CN106109497A - The stem cell medicine for the treatment of liver cirrhosis - Google Patents
The stem cell medicine for the treatment of liver cirrhosis Download PDFInfo
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- CN106109497A CN106109497A CN201610586793.9A CN201610586793A CN106109497A CN 106109497 A CN106109497 A CN 106109497A CN 201610586793 A CN201610586793 A CN 201610586793A CN 106109497 A CN106109497 A CN 106109497A
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- stem cell
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- liver cirrhosis
- oxymatrine
- cell medicine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
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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]
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Abstract
The invention discloses a kind of stem cell medicine treating liver cirrhosis, wherein, the stem cell medicine of described treatment liver cirrhosis includes: mescenchymal stem cell, IL 18 associated proteins, oxymatrine and solvent, and wherein, the concentration of described mescenchymal stem cell is (5～10) × 106Individual/ml；The described protein-bonded concentration of IL 18 is 0.1～1mg/L；The concentration of described oxymatrine is 10～100mg/L.The present invention treats the stem cell medicine of liver cirrhosis can effectively alleviate inflammation degree, reverses hepatic fibrosis, repairs liver organization, promotes liver function recovery, and effectively suppression hepatic fibrosis is formed.
Description
Technical field
The present invention relates to stem cell and tissue engineering technique field, particularly relate to a kind of stem cell system treating liver cirrhosis
Agent.
Background technology
Liver cirrhosis is a kind of thorny hepatic disease.Although liver transplantation is effective Therapeutic Method of end-age cirrhosis, but this
The prognosis of a little patients is the most very poor；And there is the problems such as donor shortage, surgical injury, immunologic rejection and somewhat expensive in liver transplantation.
Hepatocyte transplantation is considered as one of alternative method of organ transplantation, can not only maintain the liver function of the patient waiting liver transplantation temporarily
Can, and some liver metabolism disease can also be treated.But hepatocyte is a kind of akinete, the hepatocyte growth quantity of transplanting
Few, it is difficult to reach the effect of clinical treatment, and hepatocyte is difficult to cultivate and preserve.
Summary of the invention
Present invention is primarily targeted at a kind of stem cell medicine treating liver cirrhosis of offer, it is desirable to provide mesenchyme is dry thin
Born of the same parents and repair hardening hepatic tissue and suppression hepatic fibrosis.
For achieving the above object, the present invention provides a kind of stem cell medicine treating liver cirrhosis, described stem cell medicine bag
Include: mescenchymal stem cell, IL-18BP, oxymatrine and solvent, wherein, the concentration of described mescenchymal stem cell
For (5～10) × 106Individual/ml；The concentration of described IL-18BP is 0.1～1mg/L；The concentration of described oxymatrine
It is 10～100mg/L.
Preferably, the concentration of described mescenchymal stem cell is 7.5 × 106Individual/ml.
Preferably, the concentration of described IL-18BP is 0.5mg/l.
Preferably, the concentration of described oxymatrine is 50mg/l.
Preferably, described mescenchymal stem cell is mesenchymal stem cells MSCs.
Preferably, described solvent is water.
Technical solution of the present invention, by by mescenchymal stem cell, IL-18BP, oxymatrine and solvent altogether
Being blended to obtain stem cell medicine, this stem cell medicine can effectively alleviate inflammation degree, reverses hepatic fibrosis, repairs liver group
Knit, promote liver function recovery, and suppression hepatic fibrosis is formed, and then liver cirrhosis can be treated.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
In having technology to describe, the required accompanying drawing used is briefly described, it should be apparent that, the accompanying drawing in describing below is only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, it is also possible to
Other accompanying drawing is obtained according to the content shown in these accompanying drawings.
Fig. 1 is the form schematic diagram of the original cuiture 3d of the human marrow mesenchymal stem cell of the embodiment of the present invention 1 preparation；
Fig. 2 is the form schematic diagram of the original cuiture 10d of the human marrow mesenchymal stem cell of the embodiment of the present invention 1 preparation；
Fig. 3 is the 3rd generation human marrow mesenchyme stem cell surface molecular CD29-PE/CD34-of the embodiment of the present invention 1 preparation
The flow cytomery cartogram of the expression of FITC；
Fig. 4 is the 3rd generation human marrow mesenchyme stem cell surface molecular CD44-PE/CD45-of the embodiment of the present invention 1 preparation
The flow cytomery cartogram of the expression of FITC.
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Describe, it is clear that described embodiment is only a part of embodiment of the present invention rather than whole embodiments wholely.Base
Embodiment in the present invention, those of ordinary skill in the art obtained under not making creative work premise all its
His embodiment, broadly falls into the scope of protection of the invention.
The present invention provides a kind of stem cell medicine treating liver cirrhosis, and this stem cell medicine includes that raw material is as follows:
Mescenchymal stem cell (mesenchymal stem cells, MSCs), IL-18BP, oxymatrine with
And solvent.After mixing, in this stem cell medicine, the concentration of this mescenchymal stem cell is (5～10) × 106Individual/ml；This IL-
18 protein-bonded concentration are 0.1～1mg/L；The concentration of this oxymatrine is 10～100mg/L.In other words, 1L's
In stem cell medicine, this mescenchymal stem cell has (5～10) × 109Individual, the consumption of this IL-18BP is 0.1～1mg,
The consumption of this oxymatrine is 10～100mg.
The present invention treats the stem cell medicine of liver cirrhosis can effectively alleviate inflammation degree, reverses hepatic fibrosis, repaiies
The liver organization of multiple hardening, promotes liver function recovery, and suppression hepatic fibrosis is formed, and then can treat liver cirrhosis；Wherein,
MSCs is by the side of macrophage phenotype conversion in immunomodulating such as secrete cytokines (IL-4, IL-10) induced damage tissue
Formula, suppresses hepatic fibrosis, repairs hepatic tissue, and can alleviate the acute injury of multiple organ simultaneously；IL-18BP is (white
Cytokine-18 associated proteins) can affect liver microenvironment by its correlation molecule mechanism, it is possible to the propagation of promotion MSCs or
Person breaks up, thus improves curative effect；Oxymatrine participates in or has blocked the immune response of T cell in virus function,
Thus play it to viral hepatic fibrosis (hepatic fibrosis, HF) therapeutic effect.
It should be noted that this mescenchymal stem cell is derived from, growth is the most mesoblastic has height self renewal energy
Power and the pluripotent stem cell of multi-lineage potential, be widely present in the Various Tissues such as umbilical cord, bone marrow, can cultivate expansion in vitro
Increase, and hepatocyte, neurocyte, osteoblast, chondrocyte, muscle cell, adipose cell can be divided under given conditions
Deng；Specifically, mescenchymal stem cell of the present invention can derive from umbilical cord, bone marrow, umbilical blood, whole body connective tissue or organ interstitial.
As long as this solvent meets the medium of the survival filling stem cell between offer, can be specifically culture medium, buffer or water etc.,
Consumption as solvent can be allocated according to the actual requirements, is the Normal practice of those skilled in the art, does not do superfluous at this
State.
Now by embodiment, the present invention treats the stem cell medicine of liver cirrhosis to be further explained, to describe its skill in detail
Art scheme and the technique effect brought.
Embodiment 1
One, the preparation of mescenchymal stem cell
Material source: human marrow mesenchymal stem cell derives from 61 years old Operation on Esophageal Cancer male patient of 1 example, by Shenzhen
One the People's Hospital cardiothoracic surgery provides, patient for treatment and test equal informed consent, and experimental program is through hospital Medical Ethics committee member
Can ratify.
The separation of human marrow mesenchymal stem cell and cultivation: obtain bone marrow 2mL from the rib of esophageal carcinoma patient with operation excision,
After anticoagulant heparin, move into the centrifuge tube upper strata containing same volume Percoll lymphocyte separation medium, 2000r/min density gradient
Centrifugal 10min, takes middle mononuclear cell layer, rinses 2 times with PBS, adds the DMEM containing the hyclone that volume fraction is 20%
Culture medium, adjusting cell concentration is 2 × 108L-1, it is inoculated in 25cm2In culture bottle, 37 DEG C, volume fraction be the CO of 5%2Training
Supporting in case and cultivate, change liquid first, discard non-attached cell after 3d, the most every couple of days changes liquid 1 time, under inverted phase contrast microscope
Day by day observation of cell form.When cell reaches 80%～90% fusion, 2.5g/L pancreatin conventional digestion, is passed on by 1:2, passage cell
The next day change liquid first, change liquid 1 time every 3d later, when cell grow to 80%～90% fusion time continue Secondary Culture.
The detection of human marrow mesenchymal stem cell
Morphological observation: observe the shape of the primary generation human marrow mesenchyme stem cell of In vitro culture under inverted phase contrast microscope
State feature, as shown in Figures 1 and 2.According to Fig. 1 and Fig. 2: the i.e. visible more cell attachment of primary inoculation 3d, outward appearance in
Spindle, spindle shape and polygon etc., be shown in Fig. 1；4d cell quantity starts to increase, and breeds cloning colony mode, cellular morphology
In spindle shape, cell arrangement becomes swirling, and iuntercellular boundary is unclear, at the bottom of 10～14d are paved with bottle, sees Fig. 2.Still protect after passage
Holding spindle shape, growth cycle relatively primary cell shortens, and within six or seven days, the most again merges.
Cell surface molecule flow cytomery: take the 3rd generation human marrow mesenchyme stem cell, 2.5g/L trypsinization,
PBS washs 2 times, makes 1 × 109L-1Cell suspension.Take 3 clean common plastics test tubes, be separately added into fluorescent labeling mice
Anti-human monoclonal antibodies CD29-PE/CD34-FITC, CD44-PE/CD45-FITC and homotype Negative control mice anti-mouse
IgG1-PE/IgG1-FITC monoclonal antibody 20 μ L, often pipe adds cell suspension 50 μ L, mixing, incubated at room temperature 15min, PBS
Washing 2 times, 1000r/min is centrifuged 10min, abandons supernatant, and PBS 0.5mL is resuspended, and flow cytomery cell surface divides
Son；Testing result is as shown in Figures 3 and 4.According to Fig. 3 and Fig. 4: human marrow-interstitial stem cell does not express hematopoietic cell mark
CD34 (4.7%), CD45 (5.6%), strongly expressed β 1-integrin CD29 (99.4%) and matrix receptors CD44 (97.2%), table
Person of good sense's bone marrow interstital stem cell is to be different from the another kind of cell of hematopoietic cell in people's bone marrow, sees Fig. 3 and Fig. 4.
Therefore, after testing, cell prepared by the present embodiment possesses the morphological characteristic of human marrow mesenchymal stem cell, and expresses
CD29, CD44, do not express CD34, CD45, and then can be accredited as human marrow mesenchymal stem cell.
Two, the preparation of stem cell medicine
3rd generation human marrow mesenchyme stem cell 1.5 × 10 prepared by step one8Individual, with IL-18BP
0.01mg, oxymatrine 1mg, appropriate water are blended, and prepare the stem cell medicine of 20ml.And then, mescenchymal stem cell
Concentration is 7.5 × 106Individual, the concentration of IL-18BP is 0.5mg/l, and the concentration of oxymatrine is 50mg/l.
Embodiment 2
One, the preparation of mescenchymal stem cell
Refer to embodiment 1, same as in Example 1.
Two, the preparation of stem cell medicine
3rd generation human marrow mesenchyme stem cell 1 × 10 prepared by step one8Individual, and IL-18BP 0.002mg,
Oxymatrine 0.2mg, appropriate water are blended, and prepare the stem cell medicine of 20ml.And then, the concentration of mescenchymal stem cell is
5×106Individual, the concentration of IL-18BP is 0.1mg/l, and the concentration of oxymatrine is 10mg/l.
Embodiment 3
One, the preparation of mescenchymal stem cell
Refer to embodiment 1, same as in Example 1.
Two, the preparation of stem cell medicine
3rd generation human marrow mesenchyme stem cell 2 × 10 prepared by step one8Individual, and IL-18BP 0.02mg,
Oxymatrine 2mg, appropriate water are blended, and prepare the stem cell medicine of 20ml.And then, the concentration of mescenchymal stem cell is 1
×107Individual, the concentration of IL-18BP is 1mg/l, and the concentration of oxymatrine is 100mg/l.
Confirmatory experiment:
6 week old male SD rat 40 (being provided by Nanfang Medical Univ's animal experimental center), body weight 207～236g are provided.
Often to organize 10, it is randomly divided into four groups: embodiment 1 group, embodiment 2 groups, embodiment 3 groups and matched group.Sub-cage rearing, feeds bar
Part and food are the most identical.Four groups all use carbon tetrachloride (CCl4) revulsion prepares Hepatocirrhosis Model: CCl4(40ml/L, Sigma)
Press 0.2ml/100g lumbar injection, 2 times a week, continuous 6 weeks according to body weight, thus set up Hepatocirrhosis Model；
Final injection CCl4Within latter 1 week, treat as follows:
Embodiment 1 group gives the stem cell medicine tail vein injection of embodiment 1 preparation of 300 μ l；
Embodiment 2 groups gives the stem cell medicine tail vein injection of embodiment 2 preparation of 300 μ l；
Embodiment 3 groups gives the stem cell medicine tail vein injection of embodiment 3 preparation of 300 μ l；
Matched group gives the normal saline tail vein injection of 300 μ l；
The treatment of above-mentioned four groups, at least once a week, 4 weeks posterior orbit venous blood samplings, conventional method detection glutamate pyruvate transaminase
(ALT), glutamic oxaloacetic transaminase, GOT (AST) level；Use enzyme linked immunosorbent assay detection serum IL-4, IL-10 level simultaneously, strictly press
Carry out according to test kit operating procedure.Testing result sees following table:
| Group | ALT activity (U/L) | AST activity (U/L) | IL-4 | IL-10 |
| Embodiment 1 group | 31.88±1.27 | 26.99±3.02 | 24.6±0.9 | 73.8±0.7 |
| Embodiment 2 groups | 40.12±1.01 | 38.11±3.21 | 18.1±0.8 | 61.2±0.9 |
| Embodiment 3 groups | 34.10±1.12 | 31.87±3.37 | 21.7±0.8 | 69.9±0.8 |
| Matched group | 131.02±3.65 | 196.29±2.73 | 10.3±1.1 | 46.9±0.8 |
In the reversing liver cirrhosis phase, promote the conversion of fibroplastic macrophage generation phenotype, be converted to the huge of fibrosis
Phagocyte, the mononuclear cell simultaneously raised outside liver is divided into the macrophage with fibrosis function.MSCs can be by secretion
IL-4 promotes macrophage phenotype conversion, thus increases macrophages secrete anti-inflammatory cytokines, the work of phagocyte epimatrix
With.Oxymatrine is worked in coordination with MSCs treatment and be can further improve the level of IL-4 in serum.By promoting that MSCs secretes IL-4, increase
The function of strong MSCs fibrosis.Additionally, IL-10 is also proved can pass through inducing macrophage Phenotypic change, alleviates muscle and damage
Wound, promotes anathrepsis.Oxymatrine promotes that MSCs secretion IL-10 is also its collaborative machine promoting MSCs anti-fibrosis effect
One of system.
In sum, in conjunction with upper table, the stem cell medicine of the embodiment of the present invention 1 to 3 preparation can reverse liver liver effectively
Hardening, wherein, pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase, GOT (AST) secretion level all decline, and have the secretion towards normal liver
The development trend of level, and IL-4 and IL-10 secretion level all improves, and strengthens effect of anti hepatic fibrosis；Therefore, this stem cell
Preparation for treating liver cirrhosis effective, wherein, the therapeutic effect of embodiment 1 it is further preferred that.
The foregoing is only the preferred embodiments of the present invention, not thereby limit the scope of the claims of the present invention, every at this
Under the inventive concept of invention, utilize the equivalent transformation that description of the invention and accompanying drawing content are made, or directly/indirectly it is used in it
The technical field that he is correlated with is included in the scope of patent protection of the present invention.
Claims (6)
1. the stem cell medicine treating liver cirrhosis, it is characterised in that including: mescenchymal stem cell, IL-18BP,
Oxymatrine and solvent, wherein,
The concentration of described mescenchymal stem cell is (5～10) × 106Individual/ml；
The concentration of described IL-18BP is 0.1～1mg/L；
The concentration of described oxymatrine is 10～100mg/L.
2. the as claimed in claim 1 stem cell medicine treating liver cirrhosis, it is characterised in that described mescenchymal stem cell dense
Degree is 7.5 × 106Individual/ml.
3. the stem cell medicine treating liver cirrhosis as claimed in claim 1, it is characterised in that described IL-18BP
Concentration is 0.5mg/l.
4. the stem cell medicine treating liver cirrhosis as claimed in claim 3, it is characterised in that the concentration of described oxymatrine
For 50mg/l.
5. the stem cell medicine treating liver cirrhosis as claimed in claim 1, it is characterised in that described mescenchymal stem cell is bone
Bone marrow-drived mesenchymal stem.
6. the stem cell medicine treating liver cirrhosis as claimed in claim 1, it is characterised in that described solvent is water.
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| CN201610586793.9A CN106109497A (en) | 2016-07-25 | 2016-07-25 | The stem cell medicine for the treatment of liver cirrhosis |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108904533A (en) * | 2018-07-04 | 2018-11-30 | 卡替（上海）生物技术股份有限公司 | Dental pulp mescenchymal stem cell is preparing the purposes in cirrhosis treatment drug |
| CN111529753A (en) * | 2020-04-28 | 2020-08-14 | 宁夏医科大学总医院 | Oxymatrine-placenta mesenchymal stem cell hydrogel, preparation method and application |
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2016
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| CN1404400A (en) * | 2000-02-21 | 2003-03-19 | 应用研究系统Ars股份公司 | Use of IL-18 inhibitors |
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Non-Patent Citations (2)
| Title |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108904533A (en) * | 2018-07-04 | 2018-11-30 | 卡替（上海）生物技术股份有限公司 | Dental pulp mescenchymal stem cell is preparing the purposes in cirrhosis treatment drug |
| CN111529753A (en) * | 2020-04-28 | 2020-08-14 | 宁夏医科大学总医院 | Oxymatrine-placenta mesenchymal stem cell hydrogel, preparation method and application |
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