Mesenchymal stem cell composition for treating prostate disease and preparation method and application thereof

Abstract

The present application relates to the technical field of stem cell medicine, in particular to a mesenchymal stem cell composition for treating prostate diseases and a preparation method and application thereof. The mesenchymal stem cell composition for treating prostate diseases comprises mesenchymal stem cells treated by low oxygen and an anti-inflammatory peptide WYPWMKKHHRRRRRRRRR. The prostate diseases are selected from prostatitis, prostate calcification, sexual function improvement, frequent urination and urgency, etc. The mesenchymal stem cell composition can significantly improve the symptoms of prostate diseases, and the curative effect is remarkable.

Description

Technical Field

[0001] This invention relates to the field of stem cell drug technology, specifically to a mesenchymal stem cell composition for treating prostate diseases, its preparation method, and its application. Background Technology

[0002] Common prostate diseases include prostatitis, impaired sexual function, frequent urination, and urgency. The incidence of prostate diseases increases significantly with age. The pathogenesis of prostate diseases is complex, mainly related to genetic factors, environmental factors, and hormonal changes. Prostate diseases generally have a long course and require a long treatment period. Early treatment is often ineffective, and the diseases are characterized by their persistent and difficult-to-cure nature, making clinical treatment challenging.

[0003] Mesenchymal stem cells (MSCs) are pluripotent adult stem cells derived from the mesoderm and possessing self-renewal capacity. MSCs are widely distributed in tissues, possess self-renewal and differentiation capabilities, exhibit multi-lineage differentiation potential, and possess immunomodulatory properties. They are abundant in tissues such as bone marrow, umbilical cord tissue, umbilical cord blood, peripheral blood, and adipose tissue, and have become a core research direction in regenerative medicine. Under specific in vivo or in vitro induction conditions, MSCs can differentiate into various organ cells, bone cells, adipocytes, muscle cells, and other mesodermal mesenchymal tissue cells. They can also cross germ layer boundaries, differentiating into ectoderm neurons, glial cells, and endoderm hepatocytes, among others. Mesenchymal stem cells (MSCs) can be isolated from umbilical cord (UC-MSCs), adipose tissue (AD-MSCs), bone marrow (BM-MSCs), placenta (P-MSCs), and dental pulp (DP-SCs), among others. The characteristics of these different sources vary significantly: BM-MSCs are the most widely studied and have high differentiation potential, but their acquisition requires invasive procedures; AD-MSCs are easily obtained and have high yields, making them suitable for wound healing and treatment of Crohn's disease fistulas; UC-MSCs have strong proliferative capacity and low immunogenicity, making them suitable for allogeneic transplantation; P-MSCs possess unique tyrosine metabolism-related regenerative properties and show significant potential in obstetric diseases. Due to their immunomodulatory, chemotactic migration, and exosome secretion properties, mesenchymal stem cells have been considered a potential treatment strategy for prostate diseases in recent years.

[0004] In view of the fact that there are currently no effective mesenchymal stem cell preparations for treating prostate diseases, this invention provides a mesenchymal stem cell composition for treating prostate diseases, its preparation method and application, to meet the clinical treatment needs of prostate diseases. Summary of the Invention

[0005] This invention relates to a mesenchymal stem cell composition for treating prostate diseases, its preparation method, and its application. Specifically, the technical solution of this invention is implemented as follows:

[0006] In a first aspect, the present invention provides a mesenchymal stem cell composition for treating prostate diseases, characterized in that it comprises mesenchymal stem cells and anti-inflammatory peptides.

[0007] Furthermore, the mesenchymal stem cells are derived from adipose tissue or bone marrow. Preferably, the mesenchymal stem cells are derived from the umbilical cord.

[0008] Furthermore, the mesenchymal stem cells are subjected to hypoxia treatment. Preferably, the oxygen concentration during the hypoxia treatment is 1-5% (v / v).

[0009] Furthermore, the mesenchymal stem cells undergo hypoxia treatment, including the following steps:

[0010] S1. Take mesenchymal stem cells suitable for passage, remove the culture medium, wash with PBS, digest and centrifuge, and inoculate them into pretreated culture medium for 12-48 hours.

[0011] S2. Remove the culture medium, rinse with PBS, add serum-free culture medium, adjust the oxygen concentration of the culture environment to 1-5% (v / v), and treat for 12-48 hours to obtain the final product.

[0012] Furthermore, the inoculation density in step S1 is (1~10)×10 5 cells / mL.

[0013] Furthermore, the number of rinsing cycles in steps S1 and S2 is 1 to 5.

[0014] Furthermore, the culture temperature for the low-oxygen treatment was 37°C.

[0015] Furthermore, the pretreatment medium is DMEM medium containing 8-12% (v / v) FBS and 2-20 ng / mL transforming growth factor β3;

[0016] Furthermore, the serum-free culture medium is StemPro MSC SFM medium containing 1–5 mM L-glutamine.

[0017] Furthermore, the amino acid sequence of the anti-inflammatory peptide is WYPWMKKHHRRRRRRRRR.

[0018] Furthermore, the ratio of mesenchymal stem cells to anti-inflammatory peptides is (1-10) × 10⁻⁶. 6 cells / mL: 1–50 μM. Preferably, the ratio of mesenchymal stem cells to anti-inflammatory peptides is (2–8) × 10⁻⁶. 6 cells / mL: 2–10 μM.

[0019] Secondly, the present invention provides a method for preparing a mesenchymal stem cell composition for treating prostate diseases, characterized in that mesenchymal stem cells and anti-inflammatory peptides are taken and mixed at a ratio of (1-10) × 10⁻⁶. 6 Mix thoroughly at a ratio of 1 to 50 μM (cells / mL); the desired result is obtained.

[0020] Furthermore, the mesenchymal stem cells are derived from the umbilical cord, bone marrow, or adipose tissue. Preferably, the mesenchymal stem cells are derived from the umbilical cord.

[0021] Furthermore, the mesenchymal stem cells undergo hypoxia treatment, including the following steps:

[0022] S1. Take mesenchymal stem cells suitable for passage, remove the culture medium, wash with PBS, digest and centrifuge, and inoculate them into pretreated culture medium for 12-48 hours.

[0023] S2. Remove the culture medium, rinse with PBS, add serum-free culture medium, adjust the oxygen concentration of the culture environment to 1-5% (v / v), and treat for 12-48 hours to obtain the final product.

[0024] Furthermore, the pretreatment medium is DMEM medium containing 8-12% (v / v) FBS and 2-20 ng / mL transforming growth factor β3;

[0025] Furthermore, the serum-free culture medium is StemPro MSC SFM medium containing 1–5 mM L-glutamine.

[0026] Furthermore, the amino acid sequence of the anti-inflammatory peptide is WYPWMKKHHRRRRRRRRR.

[0027] Furthermore, the ratio of mesenchymal stem cells to anti-inflammatory peptides is (1-10) × 10⁻⁶. 6 cells / mL: 1–50 μM. Preferably, the ratio of mesenchymal stem cells to anti-inflammatory peptides is (2–8) × 10⁻⁶. 6 cells / mL: 2–10 μM.

[0028] Thirdly, the present invention provides a mesenchymal stem cell preparation for treating prostate diseases, characterized in that it comprises: mesenchymal stem cells, anti-inflammatory peptides, and pharmaceutically acceptable excipients.

[0029] Furthermore, the mesenchymal stem cells are derived from the umbilical cord, fat, or bone marrow. Preferably, the mesenchymal stem cells are derived from the umbilical cord.

[0030] Furthermore, the mesenchymal stem cells undergo hypoxia treatment, including the following steps:

[0031] S1. Take mesenchymal stem cells suitable for passage, remove the culture medium, wash with PBS, digest and centrifuge, and inoculate them into pretreated culture medium for 12-48 hours.

[0032] S2. Remove the culture medium, rinse with PBS, add serum-free culture medium, adjust the oxygen concentration of the culture environment to 1-5% (v / v), and treat for 12-48 hours to obtain the final product.

[0033] Furthermore, the pretreatment medium is DMEM medium containing 8-12% (v / v) FBS and 2-20 ng / mL transforming growth factor β3;

[0034] Furthermore, the serum-free culture medium is StemPro MSC SFM medium containing 1–5 mM L-glutamine.

[0035] Furthermore, the amino acid sequence of the anti-inflammatory peptide is WYPWMKKHHRRRRRRRRR.

[0036] Fourthly, the present invention provides the use of a mesenchymal stem cell composition for treating prostate diseases in the preparation of a medicament for treating prostate diseases, characterized in that the composition comprises mesenchymal stem cells and anti-inflammatory peptides.

[0037] Furthermore, the mesenchymal stem cells are derived from the umbilical cord, fat, or bone marrow. Preferably, the mesenchymal stem cells are derived from the umbilical cord.

[0038] Furthermore, the mesenchymal stem cells undergo hypoxia treatment, including the following steps:

[0039] S1. Take mesenchymal stem cells suitable for passage, remove the culture medium, wash with PBS, digest and centrifuge, and inoculate them into pretreated culture medium for 12-48 hours.

[0040] S2. Remove the culture medium, rinse with PBS, add serum-free culture medium, adjust the oxygen concentration of the culture environment to 1-5% (v / v), and treat for 12-48 hours to obtain the final product.

[0041] Furthermore, the pretreatment medium is DMEM medium containing 8-12% (v / v) FBS and 2-20 ng / mL transforming growth factor β3;

[0042] Furthermore, the serum-free culture medium is StemPro MSC SFM medium containing 1–5 mM L-glutamine.

[0043] Furthermore, the amino acid sequence of the anti-inflammatory peptide is WYPWMKKHHRRRRRRRRR.

[0044] Furthermore, the ratio of mesenchymal stem cells to anti-inflammatory peptides is (1-10) × 10⁻⁶. 6 cells / mL: 1–50 μM. Preferably, the ratio of mesenchymal stem cells to anti-inflammatory peptides is (2–8) × 10⁻⁶. 6 cells / mL: 2–10 μM.

[0045] Furthermore, the prostate disease mentioned is selected from prostatitis, prostate calcification, sexual function improvement, and urinary frequency and urgency.

[0046] Furthermore, the prostate diseases mentioned are prostate cancer and prostatitis.

[0047] The beneficial effects of this invention are as follows: the combination of hypoxic-treated umbilical cord mesenchymal stem cells (Lo-hBMSCs) and anti-inflammatory peptides (WYPWMKKHHRRRRRRRRR) can significantly inhibit the cell activity of human prostate inflammatory cells and suppress the expression of pro-inflammatory factors IL-2, IL-6, and TNF-α in prostate inflammatory cells. The combination of hypoxic-treated umbilical cord mesenchymal stem cells (Lo-hBMSCs) and anti-inflammatory peptides (WYPWMKKHHRRRRRRRRR) can improve symptoms of various prostate diseases such as prostatitis, prostate calcification, sexual function improvement, and urinary frequency and urgency, demonstrating significant therapeutic effects and representing a significant advancement. Attached Figure Description

[0048] Figure 1 A graph showing the survival rate of inflammatory cells in the human prostate.

[0049] Figure 2 This is a graph showing the relative expression levels of inflammatory factors in prostate cells. Detailed Implementation

[0050] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0051] The human umbilical cord mesenchymal stem cells (hBMSCs), P3-5 passages, were purchased from Shanghai Yubo Biotechnology Co., Ltd., catalog number AE-408. StemPro™ MSC SFM culture medium was purchased from Gibco, catalog number A1033201. The anti-inflammatory peptide (WYPWMKKHHRRRRRRRRR) in this invention was purchased from Shanghai Hongtai Biotechnology Co., Ltd.

[0052] Example 1: Preparation of hypoxia-treated mesenchymal stem cells (Lo-hBMSCs)

[0053] The method for preparing hypoxic mesenchymal stem cells includes the following steps:

[0054] S1. Obtain suitable human bone marrow mesenchymal stem cells for passage, aspirate the culture medium, wash with PBS, digest and centrifuge, and then... (The sentence is incomplete and requires more context to translate accurately). 5 The cells / mL were seeded in the pretreated medium and cultured for 24 hours.

[0055] S2. Remove the culture medium, wash with PBS, add serum-free culture medium, adjust the oxygen concentration of the culture environment to 3% (v / v), and treat for 24 h to obtain hypoxic mesenchymal stem cells (Lo-hBMSCs);

[0056] The pretreatment medium was DMEM medium containing 10% (v / v) FBS and 10 ng / mL transforming growth factor β3;

[0057] The serum-free culture medium was StemPro MSC SFM medium containing 2 mM L-glutamine.

[0058] Example 2: Detection of the viability of inflammatory cells in the human prostate

[0059] Human normal prostate epithelial cells RWPE-1 were used to simulate inflammatory prostatitis using a lipopolysaccharide (LPS)-induced inflammation model. RWPE-1 cell lines were treated with the following groups as follows. Cell viability was assessed using a CCK8 assay kit at 24 and 48 hours. Cell viability was calculated with the control group's cell viability as a reference of 100%.

[0060] Blank group: K-SFM medium.

[0061] Model group: K-SFM medium supplemented with 6 μg / mL LPS.

[0062] Experimental group: K-SFM medium supplemented with 6 μg / mL LPS, and 5 × 10⁻⁶ LPS. 6 Lo-hBMSCs per cell / mL and 5 μM anti-inflammatory peptide; the amino acid sequence of the anti-inflammatory peptide is WYPWMKKHHRRRRRRRRR.

[0063] Control group 1: K-SFM medium with 6 μg / mL LPS added, along with 5 × 10⁻⁶ LPS. 6 Human bone marrow mesenchymal stem cells (hBMSCs) at a concentration of cells / mL and 5 μM of anti-inflammatory peptide; the amino acid sequence of the anti-inflammatory peptide is WYPWMKKHHRRRRRRRRR. The difference between this group and the experimental group is that the human bone marrow mesenchymal stem cells were not subjected to the hypoxia treatment described above.

[0064] Comparison Group 2: K-SFM medium supplemented with 6 μg / mL LPS, and Lo-hBMSCs prepared in Example 1 at a concentration of 5 × 10⁻⁶. 6 cells / mL. The difference between this group and the experimental group is that this group does not contain anti-inflammatory peptides.

[0065] Figure 1 The results showed that the combination of Lo-hBMSCs and anti-inflammatory peptides significantly improved the activity of RWPE-1 cell line, resulting in increased cell survival. Comparison groups 1 and 2 also improved the cell activity of RWPE-1 cell line, but the cell survival rate was significantly different compared to the experimental group (p<0.01).

[0066] Example 3: Detection of Inflammatory Markers in Human Prostate Cells

[0067] We used normal human prostatic epithelial cells RWPE-1 and lipopolysaccharide (LPS) to induce an inflammatory model to simulate inflammatory prostatitis. After 24 hours of treatment according to the following groups, the expression levels of relevant inflammatory genes were detected by qRT-PCR.

[0068] Blank group: K-SFM medium.

[0069] Model group: K-SFM medium supplemented with 8 μg / mL LPS.

[0070] Experimental group: K-SFM medium supplemented with 8 μg / mL LPS, and 5 × 10⁻⁶ LPS. 6 Lo-hBMSCs per cell / mL and 5 μM anti-inflammatory peptide; the amino acid sequence of the anti-inflammatory peptide is WYPWMKKHHRRRRRRRRR.

[0071] Control group 1: K-SFM medium with 8 μg / mL LPS added, along with 5 × 10⁻⁶ LPS. 6 Human bone marrow mesenchymal stem cells (hBMSCs) at a concentration of cells / mL and 5 μM of anti-inflammatory peptide; the amino acid sequence of the anti-inflammatory peptide is WYPWMKKHHRRRRRRRRR. The difference between this group and the experimental group is that the human bone marrow mesenchymal stem cells were not subjected to the hypoxia treatment described above.

[0072] Comparison Group 2: K-SFM medium supplemented with 8 μg / mL LPS, and Lo-hBMSCs prepared in Example 1 at a concentration of 5 × 10⁻⁶. 6 cells / mL. The difference between this group and the experimental group is that this group does not contain anti-inflammatory peptides.

[0073] Figure 2This indicates that the combination of Lo-hBMSCs and anti-inflammatory peptides can significantly inhibit the relative expression levels of inflammatory factors IL-2, IL-6, and TNF-α in LPS-induced prostatitis cells. While groups 1-2 also exhibited anti-inflammatory effects, their efficacy was far less than that of the experimental group (p<0.05).

[0074] Example 4: Validation of the mouse model effect

[0075] 0.2 mL of a concentration of 1×10 6 RM1 cell suspension (cells / mL) was implanted subcutaneously into the axillary region of C57BL / 6 mice. Successful modeling was indicated by a tumor diameter ≥0.5cm in the mice.

[0076] Mice that successfully developed the model were randomly divided into four groups: model group, experimental group, control group 1, and control group 2. The control group and model group were given physiological saline, while the experimental group was given a saline solution with a concentration of 5 × 10⁻⁶. 6 The concentrations of Lo-hBMSCs (5 μM) and anti-inflammatory peptides were compared with those in group 1, which were 5 × 10⁻⁶ cells / mL. 6 The two groups were compared by administering 5 × 10 cells / mL, 5 μM hBMSCs, and anti-inflammatory peptides. 6 Lo-hBMSCs were administered via intraperitoneal injection at a volume of 0.2 mL, once daily for four consecutive weeks.

[0077] The prostate cancer tumor in the axillary region was surgically dissected and dissected, and its mass was measured. The spleen and thymus were dissected, and the spleen index and thymus index were calculated. The results are shown in Tables 1 and 2.

[0078] Table 1. Tumor weight in each group (unit: g, n=8)

[0079]

[0080] *: p < 0.05 compared with the model group; #: p < 0.05 compared with control group 1; &: p < 0.05 compared with control group 2.

[0081] Table 2. Spleen index and thymus index of each group (unit: mg / g, n=8)

[0082]

[0083] *: Compared with the control group, p<0.05; #: Compared with the model group, p<0.05; &: Compared with control group 1, p<0.05; $: Compared with control group 2, p<0.05.

[0084] Using a mouse model, it was confirmed that the combination of Lo-hBMSCs and anti-inflammatory peptides in this invention can improve spleen and thymus indices. Compared with control groups 1-2, the synergistic effect of hypoxic-treated hBMSCs and anti-inflammatory peptides significantly improved the therapeutic effect of prostate diseases.

[0085] The basic principles, main features, and advantages of this invention have been described above. Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made without departing from the spirit and scope of the invention, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection claimed by this invention is defined by the appended claims and their equivalents.

Claims

1. Use of a mesenchymal stem cell composition for treating prostatitis in the manufacture of a medicament for treating prostatitis, characterized in that, The mesenchymal stem cell composition comprises mesenchymal stem cells and an anti-inflammatory peptide; The amino acid sequence of the anti-inflammatory peptide is WYPWMKKHHRRRRRRRRR; The mesenchymal stem cells are derived from human bone marrow; The mesenchymal stem cells are subjected to hypoxic treatment, comprising the following steps: S1, taking human bone marrow mesenchymal stem cells suitable for subculture, aspirating the culture medium, rinsing with PBS, digesting and centrifuging, inoculating in a pretreatment culture medium, and culturing for 12-48 h; S2, aspirating the culture medium, rinsing with PBS, adding a serum-free culture medium, adjusting the oxygen concentration of the culture environment to 1-5% (v / v), and treating for 12-48 h to obtain mesenchymal stem cells subjected to hypoxic treatment; The pretreatment culture medium is a DMEM culture medium containing 8-12% (v / v) FBS and 2-20 ng / mL transforming growth factor β3; The serum-free culture medium is a StemPro MSC SFM culture medium containing 1-5 mM L-glutamine; The use amount ratio of the mesenchymal stem cells and the anti-inflammatory peptide is (1-10) x 10 6 cells / mL: 1-50 μM.

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