated umbilical cord Wolton zone tissue blocks by adopting a low-serum culture medium for 7-10 days;

(2) after the step (1) is completed, removing tissue blocks, and culturing cells by adopting a low serum culture medium to reach 80% confluency;

(3) after the step (2) is finished, collecting cells, digesting the cells by pancreatin, and collecting the cells, namely the P0 generation umbilical cord mesenchymal cells;

(4) generation of P0 umbilical cord mesenchymal cell 1: 2, passage, and culturing by adopting a low serum culture medium to reach 80% confluence;

(5) after the step (4) is finished, collecting cells, digesting the cells by pancreatin, and collecting the cells, namely the P1 generation umbilical cord mesenchymal cells;

(6) generation of P1 umbilical cord mesenchymal cell 1: 2, passage, and culturing by adopting a low serum culture medium to reach 80% confluence;

(7) after the step (6) is finished, collecting cells, digesting the cells by pancreatin, and collecting the cells, namely the P2 generation umbilical cord mesenchymal cells;

(8) generation of P2 umbilical cord mesenchymal cell 1: 2, passage, and culturing by adopting a low serum culture medium to reach 80% confluence;

(9) and (5) after the step (8) is finished, collecting cells, digesting by pancreatin, and collecting the cells to obtain the cell preparation.

The cell preparation prepared by any of the methods above also belongs to the protection scope of the invention.

The cell preparation of any is a cell preparation positive to CD126, the positive rate of CD126 is more than 95%, preferably more than 99%, the cell preparation of any is a cell preparation with the positive rates of CD29, CD44, CD90 and CD105 being more than 90%, the positive rates of CD29, CD44, CD90 and CD105 being preferably more than 95%, and further being preferably more than 99%, the positive rates of CD31 and CD34 of the cell preparation of any being less than 10%, preferably less than 7%, the positive rates of CD45 and HLA-DR of the cell preparation of any being less than 5%, preferably less than 3%, and further being preferably less than 1%, the positive rates of CD44, CD90 and CD105 being surface mesenchymal markers for identifying mesenchymal stem cells, the positive rates of CD29, CD29 and CD 36105 being high purity indicating that the cell preparation is mesenchymal stem cells and the CD29, a high purity index indicating that the CD29, a high purity index of the CD-29, a CD-29 and a HLA-29, a marker for transplanting index for transplanting a low HLA-29, and a CD-29-related marker for transplanting a CD-related marker for transplanting the three cell preparation for transplanting a.

The invention also protects the application of the cell preparation in preparing a medicament for treating allergic rhinitis.

The invention also protects medicines for treating allergic rhinitis, and the active ingredient of the medicines is mesenchymal cells.

The mesenchymal stem cells are umbilical cord mesenchymal stem cells. The umbilical cord mesenchymal stem cells are prepared by taking umbilical cord Wolton area tissue blocks as raw materials. The umbilical cord Wolton tissue block is used as a raw material to prepare the umbilical cord mesenchymal stem cells, and a low serum culture medium is adopted in the process. The umbilical cord is an in vitro umbilical cord. The umbilical cord mesenchymal stem cells can be primary umbilical cord mesenchymal stem cells and also can be umbilical cord mesenchymal stem cells after passage. The umbilical cord mesenchymal stem cells after passage can be umbilical cord mesenchymal stem cells within 20 passages, specifically umbilical cord mesenchymal stem cells within 10 passages, and more specifically umbilical cord mesenchymal stem cells within 3-5 passages.

The invention also protects medicines for treating allergic rhinitis, and the active ingredients of the medicines are the cell preparation.

The low serum culture medium of any above is a cell culture medium containing 3-5% (by volume) serum.

The low serum medium described in any of above comprises human epithelial growth factor 10ng/ml, human basic fibroblast growth factor 10ng/ml, recombinant human insulin-like growth factor 5. mu.g/ml, platelet-derived factor 10ng/ml, heparin 5. mu.g/ml, hydrocortisone 1. mu.g/ml, ascorbic acid 10. mu.g/ml, non-essential amino acid solution 1% (volume percent), L-glutamine 2mmol/L, fetal calf serum 3-5% (volume percent), penicillin 50U/ml, streptomycin 50. mu.g/ml, and the balance DMEM high glucose medium.

The non-essential amino Acid solution contained 10.0mM of Glycine, 10.0mM of L-Alanine, 10.0mM of L-Asparagine, 10.0mM of L-Aspartic Acid, 10.0mM of L-Glutamic Acid, 10.0mM of L-Proline and 10.0mM of L-Serine.

In the aspect of mesenchymal stem cell culture, 10 percent (volume percent) of fetal bovine serum is generally used, the serum concentration is as high as 20 percent (volume percent), the serum contains various plasma proteins, polypeptides, carbohydrates, growth factors, hormones and the like, the serum components are complex, and the difference between each batch of serum cannot ensure , in addition, although the serum contains a plurality of components beneficial to cell growth, components harmful to cells, such as complement, antibodies, endotoxin and the like, are inevitably contained, therefore, the cells cultured by the high-concentration serum are not suitable for clinical application, and the risk of clinical allergy is increased.

Mesenchymal cells commonly used for immunotherapy include bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose-derived mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, and the like. Compared with the common Mesenchymal Stem Cells (MSCs), the mesenchymal stem cells in the umbilical cord Wton interval have the advantages of abundant sources, no influence on donors, easy collection and transportation, low carcinogenic possibility, low virus pollution probability, weak immunogenicity, no social, ethical and legal disputes and the like. More importantly, the mesenchymal stem cells separated from the umbilical cord Wolton region have high content, higher proliferation capacity than bone marrow MSCs and lower immunogenicity than the bone marrow MSCs.

The cellular therapy is to regulate the immune balance of the patient from the perspective of cellular and humoral immunity by directly affecting the immune system of the patient, thereby achieving the goal of controlling allergic symptoms. The cell preparation provided by the invention is suitable for cell therapy of allergic rhinitis. The invention has great application value for treating allergic rhinitis.

Drawings

Fig. 1 is the results of markers (CD29, CD44, CD90, CD105, and CD126) for mesenchymal stem cells.

FIG. 2 shows the results of hematopoietic stem cell and endothelial cell markers (CD31, CD45, and CD 34).

FIG. 3 shows the results of the transplantation rejection-associated marker (HLA-DR).

FIG. 4 shows the results of phenotypic observations.

Fig. 5 shows the results of the pathological examination of the nasal mucosa.

FIG. 6 shows the results of IFN-. gamma.detection, IL-4 detection, IL-17 detection, and TNF- α detection.

FIG. 7 shows the results of IgE and histamine detection.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Low serum medium: 10ng/ml of human epithelial growth factor, 10ng/ml of human basic fibroblast growth factor, 5 mu g/ml of recombinant human insulin-like growth factor, 10ng/ml of platelet-derived factor, 5 mu g/ml of heparin, 1 mu g/ml of hydrocortisone, 10 mu g/ml of ascorbic acid, 1% (volume percent) of non-essential amino acid solution, 2mmol/L of L-glutamine, 4% (volume percent) of fetal bovine serum, 50U/ml of penicillin, 50 mu g/ml of streptomycin and the balance of DMEM high-sugar medium.

Human epithelial growth factor (hEGF): GenScript, EGF Recombinant human protein, Cat.Z 00333.

Human basic fibroblast growth factor (b-FGF): GenScript, Inc., cat # Z03116.

Recombinant human insulin-like growth factor (IGF): GenScript, Cat number Z03017.

Platelet derived factor (PDGF): GenScript, Inc., cat # Z02529.

Egg protein: sigma, cat # S7951.

Heparin: michael corporation. Hydrocortisone: michael corporation. Ascorbic acid: michael corporation. Penicillin: gibco Corp. Streptomycin: gibco Corp.

The non-essential amino acid solution is Gibco^TMMEM Non-Essential Amino Acids Solution, (100X), cat # 11140050. The website address https:// www.thermofisher.com/cn/zh/home/technical-resources/media-formulation.165. html. The product contains seven amino acids, and comprises the following components: 10.0mM of Glycine, 10.0mM of L-Alanine, 10.0mM of L-Asparagine, 10.0mM of L-Aspartic acid, 10.0mM of L-Glutamine, 10.0mM of L-Proline and 10.0mM of L-Serine.

SD rat: beijing Wintonlihua, Inc.