CN105483206B - A method of promoting endothelial cell proliferation ability with VCAM-1 detection MSC - Google Patents
A method of promoting endothelial cell proliferation ability with VCAM-1 detection MSC Download PDFInfo
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- CN105483206B CN105483206B CN201610007342.5A CN201610007342A CN105483206B CN 105483206 B CN105483206 B CN 105483206B CN 201610007342 A CN201610007342 A CN 201610007342A CN 105483206 B CN105483206 B CN 105483206B
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Abstract
The invention discloses a kind of methods for promoting endothelial cell proliferation ability with VCAM-1 detection MSC, it is characterized in that having found the new application of known albumen VCAM-1 a kind of, the protein expression of VCAM-1 is detected by EL I SA, total protein content is measured with Bradford method, reflect the rush endothelial cell proliferation ability of cell using the protein content of VCAM-1 in every 1g total protein, in this approach can the rush endothelial cell proliferation ability to MSC quantify.
Description
Technical field
The present invention relates to stem cell fields, detect MSC in particular to VCAM-1 (vascular cell adhesion molecule-1)
The method of (mescenchymal stem cell) rush endothelial cell proliferation ability.
Background technique
Vascular cell adhesion molecule-1 (VCAM-1), also referred to as CD106 are a kind of contactins
Cell surface sialoglycoprotein.VCAM-1 is there are two hypotype, and there are six tools or seven generate two kinds of shapes by alternative splicing
The extracellular Ig spline structure domain of the VCAM-1 of formula, main existence form are the hypotypes with seven extracellular Ig spline structure domains.VCAM-
1 is a kind of endothelial cell ligand, in conjunction with 7 integrin of VLA-4 antigen and 4 β of α expressed on leucocyte, thus mediated leucocytes-
Inner skin cell viscosity echos signal transduction.Inflammatory bowel disease, atherosclerosis, allograft rejection, infection and asthma
VCAM-1 is induced to express on endothelial cell in reflection.In these reflections, VCAM-1 is that leucocyte migration constitutes a bracket.
VCAM-1 can also activate caused by the signal in endothelial cell the unlatching of " endothelial cell door ", be moved by its leucocyte
It moves.VCAM-1 has been considered to be a kind of potential anti-inflammatory treatment target spot, and the expression for reducing VCAM-1 will slow down Atherosclerosis
The development of change.In addition, for the signal of the VCAM-1 activation of chrotoplast by cytokine modulating, this shows it in inflammation mistake in the blood vessels
It is critically important for the expression of endothelial cell adhesion molecule and function in journey.
VCAM-1 in most of quiescent endothelial cells low expressions, injury of blood vessel, stress when can be by IL-1, TNF-
The cytokine inductions such as α, IL-4 are mainly expressed in new vessels area.Its intracorporal soluble form is by mediating endothelial cell chemotaxis
Change and shows angiogenic activity.
Blood vessel is the important component of human body, and angiogenesis (angiogenesis) is generated newly from the germination of existing blood vessel
The process of blood vessel, the inner surface of entire blood vessel are covered by one layer of endothelial cell, and the generation of blood vessel and blood vessel endothelium are thin
Born of the same parents' migration is related to proliferation.
Experiments have shown that MSC has Therapeutic angiogenesis effect, it can be by being directly divided into endothelial cell for inside and outside
Or vascular smooth muscle cells participate in angiogenesis, and in addition it can also secrete a variety of rush angiogenesis factors, improve microenvironment,
Promote vascular endothelial cell proliferation and migration, and then promotes angiogenesis.
The characteristic of the angiogenesis promoting of MSC has very important clinical meaning to the treatment of limb ischemia.But at present
The method for lacking detection and the angiogenesis promoting ability of evaluation MSC, hinders the clinical application of cell.
The present inventor passes through the study found that in the angiogenesis promoting ability of MSC, and VCAM-1 shows the phase of height
Guan Xing has invented the present invention based on this discovery.
The purpose of the present invention is to provide a kind of methods that is completely new, can effectively detecting MSC angiogenesis promoting ability.
Summary of the invention
First technical solution of the invention is, a method of promoting endothelial cell proliferation ability with VCAM-1 detection MSC,
It is characterized in that, detects the protein content of VCAM-1 in MSC, total protein content is measured, according to the albumen of the VCAM-1 in total protein
Percentage composition detects the rush endothelial cell proliferation ability of MSC.
Second technical solution is to be based on the first technical solution, which is characterized in that the MSC is positive comprising VCAM-1
MSC。
Third technical solution is to be based on the second technical solution, which is characterized in that
Step 1, after MSC and endothelial cell being co-cultured, the proliferation number of endothelial cell is detected, MSC is obtained and promotes endothelium thin
Born of the same parents' proliferation rate,
Step 2, the total protein content of MSC is measured,
Step 3, the protein content of the VCAM-1 of MSC is detected,
Step 4, the protein content percentage of the VCAM-1 in total protein is calculated,
Step 5, the protein content percentage of Statistics Application method analysis VCAM-1 and the MSC in step 1 promote endothelium
Correlation between cell proliferation rate, the relationship between the protein content percentage to VCAM-1 and rush endothelial cell proliferation ability
It is quantified.
4th technical solution is to be based on third technical solution, which is characterized in that the endothelial cell is HUVEC cell.
5th technical solution is, based on any one of first to fourth technical solution, which is characterized in that detected by ELISA
The protein content of VCAM-1.
6th technical solution is, based on any one of first to fourth technical solution, which is characterized in that surveyed with Bradford method
Determine total protein content.
7th technical solution is to be based on the 5th technical solution, which is characterized in that with the egg of Bradford method measurement VCAM-1
Bai Hanliang.
Detailed description of the invention
By reading the following detailed description of the preferred embodiment, various other advantages and benefits are common for this field
Technical staff will become clear.The drawings are only for the purpose of illustrating a preferred embodiment, and is not considered as to the present invention
Limitation.And throughout the drawings, the same reference numbers will be used to refer to the same parts.Wherein in the accompanying drawings, reference number
Alphabetic flag later indicates that multiple identical components will omit its last alphabetic flag when referring to these components.Attached
In figure:
Fig. 1 shows the rush endothelial cell proliferation rate of different MSC samples；
Fig. 2 shows the protein contents of the VCAM-1 in every 1g total protein of different MSC samples；
Fig. 3 shows the correlation of different MSC samples promoted between endothelial cell proliferation rate and the protein content of VCAM-1；
Fig. 4 shows the blood perfusion amount of laser-Doppler detection ischemic limb.
Specific embodiment
The present invention provides many applicable creative concepts, which be can be reflected in a large number of on specific
Hereinafter.The specific embodiment described in following embodiments of the present invention is only used as a specific embodiment of the invention
Exemplary illustration, and be not meant to limit the scope of the invention.
Description of specific embodiments of the present invention below.
Step (1) cultivates MSC and HUVEC cell (Human umbilical vein endothelial cells) in transwell system, after co-cultivation
The proliferation number for detecting HUVEC cell obtains MSC and promotes endothelial cell proliferation rate.
Step (2) extracts the total protein of MSC, measures total protein concentration using Bradford method.
Step (3) detects the content of VCAM-1 in albumen sample using ELISA kit.
The protein content that step (4) calculates the VCAM-1 in every 1g total protein (calculates the albumen of the VCAM-1 in total protein
Percentage composition).
Step (5) Statistics Application method is analyzed related between the protein content of VCAM-1 and promotion HUVEC proliferation rate
Property.
Step (6) carries out in vivo functionality confirmatory experiment, determines effect of the VCAM-1 for the angiogenesis promoting ability of MSC.
Each step is described in detail by the following examples.
Embodiment
The rush endothelial cell proliferation ability of step 1. test sample cell
Umbilical vein endothelium (HUVEC) cell is planted in the cavity of resorption of transwell system, and MSC cell is planted in epicoele,
3 multiple holes of every group of setting detect the proliferation number of HUVEC after co-cultivation.Because endothelial cell is directly formed the cell of blood vessel, lead to
The effect for crossing sample cell (MSC) Human Umbilical Vein Endothelial Cells reflects the effect of its Angiogensis.8 Different Individuals are had chosen in experiment
The MSC cell in source is tested, and the cell for detection is P5 generation, and experimental result shows that different cells is different degrees of
Promotion HUVEC proliferation, but there are individual difference (Fig. 1), specific data are shown in Table one.
The promotion HUVEC proliferation rate of table one, different sample cells
Step 2,3, the VCAM-1 content of 4.ELISA test sample cell
8 MSC cell samples in collection step 1, lytic cell carry out total protein extraction.It is measured using Bradford method
Total protein concentration (step 2) detects the content (step 3,4) of VCAM-1 in each albumen sample using ELISA kit.Experiment
MSC cell expresses VCAM-1 as the result is shown, and the VCAM-1 content in every 1g total protein is all different (Fig. 2).Specific data are shown in
Table two.
The VCAM-1 content of table two, different sample cells
VCAM-1 content and its of step 5. cell promote the correlation analysis of endothelial cell proliferation ability for step 1 and 3
Data carry out correlation analysis, the results showed that the high MSC of VCAM-1 content more significantly can promote HUVEC to be proliferated in cell
Rate.There is correlation between VCAM-1 content and promotion HUVEC proliferation rate, statistical analysis shows Pearson correlation coefficient
Therebetween there is significant positive correlation (Fig. 3) in=0.9222, p=0.0031.It therefore can be in total protein
The protein content percentage of VCAM-1 quantifies and detects the rush endothelial cell proliferation ability of MSC.
Confirmatory experiment proves that VCAM-1 has angiogenesis promoting ability in step 6. body
The MSC that MSC and the VCAM-1 feminine gender of the VCAM-1 positive are obtained by airflow classification, is respectively labeled as VCAM-1+MSC
And VCAM-1-MSC, by establishing lower limb ischemia animal model, lower limb ischemia operation method is to block the left lower extremity stock of animal quiet
The blood flow of arteries and veins, big hidden arteriovenous and stock arteriovenous flesh branch.After modeling success, in art limb operative site locally injecting MSC, carry out thin
Born of the same parents' transplantation treatment, to measure its angiogenesis promoting ability.Experiment is divided into 3 groups, i.e. VCAM-1+MSC cell therapy group (1 × 106),
VCAM-1-MSC cell therapy group (1 × 106), control group (locally injecting PBS).Utilize the blood flow of laser-Doppler detection technique limb
Thus groundwater increment, restoration of blood flow situation evaluate the angiogenesis promoting ability of cell.From fig. 4, it can be seen that nude mice passes through lower limb ischemia
The obvious ischemic of its left hind (detection is shown as blue, black) after operation, the art limb still ischemic of control group is obvious after 3 weeks, and
It is restored and improves by the blood flow of VCAM-1+MSC cell and the nude mice lower limb of VCAM-1-MSC cell therapy and (detect display
For yellow, red).Thus it demonstrates, the MSC cell for expressing VCAM-1 not only has angiogenesis promoting ability, according to VCAM-1
Correlation between content and promotion HUVEC proliferation rate can also evaluate the angiogenesis promoting ability of MSC using VCAM-1.
Especially VCAM-1+MSC cell more correctly can be detected and be evaluated to the ability of angiogenesis promoting.
It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and this
Field technical staff can be designed alternative embodiment without departing from the scope of the appended claims.In claim
In, any reference symbol between parentheses should not be configured to limitations on claims.Word "comprising" is not excluded for depositing
In element or step not listed in the claims.
Claims (1)
1. a kind of method for promoting endothelial cell proliferation ability with VCAM-1 detection MSC, which is characterized in that VCAM-1 in detection MSC
Protein content, total protein content is measured, in the rush that MSC is detected according to the protein content percentage of the VCAM-1 in total protein
Epithelial cell proliferation ability.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110352240A (en) * | 2016-12-12 | 2019-10-18 | 北京汉氏联合生物技术股份有限公司 | The mescenchymal stem cell of perinatal period tissue derived: preparation method and use |
| US11179383B2 (en) | 2018-10-30 | 2021-11-23 | Gilead Sciences, Inc. | Compounds for inhibition of α4β7 integrin |
| AU2019373242B2 (en) | 2018-10-30 | 2023-07-13 | Gilead Sciences, Inc. | Compounds for inhibition of alpha 4 beta 7 integrin |
| CR20210213A (en) | 2018-10-30 | 2021-06-24 | Gilead Sciences Inc | Quinoline derivatives as alpha4beta7 integrin inhibitors |
| US11174256B2 (en) | 2018-10-30 | 2021-11-16 | Gilead Sciences, Inc. | Imidazopyridine derivatives |
| AU2020329207B2 (en) | 2019-08-14 | 2024-02-29 | Gilead Sciences, Inc. | Compounds for inhibition of alpha 4 beta 7 integrin |
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| CN102499135A (en) * | 2011-10-25 | 2012-06-20 | 澳门大学 | Zebra fish vascular injury model for screening vascular injury resisting medicament as well as building method and application thereof |
| CN102539736A (en) * | 2011-12-16 | 2012-07-04 | 北京汉氏联合生物技术有限公司 | CD106-positive cells, and identification and preparation method and application thereof |
| CN104508124A (en) * | 2011-07-06 | 2015-04-08 | 细胞治疗有限公司 | Progenitor cells of mesodermal lineage |
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| CN104508124A (en) * | 2011-07-06 | 2015-04-08 | 细胞治疗有限公司 | Progenitor cells of mesodermal lineage |
| CN102499135A (en) * | 2011-10-25 | 2012-06-20 | 澳门大学 | Zebra fish vascular injury model for screening vascular injury resisting medicament as well as building method and application thereof |
| CN102539736A (en) * | 2011-12-16 | 2012-07-04 | 北京汉氏联合生物技术有限公司 | CD106-positive cells, and identification and preparation method and application thereof |
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Inventor after: Liang Lu Inventor after: Geng Jie Inventor after: Han Zhihai Inventor after: Zhao Qinjun Inventor after: Han Zhongchao Inventor before: Liang Lu Inventor before: Zhao Qinjun Inventor before: Han Zhihai |
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Effective date of registration: 20211118 Address after: Room 105, 1st floor, block Q1, building 1, No.1 Kangding street, economic and Technological Development Zone, Daxing District, Beijing Patentee after: Beijing Hanshi Stem Cell Technology Co.,Ltd. Address before: 100176 building 3, Guosheng Science Park, yard 1, Kangding street, economic and Technological Development Zone, Daxing District, Beijing Patentee before: HEALTH AND BIOTECH FRANCE (H & B FRANCE) |
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