CN110772537A - Application of umbilical cord blood mesenchymal stem cell composition in treating chronic obstructive pulmonary disease - Google Patents

Abstract

The invention discloses an application of a mesenchymal stem cell composition of umbilical cord blood in treating chronic obstructive pulmonary disease. The umbilical cord blood mesenchymal stem cell composition comprises umbilical cord blood mesenchymal stem cells and basic fibroblast growth factor (bFGF), and is injected into a rat body with the chronic obstructive pulmonary disease through a tail vein. The umbilical cord blood mesenchymal stem cells relieve the inflammation in the lung of the chronic obstructive pulmonary disease by improving the expression of anti-inflammatory factors interleukin 10 and interleukin 4; and the lung is internally differentiated into alveolar epithelial cells or bronchial epithelial cells to relieve or reverse the pathological changes of the chronic obstructive pulmonary disease. Compared with the pure umbilical cord blood mesenchymal stem cells, the basic fibroblast growth factor transfected umbilical cord blood mesenchymal stem cells have more advantages in treatment effect.

Description

Application of umbilical cord blood mesenchymal stem cell composition in treating chronic obstructive pulmonary disease

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of a mesenchymal stem cell composition of umbilical cord blood in treating chronic obstructive pulmonary disease.

Background

Chronic obstructive pulmonary disease (chronic obstructive pulmonary disease for short) is a common airflow-limited respiratory disease, and airflow limitation usually develops gradually, and is accompanied by chronic airway inflammatory reaction formed after harmful gas or particles are inhaled.

Chronic obstructive pulmonary disease is mainly characterized by destruction of the lung structure, which is associated with inflammatory reactions, apoptosis of alveolar epithelial cells, endothelial dysfunction, protease and antitroteinase imbalance, and the like. Currently, the main treatment mode of chronic obstructive pulmonary disease is drug therapy, but the continuous worsening of symptoms and the progressive decline of lung function cannot be effectively prevented, so that a new therapeutic drug needs to be searched.

Disclosure of Invention

The invention mainly aims to provide application of an umbilical cord blood mesenchymal stem cell composition in treating chronic obstructive pulmonary disease, and aims to relieve inflammation in lung of the chronic obstructive pulmonary disease and relieve or reverse pathological changes of the chronic obstructive pulmonary disease.

In order to achieve the purpose, the invention provides an application of a umbilical cord blood mesenchymal stem cell composition in treating chronic obstructive pulmonary disease, which comprises the following technical scheme:

the umbilical cord blood mesenchymal stem cell composition comprises umbilical cord blood mesenchymal stem cells and basic fibroblast growth factor (bFGF).

Preferably, the umbilical cord blood mesenchymal stem cells reduce inflammation in the lung of the chronic obstructive pulmonary disease through the expression of interleukin 10 and interleukin 4.

Preferably, the interleukin 10 is capable of inhibiting the production of pro-inflammatory cytokines by T helper 1(Th1) cells.

Preferably, the interleukin 4 plays a role in the type 2T-helper response and isotype switching of B cells towards IgE synthesis.

Preferably, the preparation method of the umbilical cord blood mesenchymal stem cell composition comprises the following steps:

s1: culturing umbilical cord blood mesenchymal stem cells by adopting a density gradient centrifugation-adherent culture method;

s2: adopting CM-DIL to mark umbilical cord blood mesenchymal stem cells;

s3: transfecting the mesenchymal stem cells of the umbilical cord blood with the bFGF-pcDNA3.1 plasmid by using a liposome transfection reagent.

Preferably, the umbilical cord blood mesenchymal stem cell composition is injected into the body of the chronic obstructive pulmonary disease rat through the tail vein.

The invention has the beneficial effects that:

firstly, injecting the umbilical cord blood mesenchymal stem cell composition carrying the basic fibroblast growth factor gene into a rat body with the chronic obstructive pulmonary disease through a tail vein can relieve pathological changes and enhance the anti-inflammatory effect; through inhibiting alveolar macrophage COX-2/PGE2, part of the alveolar macrophage COX-2/PGE2 is mediated through p38 MAPK and ERK channels, the airway inflammation and the emphysema of a rat with the chronic obstructive pulmonary disease are relieved, meanwhile, the lung injury of a chronic obstructive pulmonary disease model can be relieved by promoting the proliferation of endogenous lung stem cells, and the apoptosis of alveolar walls is reduced; moreover, umbilical cord blood mesenchymal stem cells transplanted into the lung can secrete an important epithelial growth factor, and bone marrow mesenchymal stem cells retained in the lung may differentiate into alveolar epithelial cells and regulate other alveolar epithelial cells in a paracrine manner;

reducing inflammation in the lung of the chronic obstructive pulmonary disease by improving the expression of anti-inflammatory factors interleukin 10 and interleukin 4 by the umbilical cord blood mesenchymal stem cells; and the lung is internally differentiated into alveolar epithelial cells or bronchial epithelial cells to relieve or reverse the pathological changes of the chronic obstructive pulmonary disease.

Drawings

FIG. 1: detecting the expression of the surface marker of the umbilical cord blood mesenchymal stem cells by a flow cytometer;

FIG. 2: transfecting umbilical cord blood mesenchymal stem cells (multiplied by 40) by using the empty plasmid and the basic fibroblast growth factor plasmid;

FIG. 3: day 28 lung tissue hematoxylin-eosin staining (x 10) in each group of rats;

FIG. 4: qRT-PCR detects the expression of interleukin 10 and interleukin 4mRNA in the lung tissues of rats in each group;

FIG. 5: CM-Dil and CC16 double-labeled cells (x 40) in umbilical cord blood mesenchymal stem cell group;

FIG. 6: CM-Dil and SPC double standard cells (x 40) in umbilical cord blood mesenchymal stem cell group;

FIG. 7: the CM-Dil and CC16 double-labeled cells (x 40) of pcDNA3.1-BMSCs group;

FIG. 8: : pcDNA3.1-BMSCs group CM-Dil and SPC double standard cells (. times.40);

FIG. 9: bFGF-pcDNA3.1-BMSCs group CM-Dil and CC16 double-labeled cells (. times.40);

FIG. 10: bFGF-pcDNA3.1-BMSCs group CM-Dil and SPC double-labeled cells (. times.40).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.

It should be noted that all the directional indicators (such as up, down, left, right, front, and rear … …) in the embodiment of the present invention are only used to explain the relative position relationship between the components, the movement situation, etc. in a specific posture (as shown in the drawing), and if the specific posture is changed, the directional indicator is changed accordingly.

In addition, the descriptions related to "first", "second", etc. in the present invention are for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.

In the embodiment of the invention, the application of the umbilical cord blood mesenchymal stem cell composition in treating chronic obstructive pulmonary disease comprises the following scheme:

separating, culturing and identifying umbilical cord blood mesenchymal stem cells, and transfecting basic fibroblast growth factor genes:

taking a healthy SD rat of 6 weeks old, purifying umbilical cord blood mesenchymal stem cells of the rat by adopting a density gradient centrifugation-adherent culture method, inoculating the rat umbilical cord blood mesenchymal stem cells into a plastic culture dish, culturing the rat umbilical cord blood mesenchymal stem cells by using a DMEM-LG culture medium containing 10% fetal calf serum in volume fraction at 37 ℃ under the conditions of 5% CO2 in volume fraction and 100% saturated humidity, carrying out passage at the ratio of 1: 3 every 3d or 4d, detecting the expression of the surface marker of the umbilical cord blood mesenchymal stem cells by using a flow cytometer, and marking the umbilical cord blood mesenchymal stem cells by using CM-DIL.

When the coverage rate of the umbilical cord blood mesenchymal stem cells reaches about 90%, adding Lipofectamine2000 according to 0.5 mu L/cm2, adding bFGF-pcDNA3.1 plasmid according to 1 mu g/cm2, incubating for 6h in an original incubator, discarding an old culture medium, adding a complete culture medium, and putting into the original incubator under the same conditions for culturing for 24 h; and (3) observing visible green fluorescence by a fluorescence microscope, confirming that the transfection of the basic fibroblast growth factor is successful, screening the stably transfected cells by G418, and stably transfecting for 24h for transplantation.

Preparation and experimental grouping of a chronic obstructive pulmonary disease rat model:

150 healthy 2-month-old SD rats with unlimited male and female bodies and a body mass of 200-230g are selected, 30 rats are randomly selected as normal control groups, and the rest 120 rats are prepared into the chronic obstructive pulmonary disease model by adopting a lipopolysaccharide and fumigation combined stimulation method.

Preparing a chronic obstructive pulmonary disease model: lipopolysaccharide (200 microgram/time) is injected into trachea on day 1 and day 14, rats are placed into a special smoke box (50cm multiplied by 40cm, two smoke holes are formed in the side wall) on day 2-30 (except day 14), Lushan brand cigarette (tar content 13mg, nicotine content 1.2mg, Jiangxi Nanchang cigarette factory) smoke is injected for 2 times/d, 40min each time (8 cigarettes each time, 4 times of burning, about 9min for burning out each time, and 30s for changing the smoke) is 1 month.

Grouping experiments: the prepared chronic obstructive pulmonary disease rats are randomly divided into 4 groups: chronic obstructive pulmonary disease group, BMSCs group, pcDNA3.1-BMSCs group, bFGF-pcDNA3.1-BMSCs group, 30 each. Immediately after the above grouping (including the normal control group), the following interventions were carried out during normal feeding. Normal control group: healthy SD rats were injected with 1mLPBS via tail vein; chronic obstructive pulmonary disease group: model rats were injected 1mLPBS via tail vein; BMSCs group: model rat tail was injected intravenously with CM-Dil-labeled 3 rd generation umbilical cord blood mesenchymal stem cells (1X 10) ⁷1 mLPBS; pcDNA3.1-BMSCs group: model rat umbilical cord blood mesenchymal stem cell generation 3 of CM-Dil-labeled transfection pcDNA3.1 plasmid injected through tail vein ⁷1 mLPBS; bFGF-pcDNA3.1-BMSCs group: model rat through tail vein injection CM-Dil labeled 3 rd generation umbilical cord blood mesenchymal stem cell transfected with bFGF-pcDNA3.1 plasmid 1 x 10 ⁷/1mLPBS。

Reserving specimens, reserving 10 dead rats at each position after 7, 14 and 28 days of intervention in each group, ① reserving part of right lung middle leaves, preparing paraffin sections after fixing the rats by 10 percent of formaldehyde solution in volume fraction, preparing paraffin sections with the thickness of about 4 mu m and performing hematoxylin-eosin staining, ② reserving part of right lung middle leaves and preserving the paraffin sections in a refrigerator at the temperature of-80 ℃ for detecting the expression of interleukin 10 and interleukin 4 in lung tissues in a qRT-PCR experiment, ③ reserving peripheral blood, centrifuging for 10min at 2500r/min, taking upper layer faint yellow serum and preserving the upper layer faint yellow serum in a refrigerator at the temperature of-80 ℃ for detecting the levels of the interleukin 10 and the interleukin 4 in an ELISA experiment, ④ reserving part of right lung middle leaves, preparing fresh frozen sections, cutting the sections to the diameter of about 5 mu m, observing the distribution condition of CM-Dil staining positive cells of the frozen sections under a fluorescence microscope, selecting the sections with more CM-l positive cells for immunofluorescence staining, and observing the umbilical cord differentiation condition of the mesenchymal stem cells in the lung tissues.

Expression of interleukin 10, interleukin 4mRNA in lung tissue:

taking right lung middle lobe, homogenizing and grinding, detecting the expression of interleukin 10 and interleukin 4 in lung tissues by a qRT-PCR method, wherein the reaction conditions comprise pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, extension at 60 ℃ for 30s, and circulation for 40 times to obtain a product, namely cDNA, and storing at-20 ℃, the internal reference β -action primer F is 5'-AGC GAG CAT CCC CCAAGT T-3', R is 5'-GGG CACGAA GGC TAC ATC ATT-3', the size of an amplification product is 101bp, the interleukin 10 primer F is 5'-AAG GGT TACTTG GGT TGC CA-3', R is 5'-AGA CAC CTT TGT CTT GGA GCTT-3', the size of the amplification product is 241bp, the interleukin 4 primer F is 5'-TCT GTA GAG GTG TCA GCG GT-3', R is 5'-AGT GTT GTG AGC GTG GACTC-3', the size of the amplification product is 70 bp., and the relative multiple of each experimental group relative to the gene expression of a normal control group is calculated by a 2-delta Ct method.

Differentiation of umbilical cord blood mesenchymal stem cells in lung tissue:

fresh frozen sections with more CM-Dil positive cells are incubated overnight at 4 ℃ through primary antibody (rabbit anti-mouse SPC and CC16 polyclonal antibody are diluted 1: 200), and incubated for 30min at 37 ℃ after rewarming; after incubation with secondary antibody (diluted 1: 200 in goat anti-rabbit FIFC) at 37 ℃ for 40min, the number of double-stained cells in the same visual field was observed.

The method mainly comprises the steps of observing lung tissue pathological changes of each experimental group ①, detecting the expression of interleukin 10 and interleukin 4mRNA in lung tissue through ② qRT-PCR experiments, detecting the levels of the interleukin 10 and the interleukin 4 in peripheral blood through ③ ELISA experiments, observing the distribution condition of CM-Dil staining positive cells of a frozen section ④ under a fluorescence microscope, and observing the differentiation condition of ⑤ umbilical cord blood mesenchymal stem cells in the lung tissue.

The statistical analysis experiment shows that the measured data is expressed by x +/-s, the SSPS 17.0 software is used for statistical analysis, the test level is α -0.05, multiple comparison among sample mean numbers is carried out, the variance is judged by F test, the LSD-t test is used when the variance is qualified, the rank-sum test is used when the variance is irregular, the difference P <0.05 has significance, and the difference P <0.01 has significance.

Morphology, identification, marking and transfection results of the umbilical cord blood mesenchymal stem cells:

the primary umbilical cord blood mesenchymal stem cells are typically spindle-shaped, spindle-shaped or triangular, and local visible microcolony formation. The extracted and cultured 4 th generation cells show high expression of CD29 and CD44 through flow cytometry detection, and basically do not express markers of CD34 and CD45 of hematopoietic stem cells, and the figure is shown in figure 1. The umbilical cord blood mesenchymal stem cells are transfected by the basic fibroblast growth factor plasmid for 24-48h, and the visible part of the cells are observed to be green fluorescence under a fluorescence microscope to prompt the successful transfection of the plasmid, which is shown in figure 2 (A, C in the figure is the fourth generation umbilical cord blood mesenchymal stem cells; B is the basic fibroblast growth factor plasmid transfected umbilical cord blood mesenchymal stem cells, and the visible part of the cells are observed to be green fluorescence under the fluorescence microscope to prompt the successful transfection of the plasmid; D is the empty plasmid transfected umbilical cord blood mesenchymal stem cells, and green fluorescence is not shown). The control group showed no green fluorescence.

Pathological changes in lung tissue:

referring to FIG. 3 (in the figure, A is a normal control group; B is a chronic obstructive pulmonary disease group; C is a BMSCs group; D is pcDNA3.1-BMSCs group; E is bFGF-pcDNA3.1-BMSCs group);

and (3) reserving part of right lung lobes in each experimental group on the 28 th day of intervention to perform pathological hematoxylin-eosin staining, and observing pathological changes under an optical microscope, wherein the pathological changes of the chronic obstructive pulmonary disease group are compared with those of a normal control group: the alveolar cavities are irregularly enlarged, and a part of lungs are broken at intervals, and a plurality of alveoli are fused into a bullous lung; inflammatory cell infiltration is seen around the bronchi and pulmonary interstitial vessels, as shown in fig. 3B, which is consistent with the pathological changes of typical chronic obstructive pulmonary diseases. BMSCs group, pcDNA3.1-BMSCs group, bFGF-pcDNA3.1-BMSCs group: there are typical pathological changes of chronic obstructive pulmonary disease. Under the same visual field, the alveolar space is irregularly enlarged, a part of the alveolar space is visible to be broken, the pulmonary bulla is formed, and the tracheal wall and the pulmonary interstitial vascular wall are infiltrated by a few inflammatory cells. The BMSCs group and the pcDNA3.1-BMSCs group have no obvious difference; the bFGF-pcDNA3.1-BMSCs group showed less pathological changes compared to the BMSCs group and pcDNA3.1-BMSCs group, as shown in FIGS. 3C-E.

The peripheral blood interleukin 10 and interleukin 4 levels of rats in each group:

peripheral blood is reserved in each group at 7, 14 and 28 days of intervention, and the levels of the self-cytokine 10 and the interleukin 4 in the peripheral blood are detected by a double antibody sandwich ELISA method, and the results show that: at the same time node, the levels of interleukin 10 and interleukin 4 in peripheral blood of 3 treatment groups are higher than that of the chronic obstructive pulmonary disease group (mean P is less than 0.01), and the levels of interleukin 10 and interleukin 4 in peripheral blood of the BMSCs group and the pcDNA3.1-BMSCs group are not greatly different, but the levels of interleukin 10 and interleukin 4 in bFGF-pcDNA3.1-BMSCs group are relatively higher than those of the two groups, and the difference has significance (mean P is less than 0.05), which is shown in Table 1.

TABLE 1 Interleukin 10, Interleukin 4 levels in peripheral blood of rats in each group

Table1 Levels of interleukin-10 and interleukin-4 in rat penpheralblood

And (4) surface note: at the same time point, compared with the chronic obstructive pulmonary disease group, ^ap is less than 0.05: compared with the bone marrow mesenchymal stem cell group, ^bp is less than 0.05. BMSCs: bone marrow mesenchymal stem cells: bFGF: basic fibroblast growth factor.

Expression of mRNA from interleukin 10, interleukin 4 in rat lung tissue of each group:

in each group, partial right lung middle lobe is reserved on 7 th, 14 th and 28 th days of intervention respectively, and after homogenization and grinding, mRNA expression of interleukin 10 and interleukin 4 in lung tissues is detected through qRT-PCR. At the same time node, the interleukin 10 and interleukin 4 expression in the lung tissue of 3 treatment groups is higher than that of the chronic obstructive pulmonary disease group (mean P <0.01), and the interleukin 10 and interleukin 4 expression in the lung tissue of BMSCs group and pcDNA3.1-BMSCs group are not greatly different, but the bFGF-pcDNA3.1-BMSCs group is relatively higher than that of the two groups, and the difference has significance (mean P <0.05), which is shown in figure 4.

Differentiation of umbilical cord blood mesenchymal stem cells in lung tissue:

and (3) reserving a part of right lung middle lobes of each experimental group on the 28 th day of intervention, performing fresh frozen sections, observing the distribution condition of CM-Dil staining positive cells under a fluorescence microscope, selecting sections with more CM-Dil positive cells for immunofluorescence staining, and observing the differentiation condition of umbilical cord blood mesenchymal stem cells in lung tissues. Under a fluorescence microscope, a few cells with orange-red fluorescence are present in lung tissue sections of 3 treatment groups, and light green fluorescence (CM-Dil and SPC/CC16 double staining marks) is shown in FIGS. 5-10 (FIG. 5: A shows that CM-Dil is stained with orange-red fluorescence, B shows that CC16-FIFC is stained with light green fluorescence, FIG. 6 shows that A shows that CM-Dil is stained with orange-red fluorescence, B shows that SPC-FIFC is stained with light green fluorescence, FIG. 7 shows that A shows that CM-Dil is stained with red fluorescence, B shows that CC16-FIFC is stained with green fluorescence, FIG. 8 shows that A shows that CM-Dil is stained with red fluorescence, B shows that SPC-FIFC is stained with green fluorescence, FIG. 9 shows that CM-Dil is stained with orange-red fluorescence, B shows that CC16-FIFC is stained with green fluorescence, and FIG. 10 shows that A shows that CM-Dil is stained with orange-red fluorescence, and B shows that SPC-FIFC is stained with light green. The alveolar wall has a small amount of cells showing orange-red fluorescence (CM-Dil mark) and light green fluorescence (SPC-FIFC mark), the bronchial wall has a small amount of cells showing orange-red fluorescence (CM-Dil mark) and light green fluorescence (CC16-FIFC mark), and double-staining positive cells are more found in the bFGF-pcDNA3.1-BMSCs group compared with the BMSCs group and the pcDNA3.1-BMSCs group.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims (6)

1. The application of the umbilical cord blood mesenchymal stem cell composition in treating the chronic obstructive pulmonary disease is characterized in that the umbilical cord blood Mesenchymal Stem Cell (MSC) composition comprises umbilical cord blood mesenchymal stem cells and basic fibroblast growth factor (bFGF).

2. The umbilical cord blood mesenchymal stem cell composition for use in treating chronic obstructive pulmonary disease according to claim 1, wherein the umbilical cord blood mesenchymal stem cells reduce inflammation in lung of chronic obstructive pulmonary disease through the expression of interleukin 10 and interleukin 4.

3. The umbilical cord blood mesenchymal stem cell composition for use in treating chronic obstructive pulmonary disease according to claim 2, wherein the interleukin 10 is capable of inhibiting the production of pro-inflammatory cytokines by T helper 1(Th1) cells.

4. The umbilical cord blood mesenchymal stem cell composition for use in the treatment of chronic obstructive pulmonary disease according to claim 2, wherein the interleukin 4 plays a role in the type 2T-helper response and isotype switch of B-cells to IgE synthesis.

5. The application of the umbilical cord blood mesenchymal stem cell composition in the treatment of chronic obstructive pulmonary disease according to claim 1, wherein the preparation method of the umbilical cord blood mesenchymal stem cell composition comprises the following steps:

s1: culturing umbilical cord blood mesenchymal stem cells by adopting a density gradient centrifugation-adherent culture method;

s2: adopting CM-DIL to mark umbilical cord blood mesenchymal stem cells;

s3: transfecting the mesenchymal stem cells of the umbilical cord blood with the bFGF-pcDNA3.1 plasmid by using a liposome transfection reagent.

6. The umbilical cord blood mesenchymal stem cell composition for treating the chronic obstructive pulmonary disease according to claim 1, wherein the umbilical cord blood mesenchymal stem cell composition is injected into a rat body with the chronic obstructive pulmonary disease through a tail vein.

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