WO2022247848A1 - Preparation method for and application of hair follicle mesenchymal stem cell - Google Patents
Preparation method for and application of hair follicle mesenchymal stem cell Download PDFInfo
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- WO2022247848A1 WO2022247848A1 PCT/CN2022/094871 CN2022094871W WO2022247848A1 WO 2022247848 A1 WO2022247848 A1 WO 2022247848A1 CN 2022094871 W CN2022094871 W CN 2022094871W WO 2022247848 A1 WO2022247848 A1 WO 2022247848A1
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- hair follicle
- mesenchymal stem
- osteoporosis
- stem cells
- tissue
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0666—Mesenchymal stem cells from hair follicles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Definitions
- the application relates to a preparation method of hair follicle mesenchymal stem cells and a medical application of hair follicle mesenchymal stem cells.
- MSCs Mesenchymal Stem Cells
- Mesenchymal Stem Cells are a type of undifferentiated adult stem cells that exist in differentiated tissues. They have the potential of self-renewal and multidirectional differentiation, and can be induced to differentiate into chondrocytes, osteoblasts, smooth muscle cells, etc. It is an ideal seed cell source for regenerative medicine. Mesenchymal stem cells also have a strong paracrine ability, secreting a variety of growth factors and cytokines that can promote angiogenesis and wound repair. Mesenchymal stem cells with self-renewal and multidirectional differentiation capabilities have been isolated from bone marrow, hair follicle, fat, pancreas, periosteum, synovium, skeletal muscle, skin, umbilical cord blood and other tissues.
- Human hair follicle mesenchymal stem cells are located in the hair papilla in the middle of the hair follicle. As a type of adult stem cells derived from hair follicles, they have self-renewal and multidirectional differentiation potential. It plays an important role in the formation of the cycle. As an important source of stem cells, it also has the advantages of abundant sources, convenient acquisition, easy expansion, low immunogenicity, and less ethical controversy. It is the seed cell for vascular tissue engineering, nerve regeneration, and hair follicle reconstruction. important clinical implications. In order to obtain hair follicle-derived mesenchymal stem cells whose quality and quantity meet the needs of clinical treatment, the selection of isolation and culture methods is very important.
- Osteoporosis namely osteoporosis (osteoporosis)
- osteoporosis is a systemic bone disease in which bone density and bone quality decrease and bone microstructure is destroyed due to various reasons, resulting in increased bone fragility and prone to fracture.
- Osteoporosis is divided into primary and secondary two categories.
- Primary osteoporosis is divided into three types: postmenopausal osteoporosis (type I), senile osteoporosis (type II) and idiopathic osteoporosis; secondary osteoporosis is due to Reduced bone mass caused by various systemic or endocrine and metabolic diseases.
- postmenopausal osteoporosis generally occurs in women within 5 to 10 years after menopause; senile osteoporosis generally refers to the osteoporosis that occurs in the elderly after the age of 70; and idiopathic osteoporosis mainly occurs in adolescents.
- the serious consequence of osteoporosis is the occurrence of osteoporotic fractures (fragility fractures), that is, fractures that can occur during minor trauma or daily activities, and the most common sites are the spine, hip and forearm. Fractures can lead to significantly increased morbidity and mortality in patients with osteoporosis. Therefore, it is extremely important to find an effective method for treating osteoporosis.
- One of the contents of the present invention is a method for preparing hair follicle mesenchymal stem cells, the preparation method comprising:
- step 2) can adopt enzymatic hydrolysis method to obtain hair follicle mesenchymal stem cells by separating from hair follicle tissue, and the enzymatic hydrolysis solution used can contain collagenase, dispase II (Dispase II) and calcium chloride.
- the culture medium may contain amino acids, vitamins, organic compounds, inorganic compounds, hormones, growth factors, trace elements, fetal bovine serum and antibiotics.
- the method of the present invention may also include step 4): passage the cultured hair follicle mesenchymal stem cells.
- the culture medium is ScienCell 7501 mesenchymal stem cell culture medium.
- the enzymolysis solution may contain collagenase IV, dispase II (Dispase II) and calcium chloride.
- the content of the present invention also includes the hair follicle mesenchymal stem cells obtained by the method of the present invention.
- the content of the present invention also includes the application of the hair follicle mesenchymal stem cells in the preparation of drugs for the treatment of osteoporosis and osteopenia; preferably, the osteoporosis is primary osteoporosis; more preferably, the Described osteoporosis is middle-aged and elderly osteoporosis or postmenopausal osteoporosis.
- the drug is in a liquid dosage form, preferably an intravenous administration dosage form.
- the content of the present invention also includes a method for treating osteoporosis or osteopenia: comprising the step of administering the hair follicle mesenchymal stem cells to patients suffering from osteoporosis or osteopenia.
- the osteoporosis is primary osteoporosis; more preferably, the osteoporosis is middle-aged osteoporosis or postmenopausal osteoporosis.
- the administration method is intravenous administration, preferably intravenous drip or intravenous injection.
- FIG. 1 shows the result graph of the amplification factor of different generations of hair follicle mesenchymal stem cells prepared by the present invention in Example 1.
- FIG. 2 shows the results of the total amount of hair follicle mesenchymal stem cells prepared by the present invention in Example 1 at different generations.
- FIG. 3 shows the results of regulation of Th lymphocyte subsets by hair follicle mesenchymal stem cells cultured in different media in Example 2.
- FIG. 4 shows the results of regulation of Treg lymphocyte subsets by hair follicle mesenchymal stem cells cultured in different media in Example 2.
- Figure 5 shows the situation of human-derived hair follicle mesenchymal stem cells delaying the age-related bone loss of premature aging mice SAMP8 in Example 4.
- sub-figures A, B, C, D, and E respectively show the changes in bone volume fraction, trabecular thickness, number of trabecular bone, trabecular bone gap, and shape of cancellous bone in the proximal tibia.
- Ranges are disclosed herein in terms of lower limits and upper limits. There can be one or more lower bounds, and one or more upper bounds, respectively.
- a given range is defined by selecting a lower limit and an upper limit. Selected lower and upper limits define the boundaries of a particular range. All ranges that may be defined in this manner are inclusive and combinable, ie, any lower limit may be combined with any upper limit to form a range.
- the numerical value or numerical range covers the approximate range that can be understood by those skilled in the relevant art to be equivalent to the numerical value or numerical range, for example, the numerical value Or the range of ⁇ 10% of the end value, or the range of ⁇ 5%, ⁇ 3%, ⁇ 2%, ⁇ 1% or ⁇ 0.5%.
- the invention provides a method for preparing hair follicle mesenchymal stem cells, the preparation method comprising:
- the method of the present invention may also include step 4): passage the cultured hair follicle mesenchymal stem cells.
- step 1) obtaining the hair follicle tissue includes extracting the complete hair follicle, that is, removing the skin tissue surrounding the hair follicle, and peeling off the complete hair follicle tissue.
- the specific steps may include: rinsing the skin tissue surrounding the hair follicle stored in the tissue storage solution with a cleaning solution, using ophthalmic forceps, a scalpel and other tools to peel off the complete hair follicle tissue, and storing it in a fresh tissue storage solution. middle.
- the extraction of intact hair follicles can also be obtained directly through a hair follicle extraction machine.
- step 2) obtaining hair follicle mesenchymal stem cells from hair follicle tissue includes enzymatic hydrolysis or tissue attachment method, and the obtained hair follicle mesenchymal stem cells are also called primary hair follicle mesenchymal stem cells.
- the enzymatic hydrolysis method is to use enzymatic hydrolysis solution to enzymatically hydrolyze the hair follicle tissue to make it loose and facilitate stem cells to climb out.
- the specific steps may include adding an enzymatic hydrolysis solution to enzymatically hydrolyze the hair follicle tissue, and placing it under suitable conditions for enzymatic hydrolysis.
- the tissue-adherence method mainly allows stem cells to climb out of the hair follicle tissue through adherent culture.
- specific steps may include using a needle under a stereomicroscope to make an incision at the outer hair root sheath of the hair follicle, adding medium after the hair follicle tissue is completely adhered to the wall, and culturing statically under suitable conditions to wait for the cells around the hair follicle tissue to crawl out.
- the cultivation of the primary cells in step 3) includes resuspending the primary hair follicle mesenchymal stem cells in a culture medium, inoculating them in a culture vessel, and culturing them under suitable conditions.
- the cell subculture in step 4) includes adding digestive enzymes to digest the adherent cells from the wall of the vessel, after terminating the digestion, harvesting the cells, adding medium to resuspend, inoculating in a culture container, and placing them under suitable conditions nourish.
- the medium in step 3) and step 4), contains amino acids, vitamins, organic compounds, inorganic compounds, hormones, growth factors, trace elements, fetal bovine serum and antibiotics.
- the culture medium can be prepared by oneself or purchased through commercial channels.
- the volume percentage of fetal calf serum in the culture medium is about 5-10%, preferably about 5% or about 10%.
- the antibiotic is gentamicin, penicillin, streptomycin or a combination thereof.
- the antibiotic is gentamicin, its concentration is about 50-100U/mL, preferably about 50U/mL; if the antibiotic is penicillin, its concentration is about 100-150U/mL, preferably about 100 U/mL; or if the antibiotic is streptomycin, its concentration is about 0.10-0.15 mg/mL, preferably about 0.10 mg/mL.
- the medium is ScienCell 7501 mesenchymal stem cell medium (purchased from ScienCell Research Laboratories).
- the enzymatic hydrolysis solution used in step 2) includes collagenase, dispase II and calcium chloride.
- the collagenase may be collagenase I or collagenase IV, preferably collagenase IV.
- the concentration of collagenase is about 0.1-5 mg/mL, preferably about 1 mg/mL.
- the concentration of the neutral protease (Dispase II) is about 1-4 mg/mL, preferably about 2 mg/mL.
- the calcium chloride concentration is about 5 mM.
- the enzymatic hydrolysis time is about 1-3.5 hours, preferably about 3 hours.
- the enzymatic hydrolysis temperature is about 37°C.
- the tissue storage solution when used in the process of obtaining hair follicle tissue in step 1), can be a basal medium containing antibiotics and fetal bovine serum, such as DMEM supplemented with antibiotics and fetal bovine serum /F12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F12).
- the antibiotic is selected from gentamicin, penicillin, streptomycin or combinations thereof.
- the tissue storage solution contains gentamicin, its concentration is about 50-100 U/mL, preferably about 50 U/mL; if the tissue storage solution contains penicillin, its concentration is about 100-150 U/mL, Preferably about 100 U/mL, if the tissue storage solution contains streptomycin, its concentration is about 0.10-0.15 mg/mL, preferably about 0.10 mg/mL. In some embodiments, the fetal bovine serum volume percentage is about 5%-10%, preferably about 10%.
- the cleaning solution used for cleaning tissues or cells in the preparation method may be basal medium or phosphate buffer, preferably basal medium and phosphate buffer containing antibiotics.
- the antibiotic can be selected from gentamicin, penicillin, streptomycin or combinations thereof.
- the cleaning solution contains gentamicin its concentration is about 50-100 U/mL, preferably about 50 U/mL; if the cleaning solution contains penicillin, its concentration is about 100-150 U/mL , preferably about 100 U/mL; or if the cleaning solution contains streptomycin, its concentration is about 0.10-0.15 mg/mL, preferably about 0.10 mg/mL.
- the digestive enzyme used for digestion of adherent cells may be trypsin or a stable trypsin replacement enzyme, preferably a stable trypsin replacement enzyme (eg TrypLE TM Express Enzyme (1 ⁇ ), no phenol red).
- a stable trypsin replacement enzyme eg TrypLE TM Express Enzyme (1 ⁇ ), no phenol red.
- the primary cells in the step 2), can be obtained by enzymatic hydrolysis at 5% CO 2 at 37°C; more preferably, the primary cells can be obtained by enzymatic hydrolysis in a constant temperature oscillating metal bath at 37°C. Generation cells, and set the rotation speed to 600rpm.
- primary cell culture and subculture can be carried out under the conditions of 5% CO 2 and 37°C.
- the invention also provides hair follicle mesenchymal stem cells prepared by the method of the invention.
- the hair follicle mesenchymal stem cells obtained in the invention have enhanced cell proliferation ability and enhanced immune regulation ability.
- the present invention also includes the application of the hair follicle mesenchymal stem cells in the preparation of medicines for treating osteoporosis and osteopenia; preferably, the osteoporosis is primary osteoporosis; more preferably, the bone Osteoporosis is middle-aged and elderly osteoporosis or postmenopausal osteoporosis.
- the pharmaceutical dosage form is a liquid dosage form, preferably an intravenous dosage form.
- the content of the present invention also includes a method for treating osteoporosis or osteopenia: comprising the step of administering the hair follicle mesenchymal stem cells to patients suffering from osteoporosis or osteopenia.
- the osteoporosis is primary osteoporosis; more preferably, the osteoporosis is middle-aged osteoporosis or postmenopausal osteoporosis.
- the administration method is intravenous administration, preferably intravenous infusion or intravenous injection.
- the order of magnitude of the dosage can be, for example, 10 6 -10 9 cells/time.
- the administered dose may be a fixed dose (also known as a flat dose) or a dose based on the patient's weight.
- the frequency of administration and the period of administration are not particularly limited, for example, once every 10 days to 3 years, for a total of 1 to 10 administrations. In a preferred embodiment, the administration is administered every three months for a total of two administrations.
- Example 1 Preparation of human hair follicle mesenchymal stem cells
- tissue storage solution prepare tissue storage solution containing 50 U/mL gentamicin (purchased from Huazhong Pharmaceutical Co., Ltd.) and 10% fetal bovine serum in DMEM/F12 basal medium.
- the scalp tissue wrapped with human hair follicles into a petri dish containing 10mL of cleaning solution, rinse twice, soak the rinsed scalp tissue in the tissue storage solution, use elbow ophthalmic tweezers, and assist a scalpel to cut off
- the left hand clamps the skin tissue laterally with ophthalmic tweezers
- the right hand uses micro tweezers to carefully separate the skin tissue wrapped around the hair follicles.
- the stripped hair follicles will carry a small portion of skin connective tissue, and the connective tissue around the hair follicle tissue is carefully separated using microscopic forceps under a stereo microscope. Place the stripped complete hair follicle tissue in a petri dish containing tissue storage solution.
- step 1) After obtaining the complete hair follicle tissue according to step 1), carefully place the hair follicle tissue at the bottom of the EP tube with microscopic tweezers, and use a pipette gun to suck out the tissue storage solution carried by the hair follicle tissue as much as possible. Add 8 ⁇ L of enzymatic hydrolysis solution to each hair follicle, and let it stand in a 5% CO 2 incubator at 37°C for 3 hours, carefully flick the bottom of the tube every 1 hour, and mix gently.
- the hair follicle mesenchymal stem cells prepared through the above steps were subcultured, and each passage was inoculated at a density of 5.0 ⁇ 103 cells/ cm2 into a culture medium containing ScienCell 7501 mesenchymal stem cell medium (purchased from ScienCell Research Laboratories).
- a culture medium containing ScienCell 7501 mesenchymal stem cell medium purchased from ScienCell Research Laboratories.
- the cell density in the culture flask reaches more than 90%, discard the medium, wash twice with the cleaning solution, and discard the cleaning solution.
- Add 3 mL of stable trypsin replacement enzyme (TrypLE TM Express Enzyme (1 ⁇ ), no phenol red) to the cell pellet at the bottom of the culture flask, and place it at room temperature for 3 minutes for digestion.
- the hair follicle mesenchymal stem cells prepared by the preparation method of the present invention can harvest a total of 3 ⁇ 10 8 cells after passage P3, which meets the production requirements.
- the cell amplification factor is more than 10 times
- the cell expansion factor is more than 5 times, showing a strong cell proliferation ability.
- Example 2 Comparison of the regulatory effects of human hair follicle mesenchymal stem cells cultured in different media on immune function
- human embryonic stem cell complete medium purchased from Saiye Biotechnology Co., Ltd.
- amniotic fluid medium purchased from Guangzhou Baidi Biomedicine Co., Ltd.
- ScienCell 7501 mesenchymal stem cell medium Purchased from ScienCell Research Laboratories
- PBMC Peripheral blood mononuclear cells
- Peripheral blood mononuclear cells were co-cultured so that the ratio of hair follicle mesenchymal stem cells and peripheral blood mononuclear cells in the wells was 1:5, and they were used as the test group.
- Peripheral blood mononuclear cells were cultured separately, and the number of inoculated cells was 5 ⁇ 10 5 per well, which was used as a control group.
- hair follicle mesenchymal stem cells can effectively inhibit the proliferation of pro-inflammatory lymphocyte subsets Th1 and Th17, and the effects of hair follicle mesenchymal stem cells cultured in different media are significantly different, among which ScienCell 7501 mesenchymal stem cell medium The cultured hair follicle mesenchymal stem cells had the best inhibitory effect.
- Hair follicle mesenchymal stem cells regulate the role of regulatory T cells (Regulatory cell, Treg for short)
- Hair follicle mesenchymal stem cells obtained from different culture media were spread in twelve-well plates at a cell concentration of 1 ⁇ 10 5 /mL/well, and after 24 hours of adherent culture, 5 ⁇ 10 5 cells were inoculated in each well.
- Peripheral blood mononuclear cells so that the ratio of hair follicle mesenchymal stem cells and peripheral blood mononuclear cells in the wells is 1:5, were co-cultured as the test group.
- Peripheral blood mononuclear cells were cultured separately, and the number of inoculated cells was 5 ⁇ 10 5 per well, which was used as a control group. Place them in a 5% CO 2 , 37°C incubator for 72 hours.
- peripheral blood mononuclear cells of the test group and the control group were collected, the cells were stained according to the operation method of the Treg detection kit (purchased from Biolegend), and the Treg cell-related cytokines (CD4+, CD25+ and FoxP3+) were detected by flow cytometry , the results are shown in Table 2 and Figure 4.
- hair follicle mesenchymal stem cells can promote the proliferation of Treg lymphocyte subsets, and the effects of hair follicle mesenchymal stem cells cultured in different media are significantly different, among which hair follicle mesenchymal stem cells cultured in ScienCell 7501 mesenchymal stem cell The promotion effect of mesenchymal stem cells is the best.
- Human-derived hair follicle mesenchymal stem cells were prepared as described in Example 1, except that the enzymatic hydrolysis solutions of the following formula 1, formula 2 and formula 3 were used instead of the enzymatic hydrolysis solution in Example 1 to enzymolyze the hair follicle tissue.
- Enzyme solution formula 1 Accurately weigh 0.1 g of collagenase I and dissolve it in 5 mL of DMEM/F12 basal medium. After the collagenase I is completely dissolved, mix it upside down and filter it through a 0.22 ⁇ m filter to obtain 20 mg/mL collagenase Add 95 mL of Hank's Balanced Salt Solution (HBSS) to the I storage solution and dilute it to obtain a 1 mg/mL collagenase I hydrolysis solution.
- HBSS Hank's Balanced Salt Solution
- Enzyme solution formula 2 Accurately weigh 0.1g of collagenase IV and dissolve in 5ml of DMEM/F12 basal medium. After the collagenase IV is completely dissolved, mix upside down and filter through a 0.22 ⁇ m filter to obtain 20mg/mL collagen Add 95 mL of Hank's Balanced Salt Solution (HBSS) to the enzyme IV stock solution to dilute it to obtain a 1 mg/mL collagenase IV hydrolyzate.
- HBSS Hank's Balanced Salt Solution
- Enzymolysis Solution Formula 3 Prepare the enzymolysis solution as described in Example 1, except that collagenase IV is replaced with collagenase I, thus obtaining a solution containing 1 mg/mL collagenase I, 2 mg/mL Dispase II and 5mM calcium chloride enzymatic solution.
- the results under the microscope showed that the inner hair root sheath and the outer hair root sheath were separated from the hair shaft after 1 hour of enzymatic hydrolysis. and outer root sheath can be dispersed into single cells after blowing. The longer the time, the more sufficient the enzymatic hydrolysis, but there is little difference between 3.5 hours of enzymatic hydrolysis and 3 hours of microscopic examination.
- the hair follicle mesenchymal stem cells prepared by enzymatic hydrolysis for 3 hours were cultured in ScienCell 7501 mesenchymal stem cell medium. The growth status was observed under a microscope and the number of finally harvested cells was counted. The results are shown in Table 3.
- Table 3 The results of the number of hair follicle mesenchymal stem cells harvested by enzymolysis of hair follicle tissue with different enzymatic solutions
- Enzyme solution formula Culture days of primary hair follicle mesenchymal stem cells Harvested cell number Embodiment 1 enzymolysis solution formula 12 days 175000 Enzyme solution formula 1 12 days 55700
- Enzyme solution formula 2 12 days 98600
- Enzyme solution formula 3 12 days 91500
- the inventors selected 20-week-old progeria mice SAMP8 as the research object, and injected low-dose (1 ⁇ 10 5 cells/mouse) and high-dose (4 ⁇ 10 5 cells/mouse) human hair follicle-derived mesenchymal stem cells ( Example 1), a total of 3 injections, with an interval of 1 week between the two injections, in order to explore the effect of human hair follicle mesenchymal stem cells on delaying the age-related bone loss of premature aging mice SAMP8.
- SAMP8 mice are a commonly used animal model of rapid aging. They are used in the study of age-related primary osteoporosis and osteoporosis because they can spontaneously develop osteopenia and even osteoporosis in the early stage.
- mice were sacrificed 2 months after the last injection of cells, and the tibiae were isolated, and the effect of exogenous stem cells on the aging bone loss of proximal tibial cancellous bone in SAMP8 mice was detected by micro-computed tomography (Micro-CT) , specifically detected the improvement of bone volume fraction and other bone microstructural parameters (such as bone trabecular thickness, number and gap) and their 3D images.
- Micro-CT micro-computed tomography
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Abstract
Disclosed are a preparation method for a hair follicle mesenchymal stem cell and a hair follicle mesenchymal stem cell prepared thereby. Disclosed is a pharmaceutical application of the hair follicle mesenchymal stem cell in a drug for treating osteoporosis or diseases related to bone mass decrease.
Description
本申请涉及毛囊间充质干细胞的制备方法及毛囊间充质干细胞的医药应用。The application relates to a preparation method of hair follicle mesenchymal stem cells and a medical application of hair follicle mesenchymal stem cells.
间充质干细胞(Mesenchymal Stem Cells,MSCs)是一类存在于分化组织中的未分化的成体干细胞,具有自我更新和多向分化潜能,可诱导分化为软骨细胞、成骨细胞、平滑肌细胞等,是再生医学理想的种子细胞来源。间充质干细胞还具有强大的旁分泌能力,分泌的多种生长因子和细胞因子可促进血管生成和创伤修复。目前已从骨髓、毛囊、脂肪、胰腺、骨膜、滑膜、骨骼肌、皮肤、脐带血等组织中分离出来具有自我更新和多向分化能力的间充质干细胞。Mesenchymal Stem Cells (MSCs) are a type of undifferentiated adult stem cells that exist in differentiated tissues. They have the potential of self-renewal and multidirectional differentiation, and can be induced to differentiate into chondrocytes, osteoblasts, smooth muscle cells, etc. It is an ideal seed cell source for regenerative medicine. Mesenchymal stem cells also have a strong paracrine ability, secreting a variety of growth factors and cytokines that can promote angiogenesis and wound repair. Mesenchymal stem cells with self-renewal and multidirectional differentiation capabilities have been isolated from bone marrow, hair follicle, fat, pancreas, periosteum, synovium, skeletal muscle, skin, umbilical cord blood and other tissues.
人毛囊间充质干细胞位于毛囊毛球部中间的毛***内,作为一类来源于毛囊、具有自我更新和多向分化潜能的成体干细胞,人毛囊间充质干细胞不仅在毛囊发生、自我更新和周期形成中扮演重要角色,作为重要的干细胞来源还具有来源丰富、获取方便、易于扩增、免疫原性低、伦理争议少等优势,是血管组织工程、神经再生和毛囊重建的种子细胞,具有重要的临床治疗意义。为了获得质量和数量均满足临床治疗需要的毛囊间充质干细胞,分离和培养方法的选择十分重要,不同的分离和培养方法会导致细胞的分离成功率、增殖和分化能力以及临床治疗效果产生较大差异,因此,寻找到一种具有较好分离成功率、增殖和分化能力以及治疗效果的毛囊间充质干细胞的制备方法是必须要解决的问题。Human hair follicle mesenchymal stem cells are located in the hair papilla in the middle of the hair follicle. As a type of adult stem cells derived from hair follicles, they have self-renewal and multidirectional differentiation potential. It plays an important role in the formation of the cycle. As an important source of stem cells, it also has the advantages of abundant sources, convenient acquisition, easy expansion, low immunogenicity, and less ethical controversy. It is the seed cell for vascular tissue engineering, nerve regeneration, and hair follicle reconstruction. important clinical implications. In order to obtain hair follicle-derived mesenchymal stem cells whose quality and quantity meet the needs of clinical treatment, the selection of isolation and culture methods is very important. Different isolation and culture methods will lead to differences in the success rate of cell isolation, proliferation and differentiation capabilities, and clinical treatment effects. Therefore, it is a problem that must be solved to find a preparation method of hair follicle mesenchymal stem cells with better separation success rate, proliferation and differentiation ability and therapeutic effect.
骨质疏松,即骨质疏松症(osteoporosis),是由于多种原因导致的骨密度和骨质量下降,骨微结构破坏,造成骨脆性增加,从而容易发生骨折的全身性骨病。骨质疏松症分为原发性和继发性两大类。原发性骨质疏松症又分为绝经后骨质疏松症(Ⅰ型)、老年性骨质疏松症(Ⅱ型)和特发性骨质疏松三种;继发性骨质疏松则是由于各种全身性或内分泌代谢性疾病引起的骨组织量减少。其中,绝经后骨质疏松症一般发生在妇女绝经后5~10年内;老年性骨质疏松症一般指老人70岁后发生的骨质疏松;而特发性骨质疏松主要发生在青少年。骨质疏松的严重后果为发生骨质疏松性骨折(脆性骨折),即在受到轻微创伤时或日常活动中即可发生的骨折,好发部位为脊柱、髋部和前臂。发生骨折会导致骨质疏松症患者的病残率和死亡率明显增加。因此,寻找到一个有效治疗骨质疏松症的方法极为重要。Osteoporosis, namely osteoporosis (osteoporosis), is a systemic bone disease in which bone density and bone quality decrease and bone microstructure is destroyed due to various reasons, resulting in increased bone fragility and prone to fracture. Osteoporosis is divided into primary and secondary two categories. Primary osteoporosis is divided into three types: postmenopausal osteoporosis (type I), senile osteoporosis (type II) and idiopathic osteoporosis; secondary osteoporosis is due to Reduced bone mass caused by various systemic or endocrine and metabolic diseases. Among them, postmenopausal osteoporosis generally occurs in women within 5 to 10 years after menopause; senile osteoporosis generally refers to the osteoporosis that occurs in the elderly after the age of 70; and idiopathic osteoporosis mainly occurs in adolescents. The serious consequence of osteoporosis is the occurrence of osteoporotic fractures (fragility fractures), that is, fractures that can occur during minor trauma or daily activities, and the most common sites are the spine, hip and forearm. Fractures can lead to significantly increased morbidity and mortality in patients with osteoporosis. Therefore, it is extremely important to find an effective method for treating osteoporosis.
发明内容Contents of the invention
本发明内容之一是一种制备毛囊间充质干细胞的方法,该制备方法包括:One of the contents of the present invention is a method for preparing hair follicle mesenchymal stem cells, the preparation method comprising:
1)获得毛囊组织,优选来自人的毛囊组织;1) obtaining hair follicle tissue, preferably from human hair follicle tissue;
2)由毛囊组织获得毛囊间充质干细胞;和2) obtaining hair follicle mesenchymal stem cells from hair follicle tissue; and
3)于培养基中对所得毛囊间充质干细胞进行培养。3) culturing the obtained hair follicle mesenchymal stem cells in a culture medium.
其中,步骤2)可以采用酶解法由毛囊组织分离获得毛囊间充质干细胞,所用酶解液可包含胶原酶,中性蛋白酶Ⅱ(Dispase Ⅱ)和氯化钙。Wherein, step 2) can adopt enzymatic hydrolysis method to obtain hair follicle mesenchymal stem cells by separating from hair follicle tissue, and the enzymatic hydrolysis solution used can contain collagenase, dispase II (Dispase II) and calcium chloride.
其中,所述培养基可包含氨基酸、维生素、有机化合物、无机化合物、激素、生长因子、微量元素、胎牛血清和抗生素。Wherein, the culture medium may contain amino acids, vitamins, organic compounds, inorganic compounds, hormones, growth factors, trace elements, fetal bovine serum and antibiotics.
或选地,本发明方法还可以包括步骤4):对经培养的毛囊间充质干细胞进行传代。Alternatively, the method of the present invention may also include step 4): passage the cultured hair follicle mesenchymal stem cells.
优选实施方式之一中,所述培养基为ScienCell 7501间充质干细胞培养基。In one of the preferred embodiments, the culture medium is ScienCell 7501 mesenchymal stem cell culture medium.
优选实施方式之一中,酶解液可包含胶原酶IV,中性蛋白酶Ⅱ(Dispase Ⅱ)和氯化钙。In one of the preferred embodiments, the enzymolysis solution may contain collagenase IV, dispase II (Dispase II) and calcium chloride.
本发明的内容还包括用本发明方法获得的毛囊间充质干细胞。The content of the present invention also includes the hair follicle mesenchymal stem cells obtained by the method of the present invention.
本发明的内容也包括所述毛囊间充质干细胞在制备治疗骨质疏松和骨量减少疾病药物中的应用;优选地,所述骨质疏松为原发性骨质疏松;更优选地,所述骨质疏松为中老年骨质疏松或绝经后骨质疏松。The content of the present invention also includes the application of the hair follicle mesenchymal stem cells in the preparation of drugs for the treatment of osteoporosis and osteopenia; preferably, the osteoporosis is primary osteoporosis; more preferably, the Described osteoporosis is middle-aged and elderly osteoporosis or postmenopausal osteoporosis.
优选实施方式之一中,所述药物为液体剂型,优选静脉给药剂型。In one of the preferred embodiments, the drug is in a liquid dosage form, preferably an intravenous administration dosage form.
本发明的内容也包括一种治疗骨质疏松或骨量减少疾病的方法:包括对患有骨质疏松或骨量减少疾病的患者施用所述毛囊间充质干细胞的步骤。优选地,所述骨质疏松为原发性骨质疏松;更优选地,所述骨质疏松为中老年骨质疏松或绝经后骨质疏松。The content of the present invention also includes a method for treating osteoporosis or osteopenia: comprising the step of administering the hair follicle mesenchymal stem cells to patients suffering from osteoporosis or osteopenia. Preferably, the osteoporosis is primary osteoporosis; more preferably, the osteoporosis is middle-aged osteoporosis or postmenopausal osteoporosis.
优选实施方式之一中,所述施用方式为静脉给药,优选静脉滴注或静脉注射。In one of the preferred embodiments, the administration method is intravenous administration, preferably intravenous drip or intravenous injection.
图1显示了实施例1中本发明制备的毛囊间充质干细胞不同代次细胞的扩增倍数结果图。FIG. 1 shows the result graph of the amplification factor of different generations of hair follicle mesenchymal stem cells prepared by the present invention in Example 1.
图2显示了实施例1中本发明制备的毛囊间充质干细胞不同代次细胞总量结果图。FIG. 2 shows the results of the total amount of hair follicle mesenchymal stem cells prepared by the present invention in Example 1 at different generations.
图3显示了实施例2中不同培养基培养的毛囊间充质干细胞调节Th淋巴细胞亚群结果图。FIG. 3 shows the results of regulation of Th lymphocyte subsets by hair follicle mesenchymal stem cells cultured in different media in Example 2. FIG.
图4显示了实施例2中不同培养基培养的毛囊间充质干细胞调节Treg淋巴细胞亚群结果图。FIG. 4 shows the results of regulation of Treg lymphocyte subsets by hair follicle mesenchymal stem cells cultured in different media in Example 2. FIG.
图5显示了实施例4中人源毛囊间充质干细胞延缓早衰小鼠SAMP8增龄性骨流失的 情况。其中,分图A、B、C、D、E分别显示了胫骨近端松质骨的骨体积分数、骨小梁厚度、骨小梁数量、骨小梁间隙以及形态的变化情况。Figure 5 shows the situation of human-derived hair follicle mesenchymal stem cells delaying the age-related bone loss of premature aging mice SAMP8 in Example 4. Among them, sub-figures A, B, C, D, and E respectively show the changes in bone volume fraction, trabecular thickness, number of trabecular bone, trabecular bone gap, and shape of cancellous bone in the proximal tibia.
在本说明书中,如果没有特别的说明,所涉及的各组分或其优组分可以相互组合形成新的技术方案。In this specification, unless otherwise specified, the various components involved or their preferred components can be combined with each other to form a new technical solution.
在本说明书中,如果没有特别的说明,本文所提到的所有实施方案以及优选实施方案可以相互组合形成新的技术方案。In this specification, unless otherwise specified, all the embodiments and preferred embodiments mentioned herein can be combined with each other to form new technical solutions.
在本说明书中,如果没有特别的说明,本文所提到的所有技术特征以及优选特征可以相互组合形成新的技术方案。In this specification, if there is no special description, all the technical features and preferred features mentioned herein can be combined with each other to form a new technical solution.
如果没有特别指出,本说明书所用的术语“一种”指“至少一种”。The term "a" used in this specification means "at least one" unless otherwise specified.
本文所公开的“范围”以下限和上限的形式。可以分别为一个或多个下限,和一个或多个上限。给定范围是通过选定一个下限和一个上限进行限定的。选定的下限和上限限定了特别范围的边界。所有可以这种方式进行限定的范围是包含和可组合的,即任何下限可以与任何上限组合形成一个范围。"Ranges" are disclosed herein in terms of lower limits and upper limits. There can be one or more lower bounds, and one or more upper bounds, respectively. A given range is defined by selecting a lower limit and an upper limit. Selected lower and upper limits define the boundaries of a particular range. All ranges that may be defined in this manner are inclusive and combinable, ie, any lower limit may be combined with any upper limit to form a range.
在本说明书中,如果没有特别的说明,所述数值或数值范围不论是否带有前行词“约”均涵盖相关领域技术人员能够理解的与该数值或数值范围等同的约略范围,例如该数值或端值±10%的范围,亦可是±5%、±3%、±2%、±1%或±0.5%的范围。In this specification, if there is no special description, the numerical value or numerical range, regardless of whether it is preceded by the word "about", covers the approximate range that can be understood by those skilled in the relevant art to be equivalent to the numerical value or numerical range, for example, the numerical value Or the range of ±10% of the end value, or the range of ±5%, ±3%, ±2%, ±1% or ±0.5%.
本发明提供了一种制备毛囊间充质干细胞的方法,该制备方法包括:The invention provides a method for preparing hair follicle mesenchymal stem cells, the preparation method comprising:
1)获得毛囊组织,优选来自人的毛囊组织;1) obtaining hair follicle tissue, preferably from human hair follicle tissue;
2)由毛囊组织获得毛囊间充质干细胞;和2) obtaining hair follicle mesenchymal stem cells from hair follicle tissue; and
3)于培养基中对所得毛囊间充质干细胞进行培养。3) culturing the obtained hair follicle mesenchymal stem cells in a culture medium.
或选地,本发明方法还可以包括步骤4):对经培养的毛囊间充质干细胞进行传代。Alternatively, the method of the present invention may also include step 4): passage the cultured hair follicle mesenchymal stem cells.
一些实施方式中,步骤1)获得毛囊组织包括提取完整毛囊,即去除包裹着毛囊的皮肤组织,剥离出完整的毛囊组织。举例来说,具体的步骤可以包括:用清洗液润洗存储于组织储存液中的包裹着毛囊的皮肤组织,采用眼科镊、手术刀等工具剥离出完整的毛囊组织,存放于新鲜组织储存液中。或者,完整毛囊的提取还可以通过毛囊提取机直接获得。In some embodiments, step 1) obtaining the hair follicle tissue includes extracting the complete hair follicle, that is, removing the skin tissue surrounding the hair follicle, and peeling off the complete hair follicle tissue. For example, the specific steps may include: rinsing the skin tissue surrounding the hair follicle stored in the tissue storage solution with a cleaning solution, using ophthalmic forceps, a scalpel and other tools to peel off the complete hair follicle tissue, and storing it in a fresh tissue storage solution. middle. Alternatively, the extraction of intact hair follicles can also be obtained directly through a hair follicle extraction machine.
一些实施方式中,步骤2)由毛囊组织获得毛囊间充质干细胞包括采用酶解法或组织贴壁法,所得毛囊间充质干细胞亦称原代毛囊间充质干细胞。其中,酶解法是利用酶解液酶解毛囊组织使其疏松利于干细胞爬出。举例来说,具体步骤可包括加入酶解液酶解毛囊 组织,置于适宜条件下酶解。组织贴壁法主要通过贴壁培养使干细胞从毛囊组织中爬出。举例来说,具体步骤可包括体式显微镜下使用针头在毛囊外毛根鞘位置做切口,等待毛囊组织完全贴壁后加入培养基,于适宜条件下静置培养等待毛囊组织周围细胞爬出。In some embodiments, step 2) obtaining hair follicle mesenchymal stem cells from hair follicle tissue includes enzymatic hydrolysis or tissue attachment method, and the obtained hair follicle mesenchymal stem cells are also called primary hair follicle mesenchymal stem cells. Among them, the enzymatic hydrolysis method is to use enzymatic hydrolysis solution to enzymatically hydrolyze the hair follicle tissue to make it loose and facilitate stem cells to climb out. For example, the specific steps may include adding an enzymatic hydrolysis solution to enzymatically hydrolyze the hair follicle tissue, and placing it under suitable conditions for enzymatic hydrolysis. The tissue-adherence method mainly allows stem cells to climb out of the hair follicle tissue through adherent culture. For example, specific steps may include using a needle under a stereomicroscope to make an incision at the outer hair root sheath of the hair follicle, adding medium after the hair follicle tissue is completely adhered to the wall, and culturing statically under suitable conditions to wait for the cells around the hair follicle tissue to crawl out.
一些实施方式中,步骤3)中原代细胞的培养包括用培养基重悬原代毛囊间充质干细胞,接种于培养容器中,置于适宜条件下培养。In some embodiments, the cultivation of the primary cells in step 3) includes resuspending the primary hair follicle mesenchymal stem cells in a culture medium, inoculating them in a culture vessel, and culturing them under suitable conditions.
一些实施方式中,步骤4)中的细胞传代包括加入消化酶从器壁上消化贴壁的细胞,终止消化后,收获细胞,加入培养基重悬,接种于培养容器中,置于适宜条件下培养。In some embodiments, the cell subculture in step 4) includes adding digestive enzymes to digest the adherent cells from the wall of the vessel, after terminating the digestion, harvesting the cells, adding medium to resuspend, inoculating in a culture container, and placing them under suitable conditions nourish.
一些实施方式中,在步骤3)、步骤4)中,所述培养基包含氨基酸,维生素,有机化合物,无机化合物,激素,生长因子,微量元素,胎牛血清和抗生素。所述培养基可以自己制备获得也可以通过商业途径购买获得。In some embodiments, in step 3) and step 4), the medium contains amino acids, vitamins, organic compounds, inorganic compounds, hormones, growth factors, trace elements, fetal bovine serum and antibiotics. The culture medium can be prepared by oneself or purchased through commercial channels.
一些实施方式中,所述培养基中胎牛血清体积百分比约为5-10%,优选约5%或约10%。In some embodiments, the volume percentage of fetal calf serum in the culture medium is about 5-10%, preferably about 5% or about 10%.
一些实施方式中,所述抗生素为庆大霉素、青霉素、链霉素或其组合。In some embodiments, the antibiotic is gentamicin, penicillin, streptomycin or a combination thereof.
一些实施方式中,所述抗生素若为庆大霉素,则其浓度约为50-100U/mL,优选约50U/mL;所述抗生素若为青霉素则其浓度约为100-150U/mL,优选约100U/mL;或所述抗生素若为链霉素则其浓度约为0.10-0.15mg/mL,优选约0.10mg/mL。In some embodiments, if the antibiotic is gentamicin, its concentration is about 50-100U/mL, preferably about 50U/mL; if the antibiotic is penicillin, its concentration is about 100-150U/mL, preferably about 100 U/mL; or if the antibiotic is streptomycin, its concentration is about 0.10-0.15 mg/mL, preferably about 0.10 mg/mL.
一些实施方式中,在步骤3)、步骤4)中,所述培养基为ScienCell 7501间充质干细胞培养基(购买自ScienCell Research Laboratories)。In some embodiments, in step 3) and step 4), the medium is ScienCell 7501 mesenchymal stem cell medium (purchased from ScienCell Research Laboratories).
一些实施方式中,在所述步骤2)采用酶解法时使用的酶解液包含胶原酶、中性蛋白酶Ⅱ(Dispase Ⅱ)和氯化钙。In some embodiments, the enzymatic hydrolysis solution used in step 2) includes collagenase, dispase II and calcium chloride.
一些实施方式中,所述胶原酶可以是胶原酶Ⅰ或胶原酶Ⅳ,优选胶原酶Ⅳ。In some embodiments, the collagenase may be collagenase I or collagenase IV, preferably collagenase IV.
一些实施方式中,胶原酶的浓度约为0.1-5mg/mL,优选约1mg/mL。In some embodiments, the concentration of collagenase is about 0.1-5 mg/mL, preferably about 1 mg/mL.
一些实施方式中,所述中性蛋白酶(Dispase Ⅱ)浓度约为1-4mg/mL,优选约2mg/mL。In some embodiments, the concentration of the neutral protease (Dispase II) is about 1-4 mg/mL, preferably about 2 mg/mL.
一些实施方式中,所述氯化钙浓度约为5mM。In some embodiments, the calcium chloride concentration is about 5 mM.
一些实施方式中,在所述步骤2)采用酶解法时酶解时间约为1-3.5小时,优选约3小时。In some embodiments, when the enzymatic hydrolysis method is used in the step 2), the enzymatic hydrolysis time is about 1-3.5 hours, preferably about 3 hours.
一些实施方式中,酶解温度为约37℃。In some embodiments, the enzymatic hydrolysis temperature is about 37°C.
一些实施方式中,当步骤1)获得毛囊组织过程中用到组织储存液时,所述组织储存液可以是含有抗生素和胎牛血清的基础培养基,例如补加有抗生素和胎牛血清的DMEM/F12(Dulbecco's Modified Eagle Medium/Nutrient Mixture F12)。一些实施方式中,所述抗生素选自庆大霉素、青霉素、链霉素或其组合。一些实施方式中,组织储存液若包含庆大霉素则其浓度约为50-100U/mL,优选约50U/mL;所述组织储存液若包含青霉素则 其浓度约为100-150U/mL,优选约100U/mL,所述组织储存液若包含链霉素则其浓度约为0.10-0.15mg/mL,优选约0.10mg/mL。一些实施方式中,所述胎牛血清体积百分比约为5%-10%,优选约10%。In some embodiments, when the tissue storage solution is used in the process of obtaining hair follicle tissue in step 1), the tissue storage solution can be a basal medium containing antibiotics and fetal bovine serum, such as DMEM supplemented with antibiotics and fetal bovine serum /F12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F12). In some embodiments, the antibiotic is selected from gentamicin, penicillin, streptomycin or combinations thereof. In some embodiments, if the tissue storage solution contains gentamicin, its concentration is about 50-100 U/mL, preferably about 50 U/mL; if the tissue storage solution contains penicillin, its concentration is about 100-150 U/mL, Preferably about 100 U/mL, if the tissue storage solution contains streptomycin, its concentration is about 0.10-0.15 mg/mL, preferably about 0.10 mg/mL. In some embodiments, the fetal bovine serum volume percentage is about 5%-10%, preferably about 10%.
一些实施方式中,制备方法中用于清洗组织或细胞的清洗液可以是基础培养基或磷酸盐缓冲液,优选为含有抗生素的基础培养基和磷酸盐缓冲液。举例来说,抗生素可以选自庆大霉素、青霉素、链霉素或其组合。一些实施方式中,所述清洗液若包含庆大霉素,则其浓度约为50-100U/mL,优选约50U/mL;所述清洗液若包含青霉素则其浓度约为100-150U/mL,优选约100U/mL;或所述清洗液若包含链霉素则其浓度约为0.10-0.15mg/mL,优选约0.10mg/mL。In some embodiments, the cleaning solution used for cleaning tissues or cells in the preparation method may be basal medium or phosphate buffer, preferably basal medium and phosphate buffer containing antibiotics. For example, the antibiotic can be selected from gentamicin, penicillin, streptomycin or combinations thereof. In some embodiments, if the cleaning solution contains gentamicin, its concentration is about 50-100 U/mL, preferably about 50 U/mL; if the cleaning solution contains penicillin, its concentration is about 100-150 U/mL , preferably about 100 U/mL; or if the cleaning solution contains streptomycin, its concentration is about 0.10-0.15 mg/mL, preferably about 0.10 mg/mL.
一些实施方式中,用于贴壁细胞消化的消化酶可以是胰蛋白酶或稳定型胰蛋白酶替代酶,优选稳定型胰蛋白酶替代酶(例如TrypLE
TM Express Enzyme(1×),no phenol red)。
In some embodiments, the digestive enzyme used for digestion of adherent cells may be trypsin or a stable trypsin replacement enzyme, preferably a stable trypsin replacement enzyme (eg TrypLE ™ Express Enzyme (1×), no phenol red).
一些实施方式中,在所述步骤2)中,可以在5%CO
2,37℃条件下进行酶解获取原代细胞;更优选地,可以在37℃恒温震荡金属浴中进行酶解获取原代细胞,并且设置转速为600rpm。
In some embodiments, in the step 2), the primary cells can be obtained by enzymatic hydrolysis at 5% CO 2 at 37°C; more preferably, the primary cells can be obtained by enzymatic hydrolysis in a constant temperature oscillating metal bath at 37°C. Generation cells, and set the rotation speed to 600rpm.
一些实施方式中,在所述步骤3)和4)中,可以在5%CO
2,37℃条件下进行原代细胞培养和传代培养。
In some embodiments, in the steps 3) and 4), primary cell culture and subculture can be carried out under the conditions of 5% CO 2 and 37°C.
本发明还提供用本发明方法制得的毛囊间充质干细胞。本发明所得毛囊间充质干细胞具有增强的细胞增殖能力和增强的免疫调节能力。The invention also provides hair follicle mesenchymal stem cells prepared by the method of the invention. The hair follicle mesenchymal stem cells obtained in the invention have enhanced cell proliferation ability and enhanced immune regulation ability.
本发明还包括所述毛囊间充质干细胞在制备治疗骨质疏松和骨量减少疾病药物中的应用;优选地,所述骨质疏松为原发性骨质疏松;更优选地,所述骨质疏松为中老年骨质疏松或绝经后骨质疏松。The present invention also includes the application of the hair follicle mesenchymal stem cells in the preparation of medicines for treating osteoporosis and osteopenia; preferably, the osteoporosis is primary osteoporosis; more preferably, the bone Osteoporosis is middle-aged and elderly osteoporosis or postmenopausal osteoporosis.
一些实施方式中,所述药物剂型为液体剂型,优选静脉给药剂型。In some embodiments, the pharmaceutical dosage form is a liquid dosage form, preferably an intravenous dosage form.
本发明的内容也包括一种治疗骨质疏松或骨量减少疾病的方法:包括对患有骨质疏松或骨量减少疾病的患者施用所述毛囊间充质干细胞的步骤。优选地,所述骨质疏松为原发性骨质疏松;更优选地,所述骨质疏松为中老年骨质疏松或绝经后骨质疏松。The content of the present invention also includes a method for treating osteoporosis or osteopenia: comprising the step of administering the hair follicle mesenchymal stem cells to patients suffering from osteoporosis or osteopenia. Preferably, the osteoporosis is primary osteoporosis; more preferably, the osteoporosis is middle-aged osteoporosis or postmenopausal osteoporosis.
一些实施方式中,所述施用方式为静脉给药,优选静脉滴注或静脉注射。In some embodiments, the administration method is intravenous administration, preferably intravenous infusion or intravenous injection.
给药剂量的数量级可以是例如10
6~10
9个细胞/次。给药剂量可以是固定剂量(亦称统一剂量),也可以是基于患者体重的剂量。给药次数和给药周期无特别限制,例如每10天~3年给药一次,共给药1~10次。在一个优选的实施方案中,每三个月给药一次,共给药两次。
The order of magnitude of the dosage can be, for example, 10 6 -10 9 cells/time. The administered dose may be a fixed dose (also known as a flat dose) or a dose based on the patient's weight. The frequency of administration and the period of administration are not particularly limited, for example, once every 10 days to 3 years, for a total of 1 to 10 administrations. In a preferred embodiment, the administration is administered every three months for a total of two administrations.
下面将结合实施例进一步详细描述本发明。应当理解,提供这些实施例只是为了起说明作用,而不是用来限制本发明的范围。The present invention will be described in further detail below in conjunction with examples. It should be understood that these examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.
实施例1:人源毛囊间充质干细胞的制备Example 1: Preparation of human hair follicle mesenchymal stem cells
材料Material
1)酶解液配制:1) Preparation of enzymolysis solution:
准确称量0.1g胶原酶Ⅳ溶解于5mL的DMEM/F12基础培养基中,待胶原酶Ⅳ完全溶解后上下颠倒混匀,0.22μm过滤器过滤,得到20mg/mL胶原酶Ⅳ储存液;准确称量0.1g中性蛋白酶Ⅱ(Dispase Ⅱ)溶解于50mM的羟乙基哌嗪乙磺酸(HEPES)中,上下颠倒混匀,待中性蛋白酶Ⅱ(Dispase Ⅱ)完全溶解后,0.22μm过滤器过滤,得到40mg/mL Dispase Ⅱ储存液;准确称量0.11g氯化钙,溶解于2mL的无菌水中,上下颠倒混匀,待氯化钙完全溶解后,0.22μm过滤器过滤,得到500mM氯化钙储存液。Accurately weigh 0.1g of collagenase IV and dissolve it in 5mL of DMEM/F12 basal medium. After the collagenase IV is completely dissolved, mix it upside down and filter it through a 0.22μm filter to obtain a 20mg/mL collagenase IV stock solution; accurately weigh Dissolve 0.1g of dispase Ⅱ (Dispase Ⅱ) in 50mM hydroxyethylpiperazineethanesulfonic acid (HEPES). Filter to obtain 40mg/mL Dispase II storage solution; accurately weigh 0.11g of calcium chloride, dissolve it in 2mL of sterile water, mix it upside down, and after the calcium chloride is completely dissolved, filter through a 0.22μm filter to obtain 500mM chlorine Calcium storage solution.
分别取上述配制好的20mg/mL胶原酶Ⅳ储存液0.05mL,40mg/mL Dispase Ⅱ储存液0.05mL和500mM氯化钙储存液0.01mL与0.89mL平衡盐溶液(Hank's Balanced Salt Solution(HBSS)混匀,由此制得含1mg/mL胶原酶Ⅳ、2mg/mL Dispase Ⅱ和5mM氯化钙的酶解液。Take 0.05 mL of the prepared 20 mg/mL collagenase Ⅳ stock solution, 0.05 mL of 40 mg/mL Dispase Ⅱ stock solution, and 0.01 mL of 500 mM calcium chloride stock solution and mix them with 0.89 mL of Hank's Balanced Salt Solution (HBSS). Homogenize, and thus prepare an enzymatic hydrolysis solution containing 1 mg/mL collagenase Ⅳ, 2 mg/mL Dispase Ⅱ and 5 mM calcium chloride.
2)配制清洗液:在DMEM/F12基础培养基中配制含50U/mL的庆大霉素(购自华中药业股份有限公司)和10%胎牛血清的清洗液。2) Prepare cleaning solution: prepare a cleaning solution containing 50 U/mL gentamicin (purchased from Huazhong Pharmaceutical Co., Ltd.) and 10% fetal bovine serum in DMEM/F12 basal medium.
3)配制组织储存液:在DMEM/F12基础培养基中配制含50U/mL的庆大霉素(购自华中药业股份有限公司)和10%胎牛血清的组织储存液。3) Prepare tissue storage solution: prepare tissue storage solution containing 50 U/mL gentamicin (purchased from Huazhong Pharmaceutical Co., Ltd.) and 10% fetal bovine serum in DMEM/F12 basal medium.
方法:method:
1)完整毛囊的提取:1) Extraction of complete hair follicles:
将包裹着人毛囊的头皮组织放入装有10mL清洗液的培养皿中,润洗两次,将润洗后的头皮组织浸泡于组织储存液中,使用弯头眼科镊,辅助手术刀切去皮肤表皮组织,左手用弯头眼科镊横向夹住皮肤组织,右手使用显微镊小心分离包裹着毛囊的皮肤组织。剥离的毛囊会携带少许部分皮肤***,体视显微镜下使用显微镊小心分离毛囊组织周围的***。将剥离出的完整毛囊组织放置于含组织储存液的培养皿中。Put the scalp tissue wrapped with human hair follicles into a petri dish containing 10mL of cleaning solution, rinse twice, soak the rinsed scalp tissue in the tissue storage solution, use elbow ophthalmic tweezers, and assist a scalpel to cut off For the skin epidermis, the left hand clamps the skin tissue laterally with ophthalmic tweezers, and the right hand uses micro tweezers to carefully separate the skin tissue wrapped around the hair follicles. The stripped hair follicles will carry a small portion of skin connective tissue, and the connective tissue around the hair follicle tissue is carefully separated using microscopic forceps under a stereo microscope. Place the stripped complete hair follicle tissue in a petri dish containing tissue storage solution.
2)原代细胞获取:2) Primary cell acquisition:
按照步骤1)获得完整毛囊组织后,使用显微镊小心将毛囊组织放置在EP管底部,使用移液枪尽量吸去毛囊组织携带的组织储存液。按每根毛囊8μL量加入酶解液,37℃,5% CO
2培养箱静置3小时,每隔1小时小心轻弹管底,轻轻混匀。酶解3小时后,镜下可见毛囊的外毛根鞘层已完全酶解,毛干部分不能完全酶解。使用100-1000μL移液枪轻柔吹打30次,将酶解液完全混合,吸取全部酶解所得细胞悬液,然后通过100μm细胞筛。
After obtaining the complete hair follicle tissue according to step 1), carefully place the hair follicle tissue at the bottom of the EP tube with microscopic tweezers, and use a pipette gun to suck out the tissue storage solution carried by the hair follicle tissue as much as possible. Add 8 μL of enzymatic hydrolysis solution to each hair follicle, and let it stand in a 5% CO 2 incubator at 37°C for 3 hours, carefully flick the bottom of the tube every 1 hour, and mix gently. After 3 hours of enzymatic hydrolysis, it can be seen under the microscope that the outer hair root sheath of the hair follicle has been completely enzymatically hydrolyzed, and the hair shaft cannot be completely enzymatically hydrolyzed. Use a 100-1000μL pipette gun to gently pipette 30 times to mix the enzymolysis solution completely, draw up all the cell suspension obtained by enzymolysis, and then pass it through a 100μm cell sieve.
3)原代细胞培养:3) Primary cell culture:
在培养瓶中加入酶解后并过筛的细胞悬液,再加入ScienCell 7501间充质干细胞培养基(购买自ScienCell Research Laboratories)重悬。将培养瓶放入37℃,5%CO
2细胞培养箱中培养。于第五天和第九天更换新鲜培养基继续培养。待细胞融合度达到90%或以上,即可收获或传代。
Add the cell suspension after enzymatic hydrolysis and sieving to the culture flask, and then add ScienCell 7501 mesenchymal stem cell culture medium (purchased from ScienCell Research Laboratories) to resuspend. Place the culture flask in a 37°C, 5% CO2 cell incubator for culture. On the fifth day and the ninth day, fresh medium was replaced to continue culturing. When the cell confluency reaches 90% or above, it can be harvested or passaged.
4)细胞传代:4) Cell passage:
将通过以上步骤制备获得的毛囊间充质干细胞进行传代培养,每代以5.0×10
3个细胞/cm
2的密度接种到内含ScienCell 7501间充质干细胞培养基(购买自ScienCell Research Laboratories)的T75培养瓶中,观察培养瓶内细胞密度达90%以上,弃去培养基后,用清洗液清洗两次,弃去清洗液。向培养瓶底部的细胞沉淀加入3mL稳定型胰蛋白酶替代酶(TrypLE
TM Express Enzyme(1×),no phenol red),置于室温静置消化3分钟。使用10mL移液管向培养瓶中加入6mL PBS来稀释酶液,将上清取出置于15mL离心管中,再加入10mL PBS润洗瓶底1次,同润洗液加入该离心管中,1500r/min,离心5分钟,弃去上清,加入1mLScienCell 7501间充质干细胞培养基重悬,采用AO/PI(AO(Acridine Orange)吖啶橙,PI(Propidium Iodide)碘化丙啶)双染细胞凋亡检测试剂盒(DNA探针双染细胞核方法,购买自上海睿钰生物科技有限公司)检测每代收获的细胞数量,计算每代的扩增倍数,实验结果见图1和图2。
The hair follicle mesenchymal stem cells prepared through the above steps were subcultured, and each passage was inoculated at a density of 5.0× 103 cells/ cm2 into a culture medium containing ScienCell 7501 mesenchymal stem cell medium (purchased from ScienCell Research Laboratories). In the T75 culture flask, observe that the cell density in the culture flask reaches more than 90%, discard the medium, wash twice with the cleaning solution, and discard the cleaning solution. Add 3 mL of stable trypsin replacement enzyme (TrypLE TM Express Enzyme (1×), no phenol red) to the cell pellet at the bottom of the culture flask, and place it at room temperature for 3 minutes for digestion. Use a 10mL pipette to add 6mL of PBS to the culture bottle to dilute the enzyme solution, take out the supernatant and place it in a 15mL centrifuge tube, then add 10mL of PBS to rinse the bottom of the bottle once, add the same rinse solution to the centrifuge tube, 1500r /min, centrifuge for 5 minutes, discard the supernatant, add 1mL of ScienCell 7501 mesenchymal stem cell medium to resuspend, and use AO/PI (AO (Acridine Orange) acridine orange, PI (Propidium Iodide) propidium iodide) double staining Apoptosis detection kit (DNA probe double-stained nucleus method, purchased from Shanghai Ruiyu Biotechnology Co., Ltd.) detects the number of cells harvested at each generation, and calculates the amplification factor of each generation. The experimental results are shown in Figures 1 and 2.
实验结论:本发明制备方法制得的毛囊间充质干细胞在P3代后均可收获3×10
8个的细胞总量,满足生产需求。P1-P3代,细胞扩增倍数在10倍以上,P4-P7代,扩增倍数在5倍以上,显示出很强的细胞增殖能力。
Experimental conclusion: the hair follicle mesenchymal stem cells prepared by the preparation method of the present invention can harvest a total of 3×10 8 cells after passage P3, which meets the production requirements. In the P1-P3 generation, the cell amplification factor is more than 10 times, and in the P4-P7 generation, the cell expansion factor is more than 5 times, showing a strong cell proliferation ability.
实施例2:不同培养基培养的人源毛囊间充质干细胞对免疫功能的调节作用比较Example 2: Comparison of the regulatory effects of human hair follicle mesenchymal stem cells cultured in different media on immune function
按照实施例1所述,分别采用人胚胎干细胞完全培养基(购买自赛业生物科技有限公司),羊水培养基(购买自广州拜迪生物医药有限公司)代替ScienCell 7501间充质干细胞培养基(购买自ScienCell Research Laboratories)来制备人源毛囊间充质干细胞。According to Example 1, human embryonic stem cell complete medium (purchased from Saiye Biotechnology Co., Ltd.) and amniotic fluid medium (purchased from Guangzhou Baidi Biomedicine Co., Ltd.) were used to replace ScienCell 7501 mesenchymal stem cell medium ( Purchased from ScienCell Research Laboratories) to prepare human hair follicle mesenchymal stem cells.
根据以下实验方法比较不同培养基培养的人源毛囊间充质干细胞对免疫功能的调节作用。According to the following experimental method, the regulation effect of human hair follicle mesenchymal stem cells cultured in different media on immune function was compared.
1)毛囊间充质干细胞调节辅助性T细胞(Th淋巴细胞亚群)作用1) Hair follicle mesenchymal stem cells regulate helper T cells (Th lymphocyte subsets)
实验方法:不同培养基培养获得的毛囊间充质干细胞分别以细胞浓度1×10
5/mL/孔铺于十二孔板内,贴壁培养24小时后,再每孔接种5×10
5个外周血单个核细胞(Peripheral blood mononuclear cell,PBMC),以使孔内的毛囊间充质干细胞和外周血单个核细胞数量比例为1:5,进行共同培养,作为试验组。再单独培养外周血单个核细胞,接种的细胞数量为5×10
5个/孔,作为对照组。试验组和对照组每孔均加入植物凝集素(Phytohemagglutinin,PHA),置于5%CO
2,37℃培养箱培养67个小时,再每孔加入蛋白转运抑制剂(Protein Transport Inhibitor,购买自BD GolgiStop
TM)和离子霉素(Ionomycin),继续在培养箱中培养5小时。收集试验组和对照组的外周血单个核细胞,用流式细胞仪检测和Th1细胞相关的细胞因子(CD4+和IFN-γ+),以及和Th17细胞相关的细胞因子(CD4+和IL-17+),结果见表1和图3。
Experimental method: Hair follicle mesenchymal stem cells obtained from different culture media were spread in twelve-well plates at a cell concentration of 1×10 5 /mL/well, and after 24 hours of adherent culture, 5×10 5 cells were inoculated in each well. Peripheral blood mononuclear cells (PBMC) were co-cultured so that the ratio of hair follicle mesenchymal stem cells and peripheral blood mononuclear cells in the wells was 1:5, and they were used as the test group. Peripheral blood mononuclear cells were cultured separately, and the number of inoculated cells was 5×10 5 per well, which was used as a control group. Each well of the test group and the control group was added with phytohemagglutinin (Phytohemagglutinin, PHA), placed in 5% CO 2 , incubated at 37°C for 67 hours, and then added a protein transport inhibitor (Protein Transport Inhibitor, purchased from BD GolgiStop ™ ) and ionomycin (Ionomycin), continue to cultivate in the incubator for 5 hours. The peripheral blood mononuclear cells of the test group and the control group were collected, and the cytokines related to Th1 cells (CD4+ and IFN-γ+) and the cytokines related to Th17 cells (CD4+ and IL-17+) were detected by flow cytometry. ), the results are shown in Table 1 and Figure 3.
表1不同培养基培养的毛囊间充质干细胞调节Th淋巴细胞亚群结果Table 1 Results of hair follicle mesenchymal stem cells cultured in different media regulating Th lymphocyte subsets
培养基种类 | ScienCell 7501培养基ScienCell 7501 Medium | 完全培养基(赛业生物)Complete Medium (Saiye Biological) | 羊水培养基(拜迪生物)Amniotic fluid culture medium (Baidi Bio) | |
Th1抑制率Th1 inhibition rate | -95.8%-95.8% | -88.76%-88.76% | -79.97%-79.97% | |
Th17抑制率Th17 inhibition rate | -84%-84% | -73%-73% | -22%-twenty two% |
实验结论:毛囊间充质干细胞能有效抑制促炎性淋巴细胞亚群Th1和Th17增殖,且不同培养基培养的毛囊间充质干细胞作用效果具有显著差异,其中以ScienCell 7501间充质干细胞培养基培养的毛囊间充质干细胞抑制效果最好。Experimental conclusion: hair follicle mesenchymal stem cells can effectively inhibit the proliferation of pro-inflammatory lymphocyte subsets Th1 and Th17, and the effects of hair follicle mesenchymal stem cells cultured in different media are significantly different, among which ScienCell 7501 mesenchymal stem cell medium The cultured hair follicle mesenchymal stem cells had the best inhibitory effect.
2)毛囊间充质干细胞调节调节性T细胞(Regulatory cell,简称Treg)的作用2) Hair follicle mesenchymal stem cells regulate the role of regulatory T cells (Regulatory cell, Treg for short)
实验方法:不同培养基培养获得的毛囊间充质干细胞分别以细胞浓度1×10
5/mL/孔铺于十二孔板内,贴壁培养24小时后,再每孔接种5×10
5个外周血单个核细胞,以使孔内的毛囊间充质干细胞和外周血单个核细胞数量比例为1:5,进行共同培养,作为试验组。再单独培养外周血单个核细胞,接种的细胞数量为5×10
5个/孔,作为对照组。置于5%CO
2,37℃培养箱培养72个小时。收集试验组和对照组的外周血单个核细胞,根据Treg检测试剂盒(购买自Biolegend)操作方法对细胞进行染色,采用流式细胞仪对Treg细胞相关细胞因子(CD4+、CD25+和FoxP3+)进行检测,结果见表2和图4。
Experimental method: Hair follicle mesenchymal stem cells obtained from different culture media were spread in twelve-well plates at a cell concentration of 1×10 5 /mL/well, and after 24 hours of adherent culture, 5×10 5 cells were inoculated in each well. Peripheral blood mononuclear cells, so that the ratio of hair follicle mesenchymal stem cells and peripheral blood mononuclear cells in the wells is 1:5, were co-cultured as the test group. Peripheral blood mononuclear cells were cultured separately, and the number of inoculated cells was 5×10 5 per well, which was used as a control group. Place them in a 5% CO 2 , 37°C incubator for 72 hours. The peripheral blood mononuclear cells of the test group and the control group were collected, the cells were stained according to the operation method of the Treg detection kit (purchased from Biolegend), and the Treg cell-related cytokines (CD4+, CD25+ and FoxP3+) were detected by flow cytometry , the results are shown in Table 2 and Figure 4.
表2不同培养基培养的毛囊间充质干细胞调节Treg淋巴细胞亚群结果Table 2 Results of hair follicle mesenchymal stem cells cultured in different media regulating Treg lymphocyte subsets
培养基种类 | ScienCell 7501培养基ScienCell 7501 Medium | 完全培养基(赛业生物)Complete Medium (Saiye Biological) | 羊水培养基(拜迪生物)Amniotic fluid culture medium (Baidi Bio) |
Treg促进率Treg promotion rate | 191.78%191.78% | 94.98%94.98% | 75.34%75.34% |
实验结论:毛囊间充质干细胞能够促进Treg淋巴细胞亚群的增殖功能,且不同培养基培养的毛囊间充质干细胞作用效果具有显著差异,其中以ScienCell 7501间充质干细胞培养基培养的毛囊间充质干细胞的促进效果最好。Experimental conclusion: hair follicle mesenchymal stem cells can promote the proliferation of Treg lymphocyte subsets, and the effects of hair follicle mesenchymal stem cells cultured in different media are significantly different, among which hair follicle mesenchymal stem cells cultured in ScienCell 7501 mesenchymal stem cell The promotion effect of mesenchymal stem cells is the best.
实施例3:酶解液配方及酶解时间对分离人源毛囊间充质干细胞的影响Example 3: Effect of Enzymolysis Solution Formula and Enzymolysis Time on Isolation of Human Hair Follicle Mesenchymal Stem Cells
如实施例1所述制备人源毛囊间充质干细胞,不同的是分别用以下配方1、配方2和配方3的酶解液代替实施例1中的酶解液酶解毛囊组织。Human-derived hair follicle mesenchymal stem cells were prepared as described in Example 1, except that the enzymatic hydrolysis solutions of the following formula 1, formula 2 and formula 3 were used instead of the enzymatic hydrolysis solution in Example 1 to enzymolyze the hair follicle tissue.
酶解液配方1:准确称量0.1克胶原酶Ⅰ溶解于5mL的DMEM/F12基础培养基中,待胶原酶Ⅰ完全溶解后上下颠倒混匀,0.22μm过滤器过滤,得到20mg/mL胶原酶Ⅰ储存液,加入95mL平衡盐溶液(Hank's Balanced Salt Solution(HBSS)稀释,得到1mg/mL的胶原酶Ⅰ酶解液。Enzyme solution formula 1: Accurately weigh 0.1 g of collagenase I and dissolve it in 5 mL of DMEM/F12 basal medium. After the collagenase I is completely dissolved, mix it upside down and filter it through a 0.22 μm filter to obtain 20 mg/mL collagenase Add 95 mL of Hank's Balanced Salt Solution (HBSS) to the Ⅰ storage solution and dilute it to obtain a 1 mg/mL collagenase Ⅰ hydrolysis solution.
酶解液配方2:准确称量0.1g胶原酶Ⅳ溶解于5毫升的DMEM/F12基础培养基中,待胶原酶Ⅳ完全溶解后上下颠倒混匀,0.22μm过滤器过滤,得到20mg/mL胶原酶Ⅳ储存液,加入95mL平衡盐溶液(Hank's Balanced Salt Solution(HBSS)稀释,得到1mg/mL的胶原酶Ⅳ酶解液。Enzyme solution formula 2: Accurately weigh 0.1g of collagenase IV and dissolve in 5ml of DMEM/F12 basal medium. After the collagenase IV is completely dissolved, mix upside down and filter through a 0.22μm filter to obtain 20mg/mL collagen Add 95 mL of Hank's Balanced Salt Solution (HBSS) to the enzyme IV stock solution to dilute it to obtain a 1 mg/mL collagenase IV hydrolyzate.
酶解液配方3:按实施例1中所述配制酶解液,不同的是将其中的胶原酶Ⅳ替换成胶原酶Ⅰ,由此获得含1mg/mL胶原酶Ⅰ、2mg/mL Dispase Ⅱ和5mM氯化钙的酶解液。Enzymolysis Solution Formula 3: Prepare the enzymolysis solution as described in Example 1, except that collagenase IV is replaced with collagenase I, thus obtaining a solution containing 1 mg/mL collagenase I, 2 mg/mL Dispase II and 5mM calcium chloride enzymatic solution.
酶解1小时、1.5小时、2小时、2.5小时、3小时、3.5小时后光学显微镜下观察毛囊组织的酶解效果。After enzymatic hydrolysis for 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, and 3.5 hours, the effect of enzymatic hydrolysis on hair follicle tissue was observed under an optical microscope.
镜下结果显示,酶解1小时已可见内毛根鞘和外毛根鞘与毛干分离,酶解3小时后,内毛根鞘和外毛根鞘与毛干明显分离,且经过酶解后内毛根鞘和外毛根鞘经过吹打能分散成单个细胞。时间越长酶解越充分,但酶解3.5小时镜检与3小时差异不大。取酶解3小时制得的毛囊间充质干细胞在ScienCell 7501间充质干细胞培养基中培养,显微镜下观察生长状态并计数最终收获的细胞数量,结果见表3。The results under the microscope showed that the inner hair root sheath and the outer hair root sheath were separated from the hair shaft after 1 hour of enzymatic hydrolysis. and outer root sheath can be dispersed into single cells after blowing. The longer the time, the more sufficient the enzymatic hydrolysis, but there is little difference between 3.5 hours of enzymatic hydrolysis and 3 hours of microscopic examination. The hair follicle mesenchymal stem cells prepared by enzymatic hydrolysis for 3 hours were cultured in ScienCell 7501 mesenchymal stem cell medium. The growth status was observed under a microscope and the number of finally harvested cells was counted. The results are shown in Table 3.
表3不同酶解液酶解毛囊组织最终收获的毛囊间充质干细胞数量结果Table 3 The results of the number of hair follicle mesenchymal stem cells harvested by enzymolysis of hair follicle tissue with different enzymatic solutions
酶解液配方Enzyme solution formula | 原代毛囊间充质干细胞培养天数Culture days of primary hair follicle mesenchymal stem cells |
收获细胞数量Harvested |
实施例1酶解液配方Embodiment 1 enzymolysis solution formula | 12天12 days | 175000175000 |
酶解液配方1 |
12天12 days | 5570055700 |
酶解液配方2 |
12天12 days | 9860098600 |
酶解液配方3Enzyme solution formula 3 | 12天12 days | 9150091500 |
结论:胶原酶与中性蛋白酶Ⅱ(Dispase Ⅱ)组合的效果显著优于胶原酶单用;且胶原酶Ⅳ与中性蛋白酶Ⅱ(Dispase Ⅱ)组合的效果优于胶原酶Ⅰ与中性蛋白酶Ⅱ(Dispase Ⅱ)组合。相对而言,实施例1的酶解液配方酶解后细胞增殖迅速,相同培养时间收获的细胞数量最多。Conclusion: The effect of the combination of collagenase and dispase Ⅱ (Dispase Ⅱ) is significantly better than that of collagenase alone; and the effect of the combination of collagenase Ⅳ and dispase Ⅱ (Dispase Ⅱ) is better than that of collagenase Ⅰ and dispase Ⅱ (Dispase II) combination. Relatively speaking, the enzymolysis solution formula of Example 1 proliferated rapidly after enzymolysis, and the number of cells harvested at the same culture time was the largest.
实施例4:人源毛囊间充质干细胞制剂治疗原发性骨质疏松及骨量减少Example 4: Treatment of Primary Osteoporosis and Osteopenia with Human Hair Follicle Mesenchymal Stem Cell Preparation
发明人选用20周龄早衰小鼠SAMP8作为研究对象,通过尾静脉注射低剂量(1×10
5个/只)和高剂量(4×10
5个/只)的人源毛囊间充质干细胞(实施例1制得),共注射3次,两次注射之间间隔1周,以此探究人源毛囊间充质干细胞对延缓早衰小鼠SAMP8增龄性骨流失的效果。SAMP8小鼠为一类常用的快速老化动物模型,由于可在早期自发发生骨量减少乃至骨质疏松而被用于老龄相关原发性骨质疏松和骨量减少的研究。具体地,分为5个组进行试验:1、正常对照组,SAMR1小鼠(12只),与早衰小鼠SAMP8具有相同遗传背景,但不具备早衰表型;2、空白组,早衰小鼠SAMP8(12只),不做任何处理;3、溶媒组,早衰小鼠SAMP8(12只),接受同等剂量的生理盐水(细胞重悬溶剂)尾静脉注射,共注射3次,每两次注射之间间隔1周;4、毛囊间充质干细胞低剂量组,早衰小鼠SAMP8(12只)接受人源毛囊间充质干细胞(1×10
5个/只)尾静脉注射,共注射3次,每两次注射之间间隔1周;5、毛囊间充质干细胞高剂量组,早衰小鼠SAMP8(12只)接受人源毛囊间充质干细胞(4×10
5个/只)尾静脉注射,共注射3次,每两次注射之间间隔1周。
The inventors selected 20-week-old progeria mice SAMP8 as the research object, and injected low-dose (1×10 5 cells/mouse) and high-dose (4×10 5 cells/mouse) human hair follicle-derived mesenchymal stem cells ( Example 1), a total of 3 injections, with an interval of 1 week between the two injections, in order to explore the effect of human hair follicle mesenchymal stem cells on delaying the age-related bone loss of premature aging mice SAMP8. SAMP8 mice are a commonly used animal model of rapid aging. They are used in the study of age-related primary osteoporosis and osteoporosis because they can spontaneously develop osteopenia and even osteoporosis in the early stage. Specifically, it was divided into 5 groups for the test: 1. Normal control group, SAMR1 mice (12), which have the same genetic background as the progeria mouse SAMP8, but do not have the progeria phenotype; 2. Blank group, progeria mice SAMP8 (12 rats), without any treatment; 3. Vehicle group, progeria mice SAMP8 (12 rats), received the same dose of normal saline (cell resuspension solvent) tail vein injection, a total of 3 injections, every two injections The interval was 1 week; 4. In the low-dose hair follicle mesenchymal stem cell group, premature aging mice SAMP8 (12) received human hair follicle mesenchymal stem cell (1× 105 /mouse) tail vein injection, a total of 3 injections , 1 week between each two injections; 5. Hair follicle mesenchymal stem cell high-dose group, progeria mice SAMP8 (12) received human hair follicle mesenchymal stem cells (4×10 5 /only) tail vein injection , a total of 3 injections, with an interval of 1 week between each two injections.
最后1次注射细胞的2个月后处死小鼠,分离胫骨,借助微计算机断层扫描技术(Micro-CT)检测外源干细胞对SAMP8小鼠胫骨近端松质骨增龄性骨流失的改善作用,具体检测了骨体积分数和其它骨微结构参数(如骨小梁厚度、数量和间隙)的改善情况及其3D图像,统计结果和3D图像见附图5。The mice were sacrificed 2 months after the last injection of cells, and the tibiae were isolated, and the effect of exogenous stem cells on the aging bone loss of proximal tibial cancellous bone in SAMP8 mice was detected by micro-computed tomography (Micro-CT) , specifically detected the improvement of bone volume fraction and other bone microstructural parameters (such as bone trabecular thickness, number and gap) and their 3D images. The statistical results and 3D images are shown in Figure 5.
由附图5可知,高/低剂量的毛囊间充质干细胞可以不同程度地改善小鼠胫骨近端松质骨骨体积分数(附图5A)和其它骨微结构参数,包括骨小梁厚度(附图5B)、骨小梁数量(附图5C)和骨小梁间隙(附图5D);由附图5E可知,3D图像也显示了相同的趋势,即注射高/低剂量的毛囊间充质干细胞可以有效改善早衰小鼠的骨流失现象。早衰小鼠SAMP8中的结果显示,本发明制得的人源毛囊间充质干细胞能够有效延缓增龄性骨流失。It can be seen from accompanying drawing 5 that high/low doses of hair follicle mesenchymal stem cells can improve mouse proximal tibial cancellous bone volume fraction (accompanying drawing 5A) and other bone microstructural parameters, including bone trabecular thickness ( Accompanying drawing 5B), number of bone trabeculae (accompanying drawing 5C) and bone trabecular space (accompanying drawing 5D); It can be seen from accompanying drawing 5E, 3D image also shows the same trend, namely injection of high/low dose of interstitial hair follicle Mesenchymal stem cells can effectively improve bone loss in progeria mice. The results in premature aging mice SAMP8 show that the human hair follicle mesenchymal stem cells prepared by the present invention can effectively delay age-related bone loss.
尽管本文描述了具体的例子,但是有一点对于本领域技术人员来说是明显的,即在不脱离本发明的精神和范围的前提下可对本发明作各种变化和改动。因此,所附权利要求覆盖了所有这些在本发明范围内的变动。Although specific examples are described herein, it will be apparent to those skilled in the art that various changes and modifications of the present invention can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes which are within the scope of this invention.
Claims (10)
- 一种制备毛囊间充质干细胞的方法,该制备方法包括:A method for preparing hair follicle mesenchymal stem cells, the preparation method comprising:1)获得毛囊组织,优选来自人的毛囊组织;1) obtaining hair follicle tissue, preferably from human hair follicle tissue;2)由毛囊组织获得毛囊间充质干细胞;和2) obtaining hair follicle mesenchymal stem cells from hair follicle tissue; and3)于培养基中对所得毛囊间充质干细胞进行培养;3) culturing the obtained hair follicle mesenchymal stem cells in the culture medium;其中,步骤2)采用酶解法由毛囊组织分离获得毛囊间充质干细胞,所用的酶解液包含胶原酶,中性蛋白酶Ⅱ(Dispase Ⅱ)和氯化钙;或者/且Wherein, step 2) adopts enzymatic hydrolysis method to obtain hair follicle mesenchymal stem cells from hair follicle tissue, and the enzymatic hydrolysis solution used comprises collagenase, dispase II (Dispase II) and calcium chloride; or/and所述培养基包含氨基酸、维生素、有机化合物、无机化合物、激素、生长因子、微量元素、胎牛血清和抗生素。The medium contains amino acids, vitamins, organic compounds, inorganic compounds, hormones, growth factors, trace elements, fetal bovine serum and antibiotics.
- 根据权利要求1所述的方法,其特征在于,还包括步骤4):对经培养的毛囊间充质干细胞进行传代。The method according to claim 1, further comprising step 4): passage of the cultured hair follicle mesenchymal stem cells.
- 根据权利要求1所述的方法,其特征在于,所述胎牛血清体积百分比为5-10%,优选5%或10%;The method according to claim 1, wherein the fetal bovine serum volume percentage is 5-10%, preferably 5% or 10%;或者/并且,所述抗生素为庆大霉素、青霉素、链霉素或其组合;Or/and, the antibiotic is gentamicin, penicillin, streptomycin or a combination thereof;优选地,所述庆大霉素浓度为50-100U/mL,优选50U/mL;所述青霉素浓度为100-150U/mL,优选100U/mL;或者/并且,所述链霉素浓度为0.10-0.15mg/mL,优选0.10mg/mL。Preferably, the gentamicin concentration is 50-100U/mL, preferably 50U/mL; the penicillin concentration is 100-150U/mL, preferably 100U/mL; or/and, the streptomycin concentration is 0.10 - 0.15 mg/mL, preferably 0.10 mg/mL.
- 根据权利要求1所述的方法,其特征在于,所述培养基为ScienCell 7501间充质干细胞培养基。The method according to claim 1, wherein the culture medium is ScienCell 7501 mesenchymal stem cell culture medium.
- 根据权利要求1所述的方法,还具有一项或多项以下所述的特征:The method of claim 1, further comprising one or more of the following features:所述胶原酶为胶原酶Ⅰ或胶原酶Ⅳ,优选胶原酶Ⅳ;The collagenase is collagenase I or collagenase IV, preferably collagenase IV;所述胶原酶浓度为0.1-5mg/mL,优选1mg/mL;The collagenase concentration is 0.1-5 mg/mL, preferably 1 mg/mL;所述中性蛋白酶Ⅱ(Dispase Ⅱ)浓度为1-4mg/mL,优选2mg/mL;The neutral protease II (Dispase II) concentration is 1-4mg/mL, preferably 2mg/mL;所述氯化钙浓度为5mM;The calcium chloride concentration is 5mM;酶解法的酶解时间为1-3.5小时,优选3小时;The enzymolysis time of the enzymolysis method is 1-3.5 hours, preferably 3 hours;酶解温度为37℃。The enzymatic hydrolysis temperature is 37°C.
- 根据权利要求1所述的方法,其特征在于,在所述步骤1)中,毛囊组织包含于组织储存液中,组织储存液为含有抗生素和胎牛血清的基础培养基;The method according to claim 1, wherein in said step 1), the hair follicle tissue is contained in a tissue storage solution, and the tissue storage solution is a basal medium containing antibiotics and fetal bovine serum;优选地,所述胎牛血清体积百分比为5%-10%,优选10%;Preferably, the fetal bovine serum volume percentage is 5%-10%, preferably 10%;优选地,所述抗生素选自庆大霉素、青霉素、链霉素或其组合;Preferably, the antibiotic is selected from gentamicin, penicillin, streptomycin or a combination thereof;更优选地,所述庆大霉素浓度为50-100U/mL,优选50U/mL;所述青霉素浓度为100-150U/mL,优选100U/mL;或者/并且,所述链霉素浓度为0.10-0.15mg/mL,优选0.10mg/mL。More preferably, the gentamicin concentration is 50-100U/mL, preferably 50U/mL; the penicillin concentration is 100-150U/mL, preferably 100U/mL; or/and, the streptomycin concentration is 0.10-0.15 mg/mL, preferably 0.10 mg/mL.
- 根据权利要求2所述的方法,其特征在于,在所述步骤4)包括用消化酶进行消化,所述消化酶为胰蛋白酶或稳定型胰蛋白酶替代酶,优选稳定型胰蛋白酶替代酶。The method according to claim 2, characterized in that, in the step 4), it includes digesting with a digestive enzyme, and the digestive enzyme is trypsin or a stable trypsin replacement enzyme, preferably a stable trypsin replacement enzyme.
- 权利要求1-7任一项所述方法获得的毛囊间充质干细胞。The hair follicle mesenchymal stem cells obtained by the method according to any one of claims 1-7.
- 权利要求8所述毛囊间充质干细胞在制备治疗骨质疏松或骨量减少疾病药物中的应用;The application of hair follicle mesenchymal stem cells described in claim 8 in the preparation of medicines for the treatment of osteoporosis or osteopenia;优选地,所述药物为液体剂型,优选静脉给药剂型;Preferably, the drug is in a liquid dosage form, preferably an intravenous administration dosage form;优选地,所述骨质疏松为原发性骨质疏松;更优选地,所述骨质疏松为中老年骨质疏松或绝经后骨质疏松。Preferably, the osteoporosis is primary osteoporosis; more preferably, the osteoporosis is middle-aged osteoporosis or postmenopausal osteoporosis.
- 一种治疗骨质疏松或骨量减少疾病的方法;其特征在于,包括对患有骨质疏松或骨量减少疾病的患者施用权利要求8所述毛囊间充质干细胞的步骤;A method for treating osteoporosis or osteopenia; characterized in that, comprising the step of administering hair follicle mesenchymal stem cells according to claim 8 to patients suffering from osteoporosis or osteopenia;优选地,所述施用方式为静脉给药,优选静脉滴注或静脉注射;Preferably, the mode of administration is intravenous administration, preferably intravenous drip or intravenous injection;优选地,所述骨质疏松为原发性骨质疏松;更优选地,所述骨质疏松为中老年骨质疏松或绝经后骨质疏松。Preferably, the osteoporosis is primary osteoporosis; more preferably, the osteoporosis is middle-aged osteoporosis or postmenopausal osteoporosis.
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