KR101189655B1 - Composition for treating pulmonary disease comprising mesenchymal stem cell-conditioned media - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of lung diseases. More specifically, the present invention relates to a composition for treating pulmonary disease consisting of a culture of stem cells, and in particular, the composition of the present invention can effectively treat emphysema caused by smoking, which is useful for treating chronic obstructive disease and pulmonary hypertension. Can be used.

Description

Composition for treating pulmonary disease comprising stem cell culture medium {Composition for treating pulmonary disease comprising mesenchymal stem cell-conditioned media}

The present invention relates to a composition for the prevention and treatment of lung diseases. More specifically, the composition for treating pulmonary disease comprising a culture of stem cells, in particular, can be treated for chronic obstructive pulmonary disease or pulmonary hypertension due to smoking.

Chronic obstructive pulmonary disease (COPD) is a respiratory tract that causes abnormal inflammatory reactions in the lungs by inhalation of harmful particles or gases, which in turn leads to limited airflow, resulting in decreased lung function and respiratory distress. Diseases are accompanied by emphysema, chronic bronchitis, pulmonary hypertension and the like.

Currently, chronic obstructive pulmonary disease (COPD) is considered to be the most important risk factor for smoking, and can also be caused by pollutants and industrial pollutants. The development of chronic obstructive pulmonary disease is thought to involve a variety of etiologies in addition to direct lung injury and inflammatory responses induced by smoking (O'Byrne PM, Postma DS. Am J Respir Crit Care Med 1999; 159: S41). -S66). In particular, a large number of non-smokers develop chronic obstructive pulmonary disease, why only some smokers develop chronic obstructive pulmonary disease, and in patients with chronic obstructive pulmonary disease, airway inflammation and airway epithelial cells The reason why apoptosis persists is still unknown (Hodge S et al., Eur Respir J 2005; 25: 447-54). Therefore, chronic obstructive pulmonary disease has not been known to cause the exact cause and pathogenesis until now, it is known that the underlying treatment is difficult, and the risk of death is known to be high if the disease progresses significantly reduced lung function (GOLD workshop summary. Am J Respir Crit Care Med 2001; 163: 1256-1276).

Bronchodilators and corticosteroids are known to help relieve symptoms of chronic obstructive pulmonary disease. Currently, inhaled forms of corticosteroids, bronchodilators, or theophylline are known to help alleviate the symptoms of chronic obstructive pulmonary disease, but most of the therapeutic drugs that have been developed to cure chronic obstructive pulmonary disease. It does not change its natural course over long periods of time.

Chronic obstructive pulmonary disease is the third-largest disease worldwide by 2020, but the treatment is dependent on symptomatic therapy as described above. Therefore, it is important to reconstruct damaged lung tissue for the fundamental treatment, and research on this is urgently needed.

The present inventors have recognized the above problems and necessities, and have made diligent efforts on methods for regenerating lung tissue to effectively treat chronic obstructive pulmonary disease. As a result, cultures of mesenchymal stem cells caused lung cancer and emphysema caused by smoking. The present invention has been completed by confirming the treatment of hypertension.

Accordingly, an object of the present invention is to provide a composition for the prevention and treatment of chronic obstructive pulmonary disease.

The present invention to solve the above problems provides a composition for the prevention and treatment of chronic obstructive pulmonary disease comprising a culture of stem cells.

The stem cell culture is preferably a culture of mesenchymal stem cells.

In addition, the culture medium is characterized in that obtained by passage 3 to 5 passages mesenchymal stem cells in serum-free medium.

The serum-free medium is preferably DMEM medium.

In addition, the mesenchymal stem cells of the present invention is preferably derived from bone marrow.

The composition may be formulated in the form of an injection or inhalation.

Preferably, the lung disease of the present invention is chronic obstructive pulmonary disease or pulmonary hypertension, and the chronic obstructive pulmonary disease or pulmonary hypertension may be generated by smoking.

The chronic obstructive pulmonary disease includes emphysema or chronic bronchitis.

The composition of the present invention is characterized by regenerating lung tissue caused by smoking to treat emphysema and to treat pulmonary hypertension.

According to the present invention, it is possible to effectively regenerate the lung damaged by smoking using stem cell culture, which can be usefully used as a treatment for chronic obstructive pulmonary disease.

Figure 1A is a result of measuring the number of living cells by MTT assay after exposing lung fibroblasts to cigarette smoke extract (cigarette smoke extract, CSE). Figure 1B is the result of measuring the number of cells in the subG1 phase with a flow cytometer (flow cytometer). Figure 1C is confirmed by TUNEL staining of DNA strands appearing in apoptosis (DNA nick: red, nuclear: blue).
Figure 2 shows the proliferation effect of mesenchymal stem cell-conditioned media (MSC-CM) of the rat lung fibroblasts RFL-6 cells. 2A shows the number of living cells by MTT assay, and FIG. 2B shows cell proliferation by BrdU uptake assay. Cultures of mesenchymal stem cells have been shown to induce cell proliferation in a concentration-dependent manner in RFL-6 cells.
Figure 3 shows the effect of MSC-CM to repair lung fibroblasts damaged by CSE. 3A shows the number of living cells by MTT assay, B shows the cell morphology under microscope, C shows the number of cells in subG1 phase by flow cytometry, D shows TUNEL (red) and annexin V (green) staining results are shown, E shows the cell proliferation results measured by BrdU uptake assay. The above results show that MSC-CM inhibits apoptosis in a concentration-dependent manner and restores the cell proliferation ability inhibited by CSE.
Figure 4 shows the results confirmed by Western blot protein changes according to CSE exposure and MSC-CM treatment. The above results show that MSC-CM inhibits the expression of proteins associated with apoptosis and, conversely, increases the expression and activity of proteins involved in cell proliferation.
Figure 5 shows the collagen gel contraction recovery effect inhibited by CSE exposure of MSC-CM. 5B is the result of measuring the area of the gel shown in 5A.
Figure 6 shows the effect of restoring the expression of extracellular matrix (ECM) protein inhibited by CSE exposure of MSC-CM. 6A and 6B are the results of measurement of mRNA expression and protein expression of extracellular matrix protein, respectively, and 6C is the result of observation of protein expression in cells by immunofluorescence. 6C is the result of mRNA expression of Cox-2 and mPGES-2 which inhibit collagen gel contraction.
7 are experimental results showing that the restorative effect of MSC-CM on the inhibition of cell proliferation and tissue regeneration of CSE is through the activity of PI3-K / Akt. 7A shows that Akt phosphorylation by MSC-CM is due to the activity of PI3-K, and 7B shows that cell proliferation effect by MSC-CM is inhibited by PI3-K inhibitor. 7C shows that the increase in the expression of extracellular matrix protein by MSC-CM is also inhibited by PI3-K inhibitors, and 7D is the increase in collagen gel contractility by MSC-CM is inhibited by PI3-K inhibitors. Is showing.
Figure 8 shows the therapeutic effect of the culture medium (MSC-CM) of mesenchymal stem cells (MSC) for emphysema following exposure to tobacco smoke. 8A shows the histological findings of the lung, and 8B shows the median linear intercept (MLI) value.
9 shows the effect of mesenchymal stem cell culture (MSC-CM) on relieving pulmonary hypertension caused by smoking (9A) and on regenerating blood vessels destroyed by smoking (9B).

The present invention relates to a composition for the prevention and treatment of lung diseases, including the culture of stem cells.

The stem cells are preferably mesenchymal stem cells.

The mesenchymal stem cells include adult stem cells derived from mammals. In addition, the adult stem cells include stem cells isolated from adipose tissue, bone marrow tissue, umbilical cord blood or placenta.

Stem cells are undifferentiated cells, which can divide and self-renewal for a long time, and can differentiate into various kinds of cells given a certain condition. Stem cells are divided into embryonic stem cells and adult stem cells according to the origin of the tissue. Potential has limited disadvantages than embryonic stem cells, but many therapeutics are studied for adult stem cells without ethical problems and no side effects. It is becoming.

Specifically, in the present invention, adult stem cells are used, which may use stem cells commercially available or stem cells separated from biological tissues.

In the present invention, the term "stem cell culture medium" is a substance containing a component contained in the medium obtained by culturing stem cells, the stem cells for producing the culture medium is not limited to the type. For example, the stem cells from which the culture is made can be embryonic stem cells and can also be adult stem cells. Furthermore, adult stem cells can be derived from adult stem cells of all tissues. For example, adult stem cells may be selected from bone marrow derived, cord blood derived, blood derived, hepatic derived, skin derived, gastrointestinal tract, placental derived, nerve derived, adrenal derived, epithelial derived, uterine derived and the like, preferably bone marrow Derived adult stem cells. In a specific embodiment of the present invention, culture medium was prepared using bone marrow-derived mesenchymal stem cells.

The medium for stem cell culture can be used without limitation a basic medium known in the art. The basal medium may be prepared by artificially synthesizing, or a commercially prepared medium may be used. Examples of commercially prepared media include Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, α-MEM (α-Minimal). essential medium), G-MEM (Glasgow's Minimal Essential Medium) and Isocove's Modified Dulbecco's Medium, but are not limited thereto. In addition, one or more accessory ingredients may be added to the cell culture medium as needed. Such accessory ingredients include penicillin G, penicillin G for preventing microbial contamination, including serum of fetal calf, horse or human. , Antibiotics such as streptomycin sulfate and gentamycin, antifungal agents such as amphotericin B and nystatin, and mixtures thereof. You can use more than one.

In one embodiment of the present invention, the non-adhesive cells were removed by culturing the isolated stem cells in a non-derived medium of Dulbecco's Modified Eagle's Medium (DMEM) without serum. By culturing the stem cells separated by the above process in 3 to 5 passages, the bone marrow-derived mesenchymal stem cell culture of the present invention can be obtained.

In one embodiment of the present invention, after exposure to rats for 6 months of tobacco smoke, damaged alveolar tissues were regenerated when the culture medium (MSC-CM) of mesenchymal stem cells was administered to the rats. In addition, it induced angiogenesis and reduced pulmonary hypertension associated with chronic obstructive pulmonary disease by reducing the right ventricular pressure increased due to smoking.

Therefore, the composition containing the culture medium of the mesenchymal stem cells (MSC-CM) of the present invention can effectively treat lung diseases, particularly chronic obstructive pulmonary disease and pulmonary hypertension by regenerating lung tissue.

The chronic obstructive pulmonary disease and pulmonary hypertension may be caused by smoking.

Since the mesenchymal stem cell or the composition containing the stem cell culture medium according to the present invention is a mesenchymal stem cell isolated from the human body as a raw material is less toxic, it is judged that the possibility of carcinogenesis is less because it uses the cell itself.

The composition may be formulated and administered in a unit dosage form suitable for administration in the body of a patient according to conventional methods in the pharmaceutical field, and the preparation may be effective to develop alveoli by one or several administrations. Dosage. Formulations suitable for this purpose are preferably parenteral formulations, such as injectables such as ampoules for injection and inhalants. The injection ampoule or inhalation formulation may be mixed with the injection solution immediately before use, and as the injection solution, physiological saline, glucose, mannitol, and Ringer's solution may be used.

In the pharmaceutical preparations, in addition to the active ingredient, one or more pharmaceutically acceptable inert carriers such as preservatives, analgesic agents, solubilizers or stabilizers in the case of injections, etc. It may further include a base, excipients, lubricants or preservatives.

The composition or pharmaceutical formulation of the present invention thus prepared may be administered with other stem cells used for transplantation and other uses, or in the form of a mixture with such stem cells, using administration methods commonly used in the art. . In addition, the administration may be both non-surgical administration using a catheter and surgical administration methods such as infusion or transplantation after thoracic incision, but non-surgical administration using a catheter is more preferable.

The daily dose of the mesenchymal stem cell culture, which is an active ingredient of the composition, is preferably 20-fold concentrated 1.2ml / kg body weight, and may be administered once or divided into several times. However, it should be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and gender, and therefore, the dosage may be It is not intended to limit the scope of the present invention in terms of aspects.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are provided to illustrate the present invention more easily, and the content of the present invention is not limited to the examples.

Tobacco smoke extract ( cigarette smoke extract , CSE Ready and rat and Pulmonary Fibroblasts  (lung fibroblast Cigarette smoke exposure on)

CSE was prepared by placing 40 ml of tobacco smoke into a 50 ml plastic syringe and mixing with 10 ml of DMEM. One cigarette per 10 ml of CSE was used. CSE solution filtered through a 0.22 μm filter was considered 100%.

In addition, 20 rats of tobacco smoke were exposed to 8-week-old Lewis rats for animal experiments, which were repeated 5 days a week for 6 months. One cigarette contained 8.5 mg of tar and 0.9 mg of nicotine.

Isolation of Bone Marrow Cells

Bone marrow cells (BMC) were isolated from 7-week-old male Lewis rats. After euthanizing the mice, the femur and tibia were excised and the connective tissue attached to the bone was removed aseptically. Bone marrow was extracted from the bone, the bone marrow cells were dispersed by pipetting and the suspension was filtered through a 40 μm mesh (BD Biosciences, San Jose, CA, USA). Filtered total myeloid cells were centrifuged at 1,000 g for 5 minutes, and red blood cells (RBCs) were lysed with 150 mM ammonium chloride and 10 mM potassium bicarbonate. Bone marrow cells were washed with DMEM and suspended in DMEM at 2 × 10 ⁶ cells / ml.

Intermediate lobe  Stem Cells( Mesenchymal stem cell , MSC Culture and culture medium thereof MSC -conditioned media , MSC - CM ) Ready

Bone marrow cells were placed in a plastic cell culture vessel (SPL Lifescience, Korea) with complete medium (DMEM containing 10% FBS, penicillin, streptomycin) and cultured at 5% CO ₂ , 37 ° C.

After 3 days, unattached cells were removed using complete medium and medium was changed once or twice a week. When the culture vessel was about 90% filled with cells, the cells were detached with 0.25% trypsin-EDTA and subcultured at a 1: 3 ratio.

Mesenchymal stem cell culture (MSC-CM) was obtained from mesenchymal stem cells of the third to fifth passages. When 90% of the cells were filled in the cell culture vessel, mesenchymal stem cells in a 15 cm diameter dish were washed with PBS (phosphate-buffered saline) and cultured in 50 ml of fresh serum-free DMEM. After 24 hours, mesenchymal stem cell culture (MSC-CM) was collected, filtered through a 0.22 μm filter, frozen in liquid nitrogen and stored at −70 ° C. Immediately before use, frozen MSC-CM was thawed overnight in the refrigerator and concentrated 20 times in sterile conditions. The prepared stem cell culture solution (MSC-CM) was administered to rats exposed to tobacco smoke. Twenty-fold concentrated MSC-CM was administered through the tail vein 0.3 ml once, twice a week, over 5 weeks.

MTT  Analysis and BrdU uptake  analysis

Normal medium for normal human lung fibroblasts (NHLF, Cambrex, Walkerville, MD, USA) or rat fetal lung fibroblasts (RFL-6, ATCC, Manassas, VA, USA) (DMEM with 10% FBS and penicillin-streptomycin). Each well of a 96-well plate was dispensed with 7 or 9 passages of NHLF or 3 to 8 passages of 5,000 RFL-6 cells. MTT assay (Roche) and BrdU uptake assay (Calbiochem) were performed by the manufacturer's known method to determine the number of viable cells and proliferation of cells.

Flow cell  analysis ( flow cytometry )

The percentage of SubG1 phase cells was analyzed by flow cytometry. When the culture vessel was about 70% full of cells, lung fibroblasts were rinsed with PBS and exposed to CSE for 24 hours without serum. Lung fibroblasts were removed from the culture vessel using trypsin-EDTA (Invitrogen), fixed with 70% ethanol, treated with 5 μg / ml RNase (Roche), and the DNA of the nucleus was 0.1 mg. / ml propidium iodide (Sigma). Total 10,000 cells were analyzed by FACS Calibur flow cytometer and CELL QUEST analysis program (BD Biosciences).

TUNEL  Analysis and Annexin  V dyeing ( Annexin  V staining )

Apoptosis was analyzed by TUNEL assay and Annexin V staining. Lung fibroblasts attached to glass cover slips (22 × 22 mm, Corning, Lowell, Mass., USA) for TUNEL analysis were incubated overnight in 6-well plates of complete medium, then rinsed with PBS and CSE for 24 hours. After exposure, the cells were further incubated for 24 hours in serum-free DMEM (control medium) or MSC-CM. The cells were fixed with paraformaldehyde solution for 20 minutes, permeabilized with 0.1% Triton X-100, and reacted with 2% BSA for 1 hour. After overnight, the cells were reacted with FITC-bound anti-annexin V antibody (1: 500) at 4 ° C., washed with PBS (PBS-T) containing 0.1% Tween-20, and then TUNEL assay (Roche). ) Was performed.

Western Blot  analysis ( Western blot analysis )

Western blots were assayed to analyze expression and phosphorylation of proteins. Cell lysis solution (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% SDS, 1% NP-40, 2.5 mM sodium pyrophosphate), 1 mM Pulmonary fibroblasts were lysed for 30 minutes using β-βglycerophosphate, 1 mM Na ₃ VO ₄ , 2 mM p- nitrophenyl phosphate and protease inhibitor cocktail.

After centrifugation at 14,000 g for 20 minutes at 4 ° C., the proteins in the supernatant were separated on a 10 or 15% SDS-PAGE gel and then attached to a nitrocellulose membrane. After the nitrocellulose membrane was reacted with 5% skim milk for 1 hour, the primary antibody (1: 1,000) was added and reacted at 4 ° C. overnight. The nitrocellulose membrane was washed with a buffer containing 0.5% Tween-20 (Tris-buffered saline (TBS-T)) and then reacted with HRP-conjugated secondary antibody (1: 5,000). Then, each protein was observed and analyzed using an ECL reagent (Amersham) and LAS-3000 (Fuji, Japan).

RT - PCR in mRNA  Confirmation of expression

RT-PCR (reverse transcription-polymerase chain reaction) was performed to investigate mRNA expression of genes. Fibronectin1 (fibronectin1), elastin, collagen (collagen (COL1A1 and COL4A1), cyclooxygenase-2 (COX-2)), microsomal PGE synthase-2 (mPGES-2), and glyceraldehyde 3- The expression of phosphate dehydrogenase (GAPDH) was measured by RT-PCR The total RNA of rat lung fibroblasts was isolated by TRIzol reagent (Invitrogen), and cDNA was synthesized using 1 μg of total RNA as a substrate. Was as follows.

(1) elastin

Forward primer: 5'- TGCCAAATCTGCTGCTAAGG-3 ': SEQ ID NO: 1

Reverse primer: 5'-TTAGCAGCTTTGGATGCAGC-3 ': SEQ ID NO: 2

(2) fibronectin 1 (fibronectin1)

Forward primer: 5'-GCCACCTTAACTCCTATACC-3 ': SEQ ID NO: 3

Reverse primer: 5'-GGAATGTTCACAGAAGTGGC-3 ': SEQ ID NO: 4

(3) COL1A1

Forward primer: 5'-TGGTGAGAAAGGATCTCCTG-3 ': SEQ ID NO: 5

Reverse primer: 5'-ATGCCTCTGTCACCTTGTTC-3 ': SEQ ID NO: 6

(4) COL4A1

Forward primer: 5'-GGTGATAAAGGTGATGTCGG-3 ': SEQ ID NO: 7

Reverse primer: 5'-AATTCCAATGCCAGGGAGAC-3 ': SEQ ID NO: 8

(5) COX-2

Forward primer: 5'-CGGTGAAACTCTAGACAGAC-3 ': SEQ ID NO: 9

Reverse primer: 5'-GATACTGGAACTGCTGGTTG-3 ': SEQ ID NO: 10

(6) mPGES-2

Forward primer: 5'-TGACCCTGTACCAGTACAAG-3 ': SEQ ID NO: 11

Reverse primer: 5 'AAACCAGGTAGGTCTTGAGG-3': SEQ ID NO: 12

(7) GAPDH

Forward primer: 5'-CTCATGACCACAGTCCATGC-3 ': SEQ ID NO: 13

Reverse primer: 5'-TTCAGCTCTGGGATGACCTT-3 ': SEQ ID NO: 14

Collagen Gel Shrinkage Assay ( collagen gel contraction assay )

Lung fibroblasts were exposed to CSE for 24 hours in serum-free conditions and then mixed with liquid collagen solution to give a final cell density of 9.0 X 10 ⁵ cells / ml and a final collagen concentration of 1.5 mg / ml. The collagen solution coagulated in 24-well plates was transferred to 6-well plates and incubated for 24 hours with serum free DMEM or MSC-CM.

After gel images were taken with a digital camera (Nikon D90, Nikon, Japan), the gel area was measured using image analysis software (TINA, Germany).

Intracellular collagen and Fibronectin  dyeing

RFL-6 cells were fixed with 4% paraformaldehyde solution, permeabilized with 0.1% Triton X-100, and reacted with 2% BSA for 1 hour. Subsequently, the mixture was reacted with anti-collagen 1 (1: 1,000) or anti-fibronectin antibody (1: 500) overnight at 4 ° C, followed by binding of anti-rabbit (1: 100) or Alexa Fluor 647 with FITC. It was reacted with anti-chlorine antibody (1: 100) for 1 hour. After staining the nuclei with 1 μg / ml Hoechst 33258 (Calbiochem), intracellular proteins and nuclei were observed under a confocal laser microscope (Carl Zeiss, Germany).

Right ventricular pressure  ( right ventricular systolic pressure , RVSP ) Measure

With each rat anesthetized with ketamine / xylazine and ventilated, the right ventricular pressure was inserted directly into the right ventricle by inserting a 23G needle connected to a pressure monitor (Hewlett-Packard, USA). Right ventricular systolic pressure (RVSP) was measured.

Histological analysis

Lung sites 4 μm thick were used for histological analysis. Mean linear intercept (MLI) was measured in tissue sections stained with hematoxylin and eosin, and the degree of vascular muscularization in tissue sections stained with elastin-van Gieson method. Measured.

Data of the present invention are expressed as mean ± SEM. Data comparison between the two groups was done using One-way ANOVA and Mann-Whitney test. Cases with p-values less than 0.05 were considered significant.

Example  1: tobacco smoke extract ( CSE Induction of apoptosis

To examine the effects of tobacco smoke on lung fibroblasts, CSE was exposed to normal lung fibroblasts (NHLF) and rat fetal lung fibroblasts (RFL-6) for 24 hours. MTT assay showed that CSE reduced NHLF and RFL-6 cell numbers in a concentration dependent manner (FIG. 1A). Tobacco smoke also increased the cell ratio of subG1 phase in flow cytometry (FIG. 1B) and increased the number of TUNEL-positive cells in TUNEL staining (FIG. 1C). Compared to NHLF, the number of viable cells was more reduced in RFL-6 cells, and the increase in the number of subG1 and TUNEL-positive cells was greater. These results suggest that CSE sensitivity is higher in RFL-6 cells compared to NHLF.

Example  2: Effect of Stem Cell Culture on Pulmonary Fibroblast Proliferation

The effect of mesenchymal stem cell culture (MSC-CM) on the proliferation of RFL-6 cells was examined. RFL-6 cells cultured overnight without serum were incubated 24 hours in serum-free DMEM (control, CON) or MSC-CM. As a result of MTT analysis, MSC-CM increased the number of live RFL-6 cells in a concentration-dependent manner (FIG. 2A). BrdU uptake analysis was performed to determine the RFL-6 cell proliferation effect of MSC-CM (FIG. 2B). MSC-CM increased BrdU uptake in RFL-6 cells in a concentration-dependent manner.

Example  3: CSE In lung fibroblasts exposed to MSC - CM Cell death inhibition and cell proliferation

After 24 hours of exposure of various concentrations of CSE to RFL-6 cells, the cultures were replaced with serum-free DMEM (CON) or MSC-CM to further incubate the cells for 24 hours. All concentrations of MSC-CM (one to ten fold) increased the number of living cells concentration dependently (FIG. 3A). In addition, the cell morphology was severely disintegrated in the control group after exposure to CSE, but maintained relatively well in the cell morphology in cells treated with MSC-CM (FIG. 3B). MSC-CM also reduced the percentage increase in subG1 phase cells following CSE exposure (FIG. 3C) and decreased the frequency of TUNEL-positive and Annexin V-positive cells (FIG. 3D). In addition, MSC-CM significantly increased BrdU uptake in a dose-dependent manner despite CSE exposure (FIG. 3E). From the above results, it was confirmed that MSC-CM inhibited apoptosis due to cigarette smoke exposure and enhanced the proliferation of RFL-6 cells.

Example  4: CSE On biochemical changes in lung fibroblasts exposed to MSC - CM's effect

CSE increased active caspase-3, p21, and p27 in a concentration dependent manner, while decreasing total Akt, phosphorylated Akt (FIG. 4). MSC-CMs, on the other hand, reversed these biochemical changes following CSE exposure. In other words, MSC-CM decreased active caspase-3, p21, and p27 increased by CSE, and increased reduced total Akt and phosphorylated Akt.

Example  5: CSE By Reduced  For collagen gel shrinkage ability MSC - CM Effect

Pulmonary fibroblasts from COPD patients have been reported to have impaired recovery, including collagen gel contraction. Therefore, the present invention was examined whether the recovery function of lung fibroblasts damaged by CSE is recovered by MSE-CM using collagen gel contraction. In the absence of any stimulation, RFL-6 cells contracted collagen gel by 25%, and lung fibroblasts exposed to 4% of CSE completely lost collagen gel contractile capacity (FIG. 5). In contrast, 10-fold enriched MSC-CMs increased collagen gel contraction capacity in both CSE exposed and unexposed control cells.

Example  6: Extracellular matrix  ( extracellular matrix A) for proteins and enzymes that inhibit collagen gel contraction MSC - CM Effect

In order to determine whether MSC-CM is involved in restoring expression of ECM protein, the present invention examined the expression of ECM protein. As a result, CSE exposure reduced elastin, fibronectin 1, mRNAs of COL1A1 and COL4A1 (FIG. 6A), collagen 1 and fibronectin protein (FIG. 6B). MSC-CMs, on the other hand, increased the mRNA and protein amounts of these genes in control cells as well as in CSE-damaged RFL-6 cells. In addition, cell immunostaining for collagen 1 and fibronectin showed the same result (FIG. 6C). MSC-CM also reduced the increased expression of COX-2 and mPGES-2, enzymes that produce PGE ₂ , which are well known as inhibitors of collagen gel contraction (FIG. 6D).

Example  7: MSC - CM Signal transduction pathways

After CSE exposure, the signaling pathways were investigated for the effects of cell survival and the recovery effect of MSC-CM on ECM protein expression and collagen gel contraction. MSC-CM increased phosphorylation of Akt, whereas LY294002, a PI3-K inhibitor, inhibited Akt phosphorylation by MSC-CM (FIG. 7A). In addition, LY294002 inhibited cell survival by MSC-CM (FIG. 7B) and also inhibited recovery of ECM protein expression (FIG. 7C). In addition, LY294002 also inhibited the recovery of collagen gel contraction capacity by MSC-CM (FIG. 7D). The above results suggest that the recovery effect by MSC-CM mediates the PI3-K / Akt pathway.

Example  8: Intermediate lobe  Stem Cells ( MSC Culture medium MSC - conditioned media  ( MSC - CM Confirmation of the treatment effect of emphysema induced by smoking by)).

We observed lung tissue regeneration by administering cell-free mesenchymal stem cell culture (MSC-CM) to Lewis Lewis rats that had been smoked for 6 months. As a result, histological examination confirmed that lung tissue was regenerated by MSC-CM in lungs in which emphysema caused by smoking was generated (FIG. 8A). Was normalized by MSC-CM (71.6 ± 2.1 μm) (FIG. 8B).

In addition, stem cell culture fluid promoted angiogenesis (FIGS. 9A, B) and normalized the increased right ventricular pressure (RVSP) due to smoking (FIG. 9C).

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Claims (10)

Pharmaceutical composition for the prevention or treatment of chronic obstructive pulmonary disease comprising a culture of mesenchymal stem cells as an active ingredient. delete  The pharmaceutical composition according to claim 1, wherein the culture solution is obtained by passage 3 to 5 passages of mesenchymal stem cells in a serum-free medium. The pharmaceutical composition of claim 3, wherein the medium is DMEM medium. The pharmaceutical composition of claim 1, wherein the mesenchymal stem cells are derived from bone marrow. The pharmaceutical composition of claim 1, wherein the composition is formulated in injectable or inhalable form. delete The pharmaceutical composition according to claim 1, wherein the chronic obstructive pulmonary disease is a disease caused by smoking. The pharmaceutical composition of claim 8, wherein the chronic obstructive pulmonary disease is emphysema or chronic bronchitis. The pharmaceutical composition of claim 1, wherein the composition lowers the pressure in the lungs caused by smoking.

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