TWI625392B - Uses of ligustilide for enhancing the efficacy of stem cells on treating autoimmune diseases, cardiovascular diseases, and/or hematological diseases - Google Patents

Abstract

The use of ligustilide, including the use of ligustilide to enhance the effectiveness of stem cells in the treatment of autoimmune diseases, cardiovascular diseases, and/or blood diseases, and the use of ligustilide in the aforementioned stem cell therapy Application to stem cells treated with ligustilide. By treating the stem cells with ligustilide, the therapeutic benefit of the stem cells can be enhanced, in particular, the expression of genes for promoting differentiation of stem cells, the expression of genes that promote the homing of the stem cells, and/or the reduction of inflammation of the stem cells can be increased. Gene expression.

Description

Ligustilide in the treatment of autoimmune diseases, cardiovascular diseases, and/or Application of the benefits of blood diseases

The present invention relates to the use of ligustilide, in particular to the use of ligustilide in stem cell therapy, in particular the treatment of ligustilide in stem cell therapy of stroke. By treating the stem cells with ligustilide, the therapeutic benefit of the stem cells can be enhanced, in particular, the expression of the gene for promoting differentiation of the stem cells, the expression of the gene for promoting the homing of the stem cells, and/or the reduction of the stem cells can be increased. The performance of inflammatory genes. In addition, the combination of ligustilide and ligustilide-treated stem cells provides superior stem cell treatment benefits.

Stem cells can be divided into totipotent stem cells, pluripotent stem cells, multipotent stem cells, and unpotent stem cells according to their ability to self-renew and differentiate. According to the sequence and distribution of stem cells during development, they can be divided into two types: embryonic stem cells (ES cells) and adult stem cells. In addition, studies have confirmed that cell differentiation is reversible. Inducing mature cells to reprogram into pluripotent cells by introducing specific genes into fully differentiated mature somatic cells, showing the characteristics and functions of embryonic stem cells, namely induced pluripotent stem cells (induced Pluripotent stem cells (iPS cells), these induced pluripotent stem cells can differentiate into body tissues and can be used for disease research and treatment.

In today's medical research field, stem cell therapy offers hope for many diseases that lack effective treatments. Such diseases include, for example, diabetes, autoimmune rejection, stroke, myocardial infarction, renal failure, leukemia, muscular dystrophy, severe anemia, Alzheimer's disease, Parkinson's disease, cancer, etc. The application of pluripotency in regenerative medicine may solve the long-term difficulties faced by these diseases in treatment.

For example, stroke is the most common fatal neurological disease, which can be divided into embolic stroke and hemorrhagic stroke depending on the cause. According to statistics, stroke is one of the top ten deaths in the world. However, to date only a few anticoagulant drugs or thrombolytic agents have been used to treat stroke, and not every stroke patient has anticoagulant drugs or thrombolytic agents. Therefore, in the clinical situation, new treatment methods are urgently needed to solve the problem of patients, and stem cell therapy is currently the most promising therapy in clinical practice. In 1998, cell therapy was first applied to the treatment of stroke. In recent years, the US Food and Drug Administration has agreed to apply stem cells to the clinical treatment of stroke patients.

Studies have shown that stem cells do have a therapeutic effect on stroke treatment, but the survival rate of stem cells after transplantation into the body is not high. Therefore, continuous development of methods or drugs that can effectively improve the efficacy of stem cell therapy to improve the cure rate of the disease is quite necessary and urgent.

The inventor of the case found that the ligustilide Stem cells can increase the therapeutic benefits of stem cells. In addition, the combination of ligustilide and ligustilide-treated stem cells provides a therapeutic benefit that is higher than that of ligustilide-treated stem cells alone.

Accordingly, it is an object of the present invention to provide a method for improving the therapeutic benefit of stem cells which comprises treating the stem cells with ligustilide in a stem cell culture solution.

Another object of the present invention is to provide a composition for improving the therapeutic benefit of stem cells, which comprises ligustilide.

Another object of the present invention is to provide a stem cell kit with improved therapeutic benefit comprising a stem cell, a culture fluid for the stem cell, and a ligustilide. Preferably, the ligustilide comprises a first portion and a second portion, wherein the first portion is combined with the culture solution for pretreating the stem cells, and the second portion is associated with the pretreated stem cells And used for stem cell therapy.

A further object of the present invention is to provide a use of ligustilide for the manufacture of a medicament, wherein the medicament is treated with a ligustilide-treated stem cell and used for stem cell therapy.

Still another object of the present invention is to provide a method for treating stem cells, wherein a stem cell is administered to a subject in need thereof, and the stem cell is pretreated with ligustilide. Preferably, the ligustilide and the stem cells pretreated with the ligustilide are administered separately or simultaneously to the individual in need thereof.

The detailed technical content and some specific embodiments of the present invention will be described in the following contents, so that the person having ordinary knowledge in the field to which the present invention pertains can understand the present invention. Features of the invention.

Figures 1A and 1B show the percentage of survival of adipose-derived stem cells treated with benzalrolol for 24 hours and 48 hours, respectively, where the vertical axis represents cell viability and the horizontal axis represents the concentration of nobiphenyl lactone; The system shows that the stem cells are treated with different concentrations of terpene lactone, and the photos of different genes are analyzed by the real-time reverse transcription polymerase chain reaction, including the expression of genes (ie, BDNF, NURR1) that promote differentiation, and promote regression. The expression of the genes (ie, CXCR4, SDF1αβ) and the expression of inflammatory genes (ie, IL-6, IL-8); Figure 3 shows the analysis of mice by Rotarod analysis. Coordination after different treatments, the horizontal axis is the time after treatment, and the vertical axis is the running time of the mice; among them, the first group is the untreated mouse without stroke (ie, "sham operation group"), the second group For stroke mice with saline, group 3 with stroke mice that were not treated with ligustilide, and group 4 with stroke mice treated with ligustilide-treated stem cells; Figures 4A and 4B show the balance beam test (Beam walking analys Is), analyzing the balance ability of mice after different treatments, Figure 4A shows the time required for the mice to pass the balance beam at different times after administration, and Figure 4B shows the error scores of the mice at different times after administration; The first group was untreated non-stroke mice (ie, "sham operation group"), the second group was administered with saline stroke mice, and the third group was administered with stem cells not treated with ligustilide. Stroke mice, and group 4 were treated with ligustilide Stroke mice with stem cells; Figures 5A and 5B show the balance ability of mice after different treatments using the balance beam test, and Figure 5A shows the time for the mice to pass the balance beam at different times after administration. Figure 5B shows the misclassification of the mice at different times after administration; among them, the first group was untreated non-stroke mice (ie, "sham operation group"), and the second group was administered saline saline mice. The third group is a stroke mouse that is administered with stem cells not treated with ligustilide, the fourth group is a stroke mouse that is administered with ligustilide-treated stem cells, and the fifth group is simultaneously administered with a sputum. Lactone-treated stem cells and stroke mice of 30 mg/kg body weight of ligustilide, and group 6 were strokes treated with both ligustilide-treated stem cells and 90 mg/kg body weight of ligustilide Mice; and Figures 6A and 6B show the use of an eight-channel Locomotor activity box to analyze the exercise capacity of mice after different treatments, and Figure 6A shows the total time of mice at different times after administration. Displacement distance, Figure 6B shows that mice are different after administration The shift time between the first group is the untreated mouse (ie, "sham operation group"), the second group is the saline-supplemented mice, and the third group is the untreated Stroke mice with endogenous lactone-treated stem cells, group 4 were stroke mice administered with ligustilide-treated stem cells, group 5 was co-administered with ligustilide-treated stem cells and 30 mg/kg Stroke mice of the weight of ligustilide, and group 6 were stroke mice that were simultaneously administered with ligustilide-treated stem cells and 90 mg/kg body weight of ligustilide.

In the following, some specific embodiments of the present invention will be described; however, the present invention can be implemented in various different forms without departing from the spirit of the present invention. The scope of the present invention should not be construed as being limited to the scope of the description. In addition, the terms "a", "an" and "the" and "the" "Therapeutically effective amount" means the amount of a compound which, when administered to an individual, is effective to at least partially improve the condition of the suspected individual; the term "individual" means a mammal, which may be a human or a non-human animal.

As is well known in the art, stem cells can be used to treat autoimmune diseases (eg, diabetes, autoimmune rejection), to treat digestive tract diseases (eg, anal/digestive fistula), to treat liver diseases (eg, cirrhosis, liver fibrosis), Treatment of kidney diseases (such as kidney failure), treatment of cardiovascular diseases (such as stroke, myocardial infarction), treatment of nervous system diseases (such as Alzheimer's disease, Parkinson's disease), treatment of blood diseases (such as leukemia), treatment Skeletal degeneration (eg degenerative arthritis, degeneration of knee cartilage), treatment of periodontal disease, treatment of tendon inflammation, treatment of spinal injuries, treatment of head trauma, plastic surgery (eg unilateral atrophy, depressed scar), baldness, skin whitening, And / or remove wrinkles. See, for example, Stem cells: innovations in clinical applications. Stem Cells int. Volume 2014, Article ID 516278, 9 pages, the entire disclosure of which is hereby incorporated by reference.

However, the survival rate of stem cells after transplantation into the body is not high. It has been proved that the main reason is that stem cells will induce an immune reaction after entering the body. If the expression of inflammatory factors of stem cells can be reduced, stem cells can be caused to enter the body. Less immune response, thereby increasing its survival rate and improving the therapeutic benefits of stem cells. See, for example, Qptimizing the success of cell transplantation therapy for stroke. Neurobiol Dis. 37:275-283 (2010), which is incorporated herein by reference in its entirety.

It has also been proved that if the expression of genes promoting differentiation in stem cells can be increased, the differentiation of stem cells into tissue cells of the body can be promoted, which is beneficial to the application of stem cells in the treatment of diseases. On the other hand, even if the stem cells are administered to one body by intravenous injection, if the expression of the gene for promoting regression in the stem cells can be increased, the stem cells can be promptly found to damage the target tissue and exert its repair function, thereby achieving the treatment of the disease. purpose. See, for example, Journey of mesenchymal stem cells for homing: strategies to enhance efficacy and safety of stem cell therapy. Stem Cell Int. Volume 2012, Article ID 342968, 11 pages, and Concise review: Mesenchymal stem cell tumor-homing:detection Methods in disease model systems. Stem Cells. 29(6): 920-927 (2011), the entire disclosure of which is incorporated herein by reference.

The inventors of the present study have found that stem cells treated with ligustilide can effectively increase the expression of genes that promote differentiation, increase the performance of genes that promote regression, and reduce the performance of inflammatory genes. The gene for promoting differentiation includes, for example, a nuclear receptor-related factor (NURR1), a brain-derived neurotrophic factor (BDNF); and the gene for promoting regression includes, for example, CXC. CXC chemokine receptor type-4 (CXCR4), stromal cell-derived factor-1 αβ (SDF1αβ); the inflammatory gene includes, for example, interleukin-6 (interleukin-6) -6, IL-6), interleukin-8 (IL-8).

Further studies confirmed that pretreatment with ligustilide can effectively improve the therapeutic benefit of stem cells. Accordingly, the present invention relates to the discovery of enhancing the therapeutic benefits of stem cells, and provides a composition and method for improving the therapeutic benefits of stem cells. among them, The composition comprises terpene lactone, and the method comprises treating the stem cells with a paraphenyl lactone in a stem cell culture solution. The "treatment of stem cells with ligustilide in a stem cell culture solution" means that the stem cell line is present in the stem cell culture solution when the treatment is carried out.

The compositions and methods of the invention are applicable to any convenient stem cell including, for example, embryonic stem cells, adult stem cells, and induced pluripotent stem cells. The adult stem cells include, for example, hematopoietic stem cells, mesenchymal stem cells, cord blood stem cells, peripheral blood stem cells, neural stem cells, epidermal stem cells, muscle stem cells, adipose stem cells, pancreatic stem cells, corneal stem cells, liver stem cells, and intestinal epithelial stem cells. In one embodiment, the compositions and methods of the invention are used to enhance the therapeutic benefit of adipose stem cells, for example, to treat stroke benefits.

According to the present invention, pretreatment of stem cells with the composition containing ligustilide prior to the use of stem cells for stem cell treatment enhances the effectiveness of the stem cells in stem cell therapy. The ligustilide-containing composition may be the ligustilide itself, or may be a ligustilide and a solvent in which the ligustilide is dissolved. For example, ligustilide can be added to a stem cell culture solution corresponding to the stem cells to be treated to provide a composition of the present invention, and the stem cells are treated with the composition. As another example, ligustilide can be added to a solvent such as dimethyl hydrazine, ethanol to provide a composition of the invention, and the stem cells can be treated with the composition. Still alternatively, when the composition of the present invention is the ligustilide itself, the composition may be directly added to the culture solution containing the stem cells to be treated for pretreatment. The condition is that, in order to maintain the activity of the stem cells, the pretreatment method of the present invention must be carried out in a stem cell culture solution. Therefore, if the composition of the present invention for treating stem cells does not contain a stem cell culture solution, the stem cells to be treated must be placed. Stem cell culture medium for pre-pretreatment deal with.

Appropriate treatment conditions can be selected depending on the type of stem cells. Generally, the ligustilide is used in an amount of from about 0.1 microgram to about 40 micrograms per milliliter of stem cell culture solution, preferably from about 0.1 microgram to about 20 micrograms, most preferably from about 0.1 microgram to about 10 micrograms. For example, as shown in the appended examples, when used for pretreatment of adipose stem cells, it is preferred to use about 0.5 to about 5 micrograms of ligustilide per ml of stem cell culture solution, which can effectively increase the differentiation of adipose stem cells. The performance of the gene, the expression of genes that promote the return of stem cells, and the reduction of the expression of stem cells.

Any suitable stem cell culture solution can be used in the present invention as long as the culture solution corresponds to the stem cells to be treated. The composition of the culture solution is usually adjusted depending on the corresponding stem cells, and includes essential components such as nutrients and conditions (such as pH value) required for stem cell growth and differentiation. In general, stem cell culture fluids include basal culture fluid, animal serum (such as fetal bovine serum), non-essential amino acids (NEAA), L-glutamic acid, and the like. Among them, examples of the base culture solution which can be used in the method of the present invention include, but are not limited to, K-SFM culture medium (Kelvinocyte-serum free medium), DMEM culture medium (Dulbecco's Modifled Eagle's Medium), and MEM medium (Minimum Essential Medium). , α-MEM culture solution, BME culture solution (Basal Media Eagle), MEM/F12 culture solution, Ham's F10 culture solution, Ham's F12 culture solution, and RPMI culture solution (Rosewell Park Memorial Institute). For example, when the method of the present invention is used to enhance the therapeutic benefit of adipose stem cells, K-SFM medium can be used as a base medium for pretreatment.

Therefore, the present invention further provides a stem cell kit with improved therapeutic benefit, comprising: (1) a stem cell, and (2) a culture solution for culturing the stem cell, And (3) ligustilide. Among them, the selection of stem cells, the selection of the culture solution, and the conditions and methods for the use of ligustilide are as described above.

According to the kit of the present invention, the components are usually packaged separately and can be stored separately, and the components can be separately shipped or sold, or can be packaged and sold together in a package. At the time of use, the user then mixes the ingredients on site to perform stem cell culture, treatment, and administration according to the programmed procedures and procedures.

The inventors of the present invention have further found that the use of ligustilide and ligustilide-treated stem cells for stem cell treatment can provide better therapeutic benefits. Accordingly, the kit of the present invention may comprise a first portion of ligustilide and a second portion of ligustilide, wherein the first portion is used in combination with the stem cell culture solution to pretreat the stem cells, and the second portion is The treated stem cells are used in combination for stem cell therapy.

Accordingly, the present invention is also directed to the use of ligustilide for the manufacture of a medicament, wherein the medicament is used in combination with a ligustilide-treated stem cell, the selection of stem cells, and the conditions and methods of use of ligustilide, The above description.

The agent provided by the present invention and the stem cell treated with ligustilide may be used in combination with at least one of the following: treating an autoimmune disease (such as diabetes, autoimmune rejection), and treating a digestive tract disease (such as an anus/digestive tract).瘘), treatment of liver diseases (such as cirrhosis, liver fibrosis), treatment of kidney diseases (such as kidney failure), treatment of cardiovascular diseases (such as stroke, myocardial infarction), treatment of nervous system diseases (such as Alzheimer's disease, Parkinson's disease), treatment of blood diseases (such as leukemia), treatment of bone degeneration (such as degenerative arthritis, degeneration of knee cartilage), treatment of periodontal disease, treatment of tendon inflammation, treatment of spinal injuries, treatment of head trauma, plastic surgery (eg unilateral atrophy, depressed scar), baldness, skin whitening, and wrinkle removal. In a specific embodiment, the above combination is used for the treatment of stroke.

The medicament provided by the present invention may be in any form and administered in any convenient manner. For example, but not limited thereto, the drug can be administered to an individual by oral, subcutaneous, nasal or intravenous administration. Depending on the form of use and use, the agent may additionally comprise a pharmaceutically acceptable carrier.

In the case of a dosage form suitable for oral administration, the medicament provided by the present invention may contain any pharmaceutically acceptable carrier which does not adversely affect the desired benefit of the ligustilide, for example: solvent (water, saline, dextrose) ), glycerin, ethanol or the like, and combinations thereof, oily solvents, diluents, stabilizers, absorption delaying agents, disintegrating agents, emulsifiers, antioxidants, binders, lubricants, moisture absorbents, solids Agents (such as starch, bentonite) and the like. The agent may be provided in a dosage form suitable for oral administration by any convenient method, for example, a tablet, a capsule, a granule, a powder, a flow extract, a solution, a syrup, a suspension, an emulsion, an expectorant, and the like. Wait.

As for a dosage form suitable for subcutaneous or intravenous injection, one or more e.g. isotonic solutions, salt buffers (such as phosphate buffer or citrate buffer) may be contained in the medicament produced by the present invention using ligustilide. The liquid, the solubilizing agent, the emulsifier, the 5% sugar solution, and other carriers are provided in the form of an intravenous infusion solution, an emulsion intravenous infusion solution, a dry powder injection, a suspension injection, or a dry powder suspension injection. Alternatively, the medicament is prepared as a pre-injection solid, the pre-injection solid is provided in a dosage form, or emulsifiable, which is soluble in the other solution or suspension, and is administered prior to administration to the individual in need thereof. The solid is dissolved in other solutions or suspensions or emulsified to provide the desired injection.

Optionally, an additive such as a flavoring agent, a toner, a coloring agent, or the like may be further added to the pharmaceutical agent produced by the present invention using the ligustilide to enhance the pharmaceutical agent. The mouth feel and visual sensation during use; a suitable amount of preservative, preservative, antibacterial agent, antifungal agent, etc. may be added to improve the storage property of the drug. In addition, the agent may optionally contain one or more other active ingredients or may be used in combination with the drug containing the one or more other active ingredients to further enhance the efficacy of the agent or increase the flexibility and formulation of the formulation of the formulation, as long as the other The active ingredient does not adversely affect the desired benefit of the ligustilide.

The administration of the ligustilide of the present invention at different administration times, such as once a day, multiple times a day, or several days, may vary depending on the needs of the individual to be administered. For example, when the agent prepared by using the ligustilide of the present invention and the stem cells treated with ligustilide are used in the human body to treat a stroke, the amount of the ligustilide is about 1 mg/kg body weight per day. About 120 mg / kg body weight, preferably about 10 mg / kg body weight to about 95 mg / kg body weight per day, more preferably about 20 mg / kg body weight to about 40 mg / kg body weight per day, wherein the unit "mg / “kg body weight” means the amount of drug required per kilogram of body weight. However, for acute patients, the amount can be increased according to actual needs, for example, increased to several times or tens of times. In a specific embodiment of the present invention, the use of ligustilide for the manufacture of a therapeutic stroke agent is used in combination with a ligustilide-treated stem cell, and the amount thereof is about the total amount of the ligustilide. 30 mg / kg body weight or about 90 mg / kg body weight.

The invention further relates to a method of treating stem cells, which comprises administering to a subject in need thereof a stem cell treated with ligustilide. In addition, additional ligustilide may be administered to the individual in need thereof as needed, and the administration of the ligustilide may be carried out simultaneously or separately with the administration of the ligustilide-treated stem cells. Among them, the treatment of stem cells and the selection of related materials, the range of the amount of ligustilide, and the application of related treatments are as described above.

The invention is further illustrated by the following examples. The embodiments are provided by way of illustration only and are not intended to limit the scope of the invention. The scope of the invention is shown in the appended claims.

Example

A. Cell experiment

Example 1: Cell survival test (MTT analysis)

Adipose stem cells (ADSCs) were cultured in keratinocyte-serum free medium (K-SFM, Gibco) at 37 ° C under 5% carbon dioxide. The K-SFM culture solution is supplemented with a bovine pituitary extract (Gibco), a recombinant epidermal growth factor (Gibco), and a final concentration of 2×10 ^-3 molar concentration of N- L-ascorbic acid-2-phosphate sesquioxide salt hydrate (L-acetyl-L-cysteine, Sigma), final concentration of 2 × 10 ^-4 molar concentration -ascorbic acid 2-phosphate sesquimagnesium salt hydrate, Sigma), and 10% fetal bovine serum (FBS, Hyclone).

The above culture medium containing adipose stem cells was divided into two groups of A and B, each including 8 groups, and different concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 μg/ml) were added to each group. (Z)- ligustilide was treated, group A lasted 24 hours and group B lasted 48 hours. Next, 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide, referred to as MTT) analysis reagent for staining, and then detecting the absorbance at 570/630 nm wavelength (OD _570-630nm ) with a visible light detector, and calculating the detected absorbance to understand ligustilide Whether it will affect the survival rate of adipose stem cells. The results of the two groups A and B are shown in Figures 1A and 1B, respectively.

Example 2: Ligustilide increases gene expression for differentiation, increases gene expression for regression, and reduces the performance of inflammatory genes

(1) Treatment of stem cells with ligustilide

The adipose stem cells were cultured in the K-SFM culture solution described in Example 1 at 37 ° C under 5% carbon dioxide, and the adipose stem cells were divided into 5 groups and treated with different concentrations (0, 0.625, 1.25, 2.5, respectively). 5, and 10 μg / ml) of (Z)-decalactone, which lasted 24 hours.

(2) Preparation of stem cell whole cell RNA

For each group of stem cells provided in the above (1), the culture solution was first removed, and then 1 ml of TRIzol reagent (Invitrogen) was added to each well, and after incubation for 5 minutes at room temperature, the cells were scraped and coated with scraping. In a 1.5 ml microcentrifuge tube. After the cells were fully broken, 0.2 ml of chloroform was added to the microcentrifuge tube and shaken up and down for 15 seconds. After culturing at room temperature for about 2 to 3 minutes, centrifugation (12,000 g, 15 minutes, 4 ° C), the supernatant was transferred to another new 1.5 ml microcentrifuge tube, and 0.5 ml of isopropyl was added. Alcohol and mix well. Subsequently, the mixture was incubated at room temperature for 10 minutes, and after centrifugation (12,000 g, 10 minutes, 4 ° C), the supernatant was removed. The precipitate remaining was washed with 1 ml of 75% ethanol in DEPC water (Diethylpyrocarbonate (DEPC)-treated H ₂ O). After centrifugation (7500 g, 5 minutes, 4 ° C) again, the supernatant was removed and the remaining precipitate was dried by vacuum evacuation. The dried precipitate was dissolved in about 0.01 to 0.02 ml of DEPC water to obtain whole cell RNA of the adipose stem cells of the experimental group and the blank group, respectively, and the absorbance at a wavelength of 260 nm was measured by a spectrophotometer (DU-800, Beckman). Values were used to estimate the concentration of RNA, and the resulting RNA was stored in a -80 ° C refrigerator for experiment (3).

(3) Reverse transcription polymerase chain reaction (RT-PCR) and colloidal fraction Analysis

Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the expression of ligustilide on genes that promote differentiation (eg, BDNF, NURR1), the expression of genes that promote regression (eg, CXCR4, SDF1αβ), and inflammatory genes ( For example, the effects of IL-6, IL-8) and the expression of the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene were used as a control group, and the procedure is as follows.

The whole cell RNA provided in (2) above was reverse transcribed into cDNA, and then 2.5 μl of cDNA was taken, and 2.5 μl of the primer shown in Table 1A (10 picomolar concentration) and 7.5 μl of deionized were added. Water, and 12.5 microliters of EconoTaq ^® PLUS GREEN 2X Master Mix (Lucigen, Middleton, Wisconsin, USA), then placed the aforementioned sample in a polymerase chain reactor and set the reaction conditions as follows: i) 94 ° C, 30 seconds 55 ° C, 30 seconds, 94 ° C, 60 seconds, the above steps were carried out for 30 cycles; ii) 72 ° C, 10 minutes; iii) finally cooled to 4 ° C to end the reaction.

The resulting polymerase chain reaction (PCR) product was electrophoresed on a 1.5% agar colloid for 30 minutes (voltage: 100 volts), and the gel was then stained in ethidium bromide (EtBr) for 10 minutes. Images were taken using a photopolymerization system (DOC PRINT DP-001FDC, VilberLourmat France). The results are shown in Fig. 2. Finally, the intensity of gene expression was quantified by software (Image J), and the results are shown in Table 1B.

As can be seen from Fig. 2 and Table 1B, compared to the control group (i.e., GAPDH), The expression levels of NURR1, BDNF, CXCR4, and SDF1αβ genes increased with the administration of ligustilide, and the expression levels of IL-6 and IL-8 genes decreased with the administration of ligustilide. This result shows that ligustilide can effectively increase the gene expression of differentiation, increase the gene expression for promoting regression, and reduce the expression of inflammatory genes, thereby reducing the inflammatory response of stem cells and increasing the survival rate of stem cells.

B. Animal experiment

Example 3: Establishment of a mouse stroke model

In order to avoid the urgency and anxiety caused by environmental discomfort in mice, the experimental results and results were affected. After the BALB/c male mice were caged, the adaptation period was given for several days, and the thrombus was used to induce thrombosis in the middle cerebral artery. The day before, neurobehavioral observation and analysis were performed.

Prior to surgery, male BALB/c mice (each approximately 8 weeks old, approximately 25 grams) were anesthetized with 0.25 ml of 4% anhydrous chloral hydrate. During the operation, isoflurane was continued. Maintaining anesthesia in mice prevents the mice from waking up during surgery and affecting the results of the experiment.

After anesthesia, the mice were fixed on a small animal head stereotaxic device, and then the skin was cut 1 mm behind the right eye socket of the mouse to expose the connective tissue and muscle. Next, the connective tissue and muscles were cut open, and the skull on the right side of the mouse was exposed, and the skin and muscles of the mouse were cut to the sides to be fixed. Then, the skull was opened by a small electric drill, and a skull of a circular area was taken over the tibia of the mouse. After removing the skull, the middle cerebral artery was found under a microscope, and the probe of the Laser Doppler Flowmetry was placed 1 mm above the middle cerebral artery to measure the injection of thrombin (thrombin). A blood flow that forms a thrombus after injection. Pipette 1 μl of clotting egg with a glass needle mounted on a microinjector White enzyme, then put the syringe on the hydraulic three-way regulator, and slowly approach the middle cerebral artery. Carefully insert the glass needle through the meninges and insert it into the blood vessel, then immediately inject the coagulation protease into the middle cerebral artery with a pneumatic pump. After 10 minutes, the glass needle was slowly removed, and the resulting mouse model of stroke was referred to as "stroke group (injection thrombin)". There is also a "sham operation group" that has not injected thrombin.

Example 4: Stem cell therapy for stroke mice

The adipose stem cells were cultured in K-SFM medium at 37 ° C under 5% carbon dioxide, and the stem cells were pretreated with 2.5 μg/ml of (Z)-decanolide. After 24 hours, the result was Sterolactone-treated stem cells." In addition, adipose stem cells that have not been pretreated with ligustilide are "untreated stem cells". Before the above two kinds of stem cells were injected into the mouse brain, they were stained with Hoechst dye for 1 hour to facilitate follow-up.

The above-mentioned "saltactone-treated stem cells" were injected into three positions of the mouse brain within 2 hours after the operation of the "stroke group" mice of Example 3 (each mouse was injected a total of 1×). 10 ⁶ "Lendrolactone-treated stem cells"), the injection site can be referred to the Enhancement of neuroplasticity through upregulation of betal-integrin in human umbilical cord-derived stromal cell implanted stroke model. Neurobiol Dis. 27(3): 339 -53 (2007), the full text of which is hereby incorporated by reference, which is the "stem group of stem cells treated with stroke + ligustilide. In addition, "untreated stem cells" were injected into mice of "stroke group". In the "stroke + untreated stem cell group", the control group was injected with the same amount of saline in the stroke mice only, which was "stroke + saline group".

Example 5: Ligustilide enhances the therapeutic benefit of stem cells

"Sham operation group (ie, Group 1)" and Example 4 of Embodiment 3 "Stroke + saline group (ie, group 2)", "stroke + untreated stem cell group (ie, group 3)", and "stroke + sputum lactone-treated stem cell group (ie, The fourth group) mice were subjected to neurobehavioral observation and analysis on the first day of treatment and on days 1, 3, 7, and 14 after treatment, including Rotarod analysis and Beam walking analysis.

Among them, the rotating wheel test uses the running time of the mouse on the roller to evaluate the coordination of the mouse. The results are shown in Fig. 3, wherein the longer the running time of the mouse, the better the coordination; the balance beam test system The mice were placed on a balance beam, and the time required for the mice to pass the balance beam and the number of times the hind feet slipped (i.e., the foot error score) were recorded, thereby reflecting the balance ability of the mice, and the results are shown in Figures 4A and 4B.

As can be seen from Fig. 3, the "stroke + ligustilide-treated stem cell group" mice had a significantly longer running time on the roller than the "stroke + untreated stem cell group" mice. This result shows that the coordination of stroke mice treated with "sodium lactone-treated stem cells" is better.

As can be seen from Figure 4A, compared to the "stroke + untreated stem cell group" mice, the "stroke + ligustilide-treated stem cell group" mice required significantly less time to pass the balance beam, and On day 3 after treatment with ligustilide-treated stem cells, the time required for the mice to pass the balance beam was significantly shortened. In addition, in Fig. 4B, it was found that the stroke number of the "stroke + ligustilide-treated stem cell group" mice was significantly lower than that of the "stroke + untreated stem cell group" mice. The above results showed that the stroke ability of the mice treated with "sodium lactone-treated stem cells" was better, and there was a significant recovery on the third day after the treatment.

The above results show that ligustilide does have an effect of improving the therapeutic benefit of stem cells.

Example 6: Treating disease by using " ligustilide" and "stem cells treated with ligustilide"

Except for the above "sham operation group (ie, group 1)", "stroke + saline group (ie, group 2)", "stroke + untreated stem cell group (ie, group 3)", "stroke" + stem cell group treated with ligustilide (ie, group 4). Further, (Z)- ligustilide (30 or 90 mg/kg body weight) was used in combination with the "saltactone-treated stem cells" provided in Example 4, and simultaneously injected into the brain of a stroke mouse, and the obtained abbreviation For "stroke + ligustilide-treated stem cells + 30 mg / kg body weight of the ligustilide group (ie, group 5)", and "stroke + ligustilide-treated stem cells + 90 mg / kg The body weight of the lactone group (ie, group 6). On the day before treatment and on days 1, 3, 7, and 14 after treatment, the above six groups of mice were subjected to balance beam tests, and the results are shown in Figures 5A and 5B; and the eight-channel shuttle box (Locomotor activity box). The results are shown in Figures 6A and 6B. Among them, the eight-channel shuttle box experiment is displayed by a computer-connected sensor and records the movement data of the mouse in the eight-channel shuttle box, including horizontal displacement (for example: walking), vertical displacement (for example: head-up, climbing), and The overall total displacement data was finally collected for statistical analysis to show the mice's ability to move.

As can be seen from Figures 5A and 5B, compared with the "stroke + ligustilide-treated stem cell group" mice, "stroke + ligustilide-treated stem cells + 30 mg / kg body weight of the ligustilide group" The mice and the "stroke + ligustilide-treated stem cells + 90 mg / kg body weight of the ligustilide group" mice significantly shortened the time required to pass the balance beam, and the hind foot error score was significantly lower, of which The mice with "stroke + stem cells treated with ligustilide + 30 mg / kg body weight of the ligustilide group" were most obvious. The above results show that stem cells treated with ligustilide and ligustilide The combined treatment of stroke mice has better balance ability, especially the combination of "30 mg / kg body weight of ligustilide" and "sodium lactone-treated stem cells" combined treatment is most significant.

From Fig. 6A and Fig. 6B, the total displacement distance and movement time of the mouse in the eight-channel shuttle box compared with the "stroke + ligustilide-treated stem cell group" mouse, "stroke + ligustilide treatment" Stem cells + 30 mg/kg body weight of the ligustilide group" mice and "stroke + ligustilide-treated stem cells + 90 mg/kg body weight of the ligustilide group" in an eight-channel shuttle box The total displacement distance and movement time were significantly longer. Among them, the mice with "stroke + stem cells treated with ligustilide + 30 mg / kg body weight of the ligustilide group" were most obvious. The above results show that the combination of " ligustilide" and "sodium lactam-treated stem cells" has better activity in stroke mice, especially "30 mg/kg body weight of ligustilide" and " Stem cells treated with ligustilide showed the most significant activity recovery after combined treatment.

The above results show that the use of ligustilide and stem cells treated with ligustilide can further enhance the therapeutic benefits of stem cells.

<110> Guogan Stem Cell Application Technology Co., Ltd.

<120> Application of ligustilide to enhance the benefits of stem cells in the treatment of autoimmune diseases, cardiovascular diseases, and/or blood diseases

<130> None

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<170> PatentIn version 3.5

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Claims (9)

A method for improving the benefit of stem cells for treating autoimmune diseases, cardiovascular diseases, and/or blood diseases, which comprises treating the stem cells with a ligustilide in a stem cell culture solution, wherein the ligustilide The amount is 0.1 to 20 μg per ml of stem cell culture solution. The method of claim 1, wherein the stem cell line is an embryonic stem cell, an adult stem cell, or an induced pluripotent stem cell. The method of claim 1, wherein the stem cell is adipose stem cells. The method of any one of claims 1 to 3, wherein the benefit of the stem cells in treating stroke is enhanced. A use of a stem cell treated with ligustilide and ligustilide for the manufacture of a medicament, wherein the medicament is for use in at least one of the following: treating an autoimmune disease, treating a cardiovascular disease, and treating a blood disorder, And the amount of ligustilide used to treat the stem cells is 0.1 to 20 μg per ml of stem cell culture solution. The use of claim 5, wherein the stem cell line is selected from at least one of the group consisting of an embryonic stem cell, an adult stem cell, and an induced pluripotent stem cell. The use of claim 5, wherein the stem cell is adipose stem cells. The use of any one of claims 5 to 7, wherein the agent is for the treatment of a cardiovascular disease. The use of claim 8, wherein the agent is for treating a stroke.