Umbilical cord mesenchymal stem cell preparation and its use in treating prostate diseases

Abstract

The present invention belongs to the field of biomedicine and specifically relates to an umbilical cord mesenchymal stem cell preparation and its use in treating prostate diseases. This preparation, which utilizes 3rd to 6th generation umbilical cord mesenchymal stem cells and neurofilament peptide as active ingredients, along with pharmaceutically acceptable excipients, can significantly reduce the prostate index, PACP, and SRD5A2 concentrations, demonstrating significant therapeutic efficacy in treating prostate hyperplasia. Furthermore, the 3rd to 6th generation umbilical cord mesenchymal stem cells and neurofilament peptide complement each other, creating a synergistic effect that yields significant improvements.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to stem cell preparations, and specifically to an umbilical cord mesenchymal stem cell preparation and its use in treating prostate diseases. Background Art

[0002] Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a type of multipotent stem cell found in the umbilical cord tissue of newborns. hUC-MSCs possess multipotential differentiation potential, capable of differentiating into various cell types, including osteoblasts, chondrocytes, muscle cells, and neurons. They also regulate immune responses by secreting a variety of bioactive factors, exhibiting anti-inflammatory and immunosuppressive properties. hUC-MSCs play a role in tissue repair, promoting the regeneration and repair of damaged tissue. Compared to adult-derived mesenchymal stem cells, hUC-MSCs are less immunogenic, meaning they are less likely to provoke immune rejection in the host upon transplantation. Compared to stem cells derived from bone marrow or adipose tissue, hUC-MSCs are simpler to obtain and are typically extracted from the umbilical cord of newborns after birth, harming the donor. Umbilical cord mesenchymal stem cells are regarded as a "treasure trove of mesenchymal stem cells" due to their multidirectional differentiation potential, low immunogenicity, abundant sources and few ethical controversies. They have achieved clinical research results in the treatment of various diseases and have shown great application potential in regenerative medicine and tissue engineering.

[0003] Benign prostatic hyperplasia (BPH) refers to a non-cancerous condition in which the number of prostate cells increases, leading to an enlarged prostate. It is a common aging disease in men, primarily affecting middle-aged and older men. The exact cause of BPH is not fully understood, but it is associated with age, hormone levels, and genetic factors. Symptoms of BPH may include frequent urination, urgency, increased nocturia, difficulty urinating, a thin urine stream, and dribbling. These symptoms may worsen as the prostate enlarges. Treatment options for BPH include medication and surgery. Medication primarily includes alpha-receptor antagonists (such as terazosin) and 5α-reductase inhibitors (such as finasteride), which can relieve symptoms and reduce prostate size. If left untreated, BPH can lead to complications such as urinary tract obstruction, bladder impairment, and kidney damage.

[0004] Current research suggests that mesenchymal stem cells (MSCs) have potential therapeutic value in the treatment of benign prostatic hyperplasia (BPH). MSCs possess immunomodulatory and anti-inflammatory properties, capable of reducing inflammatory responses by secreting anti-inflammatory cytokines. MSCs can promote tissue repair and inhibit apoptosis, which may be beneficial for prostate tissue repair. MSCs may influence the development of benign prostatic hyperplasia (BPH) and prostate cancer by regulating the prostate microenvironment. Importantly, the low immunogenicity of MSCs makes them less likely to induce immune rejection upon transplantation, providing a safety guarantee for clinical application.

[0005] Currently, the treatment for benign prostatic hyperplasia (BPH) mainly relies on drugs, but the use of mesenchymal stem cell preparations for the treatment of BPH 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 BPH. Summary of the Invention

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

[0007] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0008] In a first aspect, the present invention provides 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.

[0009] Furthermore, the mass ratio of the umbilical cord mesenchymal stem cells to the neurofilament peptide is 3-5:1.

[0010] Furthermore, the amino acid sequence of the neurofilament peptide is IDKQVLSRIKLEIRCL (SEQ ID NO. 1).

[0011] Furthermore, the method for isolating umbilical cord mesenchymal stem cells comprises the following steps:

[0012] S1. Take 8-12 cm of fresh human umbilical cord and rinse it with PBS to remove residual blood and impurities;

[0013] S2. Cut the umbilical cord into 2-3 cm segments, rinse again with PBS, cut the umbilical cord longitudinally, and remove Wharton's jelly.

[0014] S3. Cut the peeled Wharton's glue into 1mm pieces 3The tissue fragments were placed in a culture flask containing culture medium and cultured in a 37°C, 5% CO2 incubator; the culture flask was pre-coated with poly-lysine; the culture medium was DMEM supplemented with 10-12% FBS, 100 U / mL penicillin, and 100 mg / L streptomycin;

[0015] S4. Observe cell growth daily under an inverted microscope. Perform the first full medium change after 5-7 days, and perform a partial medium change every 2-3 days thereafter. When the cells grow to a confluence of 80-90%, digest and passage them, and inoculate them into culture flasks containing culture medium. When the cells grow to a confluence of 80% again, the first generation of umbilical cord mesenchymal stem cells is obtained.

[0016] The first generation of umbilical cord mesenchymal stem cells were digested and passaged with 0.25% trypsin and inoculated into a culture flask containing culture medium. When the cells grew again to a confluence of 80%, the second generation of umbilical cord mesenchymal stem cells were obtained. The same culture steps as above were used to obtain the third to sixth generations of umbilical cord mesenchymal stem cells.

[0017] In a second aspect, the present invention provides an umbilical cord mesenchymal stem cell preparation, comprising: umbilical cord mesenchymal stem cells of passages 3 to 6, neurofilament peptide, and pharmaceutically acceptable excipients.

[0018] Furthermore, the mass ratio of the umbilical cord mesenchymal stem cells to the neurofilament peptide is 3-5:1.

[0019] Furthermore, the amino acid sequence of the neurofilament peptide is IDKQVLSRIKLEIRCL (SEQ ID NO. 1).

[0020] Furthermore, the method for isolating umbilical cord mesenchymal stem cells comprises the following steps:

[0021] S1. Take 8-12 cm of fresh human umbilical cord and rinse it with PBS to remove residual blood and impurities;

[0022] S2. Cut the umbilical cord into 2-3 cm segments, rinse again with PBS, cut the umbilical cord longitudinally, and remove Wharton's jelly.

[0023] S3. Cut the peeled Wharton's glue into 1mm pieces 3 The tissue fragments were placed in a culture flask containing culture medium and cultured in a 37°C, 5% CO2 incubator; the culture flask was pre-coated with poly-lysine; the culture medium was DMEM supplemented with 10-12% FBS, 100 U / mL penicillin, and 100 mg / L streptomycin;

[0024] S4. Observe cell growth daily under an inverted microscope. Perform the first full medium change after 5-7 days, and perform a partial medium change every 2-3 days thereafter. When the cells grow to a confluence of 80-90%, digest and passage them, and inoculate them into culture flasks containing culture medium. When the cells grow to a confluence of 80% again, the first generation of umbilical cord mesenchymal stem cells is obtained.

[0025] The first generation of umbilical cord mesenchymal stem cells were digested and passaged with 0.25% trypsin and inoculated into a culture flask containing culture medium. When the cells grew again to a confluence of 80%, the second generation of umbilical cord mesenchymal stem cells were obtained. The same culture steps as above were used to obtain the third to sixth generations of umbilical cord mesenchymal stem cells.

[0026] In a third aspect, the present invention provides a use of an umbilical cord mesenchymal stem cell preparation in the preparation of a drug for treating prostate diseases.

[0027] Furthermore, the umbilical cord mesenchymal stem cell preparation includes: umbilical cord mesenchymal stem cells of passages 3 to 6, neurofilament peptide, and pharmaceutically acceptable excipients.

[0028] Furthermore, the mass ratio of the umbilical cord mesenchymal stem cells to the neurofilament peptide is 3-5:1.

[0029] Furthermore, the amino acid sequence of the neurofilament peptide is IDKQVLSRIKLEIRCL (SEQ ID NO. 1).

[0030] Furthermore, the method for isolating umbilical cord mesenchymal stem cells comprises the following steps:

[0031] S1. Take 8-12 cm of fresh human umbilical cord and rinse it with PBS to remove residual blood and impurities;

[0032] S2. Cut the umbilical cord into 2-3 cm segments, rinse again with PBS, cut the umbilical cord longitudinally, and remove Wharton's jelly.

[0033] S3. Cut the peeled Wharton's glue into 1mm pieces 3 The tissue fragments were placed in a culture flask containing culture medium and cultured in a 37°C, 5% CO2 incubator; the culture flask was pre-coated with poly-lysine; the culture medium was DMEM supplemented with 10-12% FBS, 100 U / mL penicillin, and 100 mg / L streptomycin;

[0034] S4. Observe cell growth daily under an inverted microscope. Perform the first full medium change after 5-7 days, and perform a partial medium change every 2-3 days thereafter. When the cells grow to a confluence of 80-90%, digest and passage them, and inoculate them into culture flasks containing culture medium. When the cells grow to a confluence of 80% again, the first generation of umbilical cord mesenchymal stem cells is obtained.

[0035] The first generation of umbilical cord mesenchymal stem cells were digested and passaged with 0.25% trypsin and inoculated into a culture flask containing culture medium. When the cells grew again to a confluence of 80%, the second generation of umbilical cord mesenchymal stem cells were obtained. The same culture steps as above were used to obtain the third to sixth generations of umbilical cord mesenchymal stem cells.

[0036] Furthermore, the prostate disease is benign prostatic hyperplasia.

[0037] Compared with the prior art, the present invention offers the following benefits: the umbilical cord mesenchymal stem cell composition can significantly reduce the prostate index, PACP, and SRD5A2 concentrations, demonstrating significant therapeutic efficacy for benign prostatic hyperplasia (BPH). The composition comprises umbilical cord mesenchymal stem cells and neurofilament peptide as its primary active ingredients, which synergistically enhance efficacy. Combining the composition with pharmaceutically acceptable excipients to create an umbilical cord mesenchymal stem cell preparation can significantly improve the treatment of BPH by addressing the current shortage of stem cell preparations. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to specific implementation cases, but the present invention is not limited to these embodiments.

[0039] The neurofilament peptide (SEQ ID NO. 1) in the present invention is obtained through repeated improvements based on the neurofilament peptide in WO2017 / 216779 through extensive experiments.

[0040] Experimental Example 1 Isolation and Identification of Human Umbilical Cord Mesenchymal Stem Cells

[0041] The umbilical cords of healthy full-term pregnant women were obtained from the obstetrics and gynecology department of the hospital, and informed consent was obtained from the mothers and their families.

[0042] The method for isolating human umbilical cord mesenchymal stem cells comprises the following steps:

[0043] S1. Take 10 cm of fresh human umbilical cord and rinse it with PBS to remove residual blood and impurities;

[0044] S2. Cut the umbilical cord into 2 cm segments, rinse again with PBS, cut the umbilical cord longitudinally, and remove Wharton's jelly;

[0045] S3. Cut the peeled Wharton's glue into 1mm pieces 3The tissue fragments were placed in a culture flask containing culture medium and cultured in a 37°C, 5% CO2 incubator; the culture flask was pre-coated with poly-lysine; the culture medium was DMEM supplemented with 12% FBS, 100 U / mL penicillin, and 100 mg / L streptomycin;

[0046] S4. Observe cell growth daily under an inverted microscope. Perform the first full medium change after 6 days, and perform a half medium change every 2 days thereafter. When the cells grow to a confluence of 80%, digest them with 0.25% trypsin and passage them. Inoculate them into a T25 culture flask containing culture medium. When the cells grow to a confluence of 80% again, the first generation of umbilical cord mesenchymal stem cells is obtained.

[0047] The first generation of umbilical cord mesenchymal stem cells were digested and passaged with 0.25% trypsin and inoculated into a T25 culture flask containing culture medium. When the cells grew to a confluence of 80% again, the second generation of umbilical cord mesenchymal stem cells were obtained. The same culture steps were used to obtain the third to sixth generations of umbilical cord mesenchymal stem cells.

[0048] Morphological observation revealed that the isolated umbilical cord mesenchymal stem cells were adherent cells with a fibroblast-like morphology. Flow cytometry revealed that passage 1 and 2 umbilical cord mesenchymal stem cells were positive for CD29, CD90, and CD105, with some expression of CD19, CD34, and CD45. Umbilical cord mesenchymal stem cells from passages 3 to 6 were positive for CD29, CD90, and CD105, but negative for CD19, CD34, and CD45, consistent with the identification of surface markers for umbilical cord mesenchymal stem cells.

[0049] The in vitro culture cycle of primary umbilical cord mesenchymal stem cells is limited. The morphology of most umbilical cord mesenchymal stem cells beyond the sixth generation will change from fibroblast-like to irregular or polygonal, and the cells will differentiate, resulting in a significant decrease in differentiation potential.

[0050] Therefore, the present invention uses umbilical cord mesenchymal stem cells of the 3rd to 6th generations as active components to ensure therapeutic efficacy.

[0051] Experimental Example 2 Preparation of Umbilical Cord Mesenchymal Stem Cell Composition

[0052] Example 1: The third generation umbilical cord mesenchymal stem cells and neurofilament peptide prepared in Experimental Example 1 were mixed uniformly at a mass ratio of 4:1 to obtain an umbilical cord mesenchymal stem cell composition; the amino acid sequence of the neurofilament peptide was IDKQVLSRIKLEIRCL (SEQ ID NO. 1).

[0053] Comparative Example 1: The third generation umbilical cord mesenchymal stem cells and neurofilament peptide prepared in Experimental Example 1 were mixed uniformly at a mass ratio of 3:1 to obtain an umbilical cord mesenchymal stem cell composition; the amino acid sequence of the neurofilament peptide was IDQQVLSRIKLEIKRCL (SEQ ID NO. 2).

[0054] Comparative Example 2: The third generation umbilical cord mesenchymal stem cells prepared in Experimental Example 1 were taken.

[0055] Experimental Example 3: Verification of the effect in rats with benign prostatic hyperplasia model

[0056] Forty male Sprague-Dawley rats weighing 190-220 g were randomly divided into five groups of eight rats each. The rats were housed at 23 ± 2°C, 55 ± 5% humidity, and 12 hours of light per day. The rats were given free access to food and water. The experiment was conducted after one week of adaptive feeding. Each group was maintained according to the following procedures:

[0057] Blank group: 5 mg / kg normal saline was injected subcutaneously every day, and 150 mg / kg 5% PEG-400 aqueous solution was injected intraperitoneally 30 minutes later for 4 consecutive weeks;

[0058] Modeling group: testosterone propionate 5 mg / kg was injected subcutaneously every day, and 150 mg / kg of 5% PEG-400 aqueous solution was injected intraperitoneally 30 minutes later for 4 consecutive weeks;

[0059] Group 1: The umbilical cord mesenchymal stem cell composition of Example 1 was dissolved in a 5% aqueous solution of PEG-400 to a concentration of 40% (m / v), designated as the Example 1 test solution. Testosterone propionate (5 mg / kg) was subcutaneously injected daily for 4 consecutive weeks, followed 30 minutes later by an intraperitoneal injection of 150 mg / kg of the Example 1 test solution.

[0060] Comparative Example 1: The umbilical cord mesenchymal stem cell composition of Comparative Example 1 was dissolved in a 5% aqueous solution of PEG-400 to a concentration of 40% (m / v), designated as Comparative Example 1 test solution. Testosterone propionate (5 mg / kg) was subcutaneously injected daily for 4 consecutive weeks, followed by an intraperitoneal injection of 150 mg / kg of Comparative Example 1 test solution 30 minutes later.

[0061] Comparative Example 2: The umbilical cord mesenchymal stem cells from Comparative Example 2 were dissolved in a 5% PEG-400 aqueous solution to a concentration of 40% (m / v), designated as Comparative Example 2 test solution. Testosterone propionate (5 mg / kg) was subcutaneously injected daily for 4 consecutive weeks, followed 30 minutes later by an intraperitoneal injection of 150 mg / kg of Comparative Example 2 test solution.

[0062] 24 hours after the last administration, the eyeballs of the rats in each group were removed and blood was collected. The levels of prostatic acid phosphatase (PACP) and Ⅱ 5-α-reductase (SRD5A2) in the serum were measured using kits. The results are shown in Table 1. The prostate was removed and weighed, and the prostate index was calculated (prostate wet weight / body weight). The results are shown in Table 2.

[0063] Table 1 Serum PACP and SRD5A2 levels

[0064]

[0065] ## 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.

[0066] Table 2 Prostate Index

[0067]

[0068] # Compared with the modeling group, p < 0.01; * Compared with the Example 1 group, p < 0.05.

[0069] The results showed that Example 1 and Comparative Examples 1-2 significantly reduced the prostate index, PACP, and SRD5A2 concentrations, demonstrating a significant improvement in testosterone propionate-induced prostatic hyperplasia. Furthermore, the efficacy of Example 1 was significantly superior to that of Comparative Examples 1-2, demonstrating the synergistic effect of the combination of umbilical cord mesenchymal stem cells and neurofilament peptide (SEQ ID NO. 1).

[0070] The above describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and description merely illustrate the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. Use of an umbilical cord mesenchymal stem cell composition in the preparation of a medicament for treating a prostate disease; the prostate disease is benign prostatic hyperplasia; The umbilical cord mesenchymal stem cell composition comprises 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; The mass ratio of the umbilical cord mesenchymal stem cells to the neurofilament peptide is 3-5:1.

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