Application of mesenchymal stem cell spheres in preparation of medicine for treating pulmonary arterial hypertension

Abstract

The invention discloses application of mesenchymal stem cell spheres in preparation of medicines for treating pulmonary arterial hypertension, and relates to the technical field of biological medicines. According to the application of the mesenchymal stem cell spheres in preparation of the medicine for treating pulmonary arterial hypertension, the pulmonary arterial pressure of mice can be reduced, lung tissue collagen deposition can be reduced to relieve pulmonary fibrosis, myocardial hypertrophy and right ventricular remodeling are inhibited, pulmonary vascular endothelial injury is improved, and integrity is enhanced; meanwhile, infiltration of inflammatory cells such as macrophages, mononuclear cells and neutrophils in lung tissues is inhibited, and circulating inflammatory response is regulated. The invention provides a novel therapeutic scheme for repairing blood vessels, regulating immunity and improving pathological remodeling for pulmonary arterial hypertension, and has clinical application potential.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of mesenchymal stem cell spheres in the preparation of a drug for treating pulmonary hypertension. Background Art

[0002] Pulmonary arterial hypertension is a serious cardiopulmonary disease that can lead to increased pulmonary vascular resistance, right heart failure, and even death. Existing drugs (such as bosentan and sildenafil) can only relieve symptoms but cannot repair diseased blood vessels. Long-term use also has side effects, and patients have a poor prognosis. More effective treatments are urgently needed.

[0003] Mesenchymal stem cells (MSCs) can secrete growth factors to repair blood vessels and regulate immunity, showing potential in the treatment of pulmonary arterial hypertension. However, traditional single-cell transplantation suffers from short survival time, weak targeted aggregation ability, and limited repair efficacy, which restricts its clinical application.

[0004] In recent years, stem cell spheres produced using three-dimensional culture technology have been shown to mimic the in vivo microenvironment, enhancing stem cell survival and secretion of functional factors (e.g., a 30%-50% increase in factors related to vascular repair), and have demonstrated improved efficacy in other diseases. However, there has been no research on the use of mesenchymal stem cell spheres in the treatment of pulmonary arterial hypertension. This present invention, for the first time, utilizes the unique advantages of stem cell spheres, providing a novel treatment option for pulmonary arterial hypertension. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of mesenchymal stem cell spheres in the preparation of a drug for treating pulmonary hypertension, so as to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A use of mesenchymal stem cell spheres in the preparation of a drug for treating pulmonary hypertension.

[0008] Preferably, the medicine prepared from the stem cell spheres can reduce pulmonary artery pressure.

[0009] Preferably, the drug can improve pulmonary vascular remodeling and lung tissue fibrosis.

[0010] Preferably, the drug can improve right ventricular remodeling and inhibit myocardial hypertrophy and ventricular fibrosis.

[0011] Preferably, the drug can inhibit abnormal proliferation of pulmonary vascular smooth muscle cells, improve endothelial damage and increase endothelial integrity.

[0012] Preferably, the drug is capable of inhibiting the infiltration of inflammatory cells in lung tissue, including macrophages, monocytes and neutrophils.

[0013] Preferably, the drug is capable of regulating circulating inflammatory responses, reducing the expression level of the pro-inflammatory cytokine IL-6 and increasing the expression level of the anti-inflammatory cytokine IL-10.

[0014] A method for preparing mesenchymal stem cell spheres as described above comprises the following steps:

[0015] Step 1: Mesenchymal stem cell culture: Use DMEM low-glucose medium, 10% fetal bovine serum and 1% double-antibody as complete culture medium to culture and obtain mesenchymal stem cells;

[0016] Step 2: Preparation of mesenchymal stem cell sphere injection: Inoculate mesenchymal stem cells to prepare mesenchymal stem cell spheres with a diameter of 50-300 μm, wash the mesenchymal stem cell spheres with normal saline at least three times, and finally prepare the mesenchymal stem cell sphere injection with 5% human albumin injection;

[0017] Step 3: Compare the viability of mesenchymal stem cell spheres and mesenchymal stem cells.

[0018] A medicine for treating pulmonary arterial hypertension, characterized by comprising the mesenchymal stem cell spheres used as described above.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The mesenchymal stem cell spheres disclosed in the present invention are used in the preparation of drugs for treating pulmonary hypertension. The mesenchymal stem cell spheres can reduce pulmonary artery pressure in mice, reduce collagen deposition in lung tissue, significantly alleviate pulmonary fibrosis, help inhibit myocardial hypertrophy and ventricular remodeling, and can also effectively improve endothelial damage, increase endothelial integrity, inhibit inflammatory cell infiltration in the lung tissue of mice with pulmonary hypertension, and inhibit circulatory inflammatory responses. Therefore, the mesenchymal stem cell spheres have certain application value in the treatment of pulmonary hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Verification diagram of MSCs cell sphere viability; (A) MSCs cell sphere morphology in the first three days; (B) Comparison of cell viability of hUC-MSCs and MSCs cell spheres determined by Tunel staining.

[0022] Figure 2 Pulmonary artery pressure maps were measured in PAH mice using small animal echocardiography and right cardiac catheterization.

[0023] Figure 3 These are the H&E staining and Masson's staining results of lung tissue in PAH mice.

[0024] Figure 4 These are the results of H&E staining, Masson staining, and WGA staining of the heart of PAH mice.

[0025] Figure 5 These are the results of immunofluorescence staining of PCNA and α-SMA, markers of pulmonary vascular smooth muscle cell proliferation, and CD31, a marker of pulmonary vascular endothelial cells, in PAH mice.

[0026] Figure 6 The results of immunohistochemical staining of CD68, Ly6C and Ly6G in PAH mice are shown.

[0027] Figure 7 The figure shows the detection results of pro-inflammatory cytokine IL-6 and anti-inflammatory cytokine IL-10 in the circulating serum of PAH mice. DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0029] The reagents or instruments used in the embodiments of the present invention without indicating the manufacturer are all conventional reagent products available in the market.

[0030] Example 1

[0031] Preparation and viability verification of MSCs spheroids

[0032] 1. Mesenchymal stem cells (hUC-MSCs) culture: Use DMEM low-glucose medium, 10% fetal bovine serum and 1% double antibiotic (penicillin / streptomycin) as complete culture medium to obtain hUC-MSCs.

[0033] 2. Inoculation of hUC-MSCs to prepare mesenchymal stem cell spheres (MSCs spheres): Inoculate hUC-MSCs to prepare MSCs spheres with a diameter of 50-300 μm. Wash the MSCs spheres three times with normal saline and prepare MSCs sphere injection with 5% human albumin injection.

[0034] 3. The cell activity of MSCs spheres was detected by Tunel cell apoptosis assay. The results showed that the cell viability of MSCs spheres was consistent with that of Huc-MSCs prepared by cell culture flasks. The cell apoptosis rate was controlled below 5%. Figure 1 shown.

[0035] The mesenchymal stem cells in the present embodiment are human umbilical cord mesenchymal stem cells, which are prepared by collecting human umbilical cord. When inoculating hUC-MSCs, centrifugation, stirring, shaking, hanging drop method, etc. can be used to promote the hUC-MSCs to form spheres and obtain MSCs cell spheres.

[0036] To verify the efficacy of MSCs spheres in treating pulmonary hypertension, the present invention established a mouse pulmonary arterial hypertension (PAH) model by combining hypoxia with subcutaneous injection of SU5416. MSCs spheres were then subcutaneously transplanted into the left axilla of the mice for treatment. The injection was repeated once, with a total of 1×10 cells injected each time. 6 Cells. The verification results obtained include the following examples:

[0037] Example 2

[0038] MSCs spheroids reduce pulmonary artery pressure in mice

[0039] like Figure 2 As shown, pulmonary artery pressure was assessed by measuring right ventricular pressure in mice using small animal echocardiography and right cardiac catheterization, indicating that MSCs cell spheres could exert the effect of reducing pulmonary artery pressure in mice for 4 weeks.

[0040] Example 3

[0041] MSCs spheroids improve pulmonary microvascular remodeling and fibrosis in PAH mice

[0042] The main pathological manifestations of pulmonary hypertension are pulmonary vascular remodeling and lung tissue fibrosis. Figure 3 As shown, the H&E staining results of lung tissue showed that subcutaneous transplantation of MSCs cell spheres could significantly reduce the thickness and area of ​​the pulmonary arteriolar wall in mice. Masson staining showed that collagen deposition in the lung tissue of the MSCs cell sphere treatment group was reduced and pulmonary fibrosis was significantly alleviated.

[0043] Example 4

[0044] MSCs spheroids improve right ventricular remodeling in PAH mice

[0045] like Figure 4 As shown in the results of cardiac H&E staining, PAH mice had significantly thickened ventricular walls and aggravated fibrosis, but the subcutaneous injection of MSCs cell spheres treatment group was able to significantly improve the degree of right ventricular fibrosis. Cardiac WGA staining showed that myocardial cells were significantly smaller than those in the model group. MSCs cell sphere treatment helped inhibit myocardial hypertrophy and ventricular remodeling.

[0046] Example 5

[0047] MSCs spheroids inhibit abnormal proliferation of pulmonary vascular smooth muscle cells and improve endothelial integrity in PAH

[0048] like Figure 5 As shown, immunofluorescence staining of PCNA and α-SMA, markers of pulmonary vascular smooth muscle cell proliferation, showed that MSCs spheroid treatment could effectively inhibit the abnormal proliferation of smooth muscle cells. Immunofluorescence staining of CD31, a marker of pulmonary vascular endothelial cells, showed that MSCs spheroid treatment could effectively ameliorate endothelial damage and increase endothelial integrity.

[0049] Example 6

[0050] MSCs spheroids inhibit inflammatory response in lung tissue

[0051] The infiltration of inflammatory cells in lung tissue was analyzed by immunohistochemical staining of CD68, Ly6C, and Ly6G. Figure 6 As shown in the results, the infiltration of macrophages, monocytes and neutrophils in the lung tissue of mice in the PAH group was significantly increased, while MSCs cell sphere treatment could significantly inhibit the infiltration of inflammatory cells in the lung tissue of mice with pulmonary hypertension.

[0052] Example 7

[0053] MSCs spheroids inhibit circulating inflammatory responses

[0054] The pro-inflammatory cytokine IL-6 and the anti-inflammatory cytokine IL-10 in the circulating serum of PAH mice were detected. Figure 7 As shown in the results, the expression level of IL-6 was significantly decreased, while the expression level of IL-10 was significantly increased. MSCs cell spheres provide effective immunomodulatory effects for the treatment of pulmonary hypertension.

[0055] In summary, the use of the mesenchymal stem cell spheres disclosed in the present invention in the preparation of drugs for the treatment of pulmonary hypertension can reduce the pulmonary artery pressure in mice, reduce collagen deposition in lung tissue, significantly alleviate pulmonary fibrosis, help inhibit myocardial hypertrophy and ventricular remodeling, and can also effectively improve endothelial damage, increase endothelial integrity, inhibit inflammatory cell infiltration in the lung tissue of mice with pulmonary hypertension, and inhibit circulatory inflammatory responses. Therefore, it has certain application value in the treatment of pulmonary hypertension.

[0056] It should be noted that the experimental animal recipients in the present invention are preferably mice, but are not limited to mice, and may also be other small animals commonly used in biological experiments.

[0057] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention, and any contents not described in detail in this specification shall belong to the prior art known to professional and technical personnel in this field.

Claims

1. Use of mesenchymal stem cell spheres in the preparation of drugs for treating pulmonary arterial hypertension.

2. Use of a mesenchymal stem cell sphere according to claim 1 in preparing a drug for treating pulmonary hypertension, characterized in that: The medicine prepared from the stem cell spheres can reduce pulmonary artery pressure.

3. Use of a mesenchymal stem cell sphere according to claim 1 in preparing a drug for treating pulmonary hypertension, characterized in that: The drug can improve pulmonary microvascular remodeling and lung tissue fibrosis.

4. Use of a mesenchymal stem cell sphere according to claim 1 in preparing a drug for treating pulmonary hypertension, characterized in that: The drug can improve right ventricular remodeling and inhibit myocardial hypertrophy and ventricular fibrosis.

5. Use of a mesenchymal stem cell sphere according to claim 1 in preparing a drug for treating pulmonary hypertension, characterized in that: The drug can inhibit abnormal proliferation of pulmonary vascular smooth muscle cells, improve endothelial damage and increase endothelial integrity.

6. Use of a mesenchymal stem cell sphere according to claim 1 in preparing a drug for treating pulmonary arterial hypertension, characterized in that: The drug can inhibit the infiltration of inflammatory cells in lung tissue, including macrophages, monocytes and neutrophils.

7. Use of a mesenchymal stem cell sphere according to claim 1 in preparing a drug for treating pulmonary arterial hypertension, characterized in that: The drug can regulate circulating inflammatory responses, reduce the expression level of the pro-inflammatory cytokine IL-6 and increase the expression level of the anti-inflammatory cytokine IL-10.

8. A method for preparing mesenchymal stem cell spheres for use according to any one of claims 1 to 7, characterized in that: The steps include: Step 1: Mesenchymal stem cell culture: Use DMEM low-glucose medium, 10% fetal bovine serum and 1% double-antibody as complete culture medium to culture and obtain mesenchymal stem cells; Step 2: Preparation of mesenchymal stem cell sphere injection: Inoculate mesenchymal stem cells to prepare mesenchymal stem cell spheres with a diameter of 50-300 μm, wash the mesenchymal stem cell spheres with normal saline at least three times, and finally prepare the mesenchymal stem cell sphere injection with 5% human albumin injection; Step 3: Compare the viability of mesenchymal stem cell spheres and mesenchymal stem cells.

9. A drug for treating pulmonary hypertension, characterized in that: The method comprises the mesenchymal stem cell sphere according to any one of claims 1 to 7.

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