CN107349419A - A kind of cell composition and its preparation method and application - Google Patents

A kind of cell composition and its preparation method and application Download PDF

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Publication number
CN107349419A
CN107349419A CN201710581984.0A CN201710581984A CN107349419A CN 107349419 A CN107349419 A CN 107349419A CN 201710581984 A CN201710581984 A CN 201710581984A CN 107349419 A CN107349419 A CN 107349419A
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cell
cell composition
cells
intestinal
stem cell
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马颖
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Guangdong Value Technology Co Ltd
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Guangdong Value Technology Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • 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
    • 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
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
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  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Cell Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Virology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Dermatology (AREA)
  • Hematology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The application belongs to field of pharmaceutical preparations, and in particular to a kind of cell composition and its preparation method and application.Cell composition provided by the present invention includes regulatory T cells, intestinal stem cell and IL 33, competent cell is used as using regulatory T cells and intestinal stem cell, realization can also adjust immune system while Intestinal Mucosal Injury in Patients Undergoing inflammation is repaired and disinthibite itself excessive immune, prevent that sb.'s illness took a turn for the worse, be widely used in cd patient；Cell composition of the present invention each component synergy, collectively promotes the reparation that Crohn disease suffers from intestinal inflammatory, therapeutic effect is notable added with cell factors such as IL 33.

Description

A kind of cell composition and its preparation method and application
Technical field
The invention belongs to field of pharmaceutical preparations, and in particular to a kind of cell composition and its preparation method and application.
Background technology
Crohn disease is a kind of not clear bowl inflammatory diseases of reason, as the main Types of IBD, is had The course of disease is long, easily recurrence, can invade and any position of intestines and stomach and the features such as be not easy to effect a radical cure.Crohn disease is a kind of gastral slow Property, recurrent exerbation and nonspecific transmural inflammation, lesion is distributed in segmental, can involve any position of alimentary canal, wherein Most commonly seen with terminal ileum, colon and anus lesion are also more.The treatment of Crohn disease at present mainly uses glucocorticoid With immunodepressant as medicine, the method can only play a part of alleviating the state of an illness, can not effectively prevent the development of the state of an illness, Crohn disease can not be effected a radical cure；And it can be induced such as osteoporosis, hypertension, hyperglycaemia using glucocorticoid medicine for a long time With the complication such as central obesity, while the long-term use of immunodepressant can also cause body immune system unbalance.
The content of the invention
In view of this, it is an object of the invention to provide a kind of cell composition suitable for cd patient, it is used for Repair intestinal inflammatory and suppress sb.'s illness took a turn for the worse.Its concrete technical scheme is as follows：
The invention provides a kind of cell composition, including：Regulatory T cells, intestinal stem cell and IL-33.
Preferably, the cell density of the regulatory T cells is 1 × 107~5 × 107Individual/mL.
Preferably, the cell density of the intestinal stem cell is 5 × 106~1 × 107Individual/mL.
Preferably, the working concentration of the IL-33 is 10~50ng/mL.
Preferably, the intestinal stem cell is the third generation to the 5th generation intestinal stem cell.
Preferably, the formulation of the cell composition is ejection preparation.
It is furthermore preferred that the solvent of the ejection preparation is physiological saline.
Present invention also offers a kind of preparation method of above-mentioned cell composition, by regulatory T cells, intestinal stem cell and IL-33 is mixed, and obtains the cell composition.
Present invention also offers the cell composition that above-mentioned cell composition or above-mentioned preparation method obtain to prepare Crow Application in grace medicine.
In summary, cell composition provided by the present invention includes regulatory T cells, intestinal stem cell and IL-33, Using regulatory T cells and intestinal stem cell as competent cell, realize to adjust while Intestinal Mucosal Injury in Patients Undergoing inflammation is repaired and exempt from Epidemic disease system is disinthibited itself excessive immune, is prevented that sb.'s illness took a turn for the worse, is widely used in cd patient；Cell composition of the present invention Added with cell factor IL-33, each component Synergistic, the reparation that Crohn disease suffers from intestinal inflammatory, therapeutic effect are collectively promoted Significantly.
Compared with prior art, the present invention has advantages below：
1) intestinal stem cell is included in cell composition of the present invention, these cells can directly be differentiated to form new enteron aisle group Knit, replace and repair the tissue of damage, inherently treated；
2) regulatory T cells, intestinal stem cell and the IL-33 in cell composition of the present invention are used to adjust sufferer itself Immune system, avoid because autoimmunity excessively fails, the effect of ensureing cell composition of the present invention；
3) cell composition of the present invention is preferably ejection preparation, using physiological saline as solvent, physiology salt water and human body The osmotic pressure of tissue always, avoids producing damage to sufferer intestinal tissue；
4) cell composition of the present invention is Cord blood preparation, can both avoid cell factor from inactivating, and can is kept well The bioactivity of intestinal stem cell and regulatory T cells, curative effect are stable.
Embodiment
In order to provide a kind of cell composition suitable for cd patient, for repairing intestinal inflammatory and suppressing the state of an illness Deteriorate.The invention provides a kind of cell composition, including：Regulatory T cells (Treg cells), intestinal stem cell and IL-33.
Though Crohn disease is benign disease, still lack highly effective treatment means, and its specific morbidity machine so far System is not fully understood, at present what was certain was that body's immunity disorder be the main reason for causing its occurrence and development it One.The technological invention mechanism of Crohn disease is believed that Crohn disease is by Th1 cells and the activation of Th17 cell transitions from immunology angle And activation, cause the intestines problem with the characteristics of Treg cells shortage.
Intestinal stem cell is predominantly located in intestinal mucosa epithelium Lieberkunn's crypts, above paneth's cell, is possessed dry thin The essential characteristic of the lifelong self-renewing of born of the same parents and differentiation.After by the signal function of extraneous physiology or pathology, intestinal stem cell Asymmetric division is carried out, a primary stem cell is formed and one has been broken up committed progenitor, committed progenitor can further divide The terminally differentiated cells with special construction and function is turned to, such as：Absorb cell, goblet cell, paneth's cell, M cells, interior point Cell etc. is secreted, repairs and rebuilds small intestinal mucosa epithelial barrier and digestion and absorption function, this potential of intestinal stem cell is to enteron aisle Reparation after damage is very crucial.At present, domestic intestines problem is common, the structure and work(of muscle layer under intestinal mucosa, or even mucous membrane It can be destroyed, it is divided into specific cell by stem cells technology and tissue engineering technique directional induction intestinal stem cell With the cell or tissue defect of alternative functions obstacle, this is probably a kind of potential effective ways for treating Crohn disease.This hair Intestinal stem cell is included in clear-cells composition, these cells can directly be differentiated to form new intestinal tissue, replace and repair damage The tissue of wound, is inherently treated.
In order to avoid sufferer because autoimmunity excessively influences intestinal stem cell and played a role, ensure the effect of its is stable, The immune system of regulatory T cells and IL-33 for adjusting sufferer itself is also added in the present composition.
Treg cells are a kind of regulatory T cells (Regulatery T cell), and inhibitory action is played to immunity of organism, The immune response harmful to itself or exogenous antigen can be suppressed.Treg expresses CD4, CD25 and transcription factor FoxP3.
The present invention does not make special restriction to regulatory T cells, intestinal stem cell and IL-33 source, using this area Known to technical staff, such as commercial source；Or use conventional technical means system well known to those skilled in the art Standby obtained regulatory T cells, intestinal stem cell and IL-33.
Below in conjunction with the embodiment of the present invention, technical scheme is clearly and completely described, it is clear that institute The embodiment of description is only part of the embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, The every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made, belongs to this hair The scope of bright protection.
Infliximab and immunodepressant in following examples derive from affiliated hospital of Nanfang Medical Univ capital Small stream Nanfang Hospital.ELISA Kit are bought in green skies bio tech ltd.
Embodiment 1
1st, the separation and Extraction of intestinal stem cell, reference：Leaf Zhang Qun, Liu Guanlin, the structure of Chen Zhiqiang Murine Intestinal Stem Cell Populations Build《Central China University of Science and Technology's journal (medicine)》[J], 2006.
The intestinal stem cell in P2 generation to P5 generations is taken, sucks culture medium, adds 0.25% pancreatin conventional digestion, is then made thin Born of the same parents' density is 1 × 105Individual/mL cell suspension, the 5 anti-Lgr5 of μ L monoclonal antibody is then added to 500 μ L cell suspension In, then lucifuge is incubated 20min at room temperature, while sets up blank Isotype control；1500r/min is centrifuged after incubation 5min, supernatant is abandoned, then washed 2 times with the PBS containing 10%FBS, then machine testing is gone up after being resuspended with 500 μ L PBS.
The surface antigen testing result of intestinal stem cell is as shown in the following Table 1：
Table 1
Lrg5+
Detect for the first time 95.6%
Second of detection 98.4%
Third time detects 96.4%
2nd, the culture of regulatory T cells, reference：Derived from cord blood Treg cells after Ma little Qian, Li Sang, Wang Wei amplification in vitros Effectiveness and reliability research《Life science》[J],2016,20(4):333-339.
Treg cells are taken, are made 1 × 106Individual/mL cell suspension, anti-human CD25, CD4, CD45RO Dan Ke are taken respectively Grand each 5 μ L of antibody, add in 500 μ L cell suspensions, lucifuge is incubated 20min at room temperature, while sets up blank Isotype control；It is incubated After 1500r/min centrifugation 5min, abandon supernatant, then wash 2 times with the PBS containing 10%FBS, then the PBS resuspensions with 500 μ L Upper machine testing afterwards.
The surface antigen testing result of Treg cells is as shown in table 2：
Table 2
CD25+CD4+ CD45RO+
Detect for the first time 94.1% 96.8%
Second of detection 93.7% 95.8%
Third time detects 95.8% 96.5%
3rd, according to preparation tri- kinds of cell compositions of A, B and C respectively are formulated described in table 3, its specific preparation process is：Take enteron aisle Stem cell and regulatory T cells, mixed cell suspension is made as solvent using physiological saline, then sequentially adds IL-33.
Table 3
Embodiment 2
1st, the mouse of SPF levels similar in 90 body weight is randomly selected, mouse is divided into 6 groups, every group 15 (male and female half and half)； Then, TNB (2,4,6-Trinitrobenzensulfonic Acid, TNBS) bowel lavage, system are carried out to each mouse Make mouse CD models；Then, enter end of line by table 4 to each model mice to be administered along intravenous injection, inject weekly once, continuous injection 4 times, such conventinal breeding carries out dislocation execution to mouse after 1 month, detects indices.
Crow grace mouse modeling bibliography：Liu Shun, Sun Keming, Lai rapamycins are to Crohn disease mouse model after poplar Therapeutic action and Mechanism Study《Place of china disease magazine》[J].2016.
The injecting and administering preparations of table 4
Group Ejection preparation
Blank control group Physiological saline
Control group 1 Infliximab
Control group 2 Immunodepressant
Experimental group 1 Cell composition A
Experimental group 2 Cell composition B
Experimental group 3 Cell composition C
2nd, mouse Colon and rectum intestinal tube HE is dyed
Fixed 48h is soaked with 4% formalin to the rectal tissue of acquisition, rear drying, fixed embedding, paraffin is made and cuts Piece, final to carry out HE dyeing, micro- sem observation simultaneously carries out histological scores.Table 5 is mouse intestinal histopathological scores result, As shown in the results, the pathology average score of blank control group is 5.94, higher than control group 1, control group 2 and experimental group 1~3, Illustrate that this five groups of ejection preparations of control group 1, control group 2 and experimental group 1~3 can play a part for the treatment of Crohn disease.Control The pathology average score of group 1 and 2 is respectively 3.68,4.07, compared with blank control group, P<0.05, there is significant difference；It is real The pathology average score for testing group 1~3 is respectively 2.83,2.76,2.79, compared with blank control group, P<0.01, have extremely notable Sex differernce.
The mouse intestinal histopathological scores (χ ± s) of table 5
* P is represented<0.05, compared with blank control group, there is significant difference；
# represents P<0.01, compared with blank control group, there is pole significant difference.
3rd, cytokines measurement
The blood of mouse is taken in anticoagulant tube, 2500rpm centrifugation 15min, blood plasma is transferred in new preservation pipe, is placed in It is standby at 4 DEG C.According to ELISA Kit operational manuals detection IFN-r, IL-17 and OPN level (three declines).
Table 6 is the testing result of cell factor, and as shown in the results, INF-r, IL-17, OPN of blank control group are respectively 7.68pg/mL, 15.67pg/mL, 56942pg/mL, control group 1, control group 2 and experimental group 1~3 are above, illustrate this five groups Ejection preparation can play a part for the treatment of Crohn disease.Three kinds of factors of control group 1 and 2 are compared with blank control group, P< 0.05, there is significant difference；Three kinds of factors of experimental group 1~3 are compared with blank control group, P<0.01, there is pole conspicuousness Difference.
The cytokines measurement result of table 6
Group Number of elements INF-r(pg/mL) IL-17(pg/mL) OPN(pg/mL)
Blank control group 15 7.68±0.43 15.67±0.5 56942±673
Control group 1 15 6.17±0.25* 13.49±0.3* 40253±871*
Control group 2 15 5.42±0.54* 12.71±0.4* 43849±587*
Experimental group 1 15 3.56±0.33# 19.69±0.4# 29519±608#
Experimental group 2 15 3.63±0.27# 21.02±0.6# 31157±594#
Experimental group 3 15 3.59±0.22# 19.83±0.6# 30468±598#
* P is represented<0.05, compared with blank control group, there is significant difference；
# represents P<0.01, compared with blank control group, there is pole significant difference.

Claims (9)

  1. A kind of 1. cell composition, it is characterised in that including：Regulatory T cells, intestinal stem cell and IL-33.
  2. 2. cell composition according to claim 1, it is characterised in that the cell density of the regulatory T cells be 1 × 107~5 × 107Individual/mL.
  3. 3. cell composition according to claim 1, it is characterised in that the cell density of the intestinal stem cell be 5 × 106~1 × 107Individual/mL.
  4. 4. cell composition according to claim 1, it is characterised in that the working concentration of the IL-1 is 10~50ng/ mL。
  5. 5. cell composition according to claim 1, it is characterised in that the intestinal stem cell is the third generation to the 5th generation Intestinal stem cell.
  6. 6. cell composition according to claim 1, it is characterised in that the formulation of the cell composition is made for injection Agent.
  7. 7. cell composition according to claim 6, it is characterised in that the solvent of the ejection preparation is physiological saline.
  8. 8. the preparation method of cell composition described in a kind of claim 1 to 7 any one, it is characterised in that regulatory T is thin Born of the same parents, intestinal stem cell and IL-33 mixing, obtain the cell composition.
  9. 9. the groups of cells that preparation method described in the cell composition or claim 8 described in claim 1 to 7 any one obtains Application of the compound in Crohn disease medicine is prepared.
CN201710581984.0A 2017-07-17 2017-07-17 A kind of cell composition and its preparation method and application Pending CN107349419A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102549147A (en) * 2009-07-09 2012-07-04 塞拉瑞克斯公司 Methods and compositions for use in cellular therapies
CN103608452A (en) * 2011-03-25 2014-02-26 特克赛尔公司 Method for using regulatory T cells in therapy

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102549147A (en) * 2009-07-09 2012-07-04 塞拉瑞克斯公司 Methods and compositions for use in cellular therapies
CN103608452A (en) * 2011-03-25 2014-02-26 特克赛尔公司 Method for using regulatory T cells in therapy

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KREISEL W等: "Complete remission of Crohn"s disease after high-dose cyclophosphamide and autologous stem cell transplantation", 《BONE MARROW TRANSPLANT》 *
孙科明等: "IL-33 对克罗恩病小鼠肠组织细胞因子及转录因子的影响", 《山东医药》 *
无: "科学家们将肠道干细胞诱导分化成为不同类型的成熟肠道细胞", 《生物医学工程与临床》 *

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Application publication date: 20171117