Umbilical cord mesenchymal stem cell preparation and application thereof in treatment of prostate diseases

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an umbilical cord mesenchymal stem cell preparation and application thereof in treating prostate diseases. According to the umbilical cord mesenchymal stem cell preparation, the umbilical cord mesenchymal stem cells of the third generation to the sixth generation and the neurofilament peptide serve as active components, pharmaceutically acceptable auxiliary materials are matched, the prostate index can be remarkably reduced, the concentration of PACP and SRD5A2 can be reduced, and the umbilical cord mesenchymal stem cell preparation has a remarkable curative effect on prostatic hyperplasia. And the umbilical cord mesenchymal stem cells of the third generation to the sixth generation and the neurofilament peptide supplement each other and have a synergistic effect, so that a remarkable progress is generated.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, relates to stem cell preparations, and particularly relates to a human umbilical cord mesenchymal stem cell preparation and its use in the treatment of prostate diseases. Background Art

[0002] Human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs) are a type of pluripotent stem cells present in the umbilical cord tissue of newborns. hUC-MSCs have multi-directional differentiation potential and can differentiate into various cell types such as osteoblasts, chondrocytes, muscle cells, and nerve cells. They can also regulate immune responses by secreting a variety of bioactive factors and have anti-inflammatory and immunosuppressive properties. hUC-MSCs play a role in tissue injury repair and can promote the regeneration and repair of damaged tissues. Compared with adult-derived mesenchymal stem cells, hUC-MSCs have lower immunogenicity, which means they are less likely to cause immune rejection reactions in the host during transplantation. Compared with stem cells derived from bone marrow or adipose tissue, the acquisition process of hUC-MSCs is simpler and is usually extracted from the umbilical cord after the birth of a newborn, causing no harm to the donor. Due to their multi-directional differentiation potential, low immunogenicity, rich sources, and few ethical controversies, umbilical cord mesenchymal stem cells are regarded as a "treasury of mesenchymal stem cells" and have achieved clinical research results in the treatment of various diseases, showing great application potential in regenerative medicine and tissue engineering.

[0003] Benign Prostatic Hyperplasia (BPH) refers to a non-cancerous lesion in which the number of prostate cells increases, leading to an enlarged prostate volume. It is a common disease in elderly men and mainly affects middle-aged and elderly men. The exact cause of BPH is not fully understood, but it is related to age, hormone levels, and genetic factors. The symptoms of BPH may include frequent urination, urgency, nocturia, difficulty urinating, a thinner urine stream, and dribbling. These symptoms may worsen as the prostate volume increases. The treatment methods for BPH include medical treatment and surgical treatment. Medical treatment mainly includes α-receptor antagonists (such as terazosin) and 5α-reductase inhibitors (such as finasteride), which can relieve symptoms and reduce the prostate volume. If BPH is not treated in a timely manner, it may lead to complications such as urinary tract obstruction, bladder function impairment, and renal function damage.

[0004] Current research shows that mesenchymal stem cells have potential therapeutic value in the treatment of benign prostatic hyperplasia. Mesenchymal stem cells have immunomodulatory and anti-inflammatory properties and can reduce the inflammatory response by secreting anti-inflammatory cytokines. Mesenchymal stem cells can promote tissue repair and inhibit apoptosis, which may be beneficial for the repair of prostate tissue. Mesenchymal stem cells may affect the development of benign prostatic hyperplasia and prostate cancer by regulating the prostate microenvironment. Importantly, the low immunogenicity of mesenchymal stem cells makes them less likely to cause immune rejection during transplantation, providing safety assurance for clinical applications.

[0005] Currently, the treatment of benign prostatic hyperplasia mainly relies on drugs, but the treatment of benign prostatic hyperplasia with mesenchymal stem cell preparations is still in a blank stage. There is an urgent need to develop an effective mesenchymal stem cell preparation to meet the clinical treatment needs of benign prostatic hyperplasia. Summary of the Invention

[0006] Based on the deficiencies in the prior art, the present invention provides a umbilical cord mesenchymal stem cell preparation and its use in the treatment of prostate diseases, which can significantly reduce the prostate index, reduce the concentrations of PACP and SRD5A2, and have a significant therapeutic effect on benign prostatic hyperplasia.

[0007] To achieve the above object, the technical solution of the present invention is realized as follows: In a first aspect, the present invention provides a umbilical cord mesenchymal stem cell composition comprising the following active components: umbilical cord mesenchymal stem cells of passages 3-6, and neurofilament peptide.

[0008] Further, the mass ratio of the umbilical cord mesenchymal stem cells to the neurofilament peptide is 3-5:1.

[0009] Further, the amino acid sequence of the neurofilament peptide is IDKQVLSRIKLEIRCL (SEQ ID NO.1).

[0010] Further, the method for isolating the umbilical cord mesenchymal stem cells comprises the following steps: S1. Take 8-12 cm of fresh human umbilical cord, rinse it with PBS to remove residual blood and impurities; S2. Cut the umbilical cord into small segments of 2-3 cm, rinse it again with PBS, longitudinally cut open the umbilical cord, and strip Wharton's jelly; S3. Cut the stripped Wharton's jelly into tissue fragments of 1 mm 3 and place them in a culture flask containing a culture medium, and statically culture them in an incubator at 37 °C and 5% CO 2 ; the culture flask is pre-coated with polylysine; the culture medium is DMEM medium supplemented with 10-12% FBS, 100 U / mL penicillin, and 100 mg / L streptomycin. S4. Observe the cell growth condition with an inverted microscope every day. Perform a full medium change for the first time after 5 - 7 days, and then perform a half medium change every 2 - 3 days thereafter. When the cell confluence reaches 80 - 90%, digest and passage the cells, and inoculate them into a culture flask containing the culture medium. When the cells grow to a confluence of 80% again, the umbilical cord mesenchymal stem cells of the first passage are obtained.

[0011] Digest and passage the umbilical cord mesenchymal stem cells of the first passage with 0.25% trypsin, and inoculate them into a culture flask containing the culture medium. When the cells grow to a confluence of 80% again, the umbilical cord mesenchymal stem cells of the second passage are obtained. The umbilical cord mesenchymal stem cells of the 3rd - 6th passages can be obtained successively by using the same culture steps as described above.

[0012] In a second aspect, the present invention provides an umbilical cord mesenchymal stem cell preparation, comprising: umbilical cord mesenchymal stem cells of the 3rd - 6th passages, neurofilament peptide, and pharmaceutically acceptable excipients.

[0013] Furthermore, the mass ratio of the umbilical cord mesenchymal stem cells to the neurofilament peptide is 3 - 5:1.

[0014] Furthermore, the amino acid sequence of the neurofilament peptide is IDKQVLSRIKLEIRCL (SEQ ID NO.1).

[0015] Furthermore, the separation method of the umbilical cord mesenchymal stem cells comprises the following steps: S1. Take 8 - 12 cm of fresh human umbilical cord, rinse it with PBS to remove residual blood and impurities. S2. Cut the umbilical cord into small segments of 2 - 3 cm, rinse it with PBS again, longitudinally cut open the umbilical cord, and strip the Wharton's jelly. S3. Cut the stripped Wharton's jelly into tissue fragments of 1 mm 3 , place them in a culture flask containing the culture medium, and statically culture them in an incubator at 37°C and 5% CO 2 . The culture flask is pre - coated with polylysine. The culture medium is DMEM medium supplemented with 10 - 12% FBS, 100 U / mL penicillin, and 100 mg / L streptomycin. S4. Observe the cell growth condition with an inverted microscope every day. Perform a full medium change for the first time after 5 - 7 days, and then perform a half medium change every 2 - 3 days thereafter. When the cell confluence reaches 80 - 90%, digest and passage the cells, and inoculate them into a culture flask containing the culture medium. When the cells grow to a confluence of 80% again, the umbilical cord mesenchymal stem cells of the first passage are obtained.

[0016] The umbilical cord mesenchymal stem cells of the first passage were digested and passaged with 0.25% trypsin, and inoculated into a culture flask containing a culture medium. When the cells grew to a confluence of 80% again, the umbilical cord mesenchymal stem cells of the second passage were obtained; the umbilical cord mesenchymal stem cells of the 3rd to 6th passages can be obtained in turn by using the same culture steps as described above.

[0017] Thirdly, the present invention provides an application of an umbilical cord mesenchymal stem cell preparation in the preparation of a drug for treating prostate diseases.

[0018] Furthermore, the umbilical cord mesenchymal stem cell preparation includes: umbilical cord mesenchymal stem cells of the 3rd to 6th passages, neurofilament peptide, and pharmaceutically acceptable excipients.

[0019] Furthermore, the mass ratio of the umbilical cord mesenchymal stem cells to the neurofilament peptide is 3 - 5:1.

[0020] Furthermore, the amino acid sequence of the neurofilament peptide is IDKQVLSRIKLEIRCL (SEQ ID NO.1).

[0021] Furthermore, the separation method of the umbilical cord mesenchymal stem cells includes the following steps: S1. Take 8 - 12 cm of fresh human umbilical cord, rinse it with PBS to remove residual blood and impurities; S2. Cut the umbilical cord into small segments of 2 - 3 cm, rinse it with PBS again, longitudinally cut open the umbilical cord, and peel off Wharton's jelly; S3. Cut the peeled Wharton's jelly into tissue fragments of 1 mm 3 , place them in a culture flask containing a culture medium, and statically culture them in an incubator at 37°C and 5% CO 2 ; the culture flask is pre-coated with polylysine; the culture medium is DMEM medium supplemented with 10 - 12% FBS, 100 U / mL penicillin, and 100 mg / L streptomycin; S4. Observe the cell growth condition under an inverted microscope every day. Perform a full medium change for the first time after 5 - 7 days, and then perform a half medium change every 2 - 3 days; when the cells grow to a confluence of 80 - 90%, digest and passage them, and inoculate them into a culture flask containing a culture medium. When the cells grow to a confluence of 80% again, the umbilical cord mesenchymal stem cells of the first passage are obtained.

[0022] The umbilical cord mesenchymal stem cells of the first passage were digested and passaged with 0.25% trypsin, and inoculated into a culture flask containing a culture medium. When the cells grew to a confluence of 80% again, the umbilical cord mesenchymal stem cells of the second passage were obtained; the umbilical cord mesenchymal stem cells of the 3rd to 6th passages can be obtained in turn by using the same culture steps as described above.

[0023] Furthermore, the prostate disease is prostate hyperplasia.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: The umbilical cord mesenchymal stem cell composition of the present invention can significantly reduce the prostate index, reduce the concentrations of PACP and SRD5A2, and has a significant therapeutic effect on benign prostatic hyperplasia. The umbilical cord mesenchymal stem cell composition of the present invention takes umbilical cord mesenchymal stem cells and neurofilament peptide as the main active ingredients, and the two synergistically enhance the effect. Preparing the umbilical cord mesenchymal stem cell composition of the present invention into an umbilical cord mesenchymal stem cell preparation by combining with pharmaceutically acceptable excipients can supplement the shortage of drugs for the application of stem cell preparations in the treatment of benign prostatic hyperplasia, and has significant progress. Detailed implementation manners

[0025] The present invention will be further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0026] The neurofilament peptide (SEQ ID NO.1) in the present invention is obtained by repeated improvement through a large number of experiments based on the neurofilament peptide in WO2017 / 216779.

[0027] Experimental example 1 Isolation and identification of human umbilical cord mesenchymal stem cells Take the umbilical cord of a healthy full-term pregnant woman in the obstetrics and gynecology department of the hospital, and obtain the informed consent of the parturient and her family members.

[0028] The isolation method of human umbilical cord mesenchymal stem cells includes the following steps: S1. Take 10 cm of fresh human umbilical cord, rinse it with PBS to remove residual blood and impurities; S2. Cut the umbilical cord into 2-cm segments, rinse it with PBS again, longitudinally cut open the umbilical cord, and peel off Wharton's jelly; S3. Cut the peeled Wharton's jelly into 1-mm 3 tissue fragments, place them in a culture flask containing a culture medium, and statically culture them in an incubator at 37°C and 5% CO 2 ; the culture flask is pre-coated with polylysine; the culture medium is DMEM medium supplemented with 12% FBS, 100 U / mL penicillin, and 100 mg / L streptomycin; S4. Observe the cell growth condition every day under an inverted microscope. Perform a full medium change for the first time after 6 days, and then perform a half medium change every 2 days; when the cell growth reaches 80% confluence, digest and passage with 0.25% trypsin, and inoculate it into a T25 culture flask containing a culture medium. When the cells grow to 80% confluence again, the first-generation umbilical cord mesenchymal stem cells are obtained.

[0029] The umbilical cord mesenchymal stem cells of the 1st generation were digested and passaged with 0.25% trypsin and inoculated into a T25 culture flask containing a culture medium. When the cells grew to a confluence of 80% again, the umbilical cord mesenchymal stem cells of the 2nd generation were obtained. The umbilical cord mesenchymal stem cells of the 3rd - 6th generations could be obtained successively by using the same culture steps.

[0030] Through morphological observation, the isolated umbilical cord mesenchymal stem cells were adherent cells, and the cell morphology was fibroblast - like. The flow cytometry results showed that the umbilical cord mesenchymal stem cells of the 1st - 2nd generations were positive for CD29, CD90, and CD105, and expressed CD19, CD34, and CD45 to a certain extent. The umbilical cord mesenchymal stem cells of the 3rd - 6th generations were positive for CD29, CD90, and CD105 and negative for CD19, CD34, and CD45, which was in line with the identification of the surface markers of umbilical cord mesenchymal stem cells.

[0031] The in vitro culture period of primary umbilical cord mesenchymal stem cells was limited. For the umbilical cord mesenchymal stem cells beyond the 6th generation, most of the cell morphologies changed from fibroblast - like to irregular or polygonal, and cell differentiation led to a significant reduction in differentiation potential.

[0032] Therefore, the present invention uses the umbilical cord mesenchymal stem cells of the 3rd - 6th generations as the active component to ensure the curative effect.

[0033] Experimental Example 2 Preparation of Umbilical Cord Mesenchymal Stem Cell Composition Example 1: Take the umbilical cord mesenchymal stem cells of the 3rd generation prepared in Experimental Example 1 and neuropeptide, and mix them evenly according to a mass ratio of 4:1 to obtain an umbilical cord mesenchymal stem cell composition; the amino acid sequence of the neuropeptide is IDKQVLSRIKLEIRCL (SEQ ID NO.1).

[0034] Comparative Example 1: Take the umbilical cord mesenchymal stem cells of the 3rd generation prepared in Experimental Example 1 and neuropeptide, and mix them evenly according to a mass ratio of 3:1 to obtain an umbilical cord mesenchymal stem cell composition; the amino acid sequence of the neuropeptide is IDQQVLSRIKLEIKRCL (SEQ ID NO.2).

[0035] Comparative Example 2: Take the umbilical cord mesenchymal stem cells of the 3rd generation prepared in Experimental Example 1.

[0036] Experimental Example 3 Verification of the Effect on Rats with Benign Prostatic Hyperplasia Model Forty male SD rats with a body weight of 190 - 220 g were randomly divided into 5 groups, with 8 rats in each group. Breeding conditions: 23 ± 2°C, humidity 55 ± 5%, lighting time 12 hours / day, free diet and water. After 1 - week of adaptive breeding, the experiment was carried out. Each group was carried out as follows: Blank group: Subcutaneously inject 5 mg / kg of normal saline every day, and intraperitoneally inject 150 mg / kg of an aqueous solution of 5% PEG-400 30 minutes later for 4 consecutive weeks; Model group: Subcutaneously inject 5 mg / kg of testosterone propionate every day, and intraperitoneally inject 150 mg / kg of an aqueous solution of 5% PEG-400 30 minutes later for 4 consecutive weeks; Example 1 group: Dissolve the umbilical cord mesenchymal stem cell composition of Example 1 with an aqueous solution of 5% PEG-400 at a concentration of 40% (m / v), denoted as the test solution of Example 1. Subcutaneously inject 5 mg / kg of testosterone propionate every day, and intraperitoneally inject 150 mg / kg of the test solution of Example 1 30 minutes later for 4 consecutive weeks; Control group 1: Dissolve the umbilical cord mesenchymal stem cell composition of Control Example 1 with an aqueous solution of 5% PEG-400 at a concentration of 40% (m / v), denoted as the test solution of Control Example 1. Subcutaneously inject 5 mg / kg of testosterone propionate every day, and intraperitoneally inject 150 mg / kg of the test solution of Control Example 1 30 minutes later for 4 consecutive weeks; Control group 2: Dissolve the umbilical cord mesenchymal stem cells of Control Example 2 with an aqueous solution of 5% PEG-400 at a concentration of 40% (m / v), denoted as the test solution of Control Example 2. Subcutaneously inject 5 mg / kg of testosterone propionate every day, and intraperitoneally inject 150 mg / kg of the test solution of Control Example 2 30 minutes later for 4 consecutive weeks.

[0037] 24 hours after the last administration, collect blood from the eyeballs of rats in each group, and measure the levels of prostate acid phosphatase (PACP) and type II 5-α-reductase (SRD5A2) in the serum using a kit. The results are shown in Table 1; Dissect the prostate and weigh it wet, and calculate the prostate index (wet weight of the prostate / body weight). The results are shown in Table 2.

[0038] Table 1 Serum PACP and SRD5A2 levels

[0039] ## Compared with the model group, p < 0.01; # Compared with the model group, p < 0.05; * Compared with the Example 1 group, p < 0.05.

[0040] Table 2 Prostate index

[0041] # Compared with the model group, p < 0.01; * Compared with the Example 1 group, p < 0.05.

[0042] The results showed that the groups of Example 1, Comparative Examples 1-2 could significantly reduce the prostate index, lower the concentrations of PACP and SRD5A2, and had a significant improvement effect on the prostate hyperplasia caused by testosterone propionate. Moreover, the curative effect of the group of Example 1 was significantly better than that of the groups of Comparative Examples 1-2, confirming the synergistic effect of the combination of umbilical cord mesenchymal stem cells and neurofilament peptide (SEQ ID NO.1).

[0043] The basic principles, main features and advantages of the present invention have been described above. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification is only the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection required by the present invention is defined by the appended claims and their equivalents.

Claims

1. An umbilical cord mesenchymal stem cell composition, comprising the following active ingredients: umbilical cord mesenchymal stem cells of passages 3 to 6, and neurofilament peptide; the amino acid sequence of the neurofilament peptide is IDKQVLSRIKLEIRCL.

2. The composition according to claim 1, characterized in that The mass ratio of the umbilical cord mesenchymal stem cells to the neurofilament peptide is 3-5:

1.

3. The composition according to claim 1, characterized in that: The method for isolating umbilical cord mesenchymal stem cells comprises the following steps: S1. Take a fresh human umbilical cord (8-12 cm), rinse it with PBS to remove residual blood and impurities; S2. Cut the umbilical cord into small pieces of 2-3 cm, rinse with PBS again, cut the umbilical cord longitudinally, and remove Wharton's jelly; S3, cutting the peeled Wharton's jelly into 1 mm3 tissue fragments, placing them in a culture bottle containing culture medium, and culturing them in an incubator at 37°C and 5% CO2; the culture bottle is pre-coated with poly-lysine; the culture medium is DMEM medium supplemented with 10-12% FBS, 100U / mL penicillin, and 100mg / L streptomycin; S4. Observe the cell growth every day under an inverted microscope. Perform the first full medium change after 5 to 7 days, and perform half medium change every 2 to 3 days thereafter. When the cells grow to a confluence of 80 to 90%, digest and subculture them, and inoculate them into culture bottles containing culture medium. When the cells grow to a confluence of 80% again, the first generation of umbilical cord mesenchymal stem cells are obtained.

4. An umbilical cord mesenchymal stem cell preparation, comprising: Umbilical cord mesenchymal stem cells of the 3rd to 6th generations, neurofilament peptide, and pharmaceutically acceptable excipients; the amino acid sequence of the neurofilament peptide is IDKQVLSRIKLEIRCL.

5. The preparation according to claim 4, characterized in that: The mass ratio of the umbilical cord mesenchymal stem cells to the neurofilament peptide is 3-5:

1.

6. Use of the umbilical cord mesenchymal stem cell preparation according to claim 4 in the preparation of a medicament for treating prostate diseases.

7. The use according to claim 6, characterized in that: The mass ratio of the umbilical cord mesenchymal stem cells to the neurofilament peptide is 3-5:

1.

8. The use according to claim 7, characterized in that: The prostate disease is prostate hyperplasia.

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