WO2017147941A1 - 重组间充质干细胞及其制备方法和应用 - Google Patents

重组间充质干细胞及其制备方法和应用 Download PDF

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WO2017147941A1
WO2017147941A1 PCT/CN2016/075812 CN2016075812W WO2017147941A1 WO 2017147941 A1 WO2017147941 A1 WO 2017147941A1 CN 2016075812 W CN2016075812 W CN 2016075812W WO 2017147941 A1 WO2017147941 A1 WO 2017147941A1
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mesenchymal stem
messenger rna
nucleotide sequence
stem cells
seq
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曾宪卓
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深圳爱生再生医学科技有限公司
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    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/19—Cytokines; Lymphokines; Interferons
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2066—IL-10
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5428—IL-10
    • C—CHEMISTRY; METALLURGY
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    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention relates to the field of biotechnology, in particular to a recombinant mesenchymal stem cell and a preparation method and application thereof.
  • Immune diseases refer to diseases caused by imbalance of immune regulation and affecting the body's immune response.
  • Generalized immune diseases include structural or functional abnormalities of the immune system caused by congenital or acquired causes.
  • MS multiple sclerosis
  • the lesions are mostly in the white matter of the brain and spinal cord. They are multifocal inflammatory cell infiltration, demyelination and neuronal damage, which can lead to dysfunctions such as sensory, motor, consciousness and neurocognition.
  • the disease is mostly caused by young adults aged 20 to 40 years.
  • the recurrence rate and disability rate are the most common causes of non-traumatic neurological disability among young adults. So far, they have not been completely cured.
  • mesenchymal stem cells were transplanted to treat multiple inflammatory sclerosis (MS), inflammatory bowel disease, rheumatoid arthritis and other immune inflammatory diseases, and achieved remarkable therapeutic effects.
  • MS multiple inflammatory sclerosis
  • Mesenchymal stem refers to a group of mesoderm-derived cell populations that have the ability to self-renew and differentiate into bone, cartilage and fat cells.
  • MSCs not only have multi-directional differentiation ability, but also MSCs can effectively regulate the function of the immune system. Because of this, MSCs have broad therapeutic application prospects for autoimmune and inflammation-related diseases.
  • RNA a recombinant mesenchymal stem cell transfected with a first messenger RNA, a second messenger RNA and a third messenger RNA;
  • the first messenger RNA is used to express P-selectin glycoprotein ligand-1
  • the second messenger RNA is used to express a sialylated Lewis oligosaccharide-X antigen
  • the third messenger RNA is used to express interleukin-10.
  • a method for preparing recombinant mesenchymal stem cells comprising the following steps:
  • the third messenger RNA is used to express interleukin-10;
  • the mesenchymal stem cells are cultured in a cell culture fluid
  • a targeting vector for a drug which comprises the recombinant mesenchymal stem cell of any of the above.
  • the above-described recombinant mesenchymal stem cells are transfected into mesenchymal stem cells to express first messenger RNA for expressing P-selectin glycoprotein ligand-1 (PSGL-1), Second messenger RNA for expression of sialyl Lewis oligosaccharide-X antigen (Sialyl Lewis-X, SLeX) and third messenger RNA for expression of interleukin-10 (IL-10), resulting in recombination Stem cells.
  • PSGL-1 P-selectin glycoprotein ligand-1
  • Second messenger RNA for expression of sialyl Lewis oligosaccharide-X antigen Sialyl Lewis-X, SLeX
  • IL-10 interleukin-10
  • the homing promoting factors PSGL-1 and SLeX can significantly increase the efficiency of recombinant mesenchymal stem cells targeting homing to disease or inflammatory lesions, and the anti-inflammatory factor IL-10 can increase the inhibitory ability of recombinant mesenchymal stem cells.
  • FIG. 1 is a flow chart showing a method of preparing a recombinant mesenchymal stem cell according to an embodiment
  • Figure 2a is a graph showing the results of immunofluorescence detection of the expression level of PSGL-1 in recombinant mesenchymal stem cells
  • Figure 2b is a graph showing the results of immunofluorescence detection of SLeX expression of recombinant mesenchymal stem cells
  • Figure 2c is a graph showing the results of detecting the expression level of IL-10 in the supernatant of recombinant mesenchymal stem cells by Elisa;
  • Figure 3 is a graph showing the results of detecting the proliferation of recombinant TMSCs by recombinant mesenchymal stem cells by flow cytometry;
  • Figure 4a is a graph showing the results of detecting the adhesion ability of recombinant mesenchymal stem cells transfected with PSGL-1 and SLeX to endothelial cells under flow chamber 1 dyn/cm 2 ;
  • Figure 4b is a graph showing the results of detecting the adhesion ability of untransfected mesenchymal stem cells to endothelial cells under flow chamber 1 dyn/cm 2 ;
  • Figure 4c is a graph showing the results of detecting the adhesion ability of HL-60 cells to endothelial cells under flow chamber 1 dyn/cm 2 ;
  • Figure 4d is a graph showing the results of detecting the adhesion ability of recombinant mesenchymal stem cells transfected with PSGL-1 and SLeX to endothelial cells under flow chamber 2dyn/cm 2 ;
  • Figure 4e is a graph showing the results of detecting the adhesion ability of untransfected mesenchymal stem cells to endothelial cells under flow chamber 2dyn/cm 2 ;
  • Figure 4f is a graph showing the results of detecting the adhesion ability of HL-60 cells to endothelial cells under flow chamber 2dyn/cm 2 ;
  • Figure 4g is a graph showing the results of adhesion to endothelial cells between recombinant mesenchymal stem cells transfected with PSGL-1 and SLeX under flow chamber 5dyn/cm 2 ;
  • Figure 4h is a graph showing the results of detecting the adhesion ability of untransfected mesenchymal stem cells to endothelial cells in a flow chamber of 5 dyn/cm 2 ;
  • Figure 4i is a graph showing the results of detecting the adhesion ability of HL-60 cells to endothelial cells under the condition of a flow chamber of 5 dyn/cm 2 ;
  • Figure 4j is a graph showing the results of detecting the adhesion ability of recombinant mesenchymal stem cells transfected with PSGL-1 and SLeX to endothelial cells under flow chamber 10 dyn/cm 2 ;
  • Figure 4k is a graph showing the results of detecting the adhesion ability of untransfected mesenchymal stem cells to endothelial cells under flow chamber 10 dyn/cm 2 ;
  • Figure 41 is a graph showing the results of detecting the adhesion ability of HL-60 cells to endothelial cells under the condition of flow chamber 10 dyn/cm 2 ;
  • Figure 4m is a statistical result diagram of Figures 4a to 4l;
  • Figure 5a is a graph showing the results of detecting the number of cells in which the recombinant mesenchymal stem cells homing into the spinal cord by fluorescence microscopy;
  • Figure 5b is a graph showing the results of fluorescence microscopy of the number of cells in which untransfected mesenchymal stem cells homing into the spinal cord;
  • Figure 5c is a graph of the statistical results of Figures 5a and 5b;
  • Figure 6 is a graph showing the results of clinical symptom scores of MS animal models of recombinant mesenchymal stem cell transplantation
  • Figure 7 is a graph showing the results of immunofluorescence detection of the degree of inflammation in an EAE animal model of recombinant mesenchymal stem cell transplantation
  • Figure 8 is a graph showing the results of demyelination of the spinal cord of the EAE animal model of recombinant mesenchymal stem cell transplantation by Lxuol rapid blue staining.
  • the first messenger RNA is used to express P-selectin glycoprotein ligand-1 (PSGL-1), and the second messenger RNA is used to express sialylated Lewis oligosaccharide-X antigen (Sialyl Lewis-X, SLeX), a third messenger RNA was used to express interleukin-10 (IL-10).
  • PSGL-1 P-selectin glycoprotein ligand-1
  • sialylated Lewis oligosaccharide-X antigen Sialyl Lewis-X, SLeX
  • IL-10 interleukin-10
  • mRNA messenger RNA
  • mRNA carries genetic information and acts as a template for protein synthesis and is easily transfected into cells.
  • the gene sequence of the first messenger RNA comprises: (a) the nucleotide sequence shown in SEQ ID No. 1; (b) has the nucleotide sequence shown in SEQ ID No. 1. a nucleotide sequence of at least 98% homology; or (c), a nucleotide sequence represented by SEQ ID No. 1, wherein one or more bases are deleted, replaced or added to the resulting nucleotide sequence.
  • the gene sequence of the second messenger RNA comprises: (a) the nucleotide sequence shown in SEQ ID No. 2; (b) having at least 98% homology to the nucleotide sequence shown in SEQ ID No. 2. a nucleotide sequence; or (c), the nucleotide sequence shown in SEQ ID No. 2, wherein one or more bases are deleted, replaced or added to the resulting nucleotide sequence.
  • the gene sequence of the third messenger RNA comprises: (a) the nucleotide sequence shown in SEQ ID No. 3; (b) having at least 98% homology to the nucleotide sequence shown in SEQ ID No. 3. a nucleotide sequence; or (c), the nucleotide sequence shown in SEQ ID No. 3, wherein one or more bases are deleted, replaced or added to the resulting nucleotide sequence.
  • nucleotide sequences having at least 98% homology to the nucleotide sequence or at least one alteration eg A gene sequence corresponding to a deletion, insertion or substitution of one or more bases in a coding sequence of a protein, or one or more amino acid deletions, insertions or substitutions in the amino acid sequence of a protein, if transfected into mesenchymal stem cells
  • alteration eg A gene sequence corresponding to a deletion, insertion or substitution of one or more bases in a coding sequence of a protein, or one or more amino acid deletions, insertions or substitutions in the amino acid sequence of a protein
  • Recombinant mesenchymal stem cells are obtained by transfecting first messenger RNA for expressing PSGL-1, second messenger RNA for expressing SLeX, and third messenger RNA for expressing IL-10 in mesenchymal stem cells.
  • the experimental results show that the recombinant mesenchymal stem cells can express PSGL-1 and SLeX. And IL-10, the ability to secrete anti-inflammatory molecules is higher.
  • the homing promoting factors PSGL-1 and SLeX can significantly increase the efficiency of recombinant mesenchymal stem cells targeting homing to disease or inflammatory lesions, and then combined with the anti-inflammatory factor IL-10 to increase the inhibitory ability of recombinant mesenchymal stem cells. Thereby increasing the effect of the treatment.
  • a method for preparing a recombinant mesenchymal stem cell comprises the steps S110 to S130.
  • S110 providing first messenger RNA, second messenger RNA and third messenger RNA, first messenger RNA for expressing P-selectin glycoprotein ligand-1, and second messenger RNA for expressing sialylated Lewis oligosaccharide-X antigen
  • the third messenger RNA is used to express interleukin-10.
  • the first messenger RNA, the second messenger RNA, and the third messenger RNA can be synthesized by genetic engineering methods according to the corresponding protein sequences, such as ribonucleotide triphosphate, RNA polymerase, Mg 2+ , and Mn 2+ .
  • the gene sequence of the first messenger RNA comprises: (a) the nucleotide sequence shown in SEQ ID No. 1; (b) has the nucleotide sequence shown in SEQ ID No. 1. a nucleotide sequence of at least 98% homology; or (c), a nucleotide sequence represented by SEQ ID No. 1, wherein one or more bases are deleted, replaced or added to the resulting nucleotide sequence.
  • the gene sequence of the second messenger RNA comprises: (a) the nucleotide sequence shown in SEQ ID No. 2; (b) having at least 98% homology to the nucleotide sequence shown in SEQ ID No. 2. a nucleotide sequence; or (c), the nucleotide sequence shown in SEQ ID No. 2, wherein one or more bases are deleted, replaced or added to the resulting nucleotide sequence.
  • the gene sequence of the third messenger RNA comprises: (a) the nucleotide sequence shown in SEQ ID No. 3; (b) having at least 98% homology to the nucleotide sequence shown in SEQ ID No. 3. a nucleotide sequence; or (c), the nucleotide sequence shown in SEQ ID No. 3, wherein one or more bases are deleted, replaced or added to the resulting nucleotide sequence.
  • S120 providing mesenchymal stem cells, and mesenchymal stem cells are cultured in a cell culture solution.
  • Mesenchymal stem cells can be derived from bone marrow, cord blood and umbilical cord tissue, placental tissue, adipose tissue, etc., can be cultured and preserved after extraction, or from ScienCell Research Laboratories or other organisms. Reagent company to buy.
  • the cell culture medium for culturing mesenchymal stem cells may be DMEM medium.
  • the mesenchymal stem cells are human bone marrow mesenchymal stem cells or human umbilical cord mesenchymal stem cells.
  • the method for preparing human bone marrow mesenchymal stem cells may include the following steps: extracting human bone marrow 8 mL to 12 mL, adding PBS (phosphate buffered saline solution) 8 mL to 12 mL, and centrifuging (800 g to 1200 g, 18 ° C to 22 ° C, 18 min to 22 min). Go to the supernatant. Then wash again with PBS in the same way. The cells were then resuspended in 8 mL to 12 mL of PBS.
  • PBS phosphate buffered saline solution
  • the cells were added to 18 mL to 22 mL of Ficoll (specific gravity: 1.073, TBD), and centrifuged (1000 g to 1200 g, 18 to 22 ° C, 25 to 30 min). A milky white villous cell layer is obtained.
  • the cells were inhaled into 8 mL to 12 mL of PBS, and the cells were collected by centrifugation (800 g to 1000 g, 18 ° C to 22 ° C, and 18 min to 22 min).
  • the cells were resuspended in 2 mL of medium, counted, and inoculated into a culture flask at a density of 1 ⁇ 10 5 /cm 2 to 5 ⁇ 10 5 /cm 2 to obtain human bone marrow mesenchymal stem cells.
  • the method for preparing human umbilical cord mesenchymal stem cells may include the following steps: taking a healthy fetal umbilical cord 4 cm to 5 cm under aseptic conditions, washing thoroughly with PBS, and rushing away residual blood in the umbilical vein and the artery.
  • the umbilical cord tissue is obtained by separating and removing the umbilical adventitial tissue and vascular tissue.
  • the gel tissue was cut into pieces of 0.5 cm 3 to 1 cm 3 and resuspended in DMEM medium to obtain human umbilical cord mesenchymal stem cells.
  • the cell culture medium for replacing the mesenchymal stem cells is further included 0.5 h to 2 h before the operation of performing the gene transfection. Specifically, the cell culture medium of the mesenchymal stem cells is removed, and a fresh cell culture solution is added. This can ensure the activity of mesenchymal stem cells and improve the efficiency of transfection.
  • the ratio of the amount of the first messenger RNA to the cell density of the mesenchymal stem cells is 0.01 ⁇ g/cm 2 to 1 ⁇ g/cm 2
  • the ratio of the amount of the second messenger RNA to the cell density of the mesenchymal stem cells is 0.01 ⁇ g/cm 2 to 1 ⁇ g/cm 2
  • the ratio of the amount of the third messenger RNA added to the cell density of the mesenchymal stem cells is 0.01 ⁇ g/cm 2 to 1 ⁇ g/cm 2 . Since mesenchymal stem cells are adherently grown cells, the cell density can be estimated from the area of the culture dish.
  • the transfection reagent can be an RNAiMAX transfection reagent. Understandably, others can promote A gene transfection reagent can also be used as the transfection reagent of the present invention.
  • the amount of the transfection reagent is calculated according to the amount of the gene expression sequence to be transfected. Specifically, the mass ratio of the first messenger RNA to the transfection reagent is 0.1 ⁇ g/ ⁇ L to 1 ⁇ g/ ⁇ L, and the second messenger RNA and the transfection reagent are used. The mass to volume ratio is from 0.1 ⁇ g/ ⁇ L to 1 ⁇ g/ ⁇ L, and the mass to volume ratio of the third messenger RNA to the transfection reagent is from 0.1 ⁇ g/ ⁇ L to 1 ⁇ g/ ⁇ L. 1 ⁇ g to 10 ⁇ L of transfection reagent is used for 1 ⁇ g of messenger RNA. More specifically, 1 ⁇ g of messenger RNA uses 3 ⁇ L to 5 ⁇ L of RNAiMAX transfection reagent.
  • the reduced serum cell culture solution refers to a culture solution in which serum is not required to be added, such as an Opti-MEM culture solution or the like. Dissolving and mixing the gene expression sequence in the serum cell culture medium, and then transfecting the gene is beneficial to improve the efficiency of transfection.
  • the amount of the reduced serum cell culture solution is calculated according to the amount of the gene expression sequence to be transfected. Specifically, the mass ratio of the first messenger RNA to the reduced serum cell culture medium is 0.01 ⁇ g/ ⁇ L to 1 ⁇ g/ ⁇ L, and the second messenger RNA is used. The mass-to-volume ratio of the reduced serum cell culture solution is 0.01 ⁇ g/ ⁇ L to 1 ⁇ g/ ⁇ L, and the mass volume ratio of the third messenger RNA to the reduced serum cell culture solution is 0.01 ⁇ g/ ⁇ L to 1 ⁇ g/ ⁇ L. One ⁇ L to 100 ⁇ L of the reduced serum cell culture solution is used for 1 ⁇ g of messenger RNA. More specifically, 1 ⁇ g of messenger RNA uses 30 ⁇ L to 60 ⁇ L of reduced serum cell culture medium.
  • the transfection reagent, the first messenger RNA, the second messenger RNA, and the third messenger RNA are further mixed and allowed to stand for 10 min to 30 min.
  • the transfection reagent is mixed with the gene expression sequence to be transfected and then allowed to stand for a period of time, which is beneficial to improve the efficiency of transfection.
  • the method further comprises culturing the mesenchymal stem cells transfected with the first messenger RNA, the second messenger RNA and the third messenger RNA for 2 to 6 hours, removing the cell supernatant, and then adding Fresh cell culture fluid is used for cell culture. After 2h-6h incubation, ensure that the gene expression sequence has been transfected into the cell, and removal of the cell supernatant can avoid unwanted transfection reagents or gene expression sequences affecting cell growth. The fresh cell culture medium is then added to continue to culture the recombinant mesenchymal stem cells. Generally, after continuous culture for 12h to 48h, recombinant mesenchymal stem cells can be detected. Secreted expression of PSGL-1, SLeX and IL-10.
  • the recombinant mesenchymal stem cells after obtaining the recombinant mesenchymal stem cells, operations such as cell passage and cell preservation can be performed. In the future, when needed, the recombinant mesenchymal stem cells can be directly cultured for cell resuscitation.
  • the expression level of P-selectin glycoprotein ligand-1, sialylated Lewis oligosaccharide-X antigen, and interleukin-10 is also detected.
  • the expression level of PSGL-1 and the expression level of SLeX in recombinant mesenchymal stem cells can be detected by immunofluorescence detection using PSGL-1 antibody and SLeX antibody, respectively.
  • the secretion concentration of IL-10 in the culture supernatant of recombinant mesenchymal stem cells was measured by ELISA (enzyme-linked immunosorbent assay).
  • the preparation method of the above recombinant mesenchymal stem cells is simple and efficient.
  • PSGL-1 and SLeX can improve the ability of mesenchymal stem cells to target homing in transplantation therapy, and recombinant mesenchymal stem cells IL-10 molecules with high anti-inflammatory effects can be secreted at the site of inflammation to improve the therapeutic effect.
  • a targeted vector for a drug comprising the recombinant mesenchymal stem cell of any of the above is also provided.
  • the recombinant mesenchymal stem cells can increase the therapeutic effect when used for treating drugs or targeting vectors of multiple sclerosis and other diseases, and provide a new and effective therapeutic drug for treating immune diseases.
  • RNAiMAX transfection reagent purchased from Lipofectamine
  • the amount of the serum-reducing medium was calculated based on the amount of mRNA used, and 50 ⁇ l of the serum-reducing medium was used per microgram of mRNA.
  • the amount of RNAiMAX transfection reagent is also calculated based on the amount of mRNA used, using 4 microliters of RNAiMAX per microgram of mRNA.
  • the transfection mixture prepared in the step (3) is added to a cell culture medium for culturing mesenchymal stem cells for gene transfection.
  • the amount of mRNA can be calculated based on the cell density of mesenchymal stem cells (cell density can be estimated from the bottom area of the culture dish of cultured mesenchymal stem cells, 1 ⁇ g of mRNA per 10 cm 2 for each mRNA. 4 h after transfection, The supernatant of mesenchymal stem cells was removed, and culture was continued by adding normal medium to obtain human bone marrow recombinant mesenchymal stem cells which can express PSGL-1, SLeX and IL-10.
  • the transfected cells were human umbilical cord mesenchymal stem cells, and the same conditions as in Example 1 were carried out to obtain human umbilical cord recombinant mesenchymal stem cells which can express PSGL-1, SLeX and IL-10.
  • the recombinant mesenchymal stem cells were obtained by the preparation method in Example 1. After transfection for 24 hours, PSGL-1 antibody and SLeX antibody were added respectively, and PSGL-1 and SLeX were detected by immunofluorescence. The amount of expression. The results of immunofluorescence detection of the expression level of PSGL-1 are shown in Fig. 2a, and the expression level of SLeX by immunofluorescence is shown in Fig. 2b. The results indicate that more than 90% of the surface of recombinant mesenchymal stem cells express PSGL-1 or SLeX.
  • EAE encephalomyelitis
  • the mononuclear cells of the spleen were isolated and separated, and the green fluorescent dye capable of analyzing the number of cell proliferation and division was used.
  • the spleen cells were pre-labeled with CFSE and cultured in a medium of RPMI-1640 plus 10% FBS and 1% double antibody to obtain T lymphocytes. Unactivated T lymphocytes served as a blank control group. Stimulated with CD3 antibody (1 ng/ul), CD28 antibody (1 ng/ul), IL-2 antibody (2 ng/ul) cytokines in vitro to obtain activated T lymphocytes, activated T lymphocytes and untransfected Mesenchymal stem cells (MSCs) were co-cultured for 6 days, which was an experimental control group.
  • the activated T lymphocytes were co-cultured with the recombinant mesenchymal stem cells in the preparation method of Example 1, which was an experimental group.
  • Flow cytometry was used to detect the proliferation of T lymphocytes in the blank control group, the experimental control group and the experimental group.
  • the results are shown in Figure 3. The results showed that the recombinant mesenchymal stem cells were more significantly inhibited than the experimental control group. Proliferation of T lymphocytes (P ⁇ 0.01).
  • Example 5 PSGL-1, SLeX transfection significantly increased the adhesion of mesenchymal stem cells to endothelial cells
  • the in vitro Flow Chamber is a commonly used method for analyzing the ability of leukocytes to adhere to vascular endothelial cells.
  • human brain microvascular endothelial cells BMECs
  • TNF-alpha tumor necrosis factor
  • the medium of mesenchymal stem cells was passed on the BMECs at different flow rates (1 dyn/cm 2 , 2 dyn/cm 2 , 5 dyn/cm 2 or 10 dyn/cm 2 ) by Flow chamber method.
  • the experimental group was recombinant mesenchymal stem cells transfected with PSGL-1 and SLeX.
  • HL-60 cells are anterior myeloid leukocyte lines and have strong endothelial cell adhesion ability, which is a positive control in this experiment.
  • Untransfected mesenchymal stem cells were used as experimental control groups.
  • FIG. 4a ⁇ 4c are adhered several experimental group, control group, positive control group and lower 1dyn / cm 2 conditions
  • FIG. FIGS. 4d ⁇ 4f under the experimental group were 2dyn / cm 2 conditions, the control group, and adhesion of the number of positive control group
  • FIGS 4j ⁇ 4l are 10dyn / cm 2 conditions
  • Figure 4m is a statistical result diagram of Figures 4a to 4l. The results showed that the number of recombinant mesenchymal stem cells transfected with PSGL-1 and SLeX adhered to BMECs was significantly higher than that of untransfected mesenchymal stem cells at four different flow rates, and the difference was statistically significant. Learning meaning.
  • recombinant mesenchymal stem cells or untransfected mesenchymal stem cells were injected through the tail vein at a dose of 1 ⁇ 10 6 cells/mouse.
  • the cells were pre-labeled with silver dye DiD before injection.
  • the spinal cord (L3-L5) of the mice was taken to prepare 10 micron thick frozen sections, fixed with 4% paraformaldehyde and embedded in glycerol. After the slide was mounted, the number of cells homing to the spinal cord in each group of animals was observed under a fluorescence microscope.
  • the results of recombinant mesenchymal stem cells are shown in Fig.
  • Fig. 5a and the results of the control group are shown in Fig. 5b, wherein Fig. 5c It is a graph of the statistical results of Figures 5a and 5b.
  • the results showed that recombinant mesenchymal stem cells (white fluorescence for transplanted MSCs, blue for all cell nuclei) were observed to occup more than the number of untransfected mesenchymal stem cells in the control group. The difference was statistically significant (P ⁇ 0.01).
  • Example 7 Recombinant mesenchymal stem cell transplantation for the treatment of MS (metabolic syndrome) and EAE (experimental autoimmune encephalomyelitis) animal model
  • tail vein injection (iv) was carried out by using the recombinant mesenchymal stem cells prepared by the method of Example 1 and the mesenchymal stem cells of the control group of 1 ⁇ 10 6 cells/cell, respectively.
  • the cells received by some mice were traced with different fluorescent dyes, the number and proportion of cells homing to the spinal cord of the mice were analyzed 24 hours later, and the other mice were used to observe and record the changes in clinical scores. Changes in the clinical symptom scores of the mice were recorded every 2 days after cell transplantation. The higher the score, the more severe the symptoms of the surface animals.
  • the scoring criteria are as follows, normal: 0 points, tail weakness: 0.5 points, tail paralysis: 1 point, tail paralysis plus unilateral hind limb weakness: 1.5 points, unilateral hind limb paralysis: 2 points, bilateral hind limb weakness: 2.5 points, double Lateral hind limb paralysis: 3 points, bilateral hind limbs plus forelimb weakness: 3.5 points, quadriplegia or sudden death or death: 4 points.
  • the PBS control group placebo control
  • the number of recombinant mesenchymal stem cells homing to the spinal cord of MS mice was significantly higher than that of the experimental control group.
  • EAE mice received recombinant PBS (transplanted group), untransfected mesenchymal stem cells (experimental control group) or PSGL-1/SLeX/IL-10 transfected recombinants after 14 days of immunization.
  • the spinal cord (L3-L5) of the mice was taken after 3 weeks.
  • Lxuol rapid blue (LFB) staining was calculated. The proportion of total white matter in the white matter of the spinal cord. As shown in Fig. 8, wherein WT is a normal mouse control group, the spinal cord has no demyelinating damage.
  • Untransfected mesenchymal stem cells reduced the demyelination area of EAE mice relative to the PBS placebo group.
  • the recombinant mesenchymal stem cells transfected with PSGL-1/SLeX/IL-10 further reduced the degree of demyelination of the spinal cord.

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Abstract

提供了一种重组间充质干细胞及其制备方法。通过在间充质干细胞中转染用于表达P选择素糖蛋白配体-1的第一信使RNA、用于表达唾液酸化路易斯寡糖-X抗原的第二信使RNA和用于表达白介素-10的第三信使RNA，得到重组间充质干细胞。

Description

重组间充质干细胞及其制备方法和应用 技术领域：
本发明涉及生物技术领域，特别是涉及一种重组间充质干细胞及其制备方法和应用。
背景技术：
免疫性疾病(immune diseases)是指免疫调节失去平衡，影响机体的免疫应答而引起的疾病。广义的免疫性疾病包括先天或后天性原因导致的免疫系统结构上或功能上的异常。其中多发性硬化(multiple sclerosis，MS)是一种常见的由自身免疫反应介导的中枢神经系统炎性脱髓鞘疾病。病灶多发于脑和脊髓的白质，呈多灶性炎性细胞侵润、脱髓鞘及神经元损伤，可导致感觉、运动、意识和神经认知等多方面的功能障碍。该病多发于20～40岁的青壮年人，复发率和致残率高，是青壮年人非外伤性神经残疾最常见的原因，迄今为止还无法完全治愈。
其中用间充质干细胞(Mesenchymal stem cell，MSCs)移植治疗多发性硬化(multiple sclerosis，MS)、炎症性肠病，类风湿性关节炎等免疫炎症性疾病，取得了显著治疗效。间充质干细是指具有自我更新能力、并能分化为骨，软骨和脂肪细胞的一组中胚层来源的细胞群。近年来许多学者发现MSCs不仅具有多向分化能力，而且MSCs还能很有效地调节免疫系统的功能，正因为如此，MSCs对于自身免疫性和炎症相关的疾病具有广阔的治疗应用前景。
目前静脉移植干细胞是最为常用和安全方便的移植途径，但是，静脉移植间充质干细胞后，由于MSCs归巢到疾病或炎症损伤部位的效率低下，以及即使少量的MSCs在达到疾病部位后，其分泌的抗炎症分子的不可控性，大大地影响了MSCs治疗免疫炎症性疾病的效率。这也是为什么在当今已经 完成或还在进行中的数百项临床试验的结果往往得出了阴性的结果。因此，如何开发出针对于增加MSCs归巢到疾病或炎症损伤的部位和如何提高MSCs在疾病部位分泌抗炎症分子的能力的有效方法，将对促进MSCs在临床上的应用具有重要的意义。
综上所述，传统的间充质干细胞归巢到疾病或炎症损伤部位效率较低、分泌抗炎症分子的能力较低。
发明内容：
基于此，有必要提供一种归巢到疾病或炎症损伤部位效率较高、分泌抗炎症分子的能力较高的重组间充质干细胞及其制备方法和应用。
一种重组间充质干细胞，所述重组间充质干细胞中转染了第一信使RNA、第二信使RNA和第三信使RNA；
所述第一信使RNA用于表达P选择素糖蛋白配体-1，所述第二信使RNA用于表达唾液酸化路易斯寡糖-X抗原，所述第三信使RNA用于表达白介素-10。
一种重组间充质干细胞的制备方法，包括如下步骤：
提供第一信使RNA、第二信使RNA和第三信使RNA，所述第一信使RNA用于表达P选择素糖蛋白配体-1，所述第二信使RNA用于表达唾液酸化路易斯寡糖-X抗原，所述第三信使RNA用于表达白介素-10；
提供间充质干细胞，所述间充质干细胞培养在细胞培养液中；以及
将转染试剂、所述第一信使RNA、所述第二信使RNA和所述第三信使RNA加入所述培养所述间充质干细胞的所述细胞培养液中进行基因转染，得到所述重组间充质干细胞。
一种药物的靶向载体，所述靶向载体中包括上述任一项所述的重组间充质干细胞。
上述重组间充质干细胞，通过在间充质干细胞中转染用于表达P选择素糖蛋白配体-1(P-selectin glycoprotein ligand 1，PSGL-1)的第一信使RNA、 用于表达唾液酸化路易斯寡糖-X抗原(Sialyl Lewis-X，SLeX)的第二信使RNA和用于表达白介素-10(Interleukin-10，IL-10)的第三信使RNA，得到重组间充质干细胞。实验结果表明，该重组间充质干细胞可表达分泌PSGL-1、SLeX和IL-10，分泌抗炎症分子的能力较高。归巢促进因子PSGL-1以及SLeX可显著提高重组间充质干细胞靶向归巢到疾病或炎症损伤部位的效率，结合抗炎因子IL-10，可增加重组间充质干细胞的炎症抑制能力。
附图说明
图1为一实施方式的重组间充质干细胞的制备方法的流程图；
图2a为免疫荧光检测重组间充质干细胞的PSGL-1的表达量的结果图；
图2b为免疫荧光检测重组间充质干细胞的SLeX的表达量的结果图；
图2c为Elisa检测重组间充质干细胞上清中的IL-10的表达量的结果图；
图3为流式细胞仪检测重组间充质干细胞抑制T淋巴细胞的增殖情况的结果图；
图4a为流动腔1dyn/cm2条件下检测转染了PSGL-1和SLeX的重组间充质干细胞间对内皮细胞的粘附能力的结果图；
图4b为流动腔1dyn/cm2条件下检测未转染的间充质干细胞间对内皮细胞的粘附能力的结果图；
图4c为流动腔1dyn/cm2条件下检测HL-60细胞间对内皮细胞的粘附能力的结果图；
图4d为流动腔2dyn/cm2条件下检测转染了PSGL-1和SLeX的重组间充质干细胞间对内皮细胞的粘附能力的结果图；
图4e为流动腔2dyn/cm2条件下检测未转染的间充质干细胞间对内皮细胞的粘附能力的结果图；
图4f为流动腔2dyn/cm2条件下检测HL-60细胞间对内皮细胞的粘附能力的结果图；
图4g为流动腔5dyn/cm2条件下检测下转染了PSGL-1和SLeX的重组间 充质干细胞间对内皮细胞的粘附能力的结果图；
图4h为流动腔5dyn/cm2条件下检测未转染的间充质干细胞间对内皮细胞的粘附能力的结果图；
图4i为流动腔5dyn/cm2条件下检测HL-60细胞间对内皮细胞的粘附能力的结果图；
图4j为流动腔10dyn/cm2条件下检测转染了PSGL-1和SLeX的重组间充质干细胞间对内皮细胞的粘附能力的结果图；
图4k为流动腔10dyn/cm2条件下检测未转染的间充质干细胞间对内皮细胞的粘附能力的结果图；
图4l为流动腔10dyn/cm2条件下检测HL-60细胞间对内皮细胞的粘附能力的结果图；
图4m为图4a～图4l的统计结果图；
图5a为荧光显微镜检测重组间充质干细胞归巢到脊髓中的细胞数量的结果图；
图5b为荧光显微镜检测未转染的间充质干细胞归巢到脊髓中的细胞数量的结果图；
图5c为图5a和图5b的统计结果图；
图6为重组间充质干细胞移植的MS动物模型的临床症状评分的结果图；
图7为免疫荧光检测重组间充质干细胞移植的EAE动物模型的炎症程度的结果图；
图8为Lxuol速蓝染色检测重组间充质干细胞移植的EAE动物模型的脊髓的脱髓鞘程度的结果图。
具体实施方式
下面主要结合附图及具体实施例对重组间充质干细胞及其制备方法和应用做进一步的解释说明。
一种重组间充质干细胞，该重组间充质干细胞中转染了第一信使RNA、 第二信使RNA和第三信使RNA。第一信使RNA用于表达P选择素糖蛋白配体-1(P-selectin glycoprotein ligand 1，PSGL-1)，第二信使RNA用于表达唾液酸化路易斯寡糖-X抗原(Sialyl Lewis-X，SLeX)，第三信使RNA用于表达白介素-10(Interleukin-10，IL-10)。
信使RNA(message RNA，mRNA)携带遗传信息，在蛋白质合成时充当模板，易于转染到细胞中。
在一个实施例中，第一信使RNA的基因序列包括：(a)、SEQ ID No.1所示的核苷酸序列；(b)、与SEQ ID No.1所示的核苷酸序列具有至少98％同源性的核苷酸序列；或(c)、SEQ ID No.1所示的核苷酸序列，其中一个或多个碱基被缺失、替代或增加得到的核苷酸序列。
第二信使RNA的基因序列包括：(a)、SEQ ID No.2所示的核苷酸序列；(b)、与SEQ ID No.2所示的核苷酸序列具有至少98％同源性的核苷酸序列；或(c)、SEQ ID No.2所示的核苷酸序列，其中一个或多个碱基被缺失、替代或增加得到的核苷酸序列。
第三信使RNA的基因序列包括：(a)、SEQ ID No.3所示的核苷酸序列；(b)、与SEQ ID No.3所示的核苷酸序列具有至少98％同源性的核苷酸序列；或(c)、SEQ ID No.3所示的核苷酸序列，其中一个或多个碱基被缺失、替代或增加得到的核苷酸序列。
可以理解，由于编码同一种氨基酸的密码子有多种，蛋白的编码序列的多态性及变异，与核苷酸序列具有至少98％同源性的核苷酸序列或至少一种改变(如蛋白质的编码序列中一个或多个碱基的缺失、插入或取代，或者蛋白质的氨基酸序列中有一个或多个氨基酸缺失、插入或取代)对应的基因序列，如果转染到间充质干细胞中，得到重组间充质干细胞与本申请的重组间充质干细胞无明显的功能差异，也应当包括在本发明的范围内。
通过在间充质干细胞中转染用于表达PSGL-1的第一信使RNA、用于表达SLeX的第二信使RNA和用于表达IL-10的第三信使RNA，得到重组间充质干细胞。实验结果表明，该重组间充质干细胞可表达分泌PSGL-1、SLeX 和IL-10，分泌抗炎症分子的能力较高。归巢促进因子PSGL-1以及SLeX可以显著提高重组间充质干细胞靶向归巢到疾病或炎症损伤部位的效率，再结合抗炎因子IL-10，增加重组间充质干细胞的炎症抑制能力，从而增加治疗的效果。
此外，如图1所示，一种重组间充质干细胞的制备方法，包括步骤S110～S130。
S110、提供第一信使RNA、第二信使RNA和第三信使RNA，第一信使RNA用于表达P选择素糖蛋白配体-1，第二信使RNA用于表达唾液酸化路易斯寡糖-X抗原，第三信使RNA用于表达白介素-10。
第一信使RNA、第二信使RNA和第三信使RNA可以根据相应的蛋白序列，提供三磷酸核糖核苷酸、RNA聚合酶、Mg2+和Mn2+等原料，通过基因工程的方法合成。
在一个实施例中，第一信使RNA的基因序列包括：(a)、SEQ ID No.1所示的核苷酸序列；(b)、与SEQ ID No.1所示的核苷酸序列具有至少98％同源性的核苷酸序列；或(c)、SEQ ID No.1所示的核苷酸序列，其中一个或多个碱基被缺失、替代或增加得到的核苷酸序列。
第二信使RNA的基因序列包括：(a)、SEQ ID No.2所示的核苷酸序列；(b)、与SEQ ID No.2所示的核苷酸序列具有至少98％同源性的核苷酸序列；或(c)、SEQ ID No.2所示的核苷酸序列，其中一个或多个碱基被缺失、替代或增加得到的核苷酸序列。
第三信使RNA的基因序列包括：(a)、SEQ ID No.3所示的核苷酸序列；(b)、与SEQ ID No.3所示的核苷酸序列具有至少98％同源性的核苷酸序列；或(c)、SEQ ID No.3所示的核苷酸序列，其中一个或多个碱基被缺失、替代或增加得到的核苷酸序列。
S120、提供间充质干细胞，间充质干细胞培养在细胞培养液中。
间充质干细胞可以来源于骨髓、脐带血和脐带组织、胎盘组织、脂肪组织等，提取后可培养传代并保存，也可以从ScienCell研究实验室或其它生物 试剂公司购买。培养间充质干细胞的细胞培养液可以为DMEM培养基。
具体的，间充质干细胞为人骨髓间充质干细胞或人脐带间充质干细胞。
制备人骨髓间充质干细胞的方法可以包括以下步骤：提取人骨髓8mL～12mL，加入PBS(磷酸缓冲盐溶液)8mL～12mL，离心(800g～1200g、18℃～22℃、18min～22min)，去上清。然后同样方法用PBS再洗一次。之后用8mL～12mL的PBS重悬细胞。将细胞加入18mL～22mL的Ficoll(比重为1.073，TBD公司)中，离心(1000g～1200g、18℃～22℃、25min～30min)。得到乳白色绒毛状细胞层。将细胞吸入8mL～12mL的PBS中，离心收集细胞(800g～1000g、18℃～22℃、18min～22min)。然后用2mL培养基重悬细胞，计数，以1×105/cm2～5×105/cm2的密度接种到培养瓶中，培养后得到人骨髓间充质干细胞。
制备人脐带间充质干细胞的方法可以包括以下步骤：无菌条件下取健康胎儿脐带4cm～5cm，用PBS充分洗涤，冲去脐静脉及动脉内的残留血液。分离并去除脐带外膜组织和血管组织，即可获得脐带的胶组织。将胶组织剪为0.5cm3～1cm3大小的组织块，用DMEM培养基重悬，培养后得到人脐带间充质干细胞。
优选的，在进行基因转染的操作之前的0.5h～2h，还包括更换间充质干细胞的细胞培养液。具体为移除间充质干细胞的细胞培养液，加入新鲜的细胞培养液。这样可以保证间充质干细胞的活性，提高转染的效率。
S130、将转染试剂、第一信使RNA、第二信使RNA和第三信使RNA加入培养间充质干细胞的细胞培养液中进行基因转染，得到重组间充质干细胞。
具体的，第一信使RNA的加入量与间充质干细胞的细胞密度的比值为0.01μg/cm2～1μg/cm2，第二信使RNA的加入量与间充质干细胞的细胞密度的比值为0.01μg/cm2～1μg/cm2，第三信使RNA的加入量与间充质干细胞的细胞密度的比值为0.01μg/cm2～1μg/cm2。由于间充质干细胞为贴壁生长的细胞，因此细胞密度可以根据培养皿的面积估算。
具体的，转染试剂可以为RNAiMAX转染试剂。可以理解，其他可促进 基因转染的试剂也可以作为本发明的转染试剂。
转染试剂的用量根据要转染的基因表达序列的用量计算，具体的，第一信使RNA与转染试剂的质量体积比为0.1μg/μL～1μg/μL，第二信使RNA与转染试剂的质量体积比为0.1μg/μL～1μg/μL，第三信使RNA与转染试剂的质量体积比为0.1μg/μL～1μg/μL。相当于1μg信使RNA使用1μL～10μL的转染试剂。更具体的，1μg信使RNA使用3μL～5μL的RNAiMAX转染试剂。
在一个实施例中，进行基因转染的操作之前，还包括提供减血清细胞培养液，将第一信使RNA、第二信使RNA和第三信使RNA与减血清细胞培养液混匀，之后再加入培养间充质干细胞的细胞培养液中进行基因转染。减血清细胞培养液指不需要添加血清的培养液，例如Opti-MEM培养液等。将基因表达序列溶解混匀在减血清细胞培养液，再进行基因转染有利于提高转染的效率。
减血清细胞培养液的用量根据要转染的基因表达序列的用量计算，具体的，第一信使RNA与减血清细胞培养液的质量体积比为0.01μg/μL～1μg/μL，第二信使RNA与减血清细胞培养液的质量体积比为0.01μg/μL～1μg/μL，第三信使RNA与减血清细胞培养液的质量体积比为0.01μg/μL～1μg/μL。相当于1μg信使RNA使用1μL～100μL的减血清细胞培养液。更具体的，1μg信使RNA使用30μL～60μL的减血清细胞培养液。
本实施方式中，进行基因转染的操作之前，还包括将转染试剂、第一信使RNA、第二信使RNA和第三信使RNA混匀，静置10min～30min。转染试剂与要转染的基因表达序列混匀后静置一段时间，有利于提高转染的效率。
具体的，进行基因转染的操作之后，还包括将转染了第一信使RNA、第二信使RNA和第三信使RNA的间充质干细胞培养2h～6h，移除细胞上清液，再加入新鲜的细胞培养液进行细胞培养。培养2h～6h后，确保基因表达序列已经转染进入细胞中，此时移除细胞上清液可以避免多余的转染试剂或基因表达序列影响细胞的生长。之后加入新鲜的细胞培养液，继续培养重组的间充质干细胞。一般继续培养12h～48h后即可检测到重组的间充质干细胞中有 分泌表达PSGL-1、SLeX以及IL-10。
可以理解，得到重组的间充质干细胞后可以进行细胞传代、细胞保存等操作。以后需要使用时可直接拿保存的重组的间充质干细胞进行细胞复苏培养。
在一个实施例中，进行基因转染的操作之后，还包括检测P选择素糖蛋白配体-1、唾液酸化路易斯寡糖-X抗原以及白介素-10的表达含量。具体的，可以分别采用PSGL-1抗体、SLeX抗体通过免疫荧光检测的方法检测重组的间充质干细胞的PSGL-1的表达量以及SLeX的表达量。通过ELISA(酶联免疫吸附检测)检测IL-10在重组的间充质干细胞的培养上清中的分泌浓度。
结果表明，大于90％的重组的间充质干细胞的表面可表达PSGL-1或者SLeX，转染七天之后IL-10的分泌量还可以达到10ng/万细胞，表达分泌抗炎症分子的能力强。
上述重组间充质干细胞的制备方法操作步骤简单而高效，利用mRNA转染方法，PSGL-1与SLeX可提高间充质干细胞在移植治疗中细胞靶向归巢的能力，并且重组间充质干细胞可以在炎症部位分泌具有高效抗炎症作用的IL-10分子，提高治疗效果。
此外，还提供一种药物的靶向载体，靶向载体包括上述任一项所述的重组间充质干细胞。
该重组间充质干细胞用于治疗多发性硬化及其它疾病的药物或靶向载体时，可增加治疗的效果，为治疗免疫性疾病提供了一种全新的高效的治疗药物。
下面为具体实施例部分。
以下实施例中，如无特别说明，未注明具体条件的实验方法，通常按照常规条件或者试剂盒生产厂家推荐的方法实现。实验中所用的试剂未特别注明的均可购自singma公司。
实施例1 制备人骨髓重组间充质干细胞
(1)通过基因工程的方法分别合成第一mRNA、第二mRNA以及第三mRNA，得到用于表达P选择素糖蛋白配体-1的第一mRNA、用于表达唾液酸化路易斯寡糖-X抗原的第二mRNA以及用于表达所述白介素-10的第三mRNA。
(2)准备人骨髓间充质干细胞，要进行基因转染前1h，更换新鲜的已经预热好的培养基(DMEM)。
(3)准备减血清培养基(Opti-MEM，购于Gibco Life Technology)和RNAiMAX转染试剂(购于Lipofectamine)，将第一信使RNA、第二信使RNA以及第三信使RNA溶解在减血清培养基中，之后加入RNAiMAX转染试剂，混匀后静置15min，得到转染混合液。减血清培养基的用量根据mRNA的用量计算，每微克mRNA使用50微升的减血清培养基。RNAiMAX转染试剂用量也是根据mRNA的用量计算，每微克mRNA使用4微升RNAiMAX。
(4)将步骤(3)中制得的转染混合液加入培养间充质干细胞的细胞培养液中进行基因转染。mRNA的用量可根据间充质干细胞的细胞密度计算(细胞密度可根据培养间充质干细胞的培养皿的底面积估算，每一种mRNA，每10平方厘米用1微克mRNA。转染后4h，移除间充质干细胞的上清液，加入正常培养基继续培养，得到可表达PSGL-1、SLeX以及IL-10的人骨髓重组间充质干细胞。
实施例2 制备重组人脐带间充质干细胞
转染的细胞为人脐带间充质干细胞，其余条件与实施例1相同，得到可表达PSGL-1、SLeX以及IL-10的人脐带重组间充质干细胞。
实施例3 PSGL-1、SLeX以及IL-10表达量的检测
采用实施例1中的制备方法得到重组间充质干细胞，基因转染24h后，分别加入PSGL-1抗体以及SLeX抗体，通过免疫荧光检测PSGL-1以及SLeX 的表达量。免疫荧光检测PSGL-1的表达量的结果如图2a所示，免疫荧光检测SLeX的表达量的如图2b所示。结果表明大于90％的重组间充质干细胞的表面表达PSGL-1或者SLeX。通过Elisa检测转染24h后的重组间充质干细胞上清中的IL-10结果如图2c所示，发现重组间充质干细胞可以分泌的高浓度的IL-10因子，转染七天之后IL-10分泌量还可以达到10ng/万细胞。
实施例4 重组间充质干细胞抑制T淋巴细胞的增殖
(1)采用实施例1中的制备方法得到重组间充质干细胞。
(2)EAE(脑脊髓炎)小鼠模型的制作：取雌性C57BL/6小鼠麻醉后，后背部皮下注射髓鞘少突胶质细胞糖蛋白乳化液(MOG35-55)，200μg/只，注射两侧，100μg/侧，诱导MS动物模型。当天及48小时候各腹腔注射一次百日咳毒素溶液(400ng/次)。EAE小鼠在免疫14天之后，用70μm细胞滤网(BDFalcon)挤压、过滤脾脏组织制成单细胞悬液，分离脾脏的单个核细胞分离后，用可以分析细胞增殖分裂次数的绿色荧光染料CFSE预标记好脾细胞，培养在RPMI-1640加10％FBS和1％双抗的培养基中，得到T淋巴细胞。未激活的T淋巴细胞作为空白对照组。在体外用CD3抗体(1ng/ul)、CD28抗体(1ng/ul)、IL-2抗体(2ng/ul)细胞因子刺激，得到激活的T淋巴细胞，激活的T淋巴细胞与未转染的间充质干细胞(MSCs)共培养6天，此为实验对照组。激活的T淋巴细胞与实施例1中的制备方法得到重组间充质干细胞共培养6天，此为实验组。利用流式细胞仪分别检测空白对照组、实验对照组以及实验组的T淋巴细胞的增殖情况，结果如图3所示，结果表明，重组间充质干细胞相比实验对照组能够更加显著地抑制T淋巴细胞的增殖(P<0.01)。
实施例5 PSGL-1、SLeX转染显著提高间充质干细胞对内皮细胞的粘附能力
体外Flow Chamber(流动腔)是常用的分析白细胞粘附血管内皮细胞能力的方法。为了检测间充质干细胞对内皮细胞的粘附能力，在体外培养人源 脑微血管内皮细胞(BMECs)，100％融合后，用50ng/mL的肿瘤坏死因子(TNF-alpha)刺激以模拟炎症环境下的血管内皮，再用Flow chamber方法让间充质干细胞的培养基在BMECs上以不同的流速(1dyn/cm2，2dyn/cm2，5dyn/cm2或10dyn/cm2)通过。实验组为PSGL-1和SLeX转染的重组间充质干细胞。HL-60细胞为前髓系白细胞系，具有很强的内皮细胞粘附能力，在本实验中为阳性对照。未转染的间充质干细胞作为实验对照组。当总共通过10万个间充质干细胞细胞后，分别计算实验对照组、实验组以及阳性对照组粘附在BMECs单层细胞上的间充质干细胞细胞的数量。如图4a～图4c分别为1dyn/cm2条件下实验组、对照组、和阳性对照组的的粘附数，图4d～图4f分别为2dyn/cm2条件下实验组、对照组、和阳性对照组的的粘附数，图4g～图4i分别为5dyn/cm2条件下实验组、对照组、和阳性对照组的的粘附数，图4j～图4l分别为10dyn/cm2条件下实验组、对照组、和阳性对照组的的粘附数。图4m为图4a～图4l的的统计结果图。结果表明PSGL-1、SLeX转染得到的重组间充质干细胞粘附在BMECs上的数量在四种不同的流速情况下均明显多于未转染的间充质干细胞，其差别具有显著的统计学意义。
实施例6 重组间充质干细胞归巢到EAE小鼠脊髓炎症部位的效率
(1)采用实施例1中的制备方法得到重组间充质干细胞。
(2)EAE小鼠免疫14天后，通过尾静脉注射重组间充质干细胞或者未转染的间充质干细胞(对照组)，剂量为1×106细胞/只小鼠。细胞在注射之前均用银光染料DiD预标记，在注射24小时后，取小鼠的脊髓(L3-L5)，制备10微米厚的冰冻切片，用4％的多聚甲醛固定并用甘油包埋封片后，置于荧光显微镜下观察并计数各组动物中归巢到脊髓中的细胞数量，重组间充质干细胞的结果如图5a所示，对照组结果如图5b所示，其中图5c为图5a和图5b的统计结果图。结果表明，可观察到重组间充质干细胞(白色荧光为移植的MSCs，蓝色为所有细胞的细胞核)归巢到脊髓的数量明显多于对照组未转染的间充质干细胞归巢的数量，其差别经统计学分析后提示显著 (P<0.01)。
实施例7 重组间充质干细胞移植治疗MS(代谢综合征)和EAE(实验性自身免疫性脑脊髓炎)的动物模型
(1)MS动物模型的建立与骨髓或脐带源神经干细胞移植治疗：取雌性C57BL/6小鼠(8周～12周)，每组10只，麻醉后，后背部皮下注射髓鞘少突胶质细胞糖蛋白乳化液(MOG35-55)200μg/只，注射两侧，100μg/侧，诱导MS动物模型。当天及48小时候各腹腔注射一次百日咳毒素溶液(400ng/次)。MOG33-35免疫14天后分别尾静脉注射(i.v.)采用实施例1的方法制备的重组间充质干细胞与对照组的间充质干细胞各1×106个细胞/只。一部分小鼠接受的细胞用不同的荧光染料示踪后，24小时后分析归巢到小鼠脊髓中的细胞数量及比例，另一部分小鼠用来观察、记录临床评分的改变。细胞移植后每2天记录小鼠的临床症状评分的改变。分数越高，表面动物症状越严重。评分标准为如下，正常：0分，尾巴无力：0.5分，尾巴麻痹：1分，尾巴麻痹加单侧后肢无力：1.5分，单侧后肢瘫痪：2分，双侧后肢无力：2.5分，双侧后肢瘫痪：3分，双侧后肢瘫痪加前肢无力：3.5分，四肢瘫痪或濒死或死亡：4分。如图6所示，PBS对照组(安慰剂对照)呈现典型的疾病进程，动物临床症状没有显著减轻。而重组间充质干细胞归巢到MS小鼠脊髓中的数量显著多于实验对照组。说明重组间充质干细胞对疾病的抑制作用比对照组的间充质干细胞更加显著。(其中PBS对照组与实验对照组的间充质干细胞比较的P值<0.05，而PBS对照组与实验组重组间充质干细胞比较的P值<0.01)。
(2)重组间充质干细胞体内抑制炎症，促进髓鞘修复与再生作用：干细胞移植4周后，取各组小鼠脊髓，制成冰冻切片采用，免疫荧光染色剂CD45单克隆抗体荧光染色细胞，检测脊髓炎症细胞浸润程度，计算炎症面积占脊髓白质总面积的比例。结果如图7所示，其中WT为正常小鼠对照组，其脊髓没有炎症损伤。相对于PBS安慰剂组，未转染的间充质干细胞可以减少EAE小鼠的炎症程度。而PSGL-1/SLeX/IL-10转染得到的重组间充质干细胞更进 一步减轻脊髓的炎症损伤程度。
(3)EAE小鼠在免疫14天后分别接受移植PBS(安慰剂组)、未转染的间充质干细胞(实验对照组)或者PSGL-1/SLeX/IL-10转染得到的重组间充质干细胞(实验组)，3周后，取小鼠的脊髓(L3-L5),固定后用Lxuol速蓝(LFB)染色检测EAE小鼠脊髓的脱髓鞘程度，并计算脱髓鞘面积占脊髓白质总面积的比例。如图8所示，其中WT为正常小鼠对照组，其脊髓没有脱髓鞘损伤。相对于PBS安慰剂组，未转染的间充质干细胞可以减少EAE小鼠脱髓鞘面积。而PSGL-1/SLeX/IL-10转染得到的重组间充质干细胞更进一步减轻脊髓的脱髓鞘程度。
以上所述实施例仅表达了本发明的一种或几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (18)

  1. 一种重组间充质干细胞，其特征在于，所述重组间充质干细胞中转染了第一信使RNA、第二信使RNA和第三信使RNA；
    所述第一信使RNA用于表达P选择素糖蛋白配体-1，所述第二信使RNA用于表达唾液酸化路易斯寡糖-X抗原，所述第三信使RNA用于表达白介素-10。
  2. 根据权利要求1所述的重组间充质干细胞，其特征在于，所述第一信使RNA的基因序列包括：(a)、SEQ ID No.1所示的核苷酸序列；(b)、与SEQ ID No.1所示的核苷酸序列具有至少98％同源性的核苷酸序列；或(c)、SEQ ID No.1所示的核苷酸序列，其中一个或多个碱基被缺失、替代或增加得到的核苷酸序列。
  3. 根据权利要求1所述的重组间充质干细胞，其特征在于，所述第二信使RNA的基因序列包括：(a)、SEQ ID No.2所示的核苷酸序列；(b)、与SEQ ID No.2所示的核苷酸序列具有至少98％同源性的核苷酸序列；或(c)、SEQ ID No.2所示的核苷酸序列，其中一个或多个碱基被缺失、替代或增加得到的核苷酸序列。
  4. 根据权利要求1所述的重组间充质干细胞，其特征在于，所述第三信使RNA的基因序列包括：(a)、SEQ ID No.3所示的核苷酸序列；(b)、与SEQ ID No.3所示的核苷酸序列具有至少98％同源性的核苷酸序列；或(c)、SEQ ID No.3所示的核苷酸序列，其中一个或多个碱基被缺失、替代或增加得到的核苷酸序列。
  5. 一种重组间充质干细胞的制备方法，其特征在于，包括如下步骤：
    提供第一信使RNA、第二信使RNA和第三信使RNA，所述第一信使RNA用于表达P选择素糖蛋白配体-1，所述第二信使RNA用于表达唾液酸化路易斯寡糖-X抗原，所述第三信使RNA用于表达白介素-10；
    提供间充质干细胞，所述间充质干细胞培养在细胞培养液中；以及
    将转染试剂、所述第一信使RNA、所述第二信使RNA和所述第三信使 RNA加入所述培养所述间充质干细胞的所述细胞培养液中进行基因转染，得到所述重组间充质干细胞。
  6. 根据权利要求5所述的重组间充质干细胞的制备方法，其特征在于，还包括在进行所述基因转染之前0.5h～2h，更换培养所述间充质干细胞的细胞培养液。
  7. 根据权利要求5所述的重组间充质干细胞的制备方法，其特征在于，还包括在进行所述基因转染之前，提供减血清细胞培养液，将所述第一信使RNA、所述第二信使RNA和所述第三信使RNA与所述减血清细胞培养液混匀；
    所述第一信使RNA与所述减血清细胞培养液的质量体积比为0.01μg/μL～1μg/μL，所述第二信使RNA与所述减血清细胞培养液的质量体积比为0.01μg/μL～1μg/μL，所述第三信使RNA与所述减血清细胞培养液的质量体积比为0.01μg/μL～1μg/μL。
  8. 根据权利要求5所述的重组间充质干细胞的制备方法，其特征在于，所述第一信使RNA的加入量与所述间充质干细胞的细胞密度的比值为0.01μg/cm2～1μg/cm2，所述第二信使RNA的加入量与所述间充质干细胞的细胞密度的比值为0.01μg/cm2～1μg/cm2，所述第三信使RNA的加入量与所述间充质干细胞的细胞密度的比值为0.01μg/cm2～1μg/cm2。
  9. 根据权利要求5所述的重组间充质干细胞的制备方法，其特征在于，还包括在进行所述基因转染之前，将所述转染试剂、所述第一信使RNA、所述第二信使RNA和所述第三信使RNA混匀，静置10min～30min。
  10. 根据权利要求5所述的重组间充质干细胞的制备方法，其特征在于，所述转染试剂为RNAiMAX转染试剂。
  11. 根据权利要求5所述的重组间充质干细胞的制备方法，其特征在于，所述第一信使RNA与所述转染试剂的质量体积比为0.1μg/μL～1μg/μL，所述第二信使RNA与所述转染试剂的质量体积比为0.1μg/μL～1μg/μL，所述第三信使RNA与所述转染试剂的质量体积比为0.1μg/μL～1μg/μL。
  12. 根据权利要求5所述的重组间充质干细胞的制备方法，其特征在于，还包括在进行所述基因转染之后，将所述转染了所述第一信使RNA、所述第二信使RNA和所述第三信使RNA的间充质干细胞培养2h～6h，移除细胞上清液，再加入新鲜的细胞培养液进行细胞培养。
  13. 根据权利要求5所述的重组间充质干细胞的制备方法，其特征在于，还包括在进行所述基因转染之后，检测所述P选择素糖蛋白配体-1、所述唾液酸化路易斯寡糖-X抗原以及所述白介素-10的表达含量。
  14. 根据权利要求5所述的重组间充质干细胞的制备方法，其特征在于，所述第一信使RNA的基因序列包括：(a)、SEQ ID No.1所示的核苷酸序列；(b)、与SEQ ID No.1所示的核苷酸序列具有至少98％同源性的核苷酸序列；或(c)、SEQ ID No.1所示的核苷酸序列，其中一个或多个碱基被缺失、替代或增加得到的核苷酸序列。
  15. 根据权利要求5所述的重组间充质干细胞的制备方法，其特征在于，所述第二信使RNA的基因序列包括：(a)、SEQ ID No.2所示的核苷酸序列；(b)、与SEQ ID No.2所示的核苷酸序列具有至少98％同源性的核苷酸序列；或(c)、SEQ ID No.2所示的核苷酸序列，其中一个或多个碱基被缺失、替代或增加得到的核苷酸序列。
  16. 根据权利要求5所述的重组间充质干细胞的制备方法，其特征在于，所述第三信使RNA的基因序列包括：(a)、SEQ ID No.3所示的核苷酸序列；(b)、与SEQ ID No.3所示的核苷酸序列具有至少98％同源性的核苷酸序列；或(c)、SEQ ID No.3所示的核苷酸序列，其中一个或多个碱基被缺失、替代或增加得到的核苷酸序列。
  17. 根据权利要求5所述的重组间充质干细胞的制备方法，其特征在于，所述间充质干细胞为人骨髓间充质干细胞或人脐带间充质干细胞。
  18. 一种药物的靶向载体，其特征在于，所述靶向载体中包括权利要求1～5中任一项所述的重组间充质干细胞。
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