WO2016041192A1 - 藁本内酯的应用 - Google Patents

藁本内酯的应用 Download PDF

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WO2016041192A1
WO2016041192A1 PCT/CN2014/086926 CN2014086926W WO2016041192A1 WO 2016041192 A1 WO2016041192 A1 WO 2016041192A1 CN 2014086926 W CN2014086926 W CN 2014086926W WO 2016041192 A1 WO2016041192 A1 WO 2016041192A1
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stem cells
ligustilide
treatment
treating
stem cell
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PCT/CN2014/086926
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English (en)
French (fr)
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林欣荣
韩鸿志
刘诗平
傅如辉
林珀丞
庄明熙
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国玺干细胞应用技术股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones

Definitions

  • This invention relates to the use of ligustilide, particularly to the use of ligustilide in stem cell therapy, particularly the use of ligustilide in stem cell therapy of stroke.
  • stem cells by treating stem cells with ligustilide, the therapeutic benefit of stem cells can be enhanced, in particular, the expression of genes for promoting differentiation of stem cells, the expression of genes for promoting the homing of the stem cells, and/or the reduction of the stem cells can be reduced.
  • the performance of inflammatory genes can be reduced.
  • 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, and that mature cells are reprogrammed into pluripotent cells by introducing specific genes into fully differentiated mature somatic cells, showing embryonic stem cells. The characteristics and functions are 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.
  • iPS cells induced pluripotent stem cells
  • stem cell therapy offers hope for many diseases that lack effective treatments.
  • 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 plight of these diseases in treatment.
  • stroke is the most common fatal neurological disease, which can be divided into embolic stroke and hemorrhagic stroke depending on the cause.
  • stroke is one of the top ten deaths in the world.
  • 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.
  • stem cell therapy was first applied to the treatment of stroke.
  • the US Food and Drug Administration has agreed to apply stem cells to the clinical treatment of stroke patients.
  • 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, it is quite necessary and urgent to continue to develop methods or drugs that can effectively improve the therapeutic effect of stem cells in order to improve the cure rate of the disease.
  • the inventors of the present study have found that treating stem cells with ligustilide can increase the therapeutic benefit of stem cells.
  • the combination of ligustilide and ligustilide-treated stem cells provides a therapeutic benefit that is higher than that of ligustilide-treated stem cells alone.
  • It is an object of the present invention to provide a method for enhancing the therapeutic benefit of stem cells comprising treating the stem cells with ligustilide in a stem cell culture fluid.
  • Another object of the present invention is to provide a composition for enhancing the therapeutic benefit of stem cells comprising a ligustilide.
  • Another object of the present invention is to provide a stem cell kit with improved therapeutic benefits comprising a stem cell, a culture fluid for the stem cell, and a ligustilide.
  • the ligustilide comprises a first portion and a second portion, wherein the first portion is used with the culture solution to pretreat the stem cells, and the second portion is used with the pretreated stem cells and used for stem cells. treatment.
  • the ligustilide and the ligustilide-pretreated stem cells are administered separately or simultaneously to the individual in need thereof.
  • the present invention provides a method for improving the therapeutic effect of stem cells, which comprises treating the stem cells with a ligustilide in a stem cell culture solution.
  • the stem cell is an embryonic stem cell, an adult stem cell, or an induced pluripotent stem cell.
  • the stem cells are adipose stem cells.
  • the amount of ligustilide is from 0.1 to 40 micrograms per milliliter of stem cell culture.
  • the improvement of stem cells is as follows: treating autoimmune diseases, treating digestive tract diseases, treating liver diseases, treating kidney diseases, treating cardiovascular diseases, treating nervous system diseases, treating blood diseases, Treatment of bone degeneration, treatment of periodontal disease, treatment of tendon inflammation, treatment of spinal injuries, treatment of head trauma, plastic surgery, baldness, skin whitening, and removal of wrinkles.
  • the benefit of boosting stem cells in treating stroke is enhanced.
  • the invention also provides the use of a ligustilide for the manufacture of a medicament, wherein the medicament is treated with a ligustilide-treated stem cell and used for stem cell therapy.
  • the stem cell is selected from the group consisting of at least one of an embryonic stem cell, an adult stem cell, and an induced pluripotent stem cell.
  • the stem cells are adipose stem cells.
  • the agent and the stem cell are used in at least one of the following: treating an autoimmune disease, treating a digestive tract disease, treating a liver disease, treating a kidney disease, treating a cardiovascular disease, treating a nervous system disease, Treats bloody diseases, treats bone degeneration, treats periodontal disease, treats tendon inflammation, treats spinal injuries, treats head trauma, plastic surgery, baldness, skin whitening, and wrinkles removal.
  • the agent is used in combination with stem cells to treat cardiovascular disease.
  • the agent is used in conjunction with stem cells to treat a stroke.
  • the beneficial effects of the invention are: the method for improving the therapeutic effect of the stem cell of the invention and the use of the transcript
  • stem cells are treated with ligustilide to effectively increase the expression of genes that promote differentiation, increase the expression of genes that promote regression, and reduce the performance of inflammatory genes.
  • the compositions and methods of the invention are applicable to any suitable stem cell that can be used to enhance the therapeutic benefit of stem cells.
  • 1A and 1B show the percentage of survival of adipose stem cells treated with indophenol lactone for 24 hours and 48 hours, respectively, wherein the vertical axis represents cell viability and the horizontal axis represents the concentration of panthenolactone;
  • Figure 2 is a photographic diagram showing the expression of different genes by the reverse transcription polymerase chain reaction after stem cells are treated with different concentrations of nonoxynol, including the expression of genes that promote differentiation (ie, BDNF, NURR1), The amount of expression of genes that promote regression (ie, CXCR4, SDF1 ⁇ ), and the amount of expression of inflammatory genes (ie, IL-6, IL-8);
  • Figure 3 shows the Rotarod analysis, which analyzes the coordination of mice after different treatments.
  • the horizontal axis is the time after treatment, and the vertical axis is the running time of mice.
  • the first group is no drug.
  • Non-stroke mice ie, sham-operated group
  • group 2 were saline-injected stroke mice
  • group 3 was treated with stem cells that were not treated with ligustilide
  • group 4 a stroke mouse for stem cells treated with ligustilide;
  • Figure 4A and Figure 4B show the balance of mice after different treatments by Beam walking analysis.
  • Figure 4A shows the time required for mice to pass the balance beam at different times after administration.
  • Figure 4B shows the mice. Foot error scores at different times after administration; among them, group 1 was untreated non-stroke mice (ie, sham operation group), group 2 was saline-injected stroke mice, and group 3 was administered. Stroke mice of stem cells not treated with ligustilide, and group 4 of stroke mice administered stem cells treated with ligustilide;
  • Figures 5A and 5B show the balance ability of mice after different treatments using the balance beam test, and Figure 5A shows the time of the mice passing the balance beam at different times after administration, Figure 5B shows The scores of the feet of the mice at different times after administration; among them, the first group was untreated non-stroke mice (ie, the sham operation group), the second group was the saline mice administered with saline, and the third group was Stroke mice administered with stem cells not treated with ligustilide, group 4 were stroke mice administered with ligustilide-treated stem cells, and group 5 was simultaneously treated with ligustilide-treated stem cells And a stroke mouse of 30 mg/kg body weight of ligustilide, and a group 6 of stroke mice which were simultaneously administered with ligustilide-treated stem cells and 90 mg/kg body weight of ligustilide;
  • the first group was untreated non-stroke mice (ie, the sham operation group)
  • the second group was the saline mice administered with saline
  • Figure 6A and Figure 6B show the locomotor activity box experiment to analyze the exercise ability of mice after different treatments.
  • Figure 6A shows the total displacement distance of mice at different times after administration
  • Figure 6B shows small The time of movement of the rats at different times after administration; among them, the first group was untreated non-stroke mice (ie, the sham operation group), the second group was the saline mice administered with saline, and the third group was administered.
  • Stroke mice of stem cells not treated with ligustilide group 4 were stroke mice administered with ligustilide-treated stem cells, group 5 was co-administered with ligustilide-treated stem cells, and 30 Stroke mice of ligustilide in mg/kg body weight, and group 6 were stroke mice treated with both ligustilide-treated stem cells and 90 mg/kg body weight of ligustilide.
  • Therapeutically effective amount means an amount of a compound which, when administered to an individual, is effective to at least partially improve the condition of the suspected individual; the so-called "individual” means a mammal, which may be a human or a non-human animal.
  • stem cells can be used to treat autoimmune diseases (such as diabetes, autoimmune rejection), to treat digestive tract diseases (such as anal/digestive fistula), to treat liver diseases (such as cirrhosis, liver fibrosis), to treat kidneys.
  • autoimmune diseases such as diabetes, autoimmune rejection
  • digestive tract diseases such as anal/digestive fistula
  • liver diseases such as cirrhosis, liver fibrosis
  • stem cells after transplantation into the body are not high. It has been proved by research that the stem cells will induce an immune response when they enter 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 benefit 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.
  • 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.
  • the stem cells are administered to one body by intravenous injection, if the expression of the regression gene is promoted in the stem cells, the stem cells can be promptly found to damage the target tissue and exert its repair function, thereby achieving the purpose of treating the disease. . See, for example, Journey of mesenchymal stem cells for homing: strategies to enhance efficacy and safety of stem cell therapy. Stem Cell Int.
  • 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 expression 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.
  • the inflammatory gene includes, for example, interleukin-6 (IL-6) and interleukin-8 (IL-8).
  • the present invention provides a composition and method for enhancing the therapeutic benefits of stem cells in relation to the discovery of improved stem cell therapeutic benefits.
  • the composition comprises indole lactone, the method comprising treating the stem cells with a paraphenyl lactone in a stem cell culture solution.
  • the phrase "treating stem cells with ligustilide in a stem cell culture solution” means that the stem cells are present in the stem cell culture solution when the treatment is carried out.
  • 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.
  • the compositions and methods of the invention are used to enhance the therapeutic benefit of adipose stem cells, for example, to treat stroke benefits.
  • the ligustilide-containing composition may be the ligustilide itself, or may be a ligustilide and a solvent in which the ligustilide is dissolved.
  • 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, which is then treated with the composition.
  • ligustilide can be added to a solvent such as dimethyl sulfoxide, ethanol to provide a composition of the invention, and the stem cells can be treated with the composition.
  • the composition of the present invention when the composition of the present invention is the ligustilide itself, the composition may be directly added to a culture solution containing 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.
  • the stem cell culture solution is used for pretreatment.
  • Appropriate treatment conditions can be selected depending on the type of stem cells.
  • the amount of ligustilide It is 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.
  • 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.
  • 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.
  • 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). , ⁇ -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).
  • K-SFM medium Kelvinocyte-serum free medium
  • DMEM culture medium Dulbecco's Modifled Eagle's Medium
  • MEM medium Minimum Essential Medium
  • ⁇ -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).
  • K-SFM medium can be used
  • the present invention further provides a stem cell kit having improved therapeutic benefit comprising (1) a stem cell, (2) a culture solution for culturing the stem cell, and (3) ligustilide.
  • a stem cell kit having improved therapeutic benefit comprising (1) a stem cell, (2) a culture solution for culturing the stem cell, and (3) ligustilide.
  • 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.
  • the components are typically packaged separately and may be stored separately, and the components may be separately shipped or sold, or may be packaged and sold together in a kit.
  • the user then mixes the ingredients on site to perform stem cell culture, treatment, and administration according to the programmed procedures and procedures.
  • the kit of the present invention may comprise the first part ⁇ The lactone and the 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 used in combination with the treated stem cells for stem cell treatment.
  • the present invention also relates 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 are as described above. instruction of.
  • the agent provided by the present invention and the stem cell treated with ligustilide may be used in at least one of the following: treating an autoimmune disease (such as diabetes, autoimmune rejection), treating a digestive tract disease (such as an anus/digestive fistula), 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) 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 Lateral atrophy, depressed scars), baldness, skin whitening, and removal of wrinkles.
  • an autoimmune disease such as diabetes, autoimmune rejection
  • a digestive tract disease such as an anus/digestive fistula
  • liver diseases such as cirr
  • the agents provided herein can be in any form and administered in any convenient manner.
  • the drug can be administered to an individual by oral, subcutaneous, nasal or intravenous administration.
  • the agent may additionally comprise a pharmaceutically acceptable carrier.
  • the medicament provided by the present invention may contain any pharmaceutically acceptable carrier which does not adversely affect the desired benefit of 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.
  • solvent water, saline, dextrose
  • glycerin glycerin
  • ethanol glycerin
  • oily solvents oily solvents
  • diluents stabilizers
  • absorption delaying agents disintegrating agents
  • emulsifiers antioxidants
  • binders binders
  • lubricants moisture absorbents
  • moisture absorbents such as starch, bentonite
  • 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.
  • the dosage form suitable for subcutaneous or intravenous injection it can be prepared by using ligustilide in the present invention.
  • the medicament contains one or more components such as an isotonic solution, a salt buffer (such as a phosphate buffer or a citrate buffer), a solubilizer, an emulsifier, a 5% sugar solution, and other carriers.
  • the medicament is provided in the form of an intravenous infusion solution, an intravenous infusion of an emulsion, a dry powder injection, a suspension injection, or a dry powder suspension injection.
  • the medicament is prepared as a pre-injection solid
  • the pre-injection solid is provided in a dosage form or emulsifiable dosage form 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 injectables.
  • the agent produced by the use of ligustilide in the present invention may further contain an additive such as a flavoring agent, a toner or a coloring agent to improve the mouthfeel and visual feeling of the medicament when taken; A suitable amount of preservative, preservative, antibacterial, antifungal, etc., is used to improve the storage of the agent.
  • 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. As long as the other active ingredient does not adversely affect the desired benefit of the ligustilide.
  • the administration of the ligustilide of the present invention may be carried out at different administration frequencies, such as once a day, multiple times a day, or several times a day, depending on the needs of the individual to be administered.
  • administration frequencies such as once a day, multiple times a day, or several times a day, depending on the needs of the individual to be administered.
  • 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 is "mg/ "Kilogram body weight” refers to the amount of drug required per kilogram of body weight. However, for acute patients, the amount may be increased as needed, for example, by several times or tens of times.
  • 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 is about 30 mg based on the ligustilide. / kg body weight or about 90 mg / kg body weight.
  • the invention further relates to a method of stem cell treatment comprising administering to a subject in need thereof a stem cell treated with ligustilide.
  • additional sputum may be cast for the individual in need as needed.
  • the lactone, the administration of the ligustilide can be carried out simultaneously or separately with the administration of the ligustilide-treated stem cells.
  • 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.
  • Adipose stem cells were cultured in keratinocyte-serum free medium (K-SFM, Gibco) at 37 ° C under 5% carbon dioxide.
  • K-SFM keratinocyte-serum free medium
  • 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 N-acetyl groups.
  • the aforementioned culture medium containing adipose stem cells was divided into two groups of A and B, each consisting of 8 groups, and different concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 ⁇ g/ml) were added to each group.
  • the (Z)- ligustilide treatment was carried out, group A lasted 24 hours and group B lasted 48 hours.
  • 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide, referred to as MTT) analytical reagent for staining, and then detect the absorbance at 570/630 nm wavelength (OD570-630nm) with a visible light detector, and calculate the absorbance detected to see if ligustilide affects adipose stem cells Survival rate.
  • MTT 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide
  • Example 2 Ligustilide increases gene expression that promotes differentiation, increases gene expression that promotes regression, and reduces the effectiveness of inflammatory gene expression
  • 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 ⁇ g/ml of (Z)-decalactone lasted 24 hours.
  • the culture solution was first removed, and then 1 ml of TRIzol reagent (Invitrogen) was added to each well, and the cells were incubated at room temperature for 5 minutes, and the cells were scraped and smeared by 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.
  • TRIzol reagent Invitrogen
  • RT-PCR Real-time PCR analysis of the expression of ligustilide on genes that promote differentiation (eg, BDNF, NURR1) and genes that promote regression (eg, CXCR4, SDF1 ⁇ )
  • genes that promote differentiation eg, BDNF, NURR1
  • genes that promote regression eg, CXCR4, SDF1 ⁇
  • the performance, as well as the effects of inflammatory genes eg, IL-6, IL-8
  • Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as a control group. The steps are as follows.
  • the whole cell RNA provided in (2) above was reverse transcribed into cDNA, and 2.5 ⁇ l of cDNA was taken, and 2.5 ⁇ l of the primer (10 ⁇ mol) shown in Table 1A and 7.5 ⁇ l of deionized were added.
  • PCR polymerase chain reaction
  • ligustilide can effectively increase the gene expression for promoting 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.
  • Example 3 Establishment of a mouse stroke model
  • mice 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.
  • mice 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, and the procedure was continued with isoflurane ( Isoflurane) maintains the anesthesia of the mice and prevents the mice from waking up during surgery, which affects the experimental results.
  • isoflurane isoflurane
  • 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.
  • 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.
  • a small electric drill is used to perform the skull creation of the mouse on the tibia of the mouse.
  • the square takes the skull of the next circular area.
  • the middle cerebral artery was found under a microscope, and a laser Doppler Flowmetry probe was placed 1 mm above the middle cerebral artery to measure the injection of thrombin before injection. The blood flow that forms a thrombus.
  • 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 ⁇ g/ml of (Z)-decrolactone. After 24 hours, the obtained was Ligustilide-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 “sodium lactide-treated stem cells” of the "stroke group” mice of Example 3 were injected into three positions of the mouse brain within 2 hours after surgery (each mouse was co-injected 1 ⁇ ).
  • 106 "sputum lactone-treated stem cells" the injection site can be referred to the Enhancement of neuroplasticity through upregulation of beta1-integrin in human umbilical cord-derived stromal cell implanted stroke model.
  • Neurobiol Dis.27 (3): 339- 53 (2007) the entire contents of which is hereby incorporated by reference, which is incorporated herein by reference to the "study of the stem cell group treated with the ligustilide.
  • the "untreated stem cells” were injected into the "stroke group” mice, and the results were obtained.
  • the control group was only injected with the same amount of saline solution for the stroke mice, which was "stroke + saline group”.
  • Example 5 Ligustilide enhances the therapeutic benefit of stem cells
  • mice were treated the day before On the first, third, seventh, and fourth days after treatment, the four groups of mice were subjected to neurobehavioral observation analysis, including Rotarod analysis and Beam walking analysis.
  • the rotating wheel test used 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 will test the mouse.
  • the time required for the mouse to pass the balance beam and the number of times the hind foot slipped i.e., the foot error score
  • the results are shown in Fig. 4A and Fig. 4B.
  • Example 6 Combining " ligustilide” and “salt cells treated with ligustilide” to treat diseases
  • (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 weight of the ligustilide group (ie, group 6). On the day before treatment On the 1st, 3rd, 7th, and 14th day after treatment, the above six groups of mice were subjected to the balance beam test, and the results are shown in Fig. 5A and Fig.
  • 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 overall The total displacement data was finally collected for statistical analysis to show the mice's ability to move.

Abstract

本发明公开了藁本内酯(ligustilide)的应用，包括使用藁本内酯以提升干细胞的治疗效益，以及于干细胞治疗上并用藁本内酯与经藁本内酯(ligustilide)处理的干细胞的应用。经由以藁本内酯处理干细胞，可提升干细胞的治疗效益，尤其可增加干细胞的促进分化的基因的表现、增加该干细胞的促进回归(homing)的基因的表现、及/或降低该干细胞的发炎基因的表现。

Description

藁本内酯的应用 技术领域
本发明是关于藁本内酯的应用，尤其关于藁本内酯于干细胞治疗的应用，特别是藁本内酯于中风的干细胞治疗的应用。其中通过以藁本内酯处理干细胞，可以提升干细胞的治疗效益，尤其可增加干细胞的促进分化的基因的表现、增加该干细胞的促进回归(homing)的基因的表现、及/或降低该干细胞的发炎基因的表现。此外，将藁本内酯与经藁本内酯处理的干细胞并用，可提供更加优异的干细胞治疗效益。
背景技术
干细胞依其自我更新(self-renew)及分化的能力，可分成全能(totipotent)干细胞、多能(pluripotent)干细胞、多潜能(multipotent)干细胞、及单效性(unipotent)干细胞。而依干细胞发育过程中出现的先后顺序以及分布的情形，则可将其分成胚胎干细胞(embryonic stem cell，ES cells)及成体干细胞(adult stem cell)二大类。此外，已有研究证实，细胞的分化是可逆的，通过将特定基因送入已完全分化的成熟体细胞(somatic cells)，诱导成熟体细胞重新编程(reprogram)为多能细胞，表现出胚胎干细胞的特性及功能，即为诱导型多能干细胞(induced pluripotent stem cells，iPS cells)，这些诱导型多能干细胞可分化成为身体的组织而可用于疾病的研究与治疗。
在现今的医学研究领域中，干细胞疗法为许多缺乏有效的治疗方法的疾病带来希望。此等疾病包括，例如：糖尿病、自体免疫排斥、中风、心肌梗塞、肾衰竭、白血病、肌肉萎缩症、重度贫血、阿兹海默症、帕金森氏症、癌症等，干细胞所具有的多能性(pluripotency)于再生医学上的应用，可能解决这些疾病在治疗上长期面临的困境。
举例来说，中风是最常见的致命性神经系统疾病，其依发生的原因可分成栓塞性中风及出血性中风。据统计，中风是目前世界排名十大死因之一。然而，迄今只有少数抗凝血药物或血栓溶解剂可用于治疗中风，且并非每个中风病人皆适用抗凝血药物或血栓溶解剂。因此，在临床上急需新的治疗方式来解决病人的问题，干细胞疗法即为目前临床上最有希望的疗法。在1998年，细胞治疗首次被应用于中风的治疗，近几年美国食品药物管理局更同意将干细胞应用于中风患者的临床治疗。
研究指出，干细胞对于中风治疗确实具有疗效，但干细胞移植至体内后的存活率并不高。因此，持续开发可有效提高干细胞治疗效益的方法或药物，以提升疾病的治愈率，有相当的必需性及迫切性。
本案发明人研究发现，以藁本内酯(ligustilide)处理干细胞，可增加干细胞的治疗效益。此外，将藁本内酯与经藁本内酯处理的干细胞并用，所提供的治疗效益，较单独使用经藁本内酯处理的干细胞更高。
发明内容
本发明的一目的，在于提供一种提升干细胞治疗效益的方法，其包含于干细胞培养液中以藁本内酯处理该干细胞。
本发明的另一目的，在于提供一种提升干细胞治疗效益的组合物，其包含藁本内酯。
本发明的另一目的，在于提供一种具提升治疗效益的干细胞套组，其包含一干细胞、一培养液供该干细胞用、以及藁本内酯。较佳地，该藁本内酯包含一第一部分与一第二部分，其中，该第一部分与该培养液并用以预处理该干细胞，且该第二部分与该经预处理的干细胞并用于干细胞治疗。
本发明的又一目的，在于提供一种使用藁本内酯于制造药剂的用途，其中该药剂与经藁本内酯处理的干细胞并用于干细胞治疗。
本发明的再一目的，在于提供一种干细胞治疗方法，其中对一有需要的个体投予一干细胞，该干细胞经藁本内酯预处理。较佳地，对该有需要的个体分别或同时投予藁本内酯以及经藁本内酯预处理的干细胞。
为解决上述技术问题，本发明提供一种提升干细胞治疗效益的方法，包含于干细胞培养液中以藁本内酯(ligustilide)处理该干细胞。
本发明的一个实施态样中中，其中该干细胞为胚胎干细胞、成体干细胞、或诱导型多能干细胞。
本发明的一个实施态样中，其中该干细胞为脂肪干细胞。
本发明的一个实施态样中，其中藁本内酯的用量为每毫升干细胞培养液0.1至40微克。
本发明的一个实施态样中，其中提升干细胞于以下的效益：治疗自体免疫疾病、治疗消化道疾病、治疗肝脏疾病、治疗肾脏疾病、治疗心血管疾病、治疗神经系统疾病、治疗血液性疾病、治疗骨胳退化、治疗牙周病、治疗肌腱发炎、治疗脊椎损伤、治疗头部创伤、整形手术、秃头、皮肤美白、及去除皱纹。
本发明的一个实施态样中，其中提升干细胞于治疗中风的效益。
本发明还提供一种使用藁本内酯于制造药剂的用途，其中该药剂与经藁本内酯处理的干细胞并用于干细胞治疗。
本发明的一个实施态样中，其中该干细胞选自：胚胎干细胞、成体干细胞、及诱导型多能干细胞中的至少一种。
本发明的一个实施态样中，其中该干细胞为脂肪干细胞。
本发明的一个实施态样中，其中该药剂与干细胞并用于以下之至少一者：治疗自体免疫疾病、治疗消化道疾病、治疗肝脏疾病、治疗肾脏疾病、治疗心血管疾病、治疗神经系统疾病、治疗血液性疾病、治疗骨胳退化、治疗牙周病、治疗肌腱发炎、治疗脊椎损伤、治疗头部创伤、整形手术、秃头、皮肤美白、及去除皱纹。
本发明的一个实施态样中，其中该药剂与干细胞并用以治疗心血管疾病。
本发明的一个实施态样中，其中该药剂与干细胞并用以治疗中风。
本发明的有益效果为：本发明提升干细胞治疗效益的方法及使用藁本 内酯于制造药剂的用途中，干细胞经由藁本内酯处理，可有效增加促进分化的基因的表现、增加促进回归的基因的表现、以及降低发炎基因的表现。本发明组合物及方法可适用于任何合宜的干细胞，其可用于提升干细胞的治疗效益。
本发明的详细技术内容及部分具体实施态样，将描述于以下内容中，以供本发明所属领域具通常知识者据以明了本发明的特征。
附图说明
图1A及图1B分别显示脂肪干细胞以藁苯内酯处理24小时及48小时之后的存活率百分比，其中纵轴代表细胞存活率，横轴代表藁苯内酯的浓度；
图2显示干细胞经不同浓度的藁苯内酯处理后，以即时反转录聚合酶连锁反应分析不同基因的表现量的照片图，包括促进分化的基因(即，BDNF、NURR1)的表现量、促进回归的基因(即，CXCR4、SDF1αβ)的表现量、以及发炎基因(即，IL-6、IL-8)的表现量；
图3所示为以旋转轮测试(Rotarod analysis)，分析小鼠经不同处理后的协调性，横轴为处理后的时间，纵轴为小鼠跑动时间；其中，第1组为无投药的未中风小鼠(即，假手术组)、第2组为投以食盐水的中风小鼠、第3组为投以未经藁本内酯处理的干细胞的中风小鼠、以及第4组为投以经藁本内酯处理的干细胞的中风小鼠；
图4A及图4B所示为以平衡木测试(Beam walking analysis)，分析小鼠经不同处理后的平衡能力，图4A显示小鼠在投药后不同时间通过平衡木所需的时间，图4B显示小鼠在投药后不同时间的脚误分数；其中，第1组为无投药的未中风小鼠(即，假手术组)、第2组为投以食盐水的中风小鼠、第3组为投以未经藁本内酯处理的干细胞的中风小鼠、以及第4组为投以经藁本内酯处理的干细胞的中风小鼠；
图5A及图5B所示为利用平衡木测试，分析小鼠经不同处理后的平衡能力，图5A显示小鼠在投药后不同时间的通过平衡木的时间，图5B显示 小鼠在投药后不同时间的脚误分数；其中，第1组为无投药的未中风小鼠(即，假手术组)、第2组为投以食盐水的中风小鼠、第3组为投以未经藁本内酯处理的干细胞的中风小鼠、第4组为投以经藁本内酯处理的干细胞的中风小鼠、第5组为同时投以经藁本内酯处理的干细胞以及30毫克/公斤体重的藁本内酯的中风小鼠、以及第6组为同时投以经藁本内酯处理的干细胞以及90毫克/公斤体重的藁本内酯的中风小鼠；以及
图6A及图6B所示为利用八通道穿梭箱(Locomotor activity box)实验，分析小鼠经不同处理后的运动能力，图6A显示小鼠在投药后不同时间的总位移距离，图6B显示小鼠在投药后不同时间的移动时间；其中，第1组为无投药的未中风小鼠(即，假手术组)、第2组为投以食盐水的中风小鼠、第3组为投以未经藁本内酯处理的干细胞的中风小鼠、第4组为投以经藁本内酯处理的干细胞的中风小鼠、第5组为同时投以经藁本内酯处理的干细胞以及30毫克/公斤体重的藁本内酯的中风小鼠、以及第6组为同时投以经藁本内酯处理的干细胞以及90毫克/公斤体重的藁本内酯的中风小鼠。
具体实施方式
以下将描述根据本发明的部分具体实施态样；但是，在不背离本发明精神下，本发明尚可以多种不同形式得态样来实践，不应将本发明保护范围解释为限于说明书所陈述者。此外，除非文中有另外说明，于本说明书中(尤其是在专利权利要求中)所使用的“一”、“该”及类似用语应理解为包含单数及复数形式；所谓“有效量”或“治疗有效量”，指投予至个体时，可有效至少部分改善怀疑个体的病情的化合物数量；所谓“个体”指哺乳动物，哺乳动物可为人类或非人动物。
如业界所周知，干细胞可应用于治疗自体免疫疾病(例如糖尿病、自体免疫排斥)、治疗消化道疾病(例如肛门/消化道瘘管)、治疗肝脏疾病(例如肝硬化、肝纤维化)、治疗肾脏疾病(例如肾衰竭)、治疗心血管疾病(例如中风、心肌梗塞)、治疗神经系统疾病(例如阿兹海默氏症、帕金森氏症)、治疗血液性疾病(例如白血病)、治疗骨胳退化(例如退化性关 节炎、膝软骨退化)、治疗牙周病、治疗肌腱发炎、治疗脊椎损伤、治疗头部创伤、整形手术(例如偏侧萎缩、下陷型疤痕)、秃头、皮肤美白、及/或去除皱纹。此可参见例如：Stem cells:innovations in clinical applications.Stem Cells int.Volume 2014,Article ID 516278,9 pages，该文献全文并于此处以供参考。
然而，将干细胞移植至体内后其存活率并不高，业经研究证明，原因主要为干细胞进入体内后会引发免疫反应，若可降低干细胞的发炎因子的表现，则可使干细胞在进入体内后引起较少的免疫反应，从而增加其存活率，提升干细胞的治疗效益。此可参见例如：Qptimizing the success of cell transplantation therapy for stroke.Neurobiol Dis.37:275-283(2010)，该文献全文并于此处以供参考。
亦经证明，若能增加干细胞中促进分化的基因的表现，即可促进干细胞分化成为身体的组织细胞，有利于干细胞于疾病治疗的应用。另一方面，即使是通过静脉注射将干细胞施用至一个体，若能增加干细胞中促进回归基因的表现，亦可促使干细胞顺利找到损害的目标组织，发挥其修复的功能，从而达到治疗疾病的目的。此可参见例如：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、以及Concise review:Mesenchymal stem cell tumor-homing:detection methods in disease model systems.Stem Cells.29(6):920-927(2011)，该二文献的全文并于此处以供参考。
本案发明人研究发现，干细胞经由藁本内酯处理，可有效增加促进分化的基因的表现、增加促进回归的基因的表现、以及降低发炎基因的表现。其中，该促进分化的基因包括例如：核受体相关因子(Nuclear receptor-related factor 1，NURR1)、脑衍生神经滋长因子(brain-derived neurotrophic factor，BDNF)；该促进回归的基因包括例如：C-X-C趋化激素受体-4(C-X-C chemokine receptor type-4，CXCR4)、基质细胞衍生因子-αβ(stromal  cell-derived factor-1αβ，SDF1αβ)；该发炎基因包括例如：介白素-6(interleukin-6，IL-6)、介白素-8(interleukin-8，IL-8)。
进一步研究确认，以藁本内酯预处理，可以有效提升干细胞的治疗效益。因此，本发明关于提升干细胞治疗效益的发现，提供一种提升干细胞治疗效益的组合物及方法。其中，该组合物包含藁苯内酯，该方法包含于干细胞培养液中以藁苯内酯处理该干细胞。所谓“于干细胞培养液中以藁本内酯(ligustilide)处理干细胞”指于进行该处理时，干细胞存在于干细胞培养液中。
本发明组合物及方法可适用于任何合宜的干细胞，包括例如：胚胎干细胞、成体干细胞、及诱导型多能干细胞。其中，成体干细胞包括例如：造血干细胞、间质干细胞、脐带血干细胞、周边血干细胞、神经干细胞、表皮干细胞、肌肉干细胞、脂肪干细胞、胰干细胞、眼角膜干细胞、肝脏干细胞、及肠上皮干细胞。于一具体实施态样中，本发明组合物及方法用于提升脂肪干细胞的治疗效益，例如，治疗中风的效益。
根据本发明，在将干细胞使用于干细胞治疗之前，先使用该含藁本内酯的组合物对干细胞进行预处理，可提升该干细胞于干细胞治疗的效益。该含藁本内酯的组合物可以为该藁本内酯本身，亦可以为包含藁本内酯以及一溶剂，其中，藁本内酯溶解于该溶剂中。举例言之，可以将藁本内酯添加至对应于待处理干细胞的干细胞培养液中，以提供本发明组合物，再以该组合物处理干细胞。又例如，可将藁本内酯添加至例如二甲基亚砜、乙醇的溶剂中，以提供本发明组合物，再以该组合物处理干细胞。又或者，当本发明组合物为该藁本内酯本身时，可直接将该组合物添加至含有待处理干细胞的培养液中，以进行预处理。其条件为，为维持干细胞的活性，本发明预处理方法必须于干细胞培养液中进行，因此，若用以处理干细胞的本发明组合物不含干细胞培养液，则须将待处理的干细胞置于干细胞培养液中，以进行预处理。
可视干细胞种类而选用合宜的处理条件。一般而言，藁本内酯的用量 为每毫升干细胞培养液约0.1微克至约40微克，较佳约0.1微克至约20微克，最佳约0.1微克至约10微克。举例言之，如后附实施例所示，当使用于脂肪干细胞的预处理时，较佳使用每毫升干细胞培养液约0.5至约5微克藁本内酯，此可有效增加脂肪干细胞的促进分化的基因的表现、增加干细胞的促进回归的基因的表现、及降低干细胞的发炎基因的表现。
可使用任何合宜干细胞培养液于本发明，只要该培养液与所欲处理的干细胞对应即可。通常视所对应的干细胞而调整培养液的组成，包含可提供干细胞生长、分化所需养分与条件(如酸碱值)等必要成分。一般而言，干细胞培养液包含基础培养液、动物血清(如胎牛血清)、非必需氨基酸(non-essential amino acids，NEAA)、L-麸酰胺酸等。其中，可用于本发明方法的基础培养液的例子包括，但不限于，K-SFM培养液(keratinocyte-serum free medium)、DMEM培养液(Dulbecco's Modifled Eagle's Medium)、MEM培养液(Minimum Essential Medium)、α-MEM培养液、BME培养液(Basal Media Eagle)、MEM/F12培养液、Ham's F10培养液、Ham's F12培养液以及RPMI培养液(Rosewell Park Memorial Institute)。举例言之，当使用本发明方法以提升脂肪干细胞的治疗效益时，可使用K-SFM培养液作为基础培养液以进行预处理。
因此，本发明另提供一种具提升治疗效益的干细胞套组，其包含(1)一干细胞、(2)一培养液供培养该干细胞用、以及(3)藁本内酯。其中，有关干细胞的选用、培养液的选用、以及藁本内酯的使用条件与方法，均如上述。
根据本发明的套组，各成分通常分开包装且可各自储存，且各成分可各自分开运送或销售，亦可组合成套一起配送与销售。于使用时，使用者再根据所拟定的程序与流程，于现场混合各成分以进行干细胞培养、处理及施用。
本案发明人另发现，将藁本内酯与经藁本内酯处理的干细胞并用于干细胞治疗，可提供更佳的治疗效益。因此，本发明套组可包含第一部分藁 本内酯与第二部分藁本内酯，其中该第一部分与该干细胞培养液并用，以对该干细胞进行预处理，该第二部分则与经处理的干细胞并用，使用于干细胞治疗。
因此，本发明亦关于使用藁本内酯于制造药剂的用途，其中该药剂与经藁本内酯处理的干细胞并用，有关干细胞的选用、以及藁本内酯的使用条件与方法，均如上述的说明。
可并用本发明所提供的药剂与经藁本内酯处理的干细胞，于以下至少一者：治疗自体免疫疾病(例如糖尿病、自体免疫排斥)、治疗消化道疾病(例如肛门/消化道瘘管)、治疗肝脏疾病(例如肝硬化、肝纤维化)、治疗肾脏疾病(例如肾衰竭)、治疗心血管疾病(例如中风、心肌梗塞)、治疗神经系统疾病(例如阿兹海默氏症、帕金森氏症)、治疗血液性疾病(例如白血病)、治疗骨胳退化(例如退化性关节炎、膝软骨退化)、治疗牙周病、治疗肌腱发炎、治疗脊椎损伤、治疗头部创伤、整形手术(例如偏侧萎缩、下陷型疤痕)、秃头、皮肤美白、及去除皱纹。于一具体实施态样中，系将上述并用使用于中风的治疗。
本发明所提供的药剂可呈任何形式，并以任何合宜的方式施用。举例言之，但不以此为限，该药物可以口服、皮下、鼻腔或静脉内等投药方式施用至个体上。视使用形式及用途而定，该药剂可另外包含一医药上可接受的载剂。
以适于口服投药的剂型为例，本发明所提供的药剂可含有任何不会不利影响藁本内酯的所欲效益的医药可接受载剂，例如：溶剂(水、食盐水、葡萄糖(dextrose)、甘油、乙醇或其类似物、及前述的组合)、油性溶剂、稀释剂、安定剂、吸收延迟剂、崩散剂、乳化剂、抗氧化剂、粘合剂、润滑剂、吸湿剂、固体载剂(例如淀粉、皂土(bentonite))等。可利用任何合宜的方法，以适于口服投药的剂型提供该药剂，例如：锭剂、胶囊剂、颗粒剂、散剂、流浸膏剂、溶液剂、糖浆剂、悬液剂、乳剂、及酊剂等等。
至于适于皮下或静脉内注射的剂型，则可于本发明使用藁本内酯所制 造的药剂中含有一或多种例如等张溶液、盐类缓冲液(如磷酸盐缓冲液或柠檬酸盐缓冲液)、增溶剂、乳化剂、5％糖溶液、以及其他载剂等成分，以静脉输注液、乳剂静脉输注液、干粉注射剂、悬液注射剂、或干粉悬液注射剂等剂型提供该药剂。或者，将该药剂制备成一注射前固体，以可溶于其他溶液或悬浮液中的剂型、或可乳化的剂型提供该注射前固体，并于投予至该有需要的个体前将该注射前固体溶于其他溶液或悬浮液中、或将其乳化，提供所需要的注射剂。
视需要地，可于本发明使用藁本内酯所制造的药剂中另含有调味剂、调色剂、着色剂等添加剂，以提高该药剂于服用时的口适感及视觉感受；另可添加合宜用量的保存剂、防腐剂、抗菌剂、抗真菌剂等，以改善该药剂的储存性。此外，该药剂可视需要另含一或多种其他活性成分或与含该一或多种其他活性成分的药物并用，以进一步加强该药剂的功效或增加制剂配方的运用灵活性与调配度，只要该其他活性成分对藁本内酯的所欲效益没有不利的影响即可。
可以一日一次、一日多次、或数日一次等不同投药频率施用本发明藁本内酯所制造的药剂，端视投予个体的需求而异。举例言之，当并用本发明藁本内酯所制造的药剂与经藁本内酯处理的干细胞于人体以治疗中风时，以藁本内酯计，其用量为每天约1毫克/公斤体重至约120毫克/公斤体重，较佳为每天约10毫克/公斤体重至约95毫克/公斤体重，更佳为每天约20毫克/公斤体重至约40毫克/公斤体重，其中，该单位“毫克/公斤体重”指每公斤体重个体所需的投药量。但是，对急性患者而言，其用量可视实际需要而酌增，例如增加至数倍或数十倍。于本发明一具体实施态样中，使用藁本内酯于制造治疗中风药剂的用途，该药剂与经藁本内酯处理的干细胞并用，且其用量以藁本内酯计，为约30毫克/公斤体重或约90毫克/公斤体重。
本发明另关于一种干细胞治疗方法，其包含于有需要的个体投予经藁本内酯处理的干细胞。此外，可视需要对该有需要的个体，投予额外的藁 本内酯，该藁本内酯的投予可与该经藁本内酯处理的干细胞的投予同时或分别进行。其中，有关干细胞的处理与相关物料的选用、藁本内酯的用量范围、以及相关治疗的应用，均如上述。
兹以下列实施例进一步例示说明本发明。其中该等实施例仅提供作为说明，而非用以限制本发明的保护范围。本发明保护范围如权利要求所示。
实施例
A.细胞实验
实施例1：细胞存活测试(MTT分析)
于37℃、5％二氧化碳下，将脂肪干细胞(adipose stem cell，ADSC)培养于无血清角质细胞培养液(keratinocyte-serum free medium，K-SFM，Gibco)中。该K-SFM培养液系添加有牛脑下垂体萃取物(bovine pituitary extract，Gibco)、重组表皮生长因子(recombinant epidermal growth factor，Gibco)、最终浓度为2×103摩尔浓度的N-乙酰基-L-半胱氨酸(N-acetyl-L-cysteine，Sigma)、最终浓度为2×104摩尔浓度的L-抗坏血酸-2-磷酸盐倍半镁盐水合物(L-ascorbicacid 2-phosphate sesquimagnesium salthydrate，Sigma)、以及10％胎牛血清(FBS，Hyclone)。
将前述含有脂肪干细胞的培养液分为A、B二大组，各包括8小组，于各小组添加不同浓度(0、0.625、1.25、2.5、5、10、20、及40微克/毫升)的(Z)-藁本内酯处理进行，A组历时24小时且B组历时48小时。接着，使用3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide，简称MTT)分析试剂进行染色，然后以可见光检测仪检测570/630纳米波长的吸光值(OD570-630nm)，并计算所检测到的吸光值，以了解藁本内酯是否会影响脂肪干细胞的存活率。A、B二组的结果分别示于图1A和图1B中。
实施例2：藁本内酯于增加促进分化的基因表现、增加促进回归的基因表现、以及降低发炎基因表现的效益
(1)以藁本内酯处理干细胞
于37℃、5％二氧化碳下，将脂肪干细胞培养于实施例1所述的K-SFM培养液中，将前述脂肪干细胞分为5组，并分别处理不同浓度(0、0.625、1.25、2.5、5、及10微克/毫升)的(Z)-藁本内酯，历时24小时。
(2)制备干细胞的全细胞RNA
对上述(1)所提供的各组干细胞，先移除培养液，再于每个孔中加入1毫升的TRIzol试剂(Invitrogen)，于室温培养5分钟后，以刮杓将细胞刮下并置于1.5毫升的微量离心管中。充分将细胞打破后，再于微量离心管中加入0.2毫升的三氯甲烷，上下摇晃15秒。于室温下培养约2至3分钟后，进行离心处理(12,000g，15分钟，4℃)，将上清液移至另一新的1.5毫升的微量离心管中，加入0.5毫升的异丙醇，并充分混合。接着，于室温下培养10分钟，离心处理(12,000g，10分钟，4℃)后，移除上清液。以1毫升的含75％乙醇的DEPC水(Diethylpyrocarbonate(DEPC)-treated H2O)清洗所留下的沉淀物。再次进行离心处理(7500g，5分钟，4℃)后，移除上清液，以真空抽气方式将所留下的沉淀物干燥。以约0.01至0.02毫升的DEPC水溶解该干燥后的沉淀物，分别得到实验组与空白组的脂肪干细胞的全细胞RNA，以分光光度计(DU-800，Beckman)测量波长260纳米的吸光值，以估算RNA的浓度，并将所得的RNA储存于-80℃冰箱，以供实验(3)之用。
(3)反转录聚合酶连锁反应(RT-PCR)及胶体分析
以即时反转录聚合酶连锁反应(real-time PCR，RT-PCR)分析藁本内酯对促进分化的基因(例如，BDNF、NURR1)的表现、促进回归的基因(例如，CXCR4、SDF1αβ)的表现、以及发炎基因(例如，IL-6、IL-8)的表现的影响，并以甘油醛-3-磷酸脱氢酶(Glyceraldehyde 3-phosphate dehydrogenase，GAPDH)基因的表现做为对照组，步骤如下所述。
先将上述(2)所提供的全细胞RNA反转录成cDNA，再取2.5微升cDNA，并加入2.5微升如表1A所示的引物(10微微摩尔浓度)、7.5微升的去离子水、以及12.5微升的
Figure PCTCN2014086926-appb-000001
PLUS GREEN 2X Master Mix (Lucigen，Middleton，Wisconsin，USA)，然后将前述样品置于聚合酶连锁反应器中，并设定反应条件如下：i)94℃，30秒、55℃，30秒、94℃，60秒，以上步骤进行30个循环；ii)72℃，10分钟；iii)最后降温至4℃以结束反应。
将所得的聚合酶连锁反应(PCR)产物，以1.5％琼脂胶体进行30分钟电泳(电压：100伏特)，接着将该胶体置于溴化乙锭(ethidium bromide，EtBr)中染色10分钟，再利用照胶系统(DOC PRINT DP-001FDC，VilberLourmat France)拍摄影像，结果示于图2，最后以软件(Image J)定量基因表现量的强弱，结果示于表1B。
表1A
Figure PCTCN2014086926-appb-000002
表1B
Figure PCTCN2014086926-appb-000003
由图2及表1B可知，相较于对照组(即，GAPDH)，NURR1、BDNF、CXCR4、及SDF1αβ基因的表现量皆因施以藁本内酯而上升，IL-6及IL-8基因的表现量则因施以藁本内酯而下降。此结果显示，藁本内酯可有效增加促进分化的基因表现、增加促进回归的基因表现、以及降低发炎基因表现，故可降低干细胞的发炎反应并增加干细胞的存活率。
B.动物实验
实施例3：小鼠中风模型的建立
为避免小鼠因环境不适所造成的紧迫、焦虑而影响实验进行与结果，将BALB/c雄性小鼠分笼后，先给予数天的适应期，并于以凝血酶促使中大脑动脉产生血栓的前一天，先进行神经行为学观察分析。
手术前，先以0.25毫升的4％无水氯醛(chloral hydrate)对雄性BALB/c小鼠(每只约8周龄、约25克)进行麻醉，手术进行中，持续以异氟醚(isoflurane)维持小鼠的麻醉，避免小鼠于手术期间苏醒而影响实验结果。
麻醉后，将小鼠固定在小动物头部立体定位仪上，接着在小鼠右眼窝后方1毫米处的皮肤剪开，使结缔组织与肌肉暴露出来。接着，将结缔组织与肌肉剪开，使小鼠右侧的头骨显露出来，将小鼠剪开的皮肤与肌肉拨至两侧固定。然后，利用小型电钻进行小鼠的颅骨开创，在小鼠的颧骨上 方取下一个圆形区域的头骨。取下头骨后，在显微镜下找到中大脑动脉，将激光多普勒流量计(Laser Doppler Flowmetry)的探针架设在中大脑动脉上方1毫米的位置，以测定注射凝血酶(thrombin)前至注射后形成血栓的血流。以架设在显微注射器上的玻璃针吸取1微升的凝血蛋白酶，接着把注射器放至液压三向调节仪上，并缓缓的靠近中大脑动脉，小心的将玻璃针穿破脑膜并插入血管后，立刻用气动泵将凝血蛋白酶注射进入中大脑动脉，待10分钟后将玻璃针缓缓移开，所得即为中风的小鼠模型，简称“中风组(注射凝血酶)”。另有未注射凝血酶的“假手术组”。
实施例4：对中风小鼠进行干细胞治疗
于37℃、5％二氧化碳下，将脂肪干细胞培养于K-SFM培养液中，以2.5微克/毫升的(Z)-藁本内酯对干细胞进行前处理，历时24小时后，所得为“经藁本内酯处理的干细胞”。另外，未使用藁本内酯进行前处理的脂肪干细胞则为“未经处理的干细胞”。于将前述二种干细胞注射进入小鼠脑部之前，先以Hoechst染料进行染色1小时，以利后续追踪。
于实施例3的“中风组”小鼠手术后的2小时内，将上述“经藁本内酯处理的干细胞”注射至小鼠脑部的3个位置(每只小鼠皆共注射1×106个“经藁本内酯处理的干细胞”)，注射的部位可参考Enhancement of neuroplasticity through upregulation of beta1-integrin in human umbilical cord-derived stromal cell implanted stroke model.Neurobiol Dis.27(3):339-53(2007)，该文献全文并于此处以供参考，所得为「中风+经藁本内酯处理的干细胞组。另外，对“中风组”小鼠注射“未经处理的干细胞”，所得则为“中风+未经处理的干细胞组”，对照组则为仅对中风小鼠注射等量的食盐水，此为“中风+食盐水组”。
实施例5：藁本内酯提升干细胞的治疗效益
于实施3的“假手术组(即，第1组)”以及实施例4的“中风+食盐水组(即，第2组)”、“中风+未经处理的干细胞组(即，第3组)”、及“中风+经藁本内酯处理的干细胞组(即，第4组)”小鼠进行治疗的前一天以 及治疗后的第1、3、7、14天，分别对所述四组小鼠进行神经行为学观察分析，包括旋转轮测试(Rotarod analysis)以及平衡木测试(Beam walking analysis)。
其中，旋转轮测试利用小鼠在滚轮上的跑动时间来评估小鼠的协调性，结果示于图3，其中，小鼠的跑动时间越长代表协调性越佳；平衡木测试将小鼠置于平衡木上，记录小鼠通过平衡木所需的时间以及后脚滑掉的次数(即，脚误分数)，据此反应出小鼠的平衡能力，结果示于图4A和图4B。
由图3可知，相较于“中风+未经处理的干细胞组”小鼠，“中风+经藁本内酯处理的干细胞组”小鼠于滚轮上的跑动时间明显较长。此结果显示，以“经藁本内酯处理的干细胞”进行治疗的中风小鼠的协调性较佳。
由图4A可知，相较于“中风+未经处理的干细胞组”小鼠，“中风+经藁本内酯处理的干细胞组”小鼠通过平衡木所需的时间明显较少，且于以经藁本内酯处理的干细胞治疗后的第3天，小鼠通过平衡木所需的时间明显缩短。另，图4B可知，相较于“中风+未经处理的干细胞组”小鼠，“中风+经藁本内酯处理的干细胞组”小鼠的后脚误分数明显较低。前述结果显示，以“经藁本内酯处理的干细胞”进行治疗的中风小鼠的平衡能力较佳，且于治疗后的第3天即有显著性的回复。
上述结果显示，藁本内酯确实具有提升干细胞的治疗效益的效果。
实施例6：并用“藁本内酯”及“经藁本内酯处理的干细胞”以治疗疾病
除上述“假手术组(即，第1组)”、“中风+食盐水组(即，第2组)”、“中风+未经处理的干细胞组(即，第3组)”、“中风+经藁本内酯处理的干细胞组(即，第4组)”之外。另以(Z)-藁本内酯(30或90毫克/公斤体重)与实施例4所提供的“经藁本内酯处理的干细胞”并用，同时注射至中风小鼠的脑部，所得简称为“中风+经藁本内酯处理的干细胞+30毫克/公斤体重的藁本内酯组(即，第5组)”、及“中风+经藁本内酯处理的干细胞+90毫克/公斤体重的藁本内酯组(即，第6组)”。于进行治疗的前一天 以及治疗后的第1、3、7、14天，分别对前述六组小鼠进行平衡木测试，结果示于图5A和图5B；以及八通道穿梭箱(Locomotor activity box)实验，结果示于图6A和图6B。其中，八通道穿梭箱实验由连接电脑的感应器显示并记录小鼠在八通道穿梭箱中的移动数据，包括水平位移(例如：走动)、垂直位移(例如：抬头、攀爬)、及整体总位移的数据，最后汇整进行统计分析，以显示小鼠的活动能力。
由图5A和图5B可知，相较于“中风+经藁本内酯处理的干细胞组”小鼠，“中风+经藁本内酯处理的干细胞+30毫克/公斤体重的藁本内酯组”小鼠及“中风+经藁本内酯处理的干细胞+90毫克/公斤体重的藁本内酯组”小鼠通过平衡木所需的时间明显缩短，且后脚误分数明显较低，其中，又以“中风+经藁本内酯处理的干细胞+30毫克/公斤体重的藁本内酯组”小鼠最为明显。前述结果显示，以“藁本内酯”与“经藁本内酯处理的干细胞”合并治疗的中风小鼠的平衡能力较佳，尤其以“30毫克/公斤体重的藁本内酯”与“经藁本内酯处理的干细胞”合并治疗后的平衡能力回复效果最为显著。
由图6A和图6B可知，相较于“中风+经藁本内酯处理的干细胞组”小鼠于八通道穿梭箱内的总位移距离以及移动时间，“中风+经藁本内酯处理的干细胞+30毫克/公斤体重的藁本内酯组”小鼠及“中风+经藁本内酯处理的干细胞+90毫克/公斤体重的藁本内酯组”小鼠于八通道穿梭箱内的总位移距离及移动时间皆明显较长，其中，又以“中风+经藁本内酯处理的干细胞+30毫克/公斤体重的藁本内酯组”小鼠最为明显。前述结果显示，以“藁本内酯”与“经藁本内酯处理的干细胞”合并治疗的中风小鼠的活动能力较佳，尤其以“30毫克/公斤体重的藁本内酯”与“经藁本内酯处理的干细胞”合并治疗后的活动能力回复效果最为显著。
上述结果显示，并用“藁本内酯”及“经藁本内酯处理的干细胞”，可进一步提升干细胞的治疗效益。
以上仅为本发明的较佳实施例，当不得以此限定本发明实施的技术范 围，因此凡参考本发明的说明书内容所作的简单等效变化与修饰，皆应仍属本发明的保护范围。

Claims (12)

  1. 一种提升干细胞治疗效益的方法，其特征包含于干细胞培养液中以藁本内酯(ligustilide)处理该干细胞。
  2. 如权利要求1所述的方法，其特征在于，该干细胞为胚胎干细胞、成体干细胞、或诱导型多能干细胞。
  3. 如权利要求1所述的方法，其特征在于，该干细胞为脂肪干细胞。
  4. 如权利要求1所述的方法，其特征在于，藁本内酯的用量为每毫升干细胞培养液0.1至40微克。
  5. 如权利要求1至权利要求4中任一项所述的方法，其特征在于，提升干细胞于以下至少一者的效益：治疗自体免疫疾病、治疗消化道疾病、治疗肝脏疾病、治疗肾脏疾病、治疗心血管疾病、治疗神经系统疾病、治疗血液性疾病、治疗骨胳退化、治疗牙周病、治疗肌腱发炎、治疗脊椎损伤、治疗头部创伤、整形手术、秃头、皮肤美白、及去除皱纹。
  6. 如权利要求1至权利要求4中任一项所述的方法，其特征在于，提升干细胞于治疗中风的效益。
  7. 一种使用藁本内酯于制造药剂的用途，其特征在于，该药剂与经藁本内酯处理的干细胞并用于干细胞治疗。
  8. 如权利要求7所述的用途，其特征在于，该干细胞选自：胚胎干细胞、成体干细胞、及诱导型多能干细胞中的至少一种。
  9. 如权利要求7所述的用途，其特征在于，该干细胞为脂肪干细胞。
  10. 如权利要求7至权利要求9中任一项所述的用途，其特征在于，该药剂与干细胞并用于以下的至少一者：治疗自体免疫疾病、治疗消化道疾病、治疗肝脏疾病、治疗肾脏疾病、治疗心血管疾病、治疗神经系统疾病、治疗血液性疾病、治疗骨胳退化、治疗牙周病、治疗肌腱发炎、治疗脊椎损伤、治疗头部创伤、整形手术、秃头、皮肤美白、及去除皱纹。
  11. 如权利要求10所述的用途，其特征在于，该药剂与干细胞并用以治疗心血管疾病。
  12. 如权利要求11所述的用途，其特征在于，该药剂与干细胞并用以治疗中风。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111617072A (zh) * 2019-02-27 2020-09-04 苏州凯祥生物科技有限公司 一种高尿酸血症药物组合物及用于治疗高尿酸血症的药物
CN114561347A (zh) * 2022-02-25 2022-05-31 和携科技有限公司 一种脂肪间充质干细胞的培养基和培养方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104042606A (zh) * 2013-03-12 2014-09-17 国钦生物科技股份有限公司 苯酞化合物的应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104042606A (zh) * 2013-03-12 2014-09-17 国钦生物科技股份有限公司 苯酞化合物的应用

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111617072A (zh) * 2019-02-27 2020-09-04 苏州凯祥生物科技有限公司 一种高尿酸血症药物组合物及用于治疗高尿酸血症的药物
CN111617072B (zh) * 2019-02-27 2023-06-20 苏州凯祥生物科技有限公司 一种高尿酸血症药物组合物及用于治疗高尿酸血症的药物
CN114561347A (zh) * 2022-02-25 2022-05-31 和携科技有限公司 一种脂肪间充质干细胞的培养基和培养方法

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