CN111888378A

CN111888378A - Application of adult stem cells derived from neural crest

Info

Publication number: CN111888378A
Application number: CN202010785201.2A
Authority: CN
Inventors: 吴祖泽; 李时悦; 杨月峰; 陈焕杰; 孟虹芳; 罗钰龙; 王�华; 陈迪非; 孙成峰
Original assignee: Beijing Sh Bio Tech Corp; First Affiliated Hospital of Guangzhou Medical University
Current assignee: Beijing Sh Bio Tech Corp; First Affiliated Hospital of Guangzhou Medical University
Priority date: 2020-08-06
Filing date: 2020-08-06
Publication date: 2020-11-06

Abstract

The invention provides an application of adult stem cells derived from neural crest, which is an application of the adult stem cells derived from neural crest in preparing a pharmaceutical preparation for inhibiting and/or treating pulmonary fibrosis and/or improving pulmonary function, and relates to the technical field of cell biology. The dental pulp stem cells are used for treating rats with pulmonary fibrosis, and the results show that the dental pulp stem cells can inhibit and reverse pulmonary fibrosis, improve the lung function without generating adverse effects and side effects, and have good treatment effect.

Description

Application of adult stem cells derived from neural crest

Technical Field

The invention relates to the technical field of cell biology, in particular to application of adult stem cells derived from neural crest in preparation of a pharmaceutical preparation for inhibiting and/or treating pulmonary fibrosis and/or improving pulmonary function.

Background

In the rapid development of society, factors such as environmental pollutants, smoking and pathogen infection also silently damage human lung health, people with lung diseases are enlarged year by year, and idiopathic pulmonary fibrosis, radiation pulmonary fibrosis, silicosis, chronic bronchitis (chronic bronchitis), pneumonia, lung injury and the like gradually become main threats to human health. If the lung of a patient suffering from the above diseases is repeatedly injured and stimulated, the alveolar structure is seriously damaged, fibroblasts are continuously activated and generate excessive collagen fibers to be deposited in the pulmonary interstitium, and finally the lung fibrosis is often developed, so that the ventilation and/or gas diffusion function of the lung is damaged, and the patient shows progressive aggravated dyspnea and respiratory failure, seriously harms the human health and even endangers the life. Although the etiology and pathogenesis of pulmonary fibrosis are not clear at present, the mainstream view is that damaged alveolar epithelium is the initiating link for initiating fibrosis, while the presence of pulmonary interstitial fibrosis foci is a direct factor for causing respiratory dysfunction. Thus, the treatment of pulmonary fibrosis is focused on repairing the damaged alveolar epithelium, inhibiting or reversing the progression of fibrosis. However, at present, there is no specific drug capable of achieving the two purposes at the same time, and novel anti-fibrosis drugs (pirfenidone and nintedanib) can slow down the decline of forced vital capacity of a patient to a certain extent, but still can not inhibit or reverse the progress of pulmonary fibrosis, and have very limited repair effect on pulmonary alveolar epithelium; lung transplantation is difficult to benefit more patients due to the problems of the lack of supply of lung source, strict indication, immunological rejection after operation, etc. More unfortunately, the prognosis of patients with pulmonary fibrosis is poor, and most patients with idiopathic pulmonary fibrosis have a median survival time of only 4.5 years and a 5-year survival rate of only 30% -50% after being diagnosed. It is seen that the search for more effective treatments for patients is very slow.

Disclosure of Invention

In order to solve the problems in the prior art, the present invention aims to provide an application of adult stem cells derived from neural crest in the preparation of a pharmaceutical preparation for inhibiting and/or treating pulmonary fibrosis and/or improving pulmonary function.

The invention is realized by the following technical scheme:

the first purpose of the invention is to provide application of adult stem cells derived from neural crest in preparing a pharmaceutical preparation for inhibiting or treating pulmonary fibrosis and/or improving pulmonary function.

In one embodiment, the neural crest-derived adult stem cell comprises any one of a dorsal root ganglion-derived neural crest stem cell, a skin-derived neural crest stem cell, or a bone marrow neural crest stem cell.

In a specific embodiment, the neural crest-derived adult stem cells are derived from dental-derived stem cells, including one or more of adult orthodontic tooth-derived dental pulp stem cells, deciduous tooth dental pulp stem cells, apical tooth deciduous head stem cells.

The dental pulp stem cell is an adult stem cell which is derived from neural crest and has MSC characteristic, is isolated and identified in 2000, and has stronger proliferation capacity than bone marrow stem cell and stronger anti-inflammatory capacity than umbilical cord stem cell. The dental pulp stem cells are separated from clinically discarded orthodontic teeth, have wide sources and are easy to obtain. The "dental pulp stem cells" in the present invention may be derived from any tooth in the oral cavity, for example, deciduous teeth, wisdom teeth, etc.

In particular embodiments, the symptoms of pulmonary fibrosis include one or more of fibroblast hyperproliferation, thickening of alveolar spaces, increased collagen fiber deposition, elevated TGF- β 1 expression, or dysfunction in lung ventilation and gas diffusion.

In particular embodiments, the improvement in lung function comprises an improvement in lung ventilation function or an improvement in lung gas dispersion function.

In particular embodiments, the improvement in pulmonary ventilation function comprises an improvement in one or more of FEV, PEF, PIF, RI, ERV, FRC indices.

In particular embodiments, the improvement in lung gas dispersion function comprises an improvement in one or more of arterial blood gas analysis indicators SO2, PCO 2.

In an embodiment of the invention, the "improvement" refers to a tendency of the index concerned to shift towards normal or improvement compared to before treatment, or compared to the same batch of experiments with the same dose of placebo-treated model group.

In a specific embodiment, the dose required for the transplantation of dental pulp stem cells is 5 × 10⁶Individual cells/kg.

In a specific embodiment, the dental pulp stem cell is prepared by separating and culturing dental pulp mesenchymal stem cells from dental pulp, performing surface marker detection and osteogenesis and adipogenesis induced differentiation identification to obtain the dental pulp mesenchymal stem cells, and preparing the dental pulp mesenchymal stem cells into a dental pulp stem cell suspension.

In one embodiment, after obtaining the dental pulp stem cells, the method further comprises the step of performing cell counting and cell activity detection on the obtained stem cells, so that the activity and growth vigor of the adopted dental pulp stem cells are good, and the method is suitable for clinical application.

Another object of the present invention is to provide a pharmaceutical preparation for inhibiting or treating pulmonary fibrosis and/or improving pulmonary function, which comprises a therapeutically effective amount of the neural crest-derived adult stem cells of the present invention.

The pharmaceutical preparation is not particularly limited in form, and may be in various forms such as solid, liquid, gel, semifluid, aerosol, etc., preferably spray.

In a particular embodiment, the pharmaceutical formulation is administered intratracheally.

In other embodiments, the pharmaceutical formulation may include one or more pharmaceutically acceptable carriers, as needed for the dosage form.

The pharmaceutical preparation is mainly aimed at mammals, such as rodents, primates and the like.

In some embodiments, the dental pulp stem cells of the present invention may be administered simultaneously or sequentially with a therapeutically effective amount of another agent for inhibiting or treating pulmonary fibrosis.

In one embodiment, the administration is by direct administration of the dental pulp stem cells to the subject.

In one embodiment, the mode of administration is injection, preferably infusion.

Has the advantages that:

mesenchymal Stem Cells (MSCs) first home to the lungs after intravenous injection, which provides a basis for MSCs to treat lung disease. MSC can protect and repair injury and inhibit inflammatory reaction by means of intercellular direct contact, secretion of cytokines, vesicles, exosomes and the like; can improve wear of telomeres in damaged and aged cells, supplement exhausted stem cells, regulate aging-related molecules, relieve oxidative stress, and improve apoptosis and aging.

The invention tries to treat lung diseases by using the adult stem cells derived from nerve ridges with the characteristics of MSC so as to achieve the effects of inhibiting and reversing pulmonary fibrosis and improving lung functions. The inventor finds a new application of the neural crest-derived stem cells through experiments, namely a new application of inhibiting, reversing pulmonary fibrosis and/or improving pulmonary function in pulmonary diseases.

Experiments prove that in an animal model, the dental pulp stem cells can obviously inhibit the level of TGF-beta 1 in pulmonary fibrosis cells; reducing collagen fiber deposition, and improving lung function index of FEV, PEF, PIF, RI, ERV and FRC; reducing albumin content and improving SO2 and PCO₂And the pulmonary pathological changes of the pulmonary fibrosis are relieved, and the pulmonary function is improved.

One of the advantages of the dental pulp stem cells for inhibiting and reversing pulmonary fibrosis is that the dental pulp stem cells have low immunogenicity and are suitable for a wide range of people. The neural crest-derived stem cells of the present invention, particularly dental pulp stem cells, can be used for the prevention and/or treatment of pulmonary diseases including idiopathic pulmonary fibrosis, radiation pulmonary fibrosis, silicosis, chronic bronchitis, pneumonia, lung injury, and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts. The instruments and reagents and materials used in the present invention are well known to those skilled in the art and are commercially available from commercial establishments.

FIG. 1 is the measure of the TGF-beta 1 content in lung tissue of a rat with pulmonary fibrosis according to an embodiment of the present invention after treatment;

FIG. 2 is a masson staining analysis of lung tissue sections after treatment of rats with pulmonary fibrosis according to the embodiment of the present invention;

FIG. 3 is the lung function test result of the treated rats with pulmonary fibrosis according to the embodiment of the present invention; wherein, FEV 200: expiration volume of 200 milliseconds; FEV 300: a 300 millisecond expiratory volume; FEV 400: a 400 millisecond expiratory volume; PIF: a peak inspiration value; rl: airway resistance; ERV: the amount of supplemental exhalation; FRC: functional residual capacity.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Examples

Separation and identification of dental pulp stem cells

1. Cell source

Under anesthesia, the third molar of the impacted patient is removed aseptically or the tooth is required to be removed in the orthodontic treatment, the dental pulp tissue is taken after the tooth is split into the crown, and the dental pulp tissue is repeatedly cleaned by PBS and cut into pieces.

2. Isolation and culture of dental pulp stem cells

Placing the dental pulp tissue in a solution containing 3mg/ml type I collagenase and 4mg/ml Dispase, digesting the dental pulp tissue in water bath at 37 ℃ for 0.5 to 1 hour, collecting cells through a 70-micron cell sieve, centrifuging the cell sieve at 1000rpm for 10min, and resuspending the cell pulp tissue by using a proper amount of culture medium. Inoculating into 10cm culture dish, culturing in alpha-MEM medium (containing 10% fetal calf serum) at 37 deg.C and 5% CO₂Culturing, and changing the culture solution every 3-5 days. The growth of the cells was observed daily under an inverted microscope. After 1-2 weeks, the passages were digested with 0.25% trypsin. Obtaining dental pulp stem cells, namely odontogenic stem cells.

3. Immunophenotyping detection of dental pulp stem cells and identification of osteogenic induced differentiation of adipogenic bone

Immunophenotyping dental pulp stem cells: cells were labeled with FITC or PE labeled CD73, CD90, CD105, CD11b, CD19, CD34, CD45, HLA-DR antibody and corresponding isotype control antibody and detected by flow cytometry. Carrying out osteogenic induced differentiation on dental pulp stem cells by adipogenesis: identifying by alizarin red staining after osteogenesis is induced for 3 weeks; identification was performed by staining with oil Red O (oil Red O) after 2 weeks of adipogenic induction.

4. Results

The obtained dental pulp stem cells grow fusiform, iron wall and spiral; high expression of CD73, CD90 and CD105, positive cell rate not less than 95.0%, no expression of hematopoietic and immune cell surface markers CD11b, CD19, CD34 and CD45, no expression of surface marker HLA-DR related to transplant immune rejection, and no expression of marker positive cell rate not more than 2.0%; after adipogenic induction, obvious fat drop formation can be seen in oil red O staining cells; alizarin red stained positive after osteogenic induction. Indicating that the cells obtained by separation are dental pulp stem cells.

Second, rat fibro-pulmonary fibrosis model construction

A6-8 week SD rat is anesthetized by intraperitoneal injection of 4% sodium pentobarbital (1.0mL/kg), fixed in a supine position, subjected to aseptic operation, and then exposed to the trachea through an incision in the lower neck, and the space between two cartilage rings is punctured by a syringe needle. The sham operation group is given physiological saline with the same volume, and the other groups are respectively injected with bleomycin 1.0ml/kg (5mg/kg) and injected into the trachea at one time. The needle head is immediately pulled out, the rat board is erected, the rat is kept in an upright position, the rat board is rotated back and forth for 1-2 min, and the liquid medicine reaches the lungs on the two sides as far as possible and is uniformly distributed.

Bleomycin (BLM) is an inducer of animal pulmonary fibrosis models, can damage lungs and cause pulmonary fibrosis, and is administered intratracheally to establish rat pulmonary fibrosis models.

Thirdly, the administration method comprises the following steps:

one week after rat fibropulmonary fibrosis model construction surgery, divided dosing, 7 per group:

the sham operation group: physiological saline (0.4ml) was injected in one portion.

The model group is a model control group (model): disposable injection normal saline (0.4ml)

Positive treatment group (pirfenidone): pirfenidone is administered orally at 1ml/100g per day for two weeks.

Cell treatment groups (ADMSC, DPSC and UCMSC cell treatment groups), wherein the ADMSC refers to adipose mesenchymal stem cells, the DPSC refers to dental pulp stem cells, and the UCMSC refers to umbilical cord mesenchymal stem cells. ADMSC, DPSC and UCMSC cell treatment groups are respectively injected with respective cells at one time by 2 multiplied by 10⁶Cell/cell (0.4 ml).

After administration, the behavior and weight change of the mice were noted every day, the behavior changes such as diet and sleep of the mice were recorded, and the weights of the mice in each group were measured every day at regular intervals and recorded and counted.

Two weeks after dosing, all experimental rats survived with 100% survival for each group. The content of lung tissue TGF-beta 1 is measured by ELISA for all rats, lung tissue section massson staining analysis is carried out, lung function is measured, alveolar flushing fluid ALB content is measured by ELISA, and arterial blood gas is analyzed.

Fourthly, analyzing results:

1. determination of the TGF-. beta.1 content of rat Lung tissue by ELISA

The TGF-beta 1 detection method comprises the following steps:

(1) to 471ul of reagent dilution, 25ul of specimen was added.

(2) Add 10ul 1N HCl, cover tightly, mix up and down. Standing at 2-8 deg.C for 60 + -2 min.

(3) Add 10ul of 1N NaOH, cover tightly, mix up and down. (total volume 500ul i.e. 20 fold dilution).

(4) The panels required for the test were removed from the sealed bags which had been equilibrated to room temperature.

(5) Blank holes (if a dual wavelength read plate is used, blank holes may not be provided).

(6) The activated specimen or standard substance with different concentration (0pg/ml well added with reagent diluent) is added into the corresponding well (100 ul/well), the reaction well is sealed by sealing plate gummed paper, and the incubator is incubated for 90 minutes at 37 ℃.

(7) Biotinylated antibody working solution was prepared 30 minutes earlier.

(8) The plate was washed 5 times.

(9) Biotinylated antibody working solution (100 ul/well) was added in addition to the blank wells. The reaction wells were sealed with a sealing plate of gummed paper and incubated at 37 ℃ for 60 minutes in an incubator.

(10) The enzyme conjugate working solution was prepared 30 minutes in advance. Placing the mixture at room temperature in dark.

(11) The plate was washed 5 times.

(12) Except for blank wells, add enzyme conjugate working solution (100 ul/well). The reaction wells were sealed with a sealing plate gummed paper, incubated at 37 ℃ for 30 minutes in the dark.

(13) The plate was washed 5 times.

(14) Adding 100ul of chromogenic substrate (including blank holes) per hole, incubating at 37 ℃, and incubating for 10-15 minutes in a dark place.

(15) Stop solution (including blank wells) was added at 100 ul/well and OD450 values were measured immediately after mixing (within 10 min).

The results of the measurement by ELISA are shown in FIG. 1, and show that the TGF-. beta.1 level of the model control group is increased compared to that of the sham operation group. The pirfenidone, DPSC, and ADMSC groups showed a reduction in TGF- β 1 compared to the model control group, with a slightly greater reduction in the positive treatment pirfenidone and DPSC groups. TGF-beta 1 is a pro-fibrosis factor, is one of the initiating factors participating in the progress of pulmonary fibrosis, and can be used as an index for evaluating the severity of pulmonary fibrosis, so that the DPSC is considered to relieve the severity of pulmonary fibrosis.

2. Masson staining of rat pulmonary tissue

Rats were sacrificed on day 21 (14 days post-dose) and one lung tissue was collected and stained by Masson (Masson staining kit) as shown in fig. 2.

Masson staining of lung tissue for each group showed:

no obvious pathological changes were observed in the sham operated group.

In the model control group, the alveolar spaces were significantly thickened and a large amount of blue collagen fiber deposition occurred.

In the four treatment groups, the alveolar septal thickening of the pirfenidone group is improved, the excessive deposition of the collagen fibers is not obviously improved, and the alveolar septal thickening and the excessive deposition of the collagen fibers of the three cell treatment groups are improved, wherein the excessive deposition of the collagen fibers of the DPSC group is most obviously improved. Indicating that DPSC can reduce the degree of pulmonary fibrosis.

3. Determination of pulmonary function results

The lung function index of the rat is measured by the lung function detector, the result is shown in fig. 3, compared with the sham operation group, the model control group has certain lung function damage, FEV (maximum expiratory volume of forced expiration), PEF (maximum expiratory flow rate of forced expiration after deep expiration), PIF (peak flow) decrease and RI (airway resistance), ERV (respiratory supplement volume) and FRC (functional residual capacity) increase, compared with the model control group, each treatment group has the best improvement degree of DPSC, the indexes show different improvements, UCMSC, ADMSC and positive drug pirfenidone. Indicating that DPSCs can improve lung function.

4. Rat alveolar lavage fluid ALB (albumin) assay

Alveolar lavage fluid step: fixing the rat with the collected blood, inserting one section of the butterfly needle into the organ of the rat, connecting the other end of the butterfly needle with a syringe, flushing the alveolus with 5mL of physiological saline, sucking 3mL of lavage fluid, centrifuging, retaining the supernatant, and determining the ALB content by an enzyme immunoassay method.

As shown in Table 1, the ALB content of albumin in the lavage fluid in the model group was increased compared with that in the sham operation group, and the improvement of the albumin ALB in the lavage fluid in the model group was different, wherein the improvement of the DPSC is most significant and better than that in the positive drug phenidone group. Indicating that the DPSC can improve the inflammation and the damage condition of the pulmonary capillary vessel.

TABLE 1 Effect on alveolar lavage fluid ALB

^*p＜0.05，^**p < 0.01 in comparison with model groups

5. Arterial blood and qi analysis results

On day 21 (14 days after treatment), 0.3ml of blood was drawn from the abdominal aorta and analyzed by a blood gas analyzer for each group of rats. Arterial blood gas analysis results as shown in table 2, the model group SO2 (percentage of hemoglobin saturated with oxygen) was reduced and each treatment group was improved compared to the sham group, with the most significant improvement in DPSC; compared with the sham group, the model group had increased PCO2 (partial pressure of dissolved carbon dioxide alone in plasma), and the positive drug treatment did not improve compared with the phenidone group, and the other three cell treatment groups improved, with the most significant improvement in DPSC. Indicating that DPSC can improve the respiratory function of the lung.

TABLE 2 Effect on sO2 (%) and PCO2 in rat arterial blood

^*p＜0.05，^**p < 0.01 in comparison with model groups

The results show that the dental pulp stem cells adopted by the invention can improve the lung function and inhibit and reverse pulmonary fibrosis, do not produce adverse effects and side effects, and realize the treatment purpose. The dental pulp stem cells have important application prospects in repairing lung injury, improving lung function and inhibiting pulmonary fibrosis.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of the adult stem cells derived from neural crest is the application of the adult stem cells derived from neural crest in the preparation of pharmaceutical preparations for inhibiting and/or treating pulmonary fibrosis and/or improving lung function.

2. The use of claim 1, wherein said neural crest-derived adult stem cells comprise any of dorsal root ganglion-derived neural crest stem cells, skin-derived neural crest stem cells, or bone marrow neural crest stem cells.

3. The use of claim 1, wherein the neural crest-derived adult stem cells are derived from dental-derived stem cells, including one or more of adult orthodontic tooth-derived dental pulp stem cells, deciduous tooth dental pulp stem cells, apical tooth papilla stem cells.

4. The use of claim 1, wherein the symptoms of pulmonary fibrosis include one or more of fibroblast hyperproliferation, thickening of alveolar spaces, increased deposition of collagen fibers, elevated expression of TGF- β 1, or ventilatory dysfunction.

5. Use according to claim 1, wherein the improvement of lung function comprises improvement of lung ventilation function or improvement of lung gas dispersion function.

6. The use of claim 5, wherein the improvement in lung ventilation function comprises an improvement in one or more of FEV, PEF, PIF, RI, ERV, FRC indices.

7. Use according to claim 5, wherein the improvement in lung gas dispersion function comprises an improvement in one or more of arterial blood gas analysis indicators SO2, PCO 2.

8. The use according to claim 3, wherein the dental pulp stem cell is prepared by isolating and culturing dental pulp mesenchymal stem cells from dental pulp, and preparing the dental pulp mesenchymal stem cells into a dental pulp stem cell suspension after surface marker detection and osteogenesis and adipogenesis induced differentiation identification.

9. The use of any one of claims 1-8, wherein the dose required for transplantation of dental pulp stem cells is 5 x 10⁵-1×10⁷One cell/kg body weight.

10. A pharmaceutical preparation for inhibiting or treating pulmonary fibrosis and/or improving pulmonary function, comprising a therapeutically effective amount of the neural crest-derived adult stem cells according to any one of claims 1 to 8.