CN113827617A - Application of airway basal layer stem cells in benign airway stenosis treatment - Google Patents
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- CN113827617A CN113827617A CN202110713339.6A CN202110713339A CN113827617A CN 113827617 A CN113827617 A CN 113827617A CN 202110713339 A CN202110713339 A CN 202110713339A CN 113827617 A CN113827617 A CN 113827617A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/42—Respiratory system, e.g. lungs, bronchi or lung cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
- A61L27/3882—Hollow organs, e.g. bladder, esophagus, urether, uterus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/22—Materials or treatment for tissue regeneration for reconstruction of hollow organs, e.g. bladder, esophagus, urether, uterus
Abstract
The invention relates to the use of airway basal layer stem cells in the treatment of benign airway stenosis. The research of the invention shows that the airway basal layer stem cells can be used for reconstructing normal airway epithelium on the surface of the airway stenosis section and inhibiting the proliferation of granulation tissue of the stenosis section, so that the treatment method of firstly removing granulation tissue of the stenosis section and then transplanting the airway basal layer stem cells is adopted, the tracheal stenosis can be remarkably treated, the recurrence of stenosis after conventional treatment is effectively avoided, the tracheal stenosis tends to be stable, and the quality of life of a patient is greatly improved.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of airway basal layer stem cells in benign airway stenosis treatment.
Background
Benign airway stenosis is a common disease in interventional respiratory pathology, is mostly caused by tuberculosis or tracheal intubation, takes obvious granulation tissue hyperplasia as a main pathological change, seriously influences the respiratory function of a patient and can cause asphyxia. The current mainstream treatment method is surgical treatment or bronchoscopic intervention treatment.
The parallel end-to-end anastomosis of the excised narrow section is a conventional treatment means for benign airway stenosis, has obvious curative effect, can completely recover the smoothness of the tracheal lumen, but has higher requirement on the general condition of the body of a patient in surgical treatment, and has limitation on the length of the narrow section, the stenosis of more than 5cm can not be excised through the surgical treatment because of too high tension of an anastomosis opening, and only a small part of patients can tolerate the surgical treatment. On the other hand, although the surgical treatment has few complications, the significant granulation tissue hyperplasia still appears in part of patients at the anastomotic site, which leads to the occurrence of restenosis.
Bronchoscope interventional therapy mainly clears away narrow section granulation tissue through modes such as sacculus expansion, high frequency electrotome, laser ablation in order to reach the unobstructed effect of recovery lumen, is main treatment means to the benign narrow patient of air flue who can't accept the operation. The intervention treatment has obvious short-term curative effect, but cannot inhibit granulation tissue hyperplasia, restenosis occurs within weeks or months, and patients often need to receive bronchoscopic intervention regularly to maintain the diameter of the narrow-section trachea.
Generally, both surgical treatment and bronchoscopic interventional treatment are limited in many ways, and do not effectively treat benign airway stenosis.
The airway epithelium has the function of inhibiting proliferation and differentiation of fibroblasts while protecting the submucosal tissue from external stimulation. The airway basal layer cells are taken as stem cells which can be differentiated into various airway epithelial cells, and are widely concerned by tissue engineering, researchers inoculate autologous chondrocytes and autologous respiratory epithelial cells obtained by inducing differentiation of bone marrow mesenchymal stem cells into a decellularized scaffold in a bioreactor, and transplant the decellularized scaffold into a patient body to replace the left main bronchus softened by the patient. According to the report of the literature (DOI: 10.29235/1814-. But in the long term, mesenchymal stem cells are mainly relied on for their anti-inflammatory and growth-promoting effects, since they cannot form epithelial tissues. Therefore, the benign airway stenosis cannot be radically treated by using the mesenchymal stem cells, and the disease is always relapsed.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a long-acting stem cell therapy for benign airway stenosis. The treatment method firstly removes granulation tissues at the narrow section, and then transplants the airway basal layer stem cells, so that normal airway epithelium can be reconstructed on the surface of the narrow section of the airway, the proliferation of the granulation tissues at the narrow section is inhibited, the recurrence of stenosis after conventional treatment is effectively avoided, the tracheal stenosis tends to be stable, and the life quality of patients is greatly improved.
The invention aims to provide application of airway basal layer stem cells in treatment of benign airway stenosis.
The invention also aims to provide application of the airway basal layer stem cells in preparing stem cell medicines for treating benign airway stenosis.
It is another object of the present invention to provide a suspension of airway basal layer stem cells for the treatment of benign airway stenosis.
In order to achieve the purpose, the invention provides the following technical scheme:
no researchers have proposed a viable approach to the treatment of benign airway stenosis through regenerative medicine, and the feasibility and importance of reconstructing epithelial tissue to treat airway stenosis has not been appreciated by the medical community. However, through the research of the team, the airway basal layer stem cells are determined to be capable of effectively treating benign airway stenosis, and the scheme for treating the benign airway stenosis by adopting the airway basal layer stem cells is determined for the first time.
The invention therefore claims:
use of airway basal layer stem cells in the treatment of benign airway stenosis.
Application of airway basal layer stem cells in preparing stem cell medicines for treating benign airway stenosis.
Preferably, the airway basal layer stem cells are autologous.
More preferably, the airway basal layer stem cells are taken from the airway narrowing grade 2-3 bronchial epithelium.
An airway basal layer stem cell suspension for treating benign airway stenosis, wherein the cell density of the stem cell suspension is 400-600 ten thousand per mL.
Wherein, preferably, the cell density of the stem cell suspension is 500 ten thousand/mL.
Wherein, preferably, the dispersion medium of the stem cell suspension is physiological saline or phosphate buffer solution.
Wherein, preferably, the pH value of the phosphate buffer solution is 6-8.
Preferably, the stem cell suspension contains 1-5% (w/v) of human serum albumin, and more preferably 3% (w/v) of human serum albumin.
The airway basal layer stem cell suspension for treating benign airway stenosis can be applied to preparation of stem cell medicines for treating benign airway stenosis.
The invention has the following beneficial effects:
the invention provides a feasible method for treating benign airway stenosis through regenerative medicine, which combines the removal of granulation tissue in a narrow section with airway basal layer stem cell transplantation for treatment:
(1) provides an applicable stem cell for stem cell treatment of airway stenosis.
(2) Can rebuild normal airway epithelium on the surface of airway stenosis section.
(3) Can effectively reduce the restenosis of benign airway stenosis and stabilize airway granulation.
(4) Improving the life quality of the patients.
Drawings
Figure 1 shows the morphology of airway basal layer stem cells (light mirror).
FIG. 2 shows the immunofluorescence of airway basal layer stem cells to identify the expression of KRT 5/P63.
Fig. 3 shows the trachea after model canines from the placebo group were modeled.
Fig. 4 shows the treatment results of the first model dog in the experimental group for treating tracheal injury by autologous airway basal layer stem cells.
Fig. 5 shows the treatment results of the second model dog in the experimental group for treating the tracheal injury by the autologous airway basal layer stem cells.
Fig. 6 shows the treatment results of the third model dog in the experimental group for treating the tracheal injury by the autologous airway basal layer stem cells.
Fig. 7 shows the treatment results of BMSC control group model dogs for tracheal injury via treatment with mesenchymal autologous bone marrow mesenchymal stem cells.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 airway stenosis model establishment
Randomly selecting 9 healthy adult beagle dogs, establishing a benign airway stenosis model after intubation in a mode of pressing the beagle dog airway for 24 hours by a tracheal intubation saccule, and successfully modeling after examination and verification.
Successfully constructed models were randomly divided into 3 groups (experimental, blank and BMSC control), with 3 of each group.
Example 2 extraction, isolation, identification and culture of airway basal layer Stem cells
Using a cell brush to brush 2-3 grade bronchial epithelium of the stricture of the airway of a beagle dog (experimental beagle dog) under the guidance of a bronchoscope in advance for 2 weeks to obtain autologous airway basal layer stem cells;
the cultured Cells were identified by expanding the primary Cells obtained by brushing according to the culture method described in the literature (Rapid Expansion of Human epidermal Stem Cells capable of being cultured for air Tissue Engineering) and detecting the expression of the Cells krt5/p63 by immunofluorescence technique.
As a result: the morphology of the cells obtained by the culture is shown in figure 1, the immunofluorescence technology shows that the krt5/p63 is expressed positively and is shown in figure 2, and the cells are cultured to be the airway basal layer stem cells.
EXAMPLE 3 treatment of airway stenosis with airway basal layer Stem cells
A great amount of necrotic substances at the injured part can be seen 3 days after the beagle is modeled, severe tracheal stenosis appears 7 days later, and the beagle is treated by placing a metal bracket conventionally.
(1) Removing granulation tissue of the model dog in the narrow section by a conventional treatment method 3 days before cell transplantation, and measuring the length of the narrow section of the model dog;
(2) stem cell preparation:
experimental groups: on the day of cell transplantation, according to the proportion of transplanting 500 ten thousand cells per 1 centimeter of the length of a narrow section, digesting the required airway basal layer stem cells, using PBS to resuspend the cells into a cell suspension with the concentration of about 500 ten thousand/mL, and adding human serum albumin until the mass concentration is 3%;
BMSC control group: on the day of cell transplantation, according to the proportion of 500 ten thousand cells transplanted per 1 cm of the length of the narrow section, the required umbilical cord mesenchymal stem cells (derived from a BMSC control group beagle dog) are digested and resuspended into a cell suspension with the concentration of about 500 ten thousand/mL by using PBS, and human serum albumin is added to the mass concentration of 3%;
(3) before cell transplantation, the endocrine in the airway is completely sucked through a bronchoscope;
(4) cutting off a nozzle at the end of the spray tube head, and sending the spray tube into the trachea through a bronchoscope;
(5) cell transplantation:
experimental group model dogs: sucking the stem cell suspension of the autologous airway basal layer obtained in the example 2 by using a syringe, slowly pushing the stem cell suspension through a spraying tube, and uniformly coating the stem cell suspension on the surface of a narrow section;
BMSC control group model dogs: sucking the umbilical cord mesenchymal stem cell suspension by using a syringe, slowly pushing the umbilical cord mesenchymal stem cell suspension through a spraying tube, and uniformly coating the umbilical cord mesenchymal stem cell suspension on the surface of a narrow section;
blank control group model dogs: sucking equivalent PBS containing 3% human serum albumin by using an injector, slowly pushing the PBS through a spray tube, and uniformly coating the PBS on the surface of a narrow section;
(6) repeating the steps (1) to (5) two to three times at intervals of 1 week.
As a result: as shown in fig. 3, the control group model dog is expressed after modeling, after the metal stent is placed for treatment, the trachea is recovered to be unobstructed, the diameter of the trachea can be maintained in a short period, but the trachea is restenosis after about one week, and after granulation tissues are removed, the treatment by smearing physiological saline containing 3% human albumin is ineffective, so that lethal tracheal stenosis is formed.
As shown in figures 4-6, the treatment results of 3 model dogs in the experimental group for treating tracheal injury by autologous airway basal layer stem cells are shown, and a metal bracket is placed for treatment to recover the smooth trachea, but the trachea is restenosis after one week. After granulation tissues are removed, airway basal layer stem cells are transplanted, smooth airway epithelium can be seen on the surface of the narrow section after two times of transplantation, and no obvious progress can be made in the narrow section within 1 month.
And because tracheal stenosis that did not recur for 3 months tended to be clinically cured, our animal experiments were also followed for 3 months, and the results showed that the stenotic tissue remained stable for 3 months after successful cell transplantation.
The treatment of the invention thus allows complete stabilization of the stenosed part with the placement of a metal stent and ensures that it does not recur for at least 3 months, with a long period of stabilization being expected.
Meanwhile, a contrast experiment is carried out by replacing airway basal layer stem cells with bone marrow mesenchymal stem cells (BMSC), and as shown in figure 7, no obvious progress is observed in the observed stenosis after one week of umbilical cord mesenchymal stem cell transplantation treatment, but restenosis recurs after two weeks of cell transplantation, and the lumen is severely narrowed. From the results of the experiment, the mesenchymal stem cells can stabilize the narrow tissue only within one to two weeks.
Further specifically stated: the main reason why the metal stent is placed before stem cell treatment is that the current tracheal stenosis modeling means can cause tracheal cartilage destruction in different degrees, and the lumen can be narrowed along with respiration, so that the metal stent needs to be placed for support. On the other hand, however, the metal stent itself promotes the growth of granulation tissue, resulting in the most clinically intractable tracheal stenosis. Therefore, the results of the invention show that under the condition of placing a metal bracket which is a large interference factor, the airway basal layer stem cell transplantation therapy of the invention can still effectively stabilize the narrow tissue, and the curative effect is proved to be excellent.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The application of the airway basal layer stem cells in the treatment of benign airway stenosis or the application in the preparation of medicaments for treating the benign airway stenosis.
2. The use of claim 1, wherein the airway basal layer stem cells are autologous.
3. The use according to claim 1 or 2, wherein the airway basal layer stem cells are taken from the bronchial epithelium level 2-3 of airway stenosis.
4. An airway basal layer stem cell suspension for treating benign airway stenosis is characterized in that the cell density of the stem cell suspension is 400-600 ten thousand/mL.
5. The suspension of airway basal layer stem cells for treating benign airway stenosis of claim 4 wherein the cell density of said suspension of stem cells is 500 ten thousand per mL.
6. The airway basal layer stem cell suspension for treating benign airway stenosis of claim 4, wherein the dispersion medium of the stem cell suspension is normal saline or phosphate buffer.
7. The airway basal layer stem cell suspension for treating benign airway stenosis of claim 6, wherein the pH of the phosphate buffer solution is 6 to 8.
8. The airway basal layer stem cell suspension for treating benign airway stenosis of any one of claims 4 to 7, wherein the stem cell suspension comprises 1% to 5% (w/v) human serum albumin.
9. The airway basal layer stem cell suspension for treating benign airway stenosis of claim 8, wherein the stem cell suspension comprises 3% (w/v) human serum albumin.
10. Use of a suspension of airway basal layer stem cells according to any one of claims 4 to 9 for the treatment of benign airway constriction in the manufacture of a medicament for the treatment of stem cells of benign airway constriction.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114196612A (en) * | 2019-05-16 | 2022-03-18 | 苏州吉美瑞生医学科技有限公司 | Application of bronchial basal layer cells in preparation of medicines for treating COPD |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114196612A (en) * | 2019-05-16 | 2022-03-18 | 苏州吉美瑞生医学科技有限公司 | Application of bronchial basal layer cells in preparation of medicines for treating COPD |
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| CN114196613B (en) * | 2019-05-16 | 2024-03-12 | 苏州吉美瑞生医学科技有限公司 | Application of bronchial basal layer cells in preparation of medicaments for treating bronchiectasis |
| CN114196612B (en) * | 2019-05-16 | 2024-03-12 | 苏州吉美瑞生医学科技有限公司 | Application of bronchial basal layer cells in preparation of medicines for treating COPD |
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