WO2017008686A1 - 血栓烷素a2受体作为脂肪干细胞治疗缺血性疾病的靶标 - Google Patents

血栓烷素a2受体作为脂肪干细胞治疗缺血性疾病的靶标 Download PDF

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WO2017008686A1
WO2017008686A1 PCT/CN2016/089295 CN2016089295W WO2017008686A1 WO 2017008686 A1 WO2017008686 A1 WO 2017008686A1 CN 2016089295 W CN2016089295 W CN 2016089295W WO 2017008686 A1 WO2017008686 A1 WO 2017008686A1
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adipose
stem cell
stem cells
activity
calpain
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French (fr)
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余鹰
申毓军
左胜锴
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中国科学院上海生命科学研究院
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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
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material

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  • the present invention relates to a thromboxane A2 receptor as a target for the treatment of ischemic diseases by adipose stem cells.
  • Adipose-derived stem cell is a stem cell with multi-directional differentiation potential isolated from adipose tissue in recent years.
  • the adipose tissue obtained by separating ADSC was mainly derived from the adipose tissue excised during abortion and plastic surgery.
  • ADSC can be obtained from adipose tissue of different species such as human, mouse, rabbit, pig, sheep and dog; the population doubling time of ADSC derived from different individuals is basically the same, about 60h on average; studies show that ADSC has Strong self-renewal ability and low aging characteristics are ideal stem cell sources in tissue engineering [Lu Wei, "Research progress in adipose tissue-derived stem cells", Chinese Journal of Clinicians (Electronic Edition), October 2013, Volume 7, Issue 20].
  • Lu et al [Lu F, Mizuno H, Uysal AC, etc., Improved viability of random pattern skin flaps through the use of adipose-derived stem cells, Plast Reconstr Surg, 2008, 121:50-58] found that ADSC promotes angiogenesis The function.
  • ADSC Adipose tissue-derived stromal cells as a novel option for regenerative cell therapy, Ather Thromb, 2006, l3(2): 77-81] believe that ADSC promotes angiogenic ability and There is no difference in BMSC. ADSC promotes angiogenesis not only because it has the potential to differentiate into vascular endothelial cells but also participates in vascular composition. In addition, its secretion plays an important role.
  • the thromboxane A2 receptor also known as TP, belongs to the G protein-coupled receptor and has seven transmembrane features.
  • Human TP has two subtypes: TP ⁇ and TP ⁇ , which are composed of 343 and 407 amino acids, respectively.
  • TP ⁇ and TP ⁇ are structurally different only in the C-terminal tail, some studies have shown that both are functionally There are some differences, like now It has been confirmed that TP ⁇ , but not TP ⁇ plays a role in inhibiting vascular endothelial growth factor (VEGF)-induced migration and angiogenesis [Yang Guibao et al., Relationship between thromboxane A2 receptor and cardiovascular disease, Chinese Medicine, 2011, pp. Volume 6, Issue 5].
  • VEGF vascular endothelial growth factor
  • the invention adopts advanced plasmid construction technology, laser confocal microscopy technology, siRNA knockdown technology, Real-time PCR technology, flow cytometry, immunoblotting technology and laser Doppler imaging system and other advanced biological techniques to Type and thromboxane A2 receptor (TP) knockout mouse adipose-derived stem cells (ADSC) were used to study the effect of TP signaling pathway on ADSC-induced angiogenesis and its molecular mechanism.
  • TP Type and thromboxane A2 receptor
  • the results showed that the positive rate of endothelial cell marker CD31 was significantly higher than that of wild-type ADSC after TP knockdown or inhibited ADSC differentiation, and the ability of tube formation
  • TP knockout can significantly improve the therapeutic effect of ADSC, and the number of new blood vessels is significantly increased.
  • TP knockout resulted in elevated levels of ⁇ -catenin ( ⁇ -chain protein) in ADSC. Knocking down ⁇ -catenin reduced the differentiation efficiency and ADSC tube formation ability, and decreased the therapeutic effect of ADSC on lower limb ischemia.
  • ⁇ -catenin ⁇ -chain protein
  • TP knockdown leads to a decrease in intracellular calcium ion concentration, a decrease in calcium transient response ability, and inhibition of calcium ion levels after TP overexpression can restore calpain activity and ADSC differentiation ability. Therefore, our results indicate that inhibition of TP is expected to be a new strategy to improve the treatment of ischemic diseases with ADSC.
  • a first aspect of the invention provides a stem cell derived from adipose or a composition comprising the stem cell, the stem cell:
  • the gene expressing TP in the stem cell is knocked out or a mutation that results in a decrease or loss of TP activity occurs.
  • the gene expressing calpain in the stem cell is knocked out or occurs resulting in a decrease in calpain activity or a loss of mutation.
  • the stem cell contains an expression vector that expresses a ⁇ -chain protein such that the ⁇ -chain protein is overexpressed.
  • the stem cells are derived from a lipid of a mammal such as a human, a mouse, a rabbit, a pig, a sheep, and a dog. Fat tissue.
  • the present invention also provides a method of increasing the ability of adipose-derived stem cells to promote angiogenesis or to promote differentiation of adipose-derived stem cells into vascular endothelial cells in vitro, the method comprising increasing the activity of the stem cells of ⁇ -chain protein.
  • the increasing the activity of the ⁇ -chain protein of the stem cell comprises:
  • reducing, inhibiting or eliminating TP activity of said stem cells comprises: knocking out a gene expressing TP in said stem cell, or mutating a gene expressing TP in said stem cell, or causing said stem cell to Contact with TP antagonists.
  • reducing, inhibiting or eliminating calpain activity of the stem cell comprises: knocking out a gene expressing calpain in the stem cell, or mutating a gene expressing calpain in the stem cell, or The stem cells are contacted with a calpain inhibitor.
  • the TP antagonist is selected from the group consisting of AH 29848, SQ-29548, AH-23848, GR-32191, ONO-11120, BM-13177, BM-13505, SKF-88046, L-655240, S-145 , TMQ, R-68070, CV-4151 and Bay-K 8644.
  • the calpain inhibitor is selected from the group consisting of: N-acetyl-L-leucyl-L-leucyl-L-n-leucine (ALLN), fluoromethyl ketone (Z-LLY- FMK), calpastatin, alpha-ketoamide inhibitors and Calpeptin (CPT).
  • the invention also encompasses the use of a TP antagonist and/or a calpain inhibitor for the manufacture of a medicament for increasing the ability of adipose derived stem cells to differentiate into vascular endothelial cells and/or enhance their angiogenic capacity.
  • the present invention also encompasses the use of the adipose-derived stem cells of the present invention or compositions comprising the stem cells for the preparation of a medicament for the treatment of ischemic diseases.
  • the present invention also encompasses a method of preparing vascular endothelial cells or blood vessels from adipose-derived stem cells or a composition containing the stem cells in vitro, the method comprising contacting the adipose-derived stem cells of the present invention with VEGF.
  • FIG. 1 Treatment of ADSC with VEGF activates the TXA2/TP signaling pathway.
  • a flow cytometry analysis of ADSC surface markers
  • b ADSC prostaglandin receptor expression
  • c VEGF treatment, immunoblotting detection of COX-1 and COX-2 expression in ADSC
  • df VEGF After treatment, Real-time PCR detected changes in mRNA relative expression of TxB2, TxAS and TP.
  • FIG. 2 TP knockdown increases VEGF-induced differentiation of ADSCs into endothelial cells.
  • a Immunofluorescence photographs of CD34 (green, first line), CD31 (red, second line), nuclei (DAPI, blue, third line) in the VEGF-treated group and the control group;
  • b a map of CD34 Quantitative statistics of + /CD31 + cells;
  • c relative mRNA levels of endothelial markers in ADSCs in VEGF-treated and control groups;
  • d detection of CD31 + in ADSCs by flow cytometry in VEGF-treated and control groups Cell;
  • e quantitative statistics of CD31 + cells;
  • f tube formation experiments of ADSCs of WT and TP KO in VEGF-treated and control groups;
  • g quantification of tubular structures in f-graph;
  • h Relative mRNA levels of pro-angiogenic factors in WT and TP KO ADSCs in VEGF-treated and control
  • Figure 3 TP knockdown improves the therapeutic efficiency of ADSC in a mouse model of lower limb ischemia.
  • a laser Doppler photos of ADSC treatment of lower limb ischemia in mice (arrow, ischemic lower limbs);
  • b blood flow statistics of lower limbs of a picture, calculated by calculating the ratio of blood flow of ischemic lower limbs and normal lower limbs;
  • c Immunostaining of sections of ischemic lower limbs treated with ADSC in mice with lower extremity ischemia; quantitative statistics of dg, CD31 + , PCNA + , RFP + /CD31 + /PCNA + and RFP - /CD31 + /PCNA + cells in c-picture .
  • Figure 4 TP knockdown significantly upregulates the ⁇ -catenin protein after VEGF treatment.
  • a ⁇ -catenin, c-myc, CD31 immunoblotting in WT and TP KO ADSCs in VEGF-treated and control groups;
  • bd quantification of protein levels of ⁇ -catenin, c-myc, CD31 Statistics;
  • e relative mRNA levels of ⁇ -catenin target gene in ADSC in VEGF-treated group and control group;
  • f mRNA expression of TP after over-expressing TP adenovirus and empty vector in ADSC of TP KO;
  • hj quantitative analysis of protein levels of g- ⁇ -catenin, c-myc, CD31;
  • k in VEGF Immunoblotting detection of ⁇ -catenin, c
  • FIG. 5 Knockdown of ⁇ -catenin eliminates the strong angiogenic capacity of ADSCs after TP knockout.
  • a silencing ⁇ -catenin (Si- ⁇ -cat) WT and TP KO ADSC tube formation ability
  • b quantitative statistics of a diagram tubular structure
  • c silencing ⁇ -catenin (Si- ⁇ -cat) The effect of WT and TP KO ADSC on the formation of vascular capacity in Matrigel
  • d representative image of the gel in c-picture and immunofluorescence staining, EGFP + cells derived from EGFP transgenic mice
  • d Figure Quantitative statistics of EGFP + /CD31 + cells.
  • FIG. 6 Knockdown of ⁇ -catenin can reduce the therapeutic effect of TP knockdown on mouse lower limb ischemia.
  • b lower limb blood of a Flow statistic, calculated by calculating the ratio of blood flow in the ischemic lower limb to the normal lower limb;
  • c immunostaining of the ischemic lower limb section of the lower limb ischemia in ADSC-treated mice, white arrow indicating RFP + /CD31 + /PCNA + cells ;dg, quantitative statistics of CD31 + , PCNA + , RFP + /CD31 + /PCNA + and RFP - /CD31 + /PCNA + cells in c map.
  • Figure 7 Inhibition of Calpain activity enhances angiogenesis in ADSCs mediated by ⁇ -catenin.
  • b TP KO for transfected TP adenovirus (Adeno-TP) and control adenovirus (Vector) Detection of ⁇ -catenin changes in ADSC;
  • c Immunoblot analysis of ⁇ -catenin in ADSCs of WT and TP KO by ⁇ -catenin C-terminal antibody
  • b TP KO for transfected TP adenovirus (Adeno-TP) and control adenovirus (Vector) Detection of ⁇ -catenin changes in ADSC;
  • c Immunoblot analysis of ⁇ -catenin in ADSCs of WT and TP KO by ⁇ -catenin C
  • TP affects differentiation in ADSC by coupling Gq.
  • a WT and TP KO ADSC Fluo-3 (green) immunofluorescence representative map
  • b quantitative fluorescence statistics in a picture
  • c under U46619 treatment, WT and TP KO ADSC calcium Change in flow
  • d immunofluorescence photograph of Rhod (red, middle column) after infection of TP adenovirus or control virus in the presence of U46619 in ADSC of TP KO
  • e fluorescence intensity in d Quantitative statistics
  • f changes in calcium flux after infection with TP adenovirus or control virus in the presence of U46619 in ADSC of TP KO
  • g co-precipitation detection of TP and G q/11 interaction
  • h Effects of U-73122 on the proteins of m-calpain, ⁇ -catenin, cyclinD1 and CD31 after ADSC infection of TP KO AD
  • i U73122 ability to establish
  • Figure 9 Inhibition of TP enhances the differentiation of human ADSCs into endothelial cells.
  • a SQ29548 (inhibitor of TP) human ADSC, flow detection of CD31 + cells representative map; b, statistics on CD31 + cells in a picture; c, SQ29548 on human ADSC tube formation ability; d, quantitative statistics of the tubular structure in c; e, SQ29548 on the effect of human ADSC on the angiogenesis ability of matrigel; f, representative of the gel in the e-graph and immunofluorescence staining; g, f Statistical analysis of human (h) and mouse (m) CD31 + cells; h, i, SQ29548 effects on mRNA levels of the pro-angiogenic factors VEGF-A and bFGF in human ADSCs.
  • the present invention relates to the use of TP or ⁇ -chain proteins as a target for the treatment of ischemic diseases by adipose stem cells.
  • TP activity By reducing, inhibiting or eliminating TP activity, or by increasing ⁇ -chain protein activity, it can increase the ability of adipose-derived stem cells to promote angiogenesis, or promote the differentiation of adipose-derived stem cells into vascular endothelial cells, thereby improving the adipose-derived stem cell treatment.
  • the efficacy of blood diseases By reducing, inhibiting or eliminating TP activity, or by increasing ⁇ -chain protein activity, it can increase the ability of adipose-derived stem cells to promote angiogenesis, or promote the differentiation of adipose-derived stem cells into vascular endothelial cells, thereby improving the adipose-derived stem cell treatment.
  • TP activity can be reduced, inhibited, or eliminated using techniques well known in the art, including knocking out genes that express TP in adipose-derived stem cells, causing mutations in the genes that result in decreased or lost TP activity, or making adipose-derived stem cells Antagonists of TP are in contact.
  • MF site-selective knockout of adipose-derived stem cells in vitro can be performed by techniques such as CRISPR-Cas9 or TALEN. Partial mutations can also be performed on the TP to reduce or lose its activity.
  • TP antagonists known in the art can be employed to reduce, inhibit or eliminate TP activity.
  • TP antagonists include, but are not limited to, AH 29848, SQ-29548, AH-23848, GR-32191, ONO-11120, BM-13177, BM-13505, SKF-88046, L-655240, S-145, TMQ, R -68070, CV-4151 and Bay-K 8644, etc., the structure of some compounds is as follows:
  • Increasing beta-chain protein activity can be achieved by:
  • TP activity can be achieved by knocking out genes expressing TP in adipose-derived stem cells, causing mutations in the indicated genes to cause a decrease or loss of TP activity, or contacting adipose-derived stem cells with an antagonist of TP. Reduce, suppress or eliminate.
  • calpain of stem cells For the activity of calpain of stem cells, it can be contacted with an inhibitor of calpain to achieve reduction, inhibition or elimination of the calpain.
  • Inhibitors of calpain known in the art can be employed, including but not limited to N-acetyl-L-leucyl-L-leucyl-L-norleucine (ALLN), fluoromethyl ketone (Z -LLY-FMK), calpastatin, alpha-ketoamide inhibitors and calpeptin (CPT). See, for example, Zhang Yong, “Study on the Design, Synthesis and Activity of Alpha-Ketylamide Calpain Inhibitors", Ph.D. Thesis, 2008, the entire contents of which is incorporated herein by reference.
  • ALLN N-acetyl-L-leucyl-L-leucyl-L-norleucine
  • Z -LLY-FMK fluoromethyl ketone
  • CPT alpha-ketoamide inhibitors
  • Zhang Yong “Study on the Design, Synthesis and Activity of Alpha-Ketylamide Calpain Inhibitors
  • the stem cell can be achieved by knocking out or mutating a gene that expresses calpain (Calpain) Reduction, inhibition or elimination of calpain.
  • the knockout or mutation can be accomplished using techniques well known in the art. Mutations usually mutate the active domain of calpain, thereby reducing or losing the activity of calpain; or mutating the expressed gene such that its gene is not expressed or expressed.
  • expression of a gene expressing calpain or TP can be reduced using, for example, siRNA technology.
  • the adipose-derived stem cells can be transfected in vitro by constructing a plasmid containing a ⁇ -chain protein or an adenoviral vector, thereby further over-expressing the ⁇ -chain protein. See Sawant DA, Tharakan B, Hunter FA, Smythe WR, Childs EW, "Role of ⁇ -catenin in regulating microvascular endothelial cell hyperpermeability", J Trauma, February 2011, 70(2): 481-7.
  • the amount of the antagonist or inhibitor may depend on the antagonist or inhibitor selected, the amount of adipose derived stem cells, the effect of inhibition, and the like, And combined with the existing technology to determine.
  • adipose-derived stem cells having an improved ability to promote angiogenesis and/or an ability to differentiate into vascular endothelial cells can be prepared.
  • the adipose-derived stem cells are derived from adipose tissue of a mammal, especially a human, a mouse, a rabbit, a pig, a sheep and a dog, and particularly preferably a human adipose tissue.
  • the adipose derived stem cells preferably have one or more of the following characteristics:
  • a gene expressing TP is knocked out or mutated, resulting in a decrease or loss of TP activity
  • Adipose-derived stem cells of the invention can be used to treat conditions requiring angiogenesis and/or requiring vascular endothelium, including but not limited to ischemia, due to their increased ability to promote angiogenesis and/or the ability to differentiate into vascular endothelial cells.
  • Various diseases and diabetes include, but are not limited to, lower limb ischemic diseases, cerebral thrombosis, myocardial infarction, diabetes, neurodegenerative diseases, and the like.
  • the present invention encompasses the use of a TP antagonist and/or a calpain inhibitor for the manufacture of a medicament for promoting the ability of adipose derived stem cells to differentiate into vascular endothelial cells and/or enhance their angiogenic ability.
  • the present invention also encompasses a method of treating or preventing an ischemic disease, the method comprising administering an adipose-derived stem cell of the invention to a subject in need thereof.
  • the method comprises co-cultivating the adipose-derived stem cells of the invention with Matrigel, and then administering (eg, injecting to a patient) a Matrigel containing the adipose-derived stem cells of the invention.
  • VEGF can be administered simultaneously.
  • the adipose-derived stem cells of the present invention can be directly injected into a subject's patient.
  • the subject can be a mammal, such as a human, a mouse, a rabbit, a pig, a sheep, a dog, and the like.
  • the present invention also encompasses a method of increasing the ability of adipose-derived stem cells to promote angiogenesis or to promote differentiation of adipose-derived stem cells into vascular endothelial cells in vitro, the method comprising increasing the activity of the stem cells of ⁇ -chain protein.
  • the invention also encompasses the use of adipose derived stem cells of the invention in the manufacture of a medicament for the treatment of ischemic diseases.
  • the present invention also encompasses a method of preparing vascular endothelial cells or blood vessels from adipose-derived stem cells in vitro, the method comprising contacting the adipose-derived stem cells of the invention with VEGF.
  • concentration of VEGF upon contact can be determined by actual conditions, which is within the purview of those skilled in the art.
  • the invention also includes a composition comprising the adipose derived stem cells of the invention.
  • the composition may be a pharmaceutical composition which may contain, in addition to the stem cells, a suitable pharmaceutically acceptable carrier.
  • the composition may be a culture of adipose-derived stem cells of the present invention, and the culture may contain, in addition to the stem cells, a medium suitable for the stem cells to proliferate in an undifferentiated state. These media may be various media inhibited in the art for maintaining the non-differentiated proliferation of adipose-derived stem cells.
  • mice were C57BL/6 genetic background. Red or green fluorescent protein transgenic mice were mated with TP knockout mice to produce red or green fluorescent/TP knockout mice. All mice were in compliance with the regulations of the Laboratory Animal Management Committee of the Institute of Nutritional Sciences, Chinese Academy of Sciences.
  • Calpeptin, U46619, arachidonic acid was purchased from Cayman Chemicals, Inc. (Cayman Chemical, Ann Arbor, MI, USA).
  • Mouse and human vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were purchased from Peprotech (Peprotech Inc., Rocky Hill, NJ, USA).
  • Matrigel was purchased from BD Bios (San Jose, CA, USA).
  • ADSCs adipose-derived stem cells
  • Human ADSCs were purchased from Cyagen Biotech (Cat. No.: HUXMD-01001) and cultured with OriCell human ADSC growth medium (Cat. No.: HUXMD-90011).
  • Mouse ADSCs were isolated from mouse epididymal fat pad fat. Briefly described as follows: The adipose tissue was cut into small pieces, digested with 0.2% collagenase (Sigma-Aldrich, St. Louis, MO, USA) in a 37 ° C water bath for 1 hour; then the digest was washed with a 100 um sieve (BD Biosciences) Filtration to remove tissue debris; the cell suspension was then diluted with PBS and centrifuged to remove collagenase.
  • collagenase Sigma-Aldrich, St. Louis, MO, USA
  • the cells under centrifugation were resuspended in 160 mM NH 4 Cl and allowed to stand at room temperature for 10 min to remove red blood cells. Finally, the obtained cells were supplemented with 10% fetal bovine serum plus 1% penicillin/streptomycin in Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12; Invitrogen, Carlsbad, CA, USA) medium at 37 ° C under 5% CO 2 conditions. Under cultivation. Change the fluid every other day. All mouse ADSCs were used in the second generation for experiments. For endothelial differentiation experiments, 200 ul of Matrigel was plated in one well of a 12-well plate and incubated for 1 hour at 37 ° C; then 1 ⁇ 10 5 ADSCs were seeded in the medium.
  • DMEM/F12 Dulbecco's modified Eagle's medium/Ham's F12
  • the ADSCs of the mice were subjected to flow analysis using a BD flow cytometer (FACScan, BD Biosciences). Briefly, cells were harvested and incubated in PBS containing 1% calf serum containing primary antibodies for 30 minutes. CD29, CD90, Sca-1, CD34 and CD31 used in the experiments were purchased from eBioscience (eBioscience, San Diego, CA, USA). Streaming data was analyzed using FlowJo 8.3.3 software.
  • the primary antibodies used in the experiments included mouse CD34 (dilution factor 1:50; eBioscience), CD31 (dilution factor 1:50; eBioscience), CD31 (dilution factor 1:500; BD Biosciences), GFP (dilution factor 1) : 1000; Abcam, Cambridge, MA, USA), RFP (dilution factor 1:1000; Abcam), and PCNA (dilution factor 1:1000; Cell Signaling Technology, Danvers, MA, USA) and human (dilution factor 1: 200; R&D Systems, Minneapolis, MN, USA). After overnight, excess antibody was washed away with PBS and the secondary antibody was incubated for 1 hour at room temperature.
  • the secondary antibody-conjugated fluorescence used in this experiment includes Alexa Fluor 488, Alexa Fluor 594, or Alexa Fluor 633.
  • the cultured ADSCs were stimulated with 30 ⁇ mol/L of arachidonic acid. After 30 minutes, the supernatant was collected, 12000 g, and centrifuged at 4 ° C for 15 minutes to extract prostaglandins and analyze their contents by liquid chromatography-tandem mass spectrometry.
  • ADSCs 100 ⁇ L of matrix gel containing ADSCs (0.5 ⁇ 10 6 ) was injected subcutaneously into the lateral region of C57BL/6 mice.
  • Human ADSCs (1 x 10 6 ) were mixed with 500 ng/mL VEGF (Peprotech) and 2 ⁇ M SQ29548 (Cayman Chemical) and added to 100 ⁇ L Matrigel, which was then injected subcutaneously into the lateral regions of nude mice. After two weeks, take out, take a photo, and perform a morphological analysis.
  • proximal and distal femoral arteries of the left lower extremity of eight-week-old male mice were ligated separately, and the middle was cut to cause ischemia of the lower limbs.
  • 10 6 ADSCs were injected into the ischemic lower limb gastrocnemius. Blood flow recovery was measured by laser Doppler flowmetry (LDI; Moor Instruments Ltd., UK) at 7 and 14 days.
  • the adenovirus was generated using the pAd-track-CMV GFP vector containing the full-length TP cDNA encoding mouse, which was produced and amplified in the 293A virus packaging cell line. After several rounds of amplification of the recombinant adenovirus, the cesium chloride gradient was purified by centrifugation. The infection efficiency was estimated by measuring the fluorescence of green fluorescent protein.
  • Total cellular RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's protocol.
  • the extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (Takara, Dalian, China).
  • SYBR Green (Takara) mixture was used for real-time quantitative PCR.
  • the expression level of the target gene is regulated by the expression level of actin.
  • the PCR procedure was as follows: 95 ° C, 5 minutes for one cycle, followed by 40 cycles at 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 30 seconds, and the last extension of 72 ° C for 5 minutes. The number of cycles per PCR product was obtained by the dissolution curve.
  • ADSCs were transfected using RNAiFect transfection reagent (Qiagen, Crawley, UK) according to the manufacturer's protocol, including the specific sequence ⁇ -catenin (100 nM) or control-ordered siRNA (Genepharma, Shanghai, China).
  • RNAiFect transfection reagent Qiagen, Crawley, UK
  • RNAiFect transfection reagent Qiagen, Crawley, UK
  • ADSCs were infected with an empty vector carrying the empty vector or TP cDNA.
  • Proteins were extracted from ADSCs using a lysing solution containing a protease inhibitor.
  • Total cellular protein (Pierce, Rockford, IL, USA) was determined by the BCA method using the Piece BCA Protein Concentration Assay Kit. The same amount of protein was denatured, electrophoresed with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) at 10%, then transferred to a nitrocellulose membrane, and 5% skim milk was blocked for 1 hour, then The primary antibody was incubated overnight at 4 °C.
  • the primary antibodies used in this experiment included anti-mouse actin (1:2000; Sigma-Aldrich), anti-c-myc (1:1000; geneTex), anti-cyclin D1 (1:1000; Abcam), anti- CD31 (1:500; Abgent), anti-HA-tag, anti-m-calpain, anti- ⁇ -calpain, anti-VE-cadherin, anti-phospho-GSK3 ⁇ S9, anti-total-GSK3 ⁇ , anti-phospho- ⁇ -cateninS33 (1:1000; Cell Signaling Technology), anti-C-terminal ⁇ -catenin (1:1000; BD Biosciences), anti-N-terminal ⁇ -catenin (1:1000; Abcam), and anti- ⁇ -catenin ( 1:1000; Millipore).
  • the membrane was then incubated with horseradish peroxidase-labeled secondary antibody for 2 hours at room temperature.
  • the film was then developed with an enhanced luminescent reagent (Piece).
  • results are expressed as mean ⁇ standard error (SEM). Data were statistically analyzed using the 16.0 version of SPSS software (SPSS Inc., Chicago, IL, USA) using t-test or analysis of variance. A P value of less than 0.05 in the analysis was considered statistically significant.
  • TXA2 and TP signals are activated during ADSC differentiation into the endothelium.
  • FIG. 1 shows that ADSC has a dry marker and a higher expression of the prostaglandin receptor TP.
  • the upstream enzymes of prostaglandins were up-regulated by COX1, 2 and TXAS, the secretion of TXB2 was increased, and the expression of prostaglandin synthetase TxAS and receptor TP was up-regulated, indicating that TXA2/TP axis is in the process of ADSC-to-endothelial differentiation.
  • Medium is activated.
  • knockout TP accelerates the differentiation of ADSC into endothelial cells
  • TP knockdown increased the expression of endothelial cell markers CD31 and CD34 in ADSC during induction; endothelial-associated markers Tie2, CD31, etc. were also evident in mRNA levels. Elevation; flow cytometry showed that the proportion of mature endothelial cells increased significantly; the ability of tube formation increased; the mRNA levels of vascular growth factors VEGF-A and bFGF were significantly increased; the three-dimensional tube experiment in matrigel in vivo also showed TP Stronger angiogenic capacity after knockout.
  • TP knockout makes ADSC improve the efficiency of lower limb ischemia
  • FIG. 3 shows that ADSC of TP knockout mice showed a significant increase in blood perfusion at 7 and 14 days of treatment of lower limb ischemia in mice compared to WT mouse-derived ADSCs.
  • Immunofluorescence showed that TP-knocked ADSC treatment significantly increased ischemic muscle endothelial cells; using RFP-derived ADSC tracking, CD31 + endothelial cells derived from exogenous ADSCs and proliferating endothelial cells (PCNA + , CD31 + ) were evident.
  • Increase indicating that TP knockout promotes angiogenesis of ADSC, mainly by inducing differentiation of ADSC into endothelial cells.
  • ⁇ -catenin elevation is a key factor in the promotion of ADSC to vascular endothelial differentiation by TP knockout
  • TP knockout increases the level of ⁇ -catenin in ADSC, increases the level of c-myc in the downstream target molecule, and increases the level of CD31.
  • the target gene of ⁇ -catenin after mRNA knockdown is mRNA level.
  • overexpression of TP by adenovirus transfection caused loss of the above phenotype
  • knockdown of ⁇ -catenin by siRNA prevented the expression of ADSC-related molecules in endothelial cells and promoted mRNA levels of vascular growth factors VEGF-A and bFGF decline.
  • Figure 5 shows that knockdown of ⁇ -catenin by siRNA can reduce the ability to establish ADSC; reduce ADSC in the base The three-dimensional tube-forming ability in the gel; immunofluorescence showed that knocking down ⁇ -catenin significantly reduced the role of exogenous ADSC in the process of vascular formation, and decreased the ability of endothelial differentiation.
  • Knocking down ⁇ -catenin can reduce the effect of ADSC on lower limb ischemia
  • Figure 6 shows that after knocking down ⁇ -catenin, the therapeutic effect of TP-knocked ADSC is significantly reduced, and blood perfusion at 14 days is no longer restored; immunofluorescence results show that endothelial cells are significantly reduced in the ischemic area, including exogenous ADSCs.
  • Figure 7 shows that by using the C-terminal antibody, different bands of ⁇ -catenin were detected, and overexpression of TP increased the degradation; at the same time, the activity of calpain (calpain), a calcium-dependent proteolytic enzyme, was found to be significantly decreased after TP knockdown.
  • calpain calpain
  • TP replenishment can reduce the degradation of ⁇ -catenin and promote the expression of endothelial-associated protein; meanwhile, the three-dimensional tube-forming ability of ADSC in matrigel is enhanced, and the ability of endothelial differentiation Increased, mRNA levels of the pro-angiogenic factors VEGF-A and bFGF are elevated.
  • Figure 8 shows that intracellular calcium concentration decreases after TP knockout, cell calcium transients are lost under TP agonist stimulation; overexpression of TP calcium transient recovery; co-immunoprecipitation results show that TP can indeed interact with G q/11 Using the PLC inhibitor U-73122, it can significantly affect the TP-mediated changes in protein expression of related molecules and improve the ability of tube formation due to TP overexpression; therefore, TP induces calcium influx through Gq in ADSC. Activation of calpain mediates the regulation of ⁇ -catenin, which in turn affects the differentiation of ADSC into endothelial cells.
  • Figure 9 shows that treatment of human ADSC with TP inhibitor SQ2958 has enhanced ability to differentiate into endothelial cells; enhanced tube-forming ability; three-dimensional experiments with matrigel in vivo indicate enhanced angiogenic capacity of human ADSC after TP inhibitor treatment The ability of endothelial differentiation is enhanced; the mRNA levels of the pro-angiogenic factors VEGF-A and bFGF are elevated.

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Abstract

提供了一种血栓烷素A2受体（TP）失活或受到抑制、和/或钙蛋白酶活性失活或降低、和/或β-链蛋白活性提高的脂肪来源的干细胞。还提供了该脂肪来源的干细胞在制备治疗缺血性疾病用的药物中的用途以及由该脂肪来源的干细胞制备血管内皮细胞或血管的方法。还提供了一种提高脂肪来源的干细胞促血管新生的能力或促进脂肪来源的干细胞向血管内皮细胞分化的方法。还提供了TP拮抗剂和/或钙蛋白酶抑制剂在制备促进脂肪来源的干细胞向血管内皮细胞的分化能力、提高其血管新生能力的药物中的用途。

Description

血栓烷素A2受体作为脂肪干细胞治疗缺血性疾病的靶标 技术领域
本发明涉及血栓烷素A2受体作为脂肪干细胞治疗缺血性疾病的靶标。
背景技术
脂肪来源的干细胞(adipose-derived stem cell，ADSC)是近年来从脂肪组织中分离得到的一种具有多向分化潜能的干细胞。分离获得ADSC的脂肪组织最早主要来源于抽脂术后废弃和整形术中切取的脂肪组织。目前已证实，可以从人、鼠、兔子、猪、羊和狗等不同物种的脂肪组织中获得ADSC；来源于不同个体的ADSC的群体倍增时间基本一致，平均约60h；研究表明，ADSC具有较强的自我更新能力和低衰老性特征，是组织工程中理想的干细胞来源〔陆伟，“脂肪组织来源干细胞的研究进展”，《中华临床医师杂志》(电子版)，2013年10月，第7卷，第20期〕。
Lu等〔Lu F、Mizuno H、Uysal AC等，Improved viability of random pattern skin flaps through the use of adipose-derived stem cells，Plast Reconstr Surg，2008，121:50-58〕研究发现，ADSC具备促进血管新生的功能。
Nakagami等〔Hironori Nakagami、Ryuichi Morishita、Kazuhisa Maeda等，Adipose tissue-derived stromal cells as a novel option for regenerative cell therapy，Ather Thromb，2006，l3(2):77-81〕认为，ADSC促血管新生能力和BMSC没有差别。ADSC能促进血管新生不仅是由于它具有分化为血管内皮细胞的潜能并进而参与血管构成，另外，它的分泌作用也在其中起到了重要的作用。Rehman等[33]的实验显示,在低氧环境中的ADSC，其VEGF的分泌量约为正常氧含量环境中的5倍；低氧环境中的ADSC上清能明显促进血管内皮细胞的生长和减少内皮细胞凋亡。
Kim等〔Rehman J、Traktuev D、Li J等，Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells，Circulation，2004，109:1292-1298〕进行的干细胞向血管内皮分化的诱导实验发现，ADSC比BMSC能够形成更多完整的管状结构。
血栓烷素A2受体又叫TP，属于G蛋白耦联受体，具有七次跨膜特征。人类的TP有两种亚型:TPα和TPβ，分别由343个和407个氨基酸组成，虽然TPα和TPβ在结构上仅是C端尾部存在不同，但有一些研究表明，两者在功能上也存在一些不同，如现在 已经证实是TPβ，而非TPα在抑制血管内皮生长因子(VEGF)诱导的迁移和血管生成中起作用〔杨贵宝等，血栓素A2受体与心血管疾病的关系，《中国医药》，2011，第6卷，第5期〕。
发明内容
本发明采用重组质粒构建技术、激光共聚焦显微技术、siRNA knockdown技术、Real-time PCR技术、流式细胞术、免疫印迹技术以及激光多普勒成像系统等先进的生物学技术手段，以野生型以及血栓烷素A2受体(TP)敲除的小鼠脂肪来源干细胞(ADSC)为对象，研究TP信号通路对ADSC促血管新生的影响及其分子机制。研究结果表明：TP敲除或受到抑制的ADSC诱导分化后内皮细胞标志物CD31阳性率明显高于野生型来源的ADSC，且在体和离体基质胶上的成管能力明显增强，内皮细胞标志物CD31表达显著增加。同样，在下肢缺血模型中，TP敲除可以显著提高ADSC的治疗效果，新生血管明显增多。体外培养发现，TP敲除导致ADSC胞内β-catenin(β-链蛋白)水平升高，敲低β-catenin可降低分化效率和ADSC成管能力，降低ADSC对下肢缺血的治疗效果。同时发现，TP敲除后ADSC胞内钙依赖的蛋白水解酶calpain的活性显著下降，过表达TP，calpain的活性升高，β-catenin水平下降，内皮分化效率降低。进一步，TP敲除导致胞内钙离子浓度下降，钙瞬流反应能力下降，抑制TP过表达后的钙离子水平可以恢复calpain的活性和ADSC分化能力。因此，我们的结果表明抑制TP有望成为改善以ADSC为材料治疗缺血性疾病的新策略。
因此，本发明第一方面提供一种脂肪来源的干细胞或含该干细胞的组合物，所述干细胞：
(1)TP失活或其活性受到抑制；和/或
(2)钙蛋白酶活性失活或降低；和/或
(3)β-链蛋白活性提高。
在一个具体实施例中，所述干细胞中表达TP的基因被敲除或发生导致TP活性降低或丧失的突变。
在一个具体实施例中，所述干细胞中表达钙蛋白酶的基因被敲除或发生导致钙蛋白酶活性降低或丧失突变。
在一个具体实施例中，所述干细胞含有表达β-链蛋白的表达载体，从而β-链蛋白过表达。
在一个具体实施例中，所述干细胞来自哺乳动物如人、鼠、兔、猪、羊和狗等的脂 肪组织。
本发明还提供一种体外提高脂肪来源的干细胞促血管新生的能力或促进脂肪来源的干细胞向血管内皮细胞分化的方法，所述方法包括提高所述干细胞的β-链蛋白的活性。
在一个具体实施例中，所述提高所述干细胞的β-链蛋白的活性包括：
(1)降低、抑制或消除所述干细胞的TP活性；和/或
(2)降低、抑制或消除所述干细胞的钙蛋白酶的活性；和/或
(3)过表达所述干细胞中的β-链蛋白。
在一个具体实施例中，降低、抑制或消除所述干细胞的TP活性包括：敲除所述干细胞中表达TP的基因，或使所述干细胞中表达TP的基因发生突变，或使所述干细胞与TP拮抗剂接触。
在一个具体实施例中，降低、抑制或消除所述干细胞的钙蛋白酶活性包括：敲除所述干细胞中表达钙蛋白酶的基因，或使所述干细胞中表达钙蛋白酶的基因发生突变，或使所述干细胞与钙蛋白酶抑制剂接触。
在一个具体实施例中，TP拮抗剂选自AH 29848、SQ-29548、AH-23848、GR-32191、ONO-11120、BM-13177、BM-13505、SKF-88046、L-655240、S-145、TMQ、R-68070、CV-4151和Bay-K 8644。
在一个具体实施例中，钙蛋白酶抑制剂选自：N-乙酰基-L-亮氨酰-L-亮氨酰-L-正亮氨醛(ALLN)、氟甲基酮(Z-LLY-FMK)、钙蛋白酶抑素(Calpastatin)、α-酮基酰胺类抑制剂和Calpeptin(CPT)。
本发明还包括TP拮抗剂和/或钙蛋白酶抑制剂在制备提高脂肪来源的干细胞向血管内皮细胞的分化能力和/或提高其血管新生能力的药物中的用途。
本发明还包括本发明脂肪来源的干细胞或含该干细胞的组合物在制备治疗缺血性疾病用的药物中的用途。
本发明还包括一种体外由脂肪来源的干细胞或含该干细胞的组合物制备血管内皮细胞或血管的方法，所述方法包括使本发明脂肪来源的干细胞与VEGF接触。
附图说明
图1：用VEGF处理ADSC可以激活TXA2/TP信号通路。a、流式细胞术分析ADSC的表面标记物；b、ADSC中前列腺素受体的表达情况；c、VEGF处理后，免疫印迹检测COX-1和COX-2在ADSC中的表达；d-f、VEGF处理后，Real-time PCR技术检测TxB2，TxAS以及TP的mRNA相对表达变化。
图2：TP敲除可以提高VEGF诱导的ADSC向内皮细胞的分化。a、在VEGF处理组及对照组中，CD34(绿，第一行)，CD31(红，第二行)，细胞核(DAPI，蓝，第三行)的免疫荧光照片；b、对a图CD34+/CD31+细胞的定量统计；c、在VEGF处理组及对照组中，ADSC的内皮标记的相对mRNA水平；d、在VEGF处理组及对照组中，流式细胞术检测ADSC中的CD31+的细胞；e、对d图CD31+的细胞定量统计；f、在VEGF处理组及对照组中，WT以及TP KO的ADSC的成管实验；g、对f图中管状结构的定量；h、在VEGF处理组及对照组中，WT以及TP KO的ADSC中的促血管新生因子的相对mRNA水平；i、WT以及TP KO的ADSC在体内基质胶中的生成血管实验；j、对i图中的胶进行切片兵免疫荧光染色的代表图；k、对j图中EGFP+/CD31+的血管定量。
图3：TP敲除可以改善ADSC在小鼠下肢缺血模型中的治疗效率。a、ADSC治疗小鼠下肢缺血的激光多普勒照片(箭头，缺血下肢)；b、对a图下肢血流统计，通过计算缺血下肢与正常下肢的血流比例计算；c、对ADSC治疗小鼠下肢缺血的缺血下肢的切片的免疫染色；d-g、对c图中CD31+，PCNA+，RFP+/CD31+/PCNA+以及RFP-/CD31+/PCNA+细胞的定量统计。
图4：TP敲除使β-catenin蛋白在VEGF处理后明显上调。a、在VEGF处理组及对照组中，WT以及TP KO的ADSC中的β-catenin，c-myc，CD31免疫印迹检测；b-d、对a图β-catenin，c-myc，CD31的蛋白水平定量统计；e、在VEGF处理组及对照组中，ADSC中β-catenin目的基因的相对mRNA水平；f、在TP KO的ADSC中过表达TP腺病毒及空载体后TP的mRNA表达变化；g、在TP KO的ADSC中过表达TP腺病毒后的β-catenin，c-myc，CD31免疫印迹检测；h-j、对g图β-catenin，c-myc，CD31的蛋白水平定量统计；k、在VEGF处理组及对照组中，沉默β-catenin(Si-β-cat)后β-catenin，c-myc，CD31以及VE-cadherin(VE-cad)的免疫印迹检测；l-m、沉默β-catenin(Si-β-cat)后，对VEGF-A和bFGF的mRNA的相对水平检测。
图5：敲低β-catenin可以消除在TP敲除后的ADSC的较强的血管新生能力。a、沉默β-catenin(Si-β-cat)WT以及TP KO的ADSC的成管能力的影响；b、对a图管状结构的定量统计；c、沉默β-catenin(Si-β-cat)WT以及TP KO的ADSC的在基质胶中形成血管能力的影响；d、对c图中的胶进行切片并免疫荧光染色的代表图，EGFP+的细胞来源于EGFP的转基因小鼠；e、对d图EGFP+/CD31+的细胞定量统计。
图6：敲低β-catenin可以减少TP敲除后对小鼠下肢缺血的治疗效果。a、对ADSC沉默β-catenin(Si-β-cat)或者对照组(Scram)中，治疗小鼠下肢缺血的激光多普勒照片(箭头，缺血下肢)；b、对a图下肢血流统计，通过计算缺血下肢与正常下肢的血流 比例计算；c、对ADSC治疗小鼠下肢缺血的缺血下肢的切片的免疫染色，白色箭头表示RFP+/CD31+/PCNA+的细胞；d-g、对c图中CD31+，PCNA+，RFP+/CD31+/PCNA+以及RFP-/CD31+/PCNA+细胞的定量统计。
图7：抑制Calpain活性可以增强β-catenin介导的ADSC的血管新生能力。a、用β-catenin C端抗体进行免疫印迹分析β-catenin在WT以及TP KO的ADSC中的变化；b、对转染TP腺病毒(Adeno-TP)以及对照腺病毒(Vector)的TP KO的ADSC中的β-catenin变化检测；c、在是否有U46619(TP特异性的激动剂)存在的情况下，对WT以及TP KO的ADSC中Calpain活性的检测；d、在TP KO的ADSC中，对是否有U46619的情况下感染TP腺病毒或对照病毒后，Calpain活性的检测；e、在TP KO的ADSC中，用不同剂量的Calpain抑制剂(CPT)处理后，β-catenin，cyclinD1以及CD31的蛋白变化；f、过表达TP后，CPT对ADSC在基质胶中的血管新生能力的影响；g、对f图中的胶进行切片并免疫荧光染色的代表图，GFP+的细胞来源于GFP的转基因小鼠；h、对d图GFP+/CD31+的细胞定量统计；i，j、过表达TP后，CPT对ADSC表达VEGF-A和bFGF的mRNA水平的影响；k、在VEGF处理组及对照组中，WT以及TP KO的ADSC中u-calpain以及m-calpain的蛋白水平变化；l、不同剂量U46619对ADSC中u-calpain以及m-calpain的蛋白水平的影响。
图8：TP在ADSC中通过偶联Gq来影响其分化。a、WT以及TP KO的ADSC的Fluo-3(绿)的免疫荧光代表图；b、对a图中的荧光强度进行定量统计；c、在U46619处理下，对WT以及TP KO的ADSC中钙流的变化；d、在TP KO的ADSC中，对是否有U46619的情况下感染TP腺病毒或对照病毒后，Rhod(红，中间列)的免疫荧光照片；e、对d图中的荧光强度进行定量统计；f、在TP KO的ADSC中，对是否有U46619的情况下感染TP腺病毒或对照病毒后钙流的变化；g、免疫共沉淀检测TP和Gq/11相互作用；h、对TP KO的ADSC感染TP腺病毒后，U-73122对m-calpain，β-catenin，cyclinD1以及CD31的蛋白的影响；i、U73122对转染了TP腺病毒的TP KO的ADSC的成管能力的影响；j、对i图中管状结构的定量统计；k、TP通过钙依赖的calpain/β-catenin通路调节ADSC向内皮细胞分化的模式图。
图9：抑制TP可以增强人的ADSC向内皮细胞分化。a、SQ29548(TP的抑制剂)人的ADSC后，流式检测CD31+细胞的代表图；b、对a图中的CD31+细胞进行统计；c、SQ29548对人的ADSC成管能力的影响；d、对c图中管状结构的定量统计；e、SQ29548对人的ADSC在基质胶中血管形成能力的影响；f、对e图中的胶进行切片并免疫荧光染色的代表图；g、对f图中人的(h)以及小鼠的(m)CD31+细胞的统计；h，i、SQ29548 对人的ADSC中促血管生成因子VEGF-A和bFGF的mRNA水平的影响。
具体实施方式
本发明涉及以TP或β-链蛋白作为脂肪干细胞治疗缺血性疾病的靶标。通过降低、抑制或消除TP活性，或通过提高β-链蛋白活性，可提高脂肪来源的干细胞促血管新生的能力，或促进脂肪来源的干细胞向血管内皮细胞分化，从而提高脂肪来源的干细胞治疗缺血性疾病的疗效。
可采用本领域周知的技术来降低、抑制或消除TP活性，包括敲除脂肪来源的干细胞中表达TP的基因、使所示基因发生导致TP活性降低或丧失的突变、或使脂肪来源的干细胞与TP的拮抗剂接触。
例如，可通过CRISPR-Cas9或TALEN等技术，对体外的脂肪来源的干细胞进行TP定点敲除。也可对TP实施部分突变，从而使其活性降低或丧失。
或者，可采用本领域已知的TP拮抗剂来降低、抑制或消除TP活性。这些TP拮抗剂包括但不限于AH 29848、SQ-29548、AH-23848、GR-32191、ONO-11120、BM-13177、BM-13505、SKF-88046、L-655240、S-145、TMQ、R-68070、CV-4151和Bay-K 8644等，部分化合物的结构如下所示：
Figure PCTCN2016089295-appb-000001
Figure PCTCN2016089295-appb-000002
可参见，例如陈新生、万维勤，“血栓素A2及其受体拮抗剂”，《中国医药工业杂志》，1990年第21卷第2期，第88-93页；殷凯生、赵琳，“血栓素A2受体拮抗剂塞曲司特的研究进展”，《世界临床药物》，2005年01期。本文将这些文献的全部内容以引用的方式纳入本文。
提高β-链蛋白活性可通过以下方式实现：
(1)降低、抑制或消除所述干细胞的TP活性；
(2)降低、抑制或消除所述干细胞的钙蛋白酶的活性；和/或
(3)在所述干细胞中过表达β-链蛋白。
如前所述，可通过敲除脂肪来源的干细胞中表达TP的基因、使所示基因发生导致TP活性降低或丧失的突变、或使脂肪来源的干细胞与TP的拮抗剂接触来实现TP活性的降低、抑制或消除。
对于干细胞的钙蛋白酶的活性，可使其与钙蛋白酶的抑制剂接触，从而实现该钙蛋白酶的降低、抑制或消除。
可采用本领域已知的钙蛋白酶的抑制剂，包括但不限于N-乙酰基-L-亮氨酰-L-亮氨酰-L-正亮氨醛(ALLN)、氟甲基酮(Z-LLY-FMK)、钙蛋白酶抑素(Calpastatin)、α-酮基酰胺类抑制剂和calpeptin(CPT)。可参见，例如张勇，“α-酮基酰胺类Calpain抑制剂的设计、合成与活性研究”，博士论文，2008年，本文将其全部内容以引用的方式纳入本文。
作为一个例子，CPT的结构式如下所示：
Figure PCTCN2016089295-appb-000003
或者，可通过敲除或突变表达钙蛋白酶(Calpain)的基因，从而实现所述干细胞的 钙蛋白酶的降低、抑制或消除。可采用本领域周知的技术实现所述敲除或突变。突变通常是使钙蛋白酶的活性结构域发生变异，从而使其作为钙蛋白酶的活性降低或丧失；或者使其表达基因发生突变，使得其基因不表达或表达量降低。
或者，可采用例如siRNA技术降低表达钙蛋白酶或TP的基因的表达。
可通过构建含有β-链蛋白的质粒或者腺病毒载体，在体外转染脂肪来源的干细胞，进而使β-链蛋白过表达。可参见Sawant DA，Tharakan B，Hunter FA，Smythe WR，Childs EW，“Role ofβ-catenin in regulating microvascular endothelial cell hyperpermeability”，J Trauma，2011年2月，70(2):481-7。
应理解，当将脂肪来源的干细胞与各种拮抗剂或抑制剂接触时，拮抗剂或抑制剂的量可根据所选用的拮抗剂或抑制剂、脂肪来源的干细胞的量、抑制的效果等，并结合现有技术予以确定。
通过上述手段，可制备得到促血管新生的能力提高和/或向血管内皮细胞分化的能力提高的脂肪来源的干细胞。优选的是，所述脂肪来源的干细胞来自哺乳动物，尤其是人、鼠、兔子、猪、羊和狗等的脂肪组织，特别优选来自人的脂肪组织。所述脂肪来源的干细胞优选具有以下一个或多个特征：
(1)表达TP的基因被敲除或发生突变，导致TP活性降低或丧失；
(2)表达钙蛋白酶的基因被敲除或发生突变，导致钙蛋白酶活性降低或丧失；和
(3)含有表达β-链蛋白的表达载体，从而使β-链蛋白过表达。
由于其具有提高的促血管新生的能力和/或向血管内皮细胞分化的能力，本发明的脂肪来源的干细胞可用来治疗需要血管新生和/或需要血管内皮的病症，包括但不限于缺血引起的各种疾病和糖尿病。这类病症或疾病包括但不限于下肢缺血性疾病、脑血栓、心肌梗死、糖尿病、神经退行性疾病等。
因此，本发明包括TP拮抗剂和/或钙蛋白酶抑制剂在制备促进脂肪来源的干细胞向血管内皮细胞的分化能力和/或提高其血管新生能力的药物中的用途。
本发明还包括一种治疗或预防缺血性疾病的方法，所述方法包括将本发明的脂肪来源干细胞给予需要的对象。在一个具体实施例中，所述方法包括将本发明的脂肪来源干细胞与基质胶共培育，然后将含有本发明脂肪来源干细胞的基质胶给予(例如，注射至病患处)对象。注射时，可同时给予VEGF。或者，可直接将本发明的脂肪来源干细胞注射至对象的病患处。对象可以是哺乳动物，例如人、鼠、兔子、猪、羊和狗等。
本发明还包括体外提高脂肪来源的干细胞促血管新生的能力或促进脂肪来源的干细胞向血管内皮细胞分化的方法，所述方法包括提高所述干细胞的β-链蛋白的活性。
本发明还包括本发明脂肪来源的干细胞在制备治疗缺血性疾病用的药物中的用途。
本发明还包括体外由脂肪来源的干细胞制备血管内皮细胞或血管的方法，所述方法包括使本发明脂肪来源的干细胞与VEGF接触。接触时VEGF的浓度可由实际情况加以确定，这在本领域技术人员掌握的范围之内。
本发明还包括一种组合物，该组合物含有本发明的脂肪来源的干细胞。该组合物可以是一种药物组合物，除所述干细胞外还可含有合适的药学上可接受的载体。或者，该组合物可以是本发明脂肪来源干细胞的培养物，该培养物中除所述干细胞外，还可含有适合所述干细胞以不分化的状态增殖的培养基。这些培养基可以是本领域抑制的用于维持脂肪来源干细胞不分化繁殖的各种培养基。
以下将以具体实施例的方式描述本发明。应理解，这些实施例仅仅是阐述性的，并非限制本发明的保护范围。实施例中，除非另有说明，否则采用本领域常规技术手段实施各种方法、步骤和使用各种试剂。
实验材料与方法
动物
所有的小鼠都是C57BL/6遗传背景。红色或者绿色荧光蛋白的转基因小鼠与TP敲除小鼠交配产生红色或绿色荧光/TP敲除的小鼠。所有的小鼠在使用以及饲养中都遵循了中国科学院营养科学研究所实验动物管理委员会的章程。
试剂
Calpeptin，U46619，花生四烯酸购买于Cayman化学试剂公司(Cayman Chemical，Ann Arbor，MI，USA)。小鼠以及人的血管内皮生长因子(VEGF)和基本成纤维细胞生长因子(bFGF)购买于Peprotech(Peprotech Inc.，Rocky Hill，NJ，USA)。基质胶购买于BD生物公司(San Jose，CA，USA)。
脂肪来源干细胞(ADSCs)的分离和培养
人的ADSCs买自Cyagen生物公司(货号：HUXMD-01001)，用OriCell人的ADSC生长培养基(货号：HUXMD-90011)培养。小鼠的ADSCs分离自小鼠的附睾脂肪垫脂肪。简述如下：把脂肪组织剪成小块，用0.2％的胶原酶(Sigma-Aldrich，St.Louis，MO，USA)在37℃水浴中消化1小时；然后将消化液用100um筛子(BD Biosciences)过滤以 除去组织碎片；然后将细胞悬液用PBS稀释并离心以除去胶原酶。将离心下的细胞用160mM NH4Cl重悬，室温放置10min以除去红细胞。最后将所得细胞用Dulbecco’s modified Eagle’s medium/Ham’s F12(DMEM/F12；Invitrogen，Carlsbad，CA，USA)培养基加10％胎牛血清加1％青霉素/链霉素，在37℃5％CO2条件下培养。隔天换液。所有小鼠的ADSCs在第二代用于做实验。对于内皮分化实验，将200ul基质胶铺于12孔板的一个孔中，37℃孵育1小时；然后将1×105个ADSCs种于培养基中。
流式细胞分析
小鼠的ADSCs用BD流式细胞仪(FACScan，BD Biosciences)进行流式分析。简言之，收细胞后在含有一抗的含1％小牛血清的PBS中孵育30分钟。实验中用到的CD29，CD90，Sca-1，CD34和CD31购买于eBioscience(eBioscience，San Diego，CA，USA)。流式数据用FlowJo 8.3.3软件进行分析。
免疫荧光染色
染色前，将切片或者细胞爬片用冷丙固定，PBS洗去丙酮，然后用含0.25％Triton X-100的PBS透膜10分钟，然后用3％BSA/PBS封闭30分钟，之后一抗4℃孵育过夜。实验中用到的一抗包括小鼠的CD34(稀释倍数1:50；eBioscience)，CD31(稀释倍数1:50；eBioscience)，CD31(稀释倍数1:500；BD Biosciences)，GFP(稀释倍数1:1000；Abcam，Cambridge，MA，USA)，RFP(稀释倍数1:1000；Abcam)，and PCNA(稀释倍数1:1000；Cell Signaling Technology，Danvers，MA，USA)以及人的(稀释倍数1:200；R&D Systems，Minneapolis，MN，USA)。过夜后，PBS洗去多余抗体，二抗室温孵育1小时。本实验用到的二抗偶联的荧光包括Alexa Fluor 488，Alexa Fluor 594，or Alexa Fluor 633
(Invitrogen)。用含有DAPI的封片剂封片。最后用激光共聚焦显微镜(Carl Zeiss，
Oberkochen，德国)进行拍照，统计。
前列腺素的提取及分析
用30μmol/L的花生四烯酸刺激培养的ADSCs，30分钟后收上清，12000g，4℃，离心15分钟，提取前列腺素并用液相色谱-串联质谱法分析其含量。
成管实验
在12孔板每个孔中铺入200μL基质胶，37℃孵育1小时。然后每个孔种入2× 105ADSCs。细胞培养箱中培养12小时，倒置显微镜(IX51；Olympus，Center Valley，PA，USA)下观察拍照。
基质胶在体内的血管新生实验
用包含ADSCs(0.5×106)的100μL基质胶皮下注射到C57BL/6的小鼠侧面区域。人的ADSCs(1×106)与500ng/mL VEGF(Peprotech)和2μM SQ29548(Cayman Chemical)混合后加入100μL基质胶中，然后皮下注射到裸鼠侧面区域。两周后，取出，拍照，并进行形态学分析。
下肢缺血模型
对八周大的雄性小鼠的左下肢的股动脉近端和远端分别结扎，中间剪断以造成下肢缺血。一天后，在缺血下肢腓肠肌内注射106个ADSCs。7天和14天时用激光多普勒血流仪(LDI；Moor Instruments Ltd.，UK)检测血流恢复情况。
腺病毒
腺病毒是利用pAd-track-CMV GFP载体含有编码小鼠全长TPcDNA产生，，在293A病毒包装细胞系产生并扩增。经过几轮的重组腺病毒的扩增，氯化铯梯度离心纯化。通过测定绿色荧光蛋白的荧光来估计其感染效率。
RNA的提取和实时定量聚合酶链反应
采用Trizol试剂(Invitrogen)根据生产商提供方法提取细胞总RNA。将提取的RNA用反转录试剂盒(Takara公司，大连，中国)反转录为cDNA。实时定量PCR用SYBR Green(Takara)混合液。用肌动蛋白的表达水平对靶基因的表达水平进行规范。PCR程序如下：95℃，5分钟为一个周期，随后的40个周期在95℃30秒，60℃30秒，72℃30秒，最后一次延长72℃5分钟。通过溶解曲线获得每个PCR产物的循环数。
RNA干扰实验
使用RNAiFect转染试剂(Qiagen，Crawley，UK)根据生产商提供的方法对ADSCs进行转染，包括特定的序列β-catenin(100nM)或对照无序siRNA(Genepharma，上海，中国)。一微克的siRNA转染试剂和RNAiFect混合室温孵育10到15分钟，逐滴加入到培养的细胞中，48小时后通过免疫印迹分析转染效率。
激光共聚焦的钙成像
用激光扫描共聚焦显微镜(Carl Zeiss，Inc，德国)对ADSCs钙瞬变进行记录。简单说，对细胞加载6μg/ml Fluo-3或Rhod 4后，在37℃放置30分钟。经HBSS洗涤后，Fluo-3在488nm激发，Rhod 4在594nm激发。用线性扫描(X-T)512每行像素的速度对图像进行扫描。通过1μM U46619与多路快速应用系统来评价内质网上的Ca2+负载。
免疫共沉淀
用携带空载体或TP cDNA腺病毒感染ADSCs。用含有5μL HA标签抗体或正常IgG(Cell Signaling Technology)免疫共沉淀缓冲液与1毫克全细胞裂解物制混合，在4℃孵育3小时，随后与蛋白A/G琼脂糖(Invitrogen)在4℃孵育并轻轻搅拌过夜。充分洗涤后，对免疫复合物进行煮沸变性，免疫印迹分析HA标记的抗体或抗Gq/11蛋白抗体(Santa Cruz Biotechnology，Santa Cruz，CA，USA)。
免疫印迹实验
用含有蛋白酶抑制剂的裂解液对ADSCs提取蛋白。通过使用Piece BCA蛋白浓度测定试剂盒的BCA法测定细胞总蛋白(Pierce，Rockford，IL，USA)。将等量的蛋白质变性，用10％浓度由十二烷基硫酸钠-聚丙烯酰胺凝胶(SDS-PAGE)电泳，之后转移到硝酸纤维素膜上，5％脱脂牛奶封闭1小时，然后与一抗在4℃孵育过夜。本实验用到的一抗包括：抗小鼠actin(1:2000；Sigma-Aldrich)，抗-c-myc(1:1000；geneTex)，抗-cyclin D1(1:1000；Abcam)，抗-CD31(1:500；Abgent)，anti-HA-tag，抗-m-calpain，抗-μ-calpain，抗-VE-cadherin，抗-phospho-GSK3βS9，抗-total-GSK3β,抗-phospho-β-cateninS33(1:1000；Cell Signaling Technology)，抗-C-terminalβ-catenin(1:1000；BD Biosciences)，抗-N-terminalβ-catenin(1:1000；Abcam)，and抗-β-catenin(1:1000；Millipore)。之后对膜用辣根过氧化物酶标记的二抗室温孵育2小时。然后用增强型发光试剂(Piece)对膜进行显色。
数据分析
结果用平均值±标准误差(SEM)表示。采用16.0版本SPSS软件(SPSS Inc.，Chicago，IL，USA)对数据进行统计学分析，用t检验或方差分析。分析中小于0.05的P值认为有统计学意义。
结果
1、TXA2和TP信号在ADSC向内皮分化的过程中被活化
图1显示，ADSC具有干性标记，并且较高表达前列腺素受体TP。在VEGF的诱导过程中，前列腺素的上游酶系COX1，2和TXAS都上调，分泌的TXB2增加，且前列腺素合成酶TxAS以及受体TP表达上调，说明TXA2/TP轴在ADSC向内皮分化过程中是活化的。
2、敲除TP加速ADSC向内皮细胞的分化
由图2可以看出，与WT小鼠来源的ADSC相比，TP敲除使ADSC在诱导过程中的内皮细胞标记CD31和CD34都表达上升；内皮相关的标记Tie2，CD31等在mRNA水平也明显升高；流式细胞术检测表明成熟内皮细胞比例显著增加；成管能力增强；促进血管生长因子VEGF-A和bFGF的mRNA水平明显升高；体内的基质胶里的三维成管实验也表明TP敲除后有更强的血管新生能力。
3、TP敲除使ADSC治疗下肢缺血的效率提高
图3表明，与WT小鼠来源的ADSC相比，TP敲除小鼠的ADSC在治疗小鼠下肢缺血7天和14天时血流灌注程度明显增加。免疫荧光显示，TP敲除ADSC治疗后，缺血肌肉内皮细胞明显增加；使用RFP来源的ADSC追踪显示，来源于外源ADSC的CD31+内皮细胞以及增殖的内皮细胞(PCNA+，CD31+)明显增加，说明TP敲除后促进ADSC血管新生，主要是通过诱导ADSC分化为内皮细胞实现的。
4、β-catenin升高是TP敲除促进ADSC向血管内皮分化的关键因素
图4结果表明，与WT相比，TP敲除使得ADSC胞内β-catenin水平增高，其下游靶分子c-myc水平增高，CD31水平增加；TP敲除后β-catenin的目的基因在mRNA水平明显增高；通过腺病毒转染，过表达TP使上述表型丧失；通过siRNA敲低β-catenin则可阻止ADSC向内皮细胞分化相关分子的表达，促进血管生长因子VEGF-A和bFGF的mRNA水平下降。
5、敲低β-catenin可以降低ADSC血管新生能力
图5表明，通过siRNA敲低β-catenin可降低ADSC的成管能力；降低ADSC在基 质胶里的三维成管能力；免疫荧光显示，敲低β-catenin使得来自于外源ADSC在血管构成的过程中的作用明显减弱，内皮分化能力下降。
6、敲低β-catenin可以降低ADSC治疗下肢缺血的效果
图6表明，敲低β-catenin后，TP敲除的ADSC的治疗效果显著下降，14天时的血流灌注不再恢复；免疫荧光结果显示，缺血区内皮细胞明显减少，其中来自外源ADSC的细胞分化为内皮细胞(RFP+，CD31+)以及有增殖能力的内皮细胞(PCNA+，CD31+)的比例明显下降。
7、calpain活性下降使得ADSC血管新生能力增强
图7显示，通过使用C端的抗体，检测到β-catenin的不同条带，过表达TP使得降解增加；同时发现，TP敲除后依赖钙离子的蛋白水解酶calpain(钙蛋白酶)的活性明显下降，回补TP后，calpain活性上升；使用calpain抑制剂CPT后，TP回补可以减少β-catenin的降解以及促进内皮相关蛋白表达；同时ADSC在基质胶里的三维成管能力增强，内皮分化能力增强，促血管生长因子VEGF-A和bFGF的mRNA水平升高。
8、TP敲除导致胞内钙浓度下降
图8表明，TP敲除后胞内钙离子浓度下降，细胞钙瞬流在TP激动剂刺激下丧失；过表达TP钙瞬流恢复；免疫共沉淀结果显示TP确实可以和Gq/11相互作用，用PLC的抑制剂U-73122可以明显影响TP-介导的有关分子的蛋白表达变化，改善由于TP过表达造成的成管能力下降；因此，TP在ADSC中是通过Gq引发钙内流，活化calpain后，介导对β-catenin的调控，进而影响ADSC向内皮细胞的分化。
9、抑制TP可以改善人的ADSC的血管新生能力
图9表明，用TP的抑制剂SQ2958处理人的ADSC，其向内皮细胞分化的能力增强；成管能力增强；体内基质胶的三维实验表明，TP抑制剂处理后人的ADSC的血管新生能力增强；内皮分化的能力增强；促血管生长因子VEGF-A和bFGF的mRNA水平升高。

Claims (10)

  1. 一种脂肪来源的干细胞或含该干细胞的组合物，其特征在于，所述干细胞：
    (1)TP失活或其活性受到抑制；和/或
    (2)钙蛋白酶活性失活或降低；和/或
    (3)β-链蛋白活性提高。
  2. 如权利要求1所述的脂肪来源的干细胞或含该干细胞的组合物，其特征在于，所述干细胞：
    (1)表达TP的基因被敲除或发生导致TP活性降低或丧失的突变；和/或
    (2)表达钙蛋白酶的基因被敲除或发生导致钙蛋白酶活性降低或丧失突变；和/或
    (3)含有表达β-链蛋白的表达载体，从而β-链蛋白过表达。
  3. 如权利要求1或2所述的脂肪来源的干细胞或含该干细胞的组合物，其特征在于，所述干细胞来自哺乳动物如人、鼠、兔、猪、羊和狗等的脂肪组织。
  4. 一种体外提高脂肪来源的干细胞促血管新生的能力或促进脂肪来源的干细胞向血管内皮细胞分化的方法，所述方法包括提高所述干细胞的β-链蛋白的活性。
  5. 如权利要求4所述的方法，其特征在于，所述提高所述干细胞的β-链蛋白的活性包括：
    (1)降低、抑制或消除所述干细胞的TP活性；和/或
    (2)降低、抑制或消除所述干细胞的钙蛋白酶的活性；和/或
    (3)过表达所述干细胞中的β-链蛋白。
  6. 如权利要求5所述的方法，其特征在于，
    (1)降低、抑制或消除所述干细胞的TP活性包括：敲除所述干细胞中表达TP的基因，或使所述干细胞中表达TP的基因发生突变，或使所述干细胞与TP拮抗剂接触；和
    (2)降低、抑制或消除所述干细胞的钙蛋白酶活性包括：敲除所述干细胞中表达钙蛋白酶的基因，或使所述干细胞中表达钙蛋白酶的基因发生突变，或使所述干细胞与钙蛋 白酶抑制剂接触。
  7. 如权利要求6所述的方法，其特征在于，
    (1)所述TP拮抗剂选自AH 29848、SQ-29548、AH-23848、GR-32191、ONO-11120、BM-13177、BM-13505、SKF-88046、L-655240、S-145、TMQ、R-68070、CV-4151和Bay-K8644；和
    (2)所述钙蛋白酶抑制剂选自：N-乙酰基-L-亮氨酰-L-亮氨酰-L-正亮氨醛(ALLN)、氟甲基酮(Z-LLY-FMK)、钙蛋白酶抑素(Calpastatin)、α-酮基酰胺类抑制剂和Calpeptin(CPT)。
  8. TP拮抗剂和/或钙蛋白酶抑制剂在制备提高脂肪来源的干细胞向血管内皮细胞的分化能力和/或提高其血管新生能力的药物中的用途。
  9. 权利要求1－3所述脂肪来源的干细胞或含该干细胞的组合物或采用权利要求4－7中任一项所述的方法获得的脂肪来源的干细胞在制备治疗糖尿病和缺血性疾病如下肢缺血性疾病、脑血栓、心肌梗死、和神经退行性疾病用的药物中的用途。
  10. 一种体外由脂肪来源的干细胞制备血管内皮细胞或血管的方法，所述方法包括使权利要求1－3所述脂肪来源的干细胞或含该干细胞的组合物或采用权利要求4－7中任一项所述的方法获得的脂肪来源的干细胞与VEGF接触。
PCT/CN2016/089295 2015-07-10 2016-07-08 血栓烷素a2受体作为脂肪干细胞治疗缺血性疾病的靶标 WO2017008686A1 (zh)

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Citations (1)

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WO2006127007A2 (en) * 2005-05-25 2006-11-30 Cytori Therapeutics, Inc. Methods of using adipose tissue-derived cells in the treatment of cardiovascular conditions

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WO2006127007A2 (en) * 2005-05-25 2006-11-30 Cytori Therapeutics, Inc. Methods of using adipose tissue-derived cells in the treatment of cardiovascular conditions

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