CN105838670A - Cell mixture and preparation method and application thereof - Google Patents
Cell mixture and preparation method and application thereof Download PDFInfo
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- CN105838670A CN105838670A CN201510017031.2A CN201510017031A CN105838670A CN 105838670 A CN105838670 A CN 105838670A CN 201510017031 A CN201510017031 A CN 201510017031A CN 105838670 A CN105838670 A CN 105838670A
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Abstract
The invention belongs to the technical field of laboratory animals, and relates to a cell mixture and a preparation method and application thereof, especially, to an application of a mixture of adipose-derived stem cells (ADSCs) and immortalized human umbilical vein endothelial cells to preparation of a preparation for interfering and establishing a vascular malformation animal model. The mixture of adipose-derived stem cells (ADSCs) and immortalized human umbilical vein endothelial cells is used for interfering and establishing a vascular malformation animal model. The result shows that the vascular malformation animal model can be obtained at low cost, in a short period, and with high efficiency; and the established vascular malformation animal model of a nude mouse is obviously visible in vascular malformation and makes a significant practical sense in researching pathogenesis of vascular malformation and screening of clinical medicine treatment.
Description
Technical field
The invention belongs to technical field of experimental animals, relate to a kind of cell mixture and its production and use,
Particularly relate to fat stem cell (ADSCs) and immortalization huve cell mixture intervene in preparation and build
Purposes in the preparation of vertical vascular malformation animal model.
Background technology
Prior art discloses vascular malformation is that a kind of angiogenic being apt to occur in each position of infant whole body increases
Raw, the vascular malformation of the most about 71% is apt to occur in head and cervical region, often can cause serious patient's appearance deformity also
Affect its psychosomatic development.The main pathology of described vascular malformation is the hyperplasia of vascular endothelial cell and different
The often formation of lumen of vessels.Vascular malformation was probably divided into for three phases clinically: proliferative phase, resting stage and paracmasis；Its
In quite a few can be with spontaneous regression.Nineteen eighty-two, Mulliken and Glowacki foundation cyto-dynamics,
In conjunction with physical examination and clinical disease course, vascular lesion is divided into hemangioma and vascular malformation；Described hemangioma
(hemangioma) there is endothelial cell proliferation feature, vascular malformation (vascular malformations)
It it is the vascular malformation of normal endothelial cell；Venous malformation (also known as: cavernous malformations) it is that one is apt to occur in
The non-malignant vascular deformity of head neck, this kind of deformity sickness rate in child is the highest, shows according to investigations, in infant
The sickness rate of less than 1 year old is up to 12%, and clinical manifestation is the congested lump of rapid multiplication, and majority can not be natural
Disappear, and increase with advancing age.Currently, with respect to vascular malformation and vascular malformation pathogenic factor and
Mechanism is unclear, lacks effective Therapeutic Method in clinical practice always.In recent years, the biology of vascular malformation
Learn characteristic, clinic and pathological classification and treatment is always the focus that this area is studied, especially as molecular biosciences
Learning and the development of stem cell biology, Chinese scholars reports the shutdown mutually of vascular malformation morbidity and treatment successively
System；But, the shortage of suitable vascular malformation animal model, seriously constrain the research to vascular malformation, because of
This, set up a vascular malformation experimental animal model research pathogenetic to vascular malformation and clinical drug therapy
Screening there is practice significance.
Having document to report relevant hemangioma model, the foundation of vascular malformation model then rarely has report.Based on
This, present inventor intends providing cell mixture in the preparation preparing intervention vessel deformity model
Purposes, it will help set up the research animal model of a kind of more structurally sound venous malformation of report relatively before,
The pathogenetic research of venous malformation and the screening of clinical drug therapy are had great significance.
Summary of the invention
It is an object of the invention to overcome the defect of prior art or deficiency, it is provided that a kind of cell mixture and preparation thereof
Method and purposes, especially fat stem cell (ADSCs) and immortalization huve cell mixture and
Preparation method and the purposes in the preparation preparing intervention vessel deformity model.
Human umbilical vein endothelial cells system based on immortalization (Human umbilical vein endothelial
Cell, HUVEC) have the advantages that multiplication capacity is strong, in the present invention, use fat stem cell (ADSCs) and
People's immortalization huve cell (HUVEC) mixture makes seed cell preparation, for effective prevention and
Set up vascular malformation animal model.
Concrete, the purpose of the present invention is realized by following technical method:
1) fat stem cell (ADSCs) is prepared；
2) fat stem cell (ADSCs) and people's immortalization huve cell (HUVEC) mixture are prepared
Seed cell preparation；
3) vascular malformation animal model is set up；
4) histological examination；
5) identify, evaluate vascular malformation result.
In the present invention, obtain fat stem cell (ADSCs) by following method；
(1) by vitro aseptic fatty tissue, chopping is placed in DMEM culture medium cleaning, and is centrifuged and abandons supernatant；
(2) with the mass volume ratio of 5-20 times of tissue mass be 0.05～0.3% digestive enzyme solution digestion described in fat
Tissue pieces 45～90 minutes；Described digestive enzyme is ferment enzyme, collagenase NB4 or both is mixed
Close liquid；
(3) the suspension suction pipe of step (2) is blown and beaten 3～5 minutes repeatedly；
(4) cell dissociated in step (3) is centrifuged 3-10 minute at 600-1500g, abandons supernatant, obtain cell
Agglomerate；
(5) cell obtained with the resuspended above-mentioned steps of low sugar culture fluid (4), with 1x104-5x104Individual/cm2Inoculation
Secondary Culture in culture dish, obtains fat stem cell (ADSCs).
In the present invention, take immortalization huve cell and ADSCs and mix in different ratios, make seed
Cell preparation；
In the present invention, by the fat stem cell (ADSCs) made and people's immortalization huve cell (HUVEC)
Mixture, as seed cell preparation, sets up nude mice vascular malformation animal model according to a conventional method, and after 2W, row is naked
The double lower limb MRI scan of Mus, after scanning to bilateral lower hind limb musculature row HE, Masson, immunofluorescence, exempt from
The relevant dyeing such as epidemic disease group, identify, evaluate the situation of the vascular malformation of nude mice vascular malformation animal model；Result
Showing, obvious venous malformation seen from the nude mice vascular malformation animal model of foundation, this model is to venous malformation
Pathogenetic research and the screening of clinical drug therapy have important practice significance.
In the present invention, the described fatty tissue being organized as human or animal, fatty tissue fragment volume is
0.5-10mm3；In one embodiment of the present of invention, described fatty tissue fragment volume average out to 1mm3；
In the present invention, described DMEM culture volume is 5-10 times of adipose tissue volume；
In the present invention, described middle digestive enzyme can use ferment enzyme and the mixed liquor of collagenase NB4；Described mixing
In liquid, the concentration of collagenase NB4 is preferably 0.1～0.3%, such as 0.1%, 0.2%, 0.3%；Ferment enzyme dense
Degree preferably 0.05～0.2%；
In the present invention, the solution of described digestive enzyme mixed enzyme is DMEM culture medium, can contain hyclone；This
In bright, described ferment enzyme i.e. dispase, purchased from sigma company (dispase be otherwise known as Bacillus polymyxa Neutral proteinase, in
Property protease)；
In the present invention, endotheliocyte used is the endotheliocyte of immortalization, with the difference of tradition endotheliocyte
It is that the culture fluid of this cell need not add any somatomedin, it addition, this cell can infinitely pass on, expand
Increase and maintain the characteristic of endotheliocyte；
In the present invention, described immortalization huve cell and ADSCs press 1:1,1:2,1:3,2:
The ratio mixing that 1,3:1,4:1 etc. are different；
In the present invention, described experimental mouse is without immunogenicity 6-8W nude mice, and described cell mixture is trapped in
In nude mice lower hind limb musculature；
In the present invention, described histological examination include HE, Masson, immunofluorescence (CD31, VWF, SMA),
The relevant dyeing qualifications etc. such as SABC (CD31, VWF, SMA).
Fat stem cell of the present invention (ADSCs) and immortalization huve cell mixture are used for intervening and building
Vertical vascular malformation animal model, result shows, it is possible to low cost and the most efficiently acquisition vascular malformation move
Object model, obvious venous malformation seen from the nude mice vascular malformation animal model of foundation, this model is abnormal to vein
The pathogenetic research of shape and the screening of clinical drug therapy have important practice significance.
Accompanying drawing explanation
Fig. 1 shows the foundation of nude mice left lower extremity vascular malformation model；Wherein, 1 is Control group, 2,3
For left lower extremity vascular malformation model group.
Fig. 2 shows HE (A) and the Masson dyeing of nude mice left lower extremity vascular malformation.
Fig. 3 shows the CD31 immunofluorescence dyeing of nude mice left lower extremity vascular malformation；Wherein,
A is DAPI dyeing；B is CD31 dyeing；C is GFP dyeing；D is Merge figure.
Fig. 4 shows the vWF immunofluorescence dyeing of nude mice left lower extremity vascular malformation；Wherein,
A is DAPI dyeing；B is vWF dyeing；C is GFP dyeing；D is Merge figure.
Fig. 5 shows the immunohistochemical staining of nude mice left lower extremity vascular malformation；Wherein,
A is CD31 dyeing；B is vWF dyeing；C is SMA dyeing.
Detailed description of the invention
Embodiment 1
In the present embodiment, described ferment enzyme i.e. dispase, purchased from sigma company, (dispase is otherwise known as
Bacillus polymyxa Neutral proteinase, neutral protease)
In the present embodiment, use fat stem cell (ADSCs) and people's immortalization huve cell (HUVEC)
Mixture makes seed cell preparation, for effective prevention and set up vascular malformation animal model.
First, fat stem cell (ADSCs) is obtained by following method；
By in vitro aseptic fatty tissue, shred as 1mm3, it is placed in DMEM culture medium cleaning, described DMEM
Culture volume is 5-10 times of adipose tissue volume；It is centrifuged and abandons supernatant；
With the mass volume ratio of 5-20 times of tissue mass be 0.05～0.3% digestive enzyme solution digestion described in fat group
Knit fragment 45～90 minutes；Described digestive enzyme is ferment enzyme, collagenase NB4 or both mixed liquors；Institute
State digestive enzyme and can use ferment enzyme and the mixed liquor of collagenase NB4；In described mixed liquor, collagenase NB4's is dense
Degree preferably 0.1～0.3%, such as 0.1%, 0.2%, 0.3%；The concentration of ferment enzyme is preferably 0.05～0.2%；
Above-mentioned suspension suction pipe is blown and beaten 3～5 minutes repeatedly；The cell dissociated is centrifuged at 600-1500g
3-10 minute, abandon supernatant, obtain cell mass；The cell obtained by the resuspended above-mentioned steps of low sugar culture fluid, with
1x104-5x104Individual/cm2It is inoculated in Secondary Culture in culture dish, obtains fat stem cell (ADSCs)；
Further, take immortalization huve cell and ADSCs by by 1:1,1:2,1:3,2:1,
The ratio mixing that 3:1,4:1 are different, makes seed cell preparation；
The used endotheliocyte that endotheliocyte is immortalization, the difference with tradition endotheliocyte is this cell
Culture fluid need not add any somatomedin, it addition, this cell can infinitely pass on, expand and in maintaining
The characteristic of chrotoplast；
By the fat stem cell (ADSCs) made and people's immortalization huve cell (HUVEC) mixture
As seed cell preparation, 6-8W is selected to set up nude mice blood vessel according to a conventional method without immunogenicity experiment of nude mouse Mus
Deformity model, described cell mixture is trapped in nude mice lower hind limb musculature；After 2W, row nude mice is double
Lower limb MRI scan, after scanning to bilateral lower hind limb musculature row HE, Masson, immunofluorescence, immunity glimmering
The relevant dyeing such as light (CD31, VWF, SMA), SABC (CD31, VWF, SMA), identify, evaluate nude mice
The situation of the vascular malformation of vascular malformation animal model；Result shows, the nude mice vascular malformation animal model of foundation
Visible obvious venous malformation, this model is to the pathogenetic research of venous malformation and the sieve of clinical drug therapy
Choosing has important practice significance.
In the present embodiment, the solution of described digestive enzyme mixed enzyme is DMEM culture medium, can contain hyclone；Institute
State ferment enzyme i.e. dispase, purchased from sigma company (dispase be otherwise known as Bacillus polymyxa Neutral proteinase, neutral protease).
Claims (8)
1. a cell mixture, it is characterised in that quiet by fat stem cell (ADSCs) and immortalization umbilicus
Arteries and veins endotheliocyte is pressed the ratio of 1:1,1:2,1:3,2:1,3:1 or 4:1 and is mixed.
2. the cell mixture described in claim 1 is in the preparation preparing intervention vessel deformity model
Purposes.
3. the purposes as described in claim 2, it is characterised in that described cell mixture is careful as planting
Vascular malformation animal model is intervened and set up to born of the same parents' preparation by following technical method:
1) fat stem cell (ADSCs) is prepared；
2) fat stem cell (ADSCs) and people's immortalization huve cell (HUVEC) mixture are prepared
Seed cell preparation；
3) vascular malformation animal model is set up；
4) histological examination；
5) identify, evaluate vascular malformation result.
4. the purposes as described in claim 3, it is characterised in that obtain fat stem cell by following method
(ADSCs)；
(1) by vitro aseptic fatty tissue, chopping is placed in DMEM culture medium cleaning, and is centrifuged and abandons supernatant；
(2) with the mass volume ratio of 5-20 times of tissue mass be 0.05～0.3% digestive enzyme solution digestion described in fat
Tissue pieces 45～90 minutes；Described digestive enzyme is ferment enzyme, collagenase NB4 or both is mixed
Close liquid；
(3) the suspension suction pipe of step (2) is blown and beaten 3～5 minutes repeatedly；
(4) cell dissociated in step (3) is centrifuged 3-10 minute at 600-1500g, abandons supernatant, obtain cell
Agglomerate；
(5) cell obtained with the resuspended above-mentioned steps of low sugar culture fluid (4), with 1x104-5x104Individual/cm2Inoculation
Secondary Culture in culture dish, obtains fat stem cell (ADSCs).
5. the purposes as described in claim 3, it is characterised in that in described cell mixture, people's immortality
Changing huve cell and ADSCs presses 1:1, the ratio of 1:2,1:3,2:1,3:1,4:1 is mixed
Close.
6. the purposes as described in claim 3, it is characterised in that used in described vascular malformation animal model
Experimental mouse be without immunogenicity 6-8W nude mice, described cell mixture is trapped in nude mice lower hind limb musculature
In.
7. the purposes as described in claim 3, it is characterised in that described histological examination include HE,
Masson, immunofluorescence CD31, VWF, SMA, SABC CD31, the relevant dyeing of VWF, SMA is identified.
8. the purposes as described in claim 4, it is characterised in that in described step (2), digestive enzyme uses
Ferment enzyme and the mixed liquor of collagenase NB4；In described mixed liquor, the concentration of collagenase NB4 is 0.1～0.3%,
The concentration of described ferment enzyme is 0.05～0.2%.
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| CN201510017031.2A CN105838670A (en) | 2015-01-13 | 2015-01-13 | Cell mixture and preparation method and application thereof |
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| CN201510017031.2A CN105838670A (en) | 2015-01-13 | 2015-01-13 | Cell mixture and preparation method and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102239821A (en) * | 2011-05-10 | 2011-11-16 | 郑家伟 | Human hemangioma animal model and constructing method and application thereof |
| WO2012175797A1 (en) * | 2011-06-23 | 2012-12-27 | Tampereen Yliopisto | In vitro cardiovascular model |
-
2015
- 2015-01-13 CN CN201510017031.2A patent/CN105838670A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102239821A (en) * | 2011-05-10 | 2011-11-16 | 郑家伟 | Human hemangioma animal model and constructing method and application thereof |
| WO2012175797A1 (en) * | 2011-06-23 | 2012-12-27 | Tampereen Yliopisto | In vitro cardiovascular model |
| CN103814124A (en) * | 2011-06-23 | 2014-05-21 | 坦佩雷大学 | In vitro cardiovascular model |
Non-Patent Citations (2)
| Title |
|---|
| 谭玉珍等: "血管内皮生长因子对海绵状血管瘤内皮细胞血管形成的影响", 《科学通报》 * |
| 钱勇等: "重建永生化人脐静脉内皮细胞", 《信息咨询》 * |
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