Preparation method of umbilical cord mesenchymal stem cell injection

Abstract

The invention discloses a preparation method of an umbilical cord mesenchymal stem cell injection. According to the method, the umbilical cord mesenchymal stem cell injection is prepared by using a Ringer's solution and a TE buffer solution. The invention also discloses the umbilical cord mesenchymal stem cell injection prepared according to the preparation method, and application of the umbilical cord mesenchymal stem cell injection in preparation of medicines for treating Alzheimer's disease. The prepared umbilical cord mesenchymal stem cell injection can relieve or relieve the symptoms of the Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to an improved method for preparing an umbilical cord mesenchymal stem cell injection solution. Specifically, the present application relates to a method for preparing an umbilical cord mesenchymal stem cell injection solution using Ringer's solution and TE buffer. Background Art

[0002] Stem cells exist in various tissues and organs of the human body, including bone marrow, peripheral blood, embryos, umbilical cord, fat, deciduous teeth, wisdom teeth, urine, etc. Stem cells in the human body have undergone a series of rigorous and standardized preparation and quality inspection processes to become qualified stem cell preparations, which can be used in clinical trials. Umbilical cord mesenchymal stem cells (hUC-MSCs) are derived from the umbilical cord and are mainly distributed around Wharton's jelly and umbilical cord blood vessels. hUC-MSCs have the advantages of strong proliferation ability and low immunogenicity. Related studies have shown that hUC-MSCs show good safety and efficacy in the treatment of heart failure, diabetes, acute spinal cord injury, graft-versus-host disease, premature ovarian failure, Alzheimer's disease, Parkinson's disease and other diseases.

[0003] Although hUC-MSCs have shown a lot of potential in research, the preparation and application technology of its injection is still in the stage of continuous development and improvement. Some key links in the preparation process, such as cell separation, purification, amplification and preservation, still have certain technical difficulties and instability. The quality control standards for injections have not yet been fully unified. Different manufacturers and research institutions may use different methods and standards, which may lead to differences in product quality and affect its safety and effectiveness. Summary of the invention

[0004] According to one aspect of the present invention, the present invention discloses a method for preparing an umbilical cord mesenchymal stem cell injection, the method comprising: washing the umbilical cord with physiological saline, and then soaking the umbilical cord with 75% alcohol for 1-10 minutes; transferring the umbilical cord to a sterile culture dish, and washing it twice with 10-50ml physiological saline; transferring the washed umbilical cord to a new culture dish, and cutting off the two ends of the umbilical cord; cutting the umbilical cord into small pieces, removing Wharton's jelly, and discarding arterial blood vessels, venous blood vessels and amniotic membrane; mixing Ringer's solution with TE buffer at a ratio of 1:1-1. 5 Pour into a culture dish, wash the Wharton's gel, and collect the Wharton's gel; cut the Wharton's gel into pieces, divide 0.3-0.8g of the Wharton's gel evenly into 1 bottle of T75 culture bottle, add complete culture medium and culture until the cell confluence is 60-70%; rinse the T75 culture bottle once with 10-20ml of Ringer's solution and TE buffer solution in a ratio of 1:1-5, discard the rinsed Ringer's solution and TE buffer solution in a ratio of 1:1-5; treat the culture bottle with trypsin and collect the cells; prepare the collected cells with physiological saline into an injection solution.

[0005] According to some embodiments of the present invention, the ratio of Ringer's solution to TE buffer is 1:1-3.

[0006] According to some embodiments of the present invention, the ratio of Ringer's solution to TE buffer is 1:1.

[0007] According to certain embodiments of the present invention, the method includes: washing the umbilical cord with physiological saline, and then soaking the umbilical cord with 75% alcohol for 2 minutes; transferring the umbilical cord to a sterile culture dish and washing it twice with 20 ml of physiological saline; transferring the washed umbilical cord to a new culture dish, cutting off the two ends of the umbilical cord; cutting the umbilical cord into small pieces, removing the Wharton's jelly, and discarding the arterial blood vessels, venous blood vessels and amniotic membrane; pouring Ringer's solution and TE buffer into the culture dish in a 1:1 ratio, washing the Wharton's jelly, and collecting the Wharton's jelly; cutting the Wharton's jelly into pieces, dividing 0.5 g of the Wharton's jelly into 1 bottle of T75 culture bottle, adding complete culture medium and culturing to 60-70% cell confluence; rinsing the T75 culture bottle once with 10-20 ml of Ringer's solution and TE buffer in a 1:1 ratio, and discarding the rinsed Ringer's solution and TE buffer in a 1:1 ratio; treating the culture bottle with trypsin and collecting cells.

[0008] According to one aspect of the present invention, the present invention discloses an umbilical cord mesenchymal stem cell injection prepared according to the preparation method.

[0009] According to one aspect of the present invention, the present invention discloses use of the umbilical cord mesenchymal stem cell injection for preparing a drug for treating Alzheimer's disease.

[0010] The present invention provides a method for preparing an umbilical cord mesenchymal stem cell injection and an umbilical cord mesenchymal stem cell injection prepared by the method. The technology includes the following beneficial effects:

[0011] 1) The hUC-MSC is derived from the umbilical cord. Umbilical cord tissue is easy to obtain without ethical controversy and has all the advantages of mesenchymal stem cells, such as self-replication, multidirectional differentiation, homing characteristics, repair ability, etc. Compared with other origins of adult tissue stem cells, it is more primitive, has stronger proliferation ability, has lower immunogenicity, and has stronger immunoregulatory effects.

[0012] 2) The selected Ringer's solution can maintain the osmotic pressure balance inside and outside the cells, providing a suitable living environment for hUC-MSCs.

[0013] 3) The selected TE buffer has a stable pH value and buffering capacity, and can protect the biological activity of hUC-MSCs.

[0014] 4) The Ringer's solution and TE buffer described above have good safety and biocompatibility, and can be used to perform strict quality testing and screening on hUC-MSCs.

[0015] 5) The hUC-MSCs injection preparation method, by comparing different types of rinsing solutions for rinsing cells, selects Ringer's solution:TE buffer solution at a ratio of 1:1-5, which helps to improve cell activity.

[0016] 6) The hUC-MSC injection was administered via the tail vein.

[0017] 7) The hUC-MSCs in the hUC-MSC injection were identified by PerkinElmer small animal in vivo imaging, indicating that hUC-MSCs can reach the brain tissue.

[0018] 8) The hUC-MSC injection can improve the learning and memory ability of AD model mice after being transplanted through the tail vein; enhance the motor ability of AD mice and relieve the anxiety of AD mice.

[0019] 9) The hUC-MSC injection can significantly improve hippocampal tissue pathology after transplantation through the tail vein, has a certain protective effect on neurons, and alleviates AD damage.

[0020] 10) The hUC-MSC injection can alleviate pathological characteristics after being transplanted through the tail vein, and hUC-MSCs can significantly reduce the protein expressions of Aβ40, Aβ42 and P-Tau in the cortex and hippocampus tissues of AD model mice.

[0021] 11) The hUC-MSC injection can reduce the fluorescence density of GFAP and Iba1 in the hippocampus tissue of AD mice after transplantation through the tail vein, thereby reducing the inflammatory response in the brain of AD mice.

[0022] 12) After the hUC-MSC injection was transplanted through the tail vein, compared with the model group, 2 differential proteins in the hUC-MSCs group were adjusted back and 19 differential proteins were downregulated, all tending to the control group; at the same time, it was found that the therapeutic effect of hUC-MSCs was related to multiple regulatory pathways. Among them, 9 proteins, Ndufaf2, Ndufab1, Sdhd, Ly6h, Sf3a2, Tln2, Fbxo9, Nosip, and Chtop, were found to be involved in the occurrence of AD.

[0023] 13) After the hUC-MSC injection was transplanted through the tail vein, hUC-MSC had a certain effect on the intestinal microorganisms of mice, and may regulate [L] Replication; recombination and repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 (A) shows the morphology of hUC-MSCs (left: 4×, right: 10×; scale bar = 100 μm); Figure 1 (B) shows the flow cytometry identification results of hUC-MSCs; Figure 1 (C) shows the in vivo imaging of mice at different time points after tail vein injection of DiR-labeled hUC-MSCs (the upper part is the back, and the lower part is the abdomen); Figure 1 (D) shows brain tissue imaging at different time points after tail vein injection of DiR-labeled hUC-MSCs; Figure 1 (E) shows a mouse gene identification diagram;

[0025] Figure 2 (A) shows the representative swimming paths of mice in each group; Figure 2 (B) shows the latency of each group of mice; Figure 2 (C) shows the number of platform crossings by mice in each group. Figure 2 (D) shows the results of the rotarod fatigue test, hUC-MSCs can enhance the exercise capacity of AD mice; Figure 2 (E) shows the results of the locomotor activity experiment, hUC-MSCs can alleviate the anxiety of AD mice;

[0026] Figure 3 The results of HE staining and Congo red staining of the mouse hippocampus are shown. (A) shows the morphological HE staining of the DG and CA1 regions of the hippocampus of each group of mice (×20, scale bar = 250 μm); (B) shows the representative images of Congo red staining of each group of mice (arrows marked pink indicate positive results, ×20, scale bar = 250 μm) and the number of Aβ deposits stained with Congo red;

[0027] Figure 4 The ELISA method was used to detect the amount of Aβ40, Aβ42, and P-Tau proteins in the cortex and hippocampus tissues; Figure 4 (A) Protein expression of Aβ40 (P = 0.001, P = 0.035) and Aβ42 (P = 0.001, P = 0.002) in the cortex and hippocampus of AD model mice; Figure 4 (B) Protein expression of P-Tau in the cortex and hippocampus of AD mice (P = 0.002, P = 0.001);

[0028] Figure 5 (A) shows the schematic diagram of GFAP and Iba1 expression in the hippocampus of mice in each group (immunofluorescence in DG region, ×20 / 40, scale bar = 200 μm / 100 μm); Figure 5 (B) shows the statistical histogram of fluorescence intensity;

[0029] Figure 6 (A) shows the PCA analysis graph (WT represents the control group, M represents the AD model group, and H represents the hUC-MSCs group); Figure 6 (B) shows the heat map analysis of differentially expressed proteins in the hippocampus (FC>1.5 / FC<0.67; P<0.05); Figure 6 (C) shows the GO analysis of differentially expressed proteins; Figure 6 (D) shows the KEGG analysis of differentially expressed proteins;

[0030] Figure 7 The differential protein content and possible mechanism analysis are shown. (A) shows the changes in the content of 9 differential proteins in the hippocampus of mice in each group; (B) shows the possible mechanism of action of hUC-MSCs transplantation in intervening AD;

[0031] Figure 8 The sequencing data quality assessment and microbiota composition are shown. (A) The Shannon curves of the intestinal flora of each group of mice are shown. The curves of the intestinal flora of each group tend to be flat, indicating that the amount of sequencing data is reasonable and sufficient; (B) The Rank-abundance curves of the intestinal flora of each group of mice are shown. The curve width on the horizontal axis of the intestinal flora curve of each group is large and gentle, indicating that the species abundance and uniformity are sufficient and reasonable; (C) The OTUs Venn diagram of the intestinal flora of each group of mice is shown; (D) The community structure component diagram of the class level of the intestinal flora of each group of mice is shown. Species with different metabolisms in different colors correspond to the legend on the right;

[0032] Fig. 9 The differential analysis and differential function prediction of the mouse intestinal microbiome are shown. (A) The PCA analysis result graph is shown. The horizontal axis represents the first principal component, the vertical axis represents the second principal component, and the percentage represents the contribution value to the sample difference; (B) The PCoA analysis result graph is shown. The horizontal axis represents the first principal component, the vertical axis represents the second principal component, and the percentage represents the contribution value to the sample difference; (C) The STAMP differential analysis of the microbial function of the control group (Control) and the AD model (model) is shown. The left figure shows the abundance ratio of different functional items in two samples or two groups of samples, the middle shows the difference ratio within the 95% confidence interval, and the rightmost value is the p value. The p value <0.05 indicates a significant difference; (D) The STAMP differential analysis of the microbial function of the AD model (model) and hUC-MSCs is shown. DETAILED DESCRIPTION OF THE INVENTION

[0034] definition

[0035] Umbilical cord mesenchymal stem cells (hUC-MSCs)

[0036] Human umbilical cord mesenchymal stem cells (HUC-MSCs) refer to a type of multifunctional stem cells existing in the umbilical cord tissue of newborns. They have strong proliferation and self-renewal abilities and can differentiate into one or more human tissues or organs under certain conditions. They have broad prospects for clinical application.

[0037] Alzheimer's disease (AD)

[0038] Alzheimer's disease (AD) is a degenerative disease of the central nervous system that occurs primarily in old age or pre-old age. The main features of the disease include progressive cognitive dysfunction and behavioral impairment. AD is the most common form of dementia, accounting for 60-70% of cases. AD is the most common type of dementia in old age, and the risk of developing the disease increases with age. Symptoms of AD include memory impairment, aphasia, apraxia, agnosia, and impairment of visual-spatial abilities.

[0039] AD mice

[0040] It is an experimental animal model, which is a transgenic mouse that overexpresses human genes associated with familial AD and simulates certain lesions or symptoms of AD.

[0041] Amyloid beta (Aβ or Abeta)

[0042] Amyloid-β (Aβ or Abeta) is a series of protein peptides formed after the cleavage of amyloid precursor protein (APP) by β- and γ-secretases, which are believed to be involved in the pathogenesis of Alzheimer's disease. The appearance of β-amyloid plaques, which are extracellular protein deposits, in the brain is a major hallmark of Alzheimer's disease.

[0043] Tau Protein and Tau Phosphorylation

[0044] Tau (microtubule-associated protein tau) is a microtubule-associated protein that is primarily expressed in brain cells and functions to stabilize axonal microtubules. Tau exists in eight different isoforms and is phosphorylated at multiple unique sites, which in turn reduces its ability to bind to microtubules. Although Tau is phosphorylated in normal, healthy brain cells, it is hyperphosphorylated in neurodegenerative diseases such as AD.

[0045] Gut Microbiome

[0046] Intestinal microorganisms refer to the large number of microorganisms that exist in the intestines of animals. These microorganisms rely on the intestines of animals to survive and help the host complete a variety of physiological and biochemical functions. The intestine is not only an important place for human digestion and absorption, but also the largest immune organ, playing an extremely important role in maintaining normal immune defense functions. The human intestine provides a good habitat for microorganisms and has metabolic functions that the human body does not have.

[0047] Hippocampal proteome

[0048] By studying the hippocampal proteome and its protein composition and its changing patterns, we can find certain "disease-specific protein molecules" that can become molecular targets for the design of new drugs or provide molecular markers for the early diagnosis of diseases.

[0049] Ringer's solution

[0050] Adding potassium chloride and calcium chloride to normal saline will form Ringer's solution. Because it was invented by the British physiologist "Ringer", it is called Ringer's solution, which is actually the commonly known compound sodium chloride injection. Ringer's solution is also called compound sodium chloride injection because in addition to sodium chloride, Ringer's solution also contains sodium ions, potassium ions, calcium ions, magnesium ions, chloride ions and lactate ions.

[0051] Ringer's solution is more complete than normal saline and can be used instead of normal saline to regulate body fluid, electrolyte and acid-base balance.

[0052] Ringer's solution is a compound sodium chloride solution that is close to the physiological environment of the human body. It contains a variety of ion components, such as sodium ions, potassium ions, calcium ions, etc., which can maintain the osmotic pressure balance inside and outside the cells and provide a suitable living environment for hUC-MSCs. This helps to maintain the normal morphology and function of cells and reduce cell damage and death.

[0053] TE buffer is usually composed of Tris (tris(hydroxymethylaminomethane)) and EDTA (ethylenediaminetetraacetic acid), and has a stable pH value and buffering capacity. It can prevent cells from being affected by changes in pH during the preparation process and protect the biological activity of cells.

[0054] This patent uses Ringer's solution and TE buffer for washing and rinsing. The combination of Ringer's solution and TE buffer can provide hUC-MSCs with necessary nutrients and growth factors to promote cell proliferation and differentiation. This helps to improve cell activity and function; at the same time, it enhances the therapeutic effect of the injection.

[0055] This preparation method can reduce mechanical damage and chemical stimulation to hUC-MSCs and reduce the apoptosis rate. At the same time, they can also remove impurities and harmful substances in the cell culture process to improve purity and quality. Ringer's solution and TE buffer are both commonly used solutions in clinical practice with good safety and biocompatibility. Their metabolism and excretion pathways in the human body are relatively clear and will not cause serious adverse reactions to the human body. This preparation method can strictly control the content of harmful substances such as bacteria, fungi and endotoxins in the injection solution to ensure the safety of the injection solution. At the same time, it can also conduct strict quality testing and screening of cells to exclude potential pathogens and mutant cells. Overall, Ringer's solution and TE buffer are easy to use and do not require complex preparation and adjustment. This can greatly simplify the preparation process of hUC-MSCs injection and improve production efficiency.

[0056] Preparation method

[0057] According to certain embodiments of the present invention, the preparation method comprises washing the umbilical cord with physiological saline, and then soaking the umbilical cord with 75% alcohol for 1-10 minutes; transferring the umbilical cord to a sterile culture dish, and washing it twice with 10-50 ml of physiological saline; transferring the washed umbilical cord to a new culture dish, and cutting off the two ends of the umbilical cord; cutting the umbilical cord into small pieces, removing Wharton's jelly, discarding arterial blood vessels, venous blood vessels and amniotic membrane; pouring Ringer's solution and TE buffer solution in a ratio of 1:1-5 into the culture dish, and performing Wharton's jelly. The Wharton's gel was washed and collected; the Wharton's gel was cut into pieces, and 0.3-0.8 g of the Wharton's gel was evenly divided into a T75 culture bottle, and complete culture medium was added to culture until the cell confluence was 60-70%; the T75 culture bottle was rinsed once with 10-20 ml of Ringer's solution and TE buffer solution in a ratio of 1:1-5, and the rinsed Ringer's solution and TE buffer solution in a ratio of 1:1-5 were discarded; the culture bottle was treated with trypsin and the cells were collected; the collected cells were prepared into an injection solution with physiological saline.

[0058] According to some embodiments of the present invention, the 75% alcohol soaks the umbilical cord for 1-10 minutes, such as 1-6, 2-8 or 3-5 minutes. According to some embodiments of the present invention, the 75% alcohol soaks the umbilical cord for 2 minutes.

[0059] According to some embodiments of the present invention, 10-50 ml of normal saline is used for washing, such as 10-30 ml of normal saline, 20-50 ml of normal saline or 20-40 ml of normal saline. According to some embodiments of the present invention, 20 ml of normal saline is used for washing.

[0060] According to some embodiments of the present invention, the ratio of Ringer's solution to TE buffer is 1:1-5, such as 1:1-4, 1:1-3, 1:1-2, 1:2-5, 1:2-4, 1:2-3, 1:3-5, 1:3-4 or 1:4-5. According to some embodiments of the present invention, the ratio of Ringer's solution to TE buffer is 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5. According to some embodiments of the present invention, the ratio of Ringer's solution to TE buffer is 1:1-3. According to some embodiments of the present invention, the ratio of Ringer's solution to TE buffer is 1:1.

[0061] According to some embodiments of the present invention, 0.3-0.8 g of Wharton's jelly is divided equally into one T75 culture bottle, for example, 0.3-0.6 g, 0.4-0.7 g or 0.5-0.8 g. According to some embodiments of the present invention, 0.5 g of Wharton's jelly is divided equally into one T75 culture bottle.

[0062] According to some embodiments of the present invention, the T75 culture flask is rinsed with 10-20 ml of Ringer's solution and TE buffer in a 1:1 ratio, for example, 10-15 ml or 15-20 ml.

[0063] Complete medium

[0064] According to some embodiments of the present invention, the complete culture medium comprises 5% serum replacement and 95% MEM culture medium. DETAILED DESCRIPTION

[0065] Example 1: Preparation method of umbilical cord mesenchymal stem cells (hUC-MSCs) injection

[0066] Pour the preservation solution from the preservation bottle into a new 50ml centrifuge tube for preservation, and discard the remaining preservation solution (only the umbilical cord is in the preservation bottle). Wash the umbilical cord twice with saline in the same bottle, about 30ml of saline each time, and gently shake the bottle. After washing with saline, pour 75% alcohol until the umbilical cord is completely soaked and disinfected for 2 minutes. Use sterile tweezers to transfer the umbilical cord to a sterile culture dish and wash it with 20ml of saline for the first time. Use sterile tweezers to transfer the umbilical cord to a sterile culture dish again for a second wash with 20ml of saline (to minimize the amount of blood in the umbilical cord). Transfer the rinsed umbilical cord to a new culture dish. Cut off the two ends of the umbilical cord and discard them. Use medical sterile scissors and tweezers to cut the umbilical cord into small pieces (about 2cm). Remove the Wharton's jelly and discard the arteries, veins and amniotic membrane. Pour Ringer's solution and TE buffer 1:1 into the culture dish, wash the Wharton's jelly, and inject the washing supernatant into the bacterial detection bottle with a syringe for sterility testing. Collect Wharton's jelly in a 50ml centrifuge tube. Weigh the Wharton's jelly and record it on the processing form. Use sterile medical scissors to cut the Wharton's jelly into pieces of tissue about 1-4mm. 3. Divide about 0.5g of Wharton's jelly into 1 T75 culture flask. Add 10ml of complete culture medium to each culture flask. Culture horizontally in a 37℃, 5% carbon dioxide incubator. Change the culture medium every 5 days until the cell confluence reaches 60-70%. Transfer the culture medium to a 50ml centrifuge tube. Centrifuge at 2000rpm for 5min. Discard the supernatant (useless culture medium). Add 10ml of fresh complete culture medium to each centrifuge tube and mix well. Transfer the culture medium from the 50ml centrifuge tube to a T75 culture flask and mark the date. Culture horizontally in a 37℃, 5% carbon dioxide incubator. Observe the cell status closely under a microscope every 4-5 days, and collect the cells once the cell confluence reaches 60-70%. Collect the useless culture medium into a 50ml centrifuge tube. Centrifuge at 2000rpm for 5min and set aside. Rinse the T75 culture flask once with 10-20ml of Ringer's solution and TE buffer 1:1. Discard the rinsed Ringer's solution and TE buffer 1:1. Add 3ml of warm 0.125% trypsin to each culture flask. Digest at room temperature for 4min until 80% of the cells are detached and floating. Stop the digestion of the cell suspension with useless culture medium. Collect the terminated cell suspension into a 50ml centrifuge tube. Rinse the T75 culture flask once with 10-20ml of Ringer's solution and TE buffer 1:1, and pour the rinse liquid into a 50ml centrifuge tube. Filter the terminated cell suspension to remove umbilical cord tissue fragments. Collect the filtered cell suspension into a new 50ml centrifuge tube. Centrifuge at 1000rpm for 10min. Remove the supernatant. Add 25ml of fresh complete culture medium to the centrifuge tube and mix well. Transfer the culture medium with cells to a T175 culture flask. Place it horizontally in a 37℃, 5% carbon dioxide incubator for incubation. Observe the culture flask closely under a microscope every day, and collect the cells once the cell confluence reaches 80-90%.

[0067] Example 2: Quality testing of umbilical cord mesenchymal stem cells (hUC-MSCs) injection

[0068] This example compares the cell viability of umbilical cord mesenchymal stem cells (hUC-MSCs) injection prepared using different rinse solutions.

[0069] Cell morphology standard: The cells were uniformly spindle-shaped and well refractive under microscope before collection. Cell viability: Cell viability counter, cell viability ≥ 90.0%, double-blind double-test error rate ≤ 20%. Cell volume standard: Intravenous injection standard: Use physiological saline to make up to 100 ml, mix the cells, and fill the cell suspension into a 100 ml transfer bag. Number of cells per unit: 1.0×10 7For one unit, 2 units are issued each time. Endotoxin detection: intravenous injection ≤0.25EU. Bacterial detection: blood culture instrument, negative. Flow cytometry standard: CD105 positive expression rate: ≥95.00%; CD73 positive expression rate: ≥90.00%; CD90 positive expression rate: ≥95.00%; CD45 positive expression rate: ≤5.00%; CD34 positive expression rate: ≤5.00%. Cell generation>2≤5 generations.

[0070] The following table shows the cell viability of umbilical cord mesenchymal stem cell (hUC-MSCs) injection prepared with different rinse solutions. It can be seen from the table below that the cell viability is highest when the Ringer's solution: TE buffer is 1:1-5. Therefore, this example shows that the umbilical cord mesenchymal stem cell injection prepared by the preparation method of Ringer's solution: TE buffer of 1:1-5 is significantly better than the umbilical cord mesenchymal stem cell injection prepared by other methods (such as normal saline, Ringer's solution, TE buffer and Ringer's solution: TE buffer of 1:6).

[0071] Comparison of cell viability after rinsing

[0072]

[0073]

[0074] Example 3: hUC-MSCs culture identification and in vivo tracing

[0075] The umbilical cord mesenchymal stem cells (hUC-MSCs) used in Examples 3-9 were prepared using the method of Example 1, wherein the ratio of Ringer's solution to TE buffer was 1:1.

[0076] After stable passage, hUC-MSCs were observed under an inverted microscope and the cells were attached to the surface of the culture flask and had a fibroblast-like morphology ( Figure 1 A); Flow cytometry results showed that hUC-MSCs surface markers: CD73+, CD90+ and CD105+≥95% while CD34+≤5% ( Figure 1 B).

[0077] The production and deposition of Aβ are important factors affecting the progression and prognosis of AD. The deposition of Aβ can lead to damage to the blood-brain barrier (BBB). The BBB plays a vital role in maintaining the normal metabolism of Aβ. The BBB dysfunction of AD patients is manifested as a large amount of Aβ deposition

[16] . Studies have shown that MSCs have low immunogenicity, long-distance migration, homing, cross-BBB and innate ability to communicate with surrounding immune cells

[17] . This study considered whether hUC-MSCs can reach the brain to exert their effects, and used DiR to label hUC-MSCs. DiR is an important near-infrared fluorescent dye with good continuous excitation light stability and is widely used for in vivo monitoring of cells

[18] . We injected the labeled hUC-MSCs into mice through the tail vein, and the control group was injected with an equal amount of normal saline. The PerkinElmer small animal in vivo imager was used to perform in vivo animal imaging at different time points after injection. The results are shown in the figure. Figure 1 C; and brain tissue organ imaging results at different time points Figure 2 D. Small animal in vivo imaging results showed that hUC-MSCs could reach the brain tissue after tail vein infusion, which is similar to the study of Yuan et al.

[19] .

[0078] The mouse genotype identification results are shown in Figure 2 E, excluding the positive and negative controls, the mice showing bright double bands (1, 3, 4, 5, 7, 11, 13, 14, 15, 16, 20, 23, 25) are APP / PS1 double transgenic male mice, totaling 13 mice; the mice without bands (2, 6, 8, 9, 10, 12, 17, 18, 19, 21, 22, 24, 26) are C57BL / 6J male mice, totaling 13 mice.

[0079] Example 4: Effect of hUC-MSCs transplantation on learning and memory ability of AD mice

[0080] Morris water maze is a classic experiment to evaluate the spatial learning and memory ability of mice. After each group of mice was trained in the positioning navigation experiment, the spatial exploration ability of the mice was examined. The swimming trajectories of each group of mice ( Figure 2 A), indicating that after the platform was removed after training, the mice in the control group and hUC-MSCs group had a larger range of activities, while the mice in the model group moved around the edge of the pool with a more limited range of activities. The number of platform crossings of each group of mice was counted ( Figure 2 B) and incubation period ( Figure 2C), the results showed that the number of platform crossings of mice in the hUC-MSCs administration group was higher than that in the model group, with a significant difference (P=0.047). In addition, the escape latency of mice in the AD model group was significantly higher than that in the hUC-MSCs treatment group, with a significant difference (P=0.001). In summary, hUC-MSCs effectively improved the learning and memory ability of AD model mice. Rotarod fatigue apparatus is often used to detect the motor coordination ability of animals. The experimental results showed that compared with the control group, the time on the rod of mice in the AD model group was significantly shortened, with a significant difference (P=0.001); compared with the model group, the time on the rod of mice in the hUC-MSCs administration group was significantly prolonged, with a significant difference (P=0.001). The above results show that hUC-MSCs can improve AD motor ability ( Figure 2 D).

[0081] The comparison results of the activity counts of mice in each group within 3 minutes are as follows Figure 3 E. The results showed that the AD model group mice showed a higher number of activities than the control group mice, and the difference was significant (P = 0.001). Compared with the AD model group, the number of activities of the mice in the hUC-MSCs administration group was significantly reduced, and the difference was significant (P = 0.001). hUC-MSCs can improve the anxiety of AD mice ( Figure 2 E).

[0082] Example 5: Effect of hUC-MSCs transplantation on AD mouse tissue pathology

[0083] HE staining and Congo red staining were used to further study the effect of hUC-MSCs transplantation on pathological changes in hippocampal tissue. HE staining results showed that neurons in the DG and CA1 regions of the hippocampus of mice in the normal group were arranged in an orderly manner, with normal structure, clear nuclei, obvious nucleoli, and abundant cytoplasm. Neurons in the DG and CA1 regions of the hippocampus of mice in the model group were swollen in large quantities, with loose structure, nuclear condensation, and certain vacuolar structures. Compared with the model group, the pathology of hippocampal neurons in the hUC-MSCs group was improved ( Figure 3 A). Congo red is an anisotropic organic stain used to mark Aβ plaques in the brain. Congo red staining can dye amyloid substances red, showing the pathological changes of amyloidosis. Figure 3 As shown in B, the arrow indicates that pink amyloid deposits can be seen in the hippocampus of the model group under the microscope, and the pink amyloid deposits are significantly reduced after hUC-MSCs transplantation (P=0.001). The above results suggest that hUC-MSCs transplantation has a protective effect on neurons in the hippocampus of AD mice and reduces damage.

[0084] Example 6: Effects of hUC-MSCs transplantation on Aβ deposition and Tau phosphorylation in AD mice

[0085] Aβ deposition is an important pathological feature of AD and is related to synaptic damage. In order to explore the mechanism by which hUC-MSCs improve cognitive dysfunction in APP / PS1 transgenic mice, we used ELISA to detect the expression of Aβ40, Aβ42, and P-Tau in the cortex and hippocampus. The ELISA results showed that ( Figure 4 ), hUC-MSCs can significantly reduce the protein expression of Aβ40 (P=0.001, P=0.035) and Aβ42 (P=0.001, P=0.002) in the cortex and hippocampus of AD model mice ( Figure 4 A). There is a close connection between Tau protein and Aβ. Tau protein is a soluble microtubule-associated protein. Under pathological conditions, excessive phosphorylation will lose the ability to bind to microtubules to form neurofibrillary tangles and cannot play a normal physiological role. hUC-MSCs can also significantly reduce the protein expression of P-Tau in the cortex and hippocampus of AD mice (P=0.002, P=0.001), thereby inhibiting the excessive phosphorylation of Tau protein ( Figure 4 B).

[0086] Example 7: Effects of hUC-MSCs transplantation on hippocampal neuroinflammation in AD mice

[0087] Multiple studies have shown that neuroinflammation plays an important role in the development of AD. Inflammation in the brain is a double-edged sword. The activation of microglia (MG) and astrocytes (AST) can clear debris and pathogens from the brain, but this activation may also have harmful effects on the neuronal system. In neurodegenerative diseases, this balance is disrupted, leading to chronic inflammation and tissue atrophy. Inflammation is thought to contribute to and exacerbate AD pathology, and may even precede AD. GFAP is an intermediate filament protein and a marker for AST; MG is a type of glial cell found throughout the brain and spinal cord, and Iba1 is a marker for MG, which are known to be activated when the brain is damaged or degenerated

[20] . In this study, the optical density of GFAP and Iba1 in the DG region of the mouse hippocampus was statistically analyzed by immunofluorescence. The experimental results showed that the fluorescence density of GFAP and Iba1 in the hippocampus of the control group and hUC-MSCs group was weak, while the fluorescence density of the model group was strong, which was significantly different from the hUC-MSCs group (P<0.05). GFAP and Iba1 were obviously activated, so hUC-MSCs transplantation can reduce the inflammatory response in the brain of AD mice ( Figure 5 ).

[0088] Example 8: Effect of hUC-MSCs transplantation on the hippocampal proteome of AD mice

[0089] Proteomics results showed that 5296 proteins were identified in the mouse hippocampus. PCA analysis results showed that ( Figure 6A), the differences within each group were small, with a certain range of aggregation; while the model group (M) was clearly distinguished from the other two groups, with large differences; the overall experimental results are referenceable. A fold change > 1.5 indicates that the expression of the protein is upregulated, and a fold change < 0.67 indicates that the expression of the protein is downregulated. After treatment with hUC-MSCs via tail vein infusion, a total of 21 differential proteins were screened out by the difference fold method (fold change > 1.5 indicates protein upregulation, fold change < 0.67 indicates protein downregulation) and t-test (P < 0.05). Compared with the model group, 2 differential proteins in the hUC-MSCs group were adjusted back and 19 differential proteins were downregulated, all tending to the control group. For details, see the heat map analysis ( Figure 6 B).

[0090] In order to better analyze the intrinsic connection of the above differentially expressed proteins, we performed GO enrichment analysis on the 21 proteins, such as Figure 6 C. The results showed that after hUC-MSCs treatment, the differential proteins were mainly enriched in: negative regulation of catalytic activity, mitochondrial ATP synthesis coupled proton transport, mitochondrial respiratory chain complex I assembly, aerobic respiration, etc. at the BP level; and mainly enriched in: mitochondrial respiratory chain complex I, U2-type precatalytic spliceosome, respiratory chain, mitochondrial inner membrane, mitochondrion, etc. at the CC level.

[0091] The differentially expressed proteins were further enriched by KEGG. Figure 6D. The results showed that the therapeutic effect of hUC-MSCs was associated with multiple regulatory pathways, including: oxidative phosphorylation, thermogenesis, non-alcoholic fatty liver disease, diabetic cardiomyopathy, chemical carcinogenesis-reactive oxygen species, Parkinson disease, prion disease, Huntington disease, amyotrophic lateral sclerosis and Alzheimer disease; the proteins enriched in AD included: Ndufa2, Ndufab1, and Sdhd.

[0092] The pathogenesis of AD mainly includes Aβ hypothesis, Tau protein hypothesis, cholinergic hypothesis, neuroinflammation hypothesis, mitochondrial dysfunction and oxidative stress hypothesis; this study analyzed the hippocampal tissues of each group of mice by LC-MS / MS, and then conducted bioinformatics analysis, and found that three proteins, Ndufaf2, Ndufab1, and Sdhd, were related to AD; further literature review found that among the 21 proteins, Ly6h, Sf3a2, Tln2, Fbxo9, Nosip, and Chtop may be involved in the occurrence of AD, and the possible mechanisms of action are shown in Table 1. The 9 differentially expressed proteins that may be related to AD found in this study, after hUC-MSCs transplantation, the hippocampal protein content of the treated mice tended to the control group, and the differences were significant compared with the model group (P<0.05) ( Figure 7 A) The possible mechanism of hUC-MSCs transplantation in the brain proteome of AD transgenic mice is shown in Figure 7 B.

[0093] Table 1 Differential protein analysis

[0094]

[0095] Example 9: Effect of hUC-MSCs transplantation on the intestinal microbiome of AD mice

[0096] The Shannon curve is constructed based on the microbial diversity index of each sample at different sequencing depths, such as Figure 8 As shown in A, the intestinal flora curves of each group of mice tend to be flat, indicating that the amount of sequencing data is reasonable and sufficient; the Rank-abundance curve reflects the species abundance and species uniformity, as shown in Figure 8As shown in B, the curve width on the horizontal axis of each group is large and gentle, indicating that the species abundance and uniformity are sufficient and reasonable.

[0097] In order to study the species composition diversity of the samples, the clean reads of all samples were clustered, and the sequences were clustered into Operational Taxonomic Units (OTUs) with 97% consistency, and then the species annotation was performed on the representative sequences of OTUs. A total of 3188 OTUs were identified in the three groups of samples, of which 895 OTUs were found in the three groups. The number of unique OTUs in the control group (Control), AD model (model), and hUC-MSCs was 592, 474, and 575, respectively, indicating that there were certain differences in the number of intestinal flora OTUs between the groups ( Figure 8 C).

[0098] According to the species annotation results, the top 10 species with the highest abundance at each class level in each group were selected to generate a species relative abundance column cumulative graph. Fig. 9 As shown in Figure 2, from the perspective of the richness of microbial species composition, the dominant genera at the class level mainly include c__Bacteroidia, c__Clostridia, c__Bacilli, c__Erysipelotrichia, c__Campylobacteria, c__Gammaproteobacteria, c__Deltaproteobacteria, c__Alphaproteobacteria, c__Coriobacteriia, and c__Deferribacteres, and there are significant differences among the groups. The overall AD model deviates from the control group (Control), and the hUC-MSCs group has a callback effect.

[0099] Principal Component Analysis (PCA) is a technique for simplifying data analysis and reducing the dimensionality of multidimensional data to extract the most important elements and structures in the data. Fig. 9A shows that the gap between the control group (Control) and the AD model (model) is relatively large, indicating that there are significant differences in the intestinal flora between mice; the hUC-MSCs group is closer to the control group (Control), and there is some overlap, indicating that the intestinal flora composition of the hUC-MSCs group is gradually tending towards the control group (Control). Principal Co-ordinates Analysis (PCoA), similar to PCA analysis, is used to study the similarities or differences in sample community composition. The closer the sample distance, the more similar the species composition structure. Similarly, similar to the PCA results, Fig. 9 B shows that compared with the AD model (model), the bacterial flora composition structure of hUC-MSCs and the control group (Control) is more similar.

[0100] The species composition information obtained by comparing the 16S sequencing data of four groups of intestinal microorganisms was used by PICRUSt software to infer the functional gene composition in the samples, and the functional composition of each group of flora was revealed by Clusters of Orthologous Groups of proteins (COG). Based on the results of PICRUSt functional secondary classification, STAMP difference analysis was used to compare the functional abundance between the two groups, such as Fig. 9As shown in C and D, compared with the control group, the microbiota composition of the AD model was significantly different in [R] General function prediction only; [G] Carbohydrate transport and metabolism; [E] Amino acid transport and metabolism; [M] Cell wall / membrane / envelope biogenesis; [S] Function unknown; [L] Replication; recombination and repair; [C] Energy production and conversion; [P] Inorganic ion transport and metabolism; [H] Coenzyme transport and metabolism; [O] Post-translational modification; protein turnover; and chaperones; [V] Defense mechanisms; [F] Nucleotide transport and metabolism. There are significant differences in the regulatory functions of the two groups, including [I] lipid transport and metabolism; [U] intracellular trafficking, secretion and vesicular transport; [D] cell cycle control, cell division, chromosome partitioning; [Q] secondary metabolites biosynthesis, transport; and catabolism. Fig. 9C). Compared with the AD model, the bacterial community of hUC-MSCs has significant differences in [L]Replication; recombination and repair, and the trend is closer to the control group (Control) ( Fig. 9 D).

[0101] The embodiments disclosed in the present invention are only for detailed description of the present invention, but are not sufficient to limit the present invention. A person skilled in the art may replace the preparations described herein with obvious similar substitutes or modifications, or replace the preparations with certain chemically or biologically structurally related preparations, or make changes to the relevant contents of the present invention, but they shall not exceed the spirit, scope and idea of ​​the present invention, and shall fall within the scope of protection claimed by the present invention.

[0102] References

[0103] [1] Chu Wanglong, Li Tongjing, Shangguan Yan, et al. Stability study of human umbilical cord mesenchymal stem cell preparations in large-scale production[J]. Acta Pharmaceutica Sinica. 2024, 59(03): 743-750.

[0104] [2] Xu Yan, Ma Rui, Lu Xianda, et al. Effects of intravenous administration of human umbilical cord mesenchymal stem cell injection on the central nervous system of experimental animals [C]. Proceedings of the 10th National Toxicology Conference of the Chinese Society of Toxicology.

[0105] [3] Wang Xiujuan, Li Zhengnan, Li Wei, et al. Preparation and stability study of human umbilical cord mesenchymal stem cell compound electrolyte injection [J]. Drug Evaluation Research. 2020, 43(12): 2394-2397.

[0106] [4] Lin Chunhua, Xiao Min, Dong Runcong, et al. Toxicity test of umbilical cord mesenchymal stem cell injection on SD rats after 24 weeks of repeated administration via tail vein injection [C]. Proceedings of the Academic Conference on Drug Toxicology and Safety Evaluation of the Chinese Society of Toxicology (2019) and the First Summit Forum on the Biomedical Industry in the Guangdong-Hong Kong-Macao Greater Bay Area

[0107] [5] Geng Jie, Zhang Lei, Wang Bin, et al. Study on the long-term stability of human umbilical cord mesenchymal stem cell injection [J]. Chinese Journal of Pharmaceutical Biotechnology. 2013, 8(03): 230-234.

Claims

1. A method for preparing an umbilical cord mesenchymal stem cell injection, the method comprising: Clean the umbilical cord with saline solution, then soak the umbilical cord with 75% alcohol for 1-10 minutes; Transfer the umbilical cord to a sterile culture dish and wash it twice with 10-50ml of saline; transfer the washed umbilical cord to a new culture dish and cut off the two ends of the umbilical cord; cut the umbilical cord into small pieces, remove the Wharton's jelly, discard the arteries, veins and amniotic membrane; pour Ringer's solution and TE buffer solution in a ratio of 1:1-5 into the culture dish and wash with Wharton's jelly. Collect Wharton's jelly; cut the Wharton's jelly into pieces, divide 0.3-0.8g of Wharton's jelly evenly into a T75 culture bottle, add complete culture medium and culture until the cell confluence is 60-70%; rinse the T75 culture bottle once with 10-20ml of Ringer's solution and TE buffer solution in a ratio of 1:1-5, discard the rinsed Ringer's solution and TE buffer solution in a ratio of 1:1-5; treat the culture bottle with trypsin and collect the cells; prepare the collected cells with physiological saline into an injection solution.

2. The preparation method according to claim 1, wherein The ratio of the Ringer's solution to the TE buffer is 1:1-3.

3. The preparation method according to claim 2, wherein The ratio of the Ringer's solution to the TE buffer was 1:

1.

4. The preparation method according to claim 1, wherein The method comprises: washing the umbilical cord with physiological saline, and then soaking the umbilical cord with 75% alcohol for 2 minutes; transferring the umbilical cord to a sterile culture dish, and washing it twice with 20 ml of physiological saline; transferring the washed umbilical cord to a new culture dish, and cutting off the two ends of the umbilical cord; cutting the umbilical cord into small pieces, removing the Wharton's jelly, and discarding the arterial blood vessels, venous blood vessels and amniotic membrane; pouring Ringer's solution and TE buffer solution into the culture dish in a ratio of 1:1, washing the Wharton's jelly, and collecting the Wharton's jelly; cutting the Wharton's jelly into pieces, dividing 0.5 g of the Wharton's jelly into a T75 culture bottle, adding complete culture medium and culturing until the cell confluence is 60-70%; rinsing the T75 culture bottle once with 10-20 ml of Ringer's solution and TE buffer solution in a ratio of 1:1, and discarding the rinsed Ringer's solution and TE buffer solution in a ratio of 1:1; treating the culture bottle with trypsin and collecting cells.

5. An umbilical cord mesenchymal stem cell injection prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the umbilical cord mesenchymal stem cell injection according to claim 5 for preparing a drug for treating Alzheimer's disease.

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