CN102321568A - New vascular tissue generation method - Google Patents
New vascular tissue generation method Download PDFInfo
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- CN102321568A CN102321568A CN201110231591A CN201110231591A CN102321568A CN 102321568 A CN102321568 A CN 102321568A CN 201110231591 A CN201110231591 A CN 201110231591A CN 201110231591 A CN201110231591 A CN 201110231591A CN 102321568 A CN102321568 A CN 102321568A
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Abstract
The invention relates to a new vascular tissue generation method. The method comprises, removing thoracic aorta, heart tissue, liver tissue, spleen tissue, lung tissue, kidney tissue and colon tissue from a mouse or a rat under a sterile condition; carrying out a combination of the removed tissues and the culture plate to generate the new vascular tissues respectively corresponding to the aorta, the heart tissue, the liver tissue, the spleen tissue, the lung tissue, the kidney tissue and the colon tissue. With the method provided by the present invention, disadvantages of cumbersome quantity and not high survival rate are overcome; a simple and convenient model of the new vascular tissue generating is established, wherein the model can be widely used; the method further can be applicable for screening studies of the tumor angiogenesis inhibitor.
Description
Technical field
The invention belongs to compound pharmacological action and field of molecular biotechnology, be specifically related to a kind of new organization vasculogenesis method.
Background technology
Vasculogenesis (angiogenesis) is meant regeneration neovascularity on the basis of original vescular bed, is the process that receives multifactor regulation and control of a complicacy, and it is different from the blood vessel generating process.Vasculogenesis be one by the various kinds of cell factor and various kinds of cell composition participate in, dynamic, the complex process coordinated; Migration, division, differentiation that its initial key link is vascular endothelial cell or stroma stem cell; And tube chamberization subsequently, receive the minute adjustment of vasculogenesis inducible factor and supressor.Physiological vasculogenesis is more common in wound healing, or endometrial vasculogenesis in the menstrual cycle, but in some processes such as tumour, arteriosclerosis, psoriatic, diabetic retinopathy and endometriosis, then is a kind of pathologic phenomenon.Vasculogenesis is absolutely necessary in the generation of tumour and transfer process, the growth of most malignant tumours and to shift all be that blood vessel relies on, and promptly tumor tissues can be induced the generation of neovascularity in its process of growth.The vasculogenesis phenomenon that this is brought out in the tumor growth process is referred to as tumor neogenetic blood vessels and generates.The blocking-up vasculogenesis is a kind of means and approach of new treatment tumour.The new vessel that destroys or suppress tumour generates, stop effectively the medicine of growth of tumor and transfer be referred to as the tumor neogenetic blood vessels formation inhibitor (tumorangiogenesis inhibitor, TAI).
The foundation of angiogenesis model is the prerequisite of screening anti-angiogenic medicaments, and the model of having reported has mouse ear model, chick embryo allantois membrane modle etc., but these models all have its shortcoming, generally have quantitatively loaded down with trivial details, the shortcoming that surviving rate is not high.Therefore it is very necessary setting up a kind of model simple, convenient and the new organization vasculogenesis that can be widely used.
Summary of the invention
In order to overcome the deficiency that above-mentioned prior art exists; The object of the present invention is to provide a kind of new organization vasculogenesis method; Overcome the quantitative loaded down with trivial details and not high shortcoming of surviving rate; Set up a kind of model of simple, the convenient and new organization vasculogenesis that can be widely used, this method can also be applied to the screening study of angiogenesis inhibitor.
In order to achieve the above object, the technical scheme that the present invention adopted is:
A kind of new organization vasculogenesis method; Get mouse or rat chest aorta, the heart, liver, spleen, lung, blood vessel, kidney and colon under the aseptic condition; The PBS liquid of putting into precooling immediately cleans; The treatment process of thoracic aorta is for cutting with ophthalmologic operation and outer fiber and the fatty tissue of tweezers removal arterial, after the cleaning of PBS liquid, under reading lens; In the DMEM of precooling substratum, cut into length and be the arterial ring of 0.3mm or be cut into the artery piece that length is 0.3mm, and the treatment process of the heart, liver, spleen, lung, kidney and colon is for cutting with ophthalmologic operation and tweezers are cut into 1mm after removing the adhesion organization of tissue periphery
3The tissue block of size is got fibrinogen solution and is tiled in the culture plate, and every pore capacities of culture plate is 200 μ L; Add zymoplasm 1 μ L in every hole of culture plate; Shake up the back and form gel, the tissue block that cuts is planted on the gel of culture plate, in every hole of culture plate, add Fibrinogen solvent 200 μ L and zymoplasm 1 μ L again; Shake up the back and form gel; So just form the sandwich structure of sandwich style, on this sandwich structure, added the DMEM substratum 1mL that contains mass concentration 3-10% foetal calf serum, put into volumetric concentration and be 5% CO
2With temperature is to cultivate in 37 ℃ the incubator, just can generate each self-corresponding new organization blood vessel of aorta, the heart, liver, spleen, lung, kidney and colon with this.
The speed speed of described generation aorta, the heart, liver, spleen, lung, kidney and each self-corresponding new organization blood vessel of colon is followed successively by: the new organization blood vessel of the new organization blood vessel＞blood vessel of the new organization blood vessel >=colon of the new organization blood vessel＞kidney of new organization blood vessel >=heart of the new organization blood vessel＞liver of the new organization blood vessel >=spleen of lung, the process of its Midcolic new organization vasculogenesis are divided into endotheliocyte and move out and form two kinds of blood vessel and vasculogenesis.
Comprise VEGF or bFGF growth factor in the described DMEM substratum.
Add thaspine behind the new organization vasculogenesis of described lung, can be inhibited for the new organization blood vessel of lung, and 1822 pairs of colon's vasculogenesis of adding thaspine verivate are inhibited behind the new organization vasculogenesis of described colon.
Through a kind of new organization vasculogenesis method; Combine culture plate to generate aorta, the heart, liver, spleen, lung, kidney and each self-corresponding new organization blood vessel of colon through under aseptic condition, getting mouse or rat chest aorta, the heart, liver, spleen, lung, kidney and colon; Overcome the quantitative loaded down with trivial details and not high shortcoming of surviving rate; Set up a kind of model of simple, the convenient and new organization vasculogenesis that can be widely used, this method can also be applied to the screening study of angiogenesis inhibitor.
Description of drawings
The angiogenic growth speed reduced co-ordinates figure of the various tissues of Fig. 1;
Fig. 2 thaspine is to the coordinate diagram that influences of lung tissue vasculogenesis;
1822 pairs of colon's vasculogenesis of Fig. 3 thaspine verivate influence coordinate diagram.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is done more detailed explanation.
New organization vasculogenesis method; Get mouse or rat chest aorta, the heart, liver, spleen, lung, kidney and colon under the aseptic condition; The PBS liquid of putting into precooling immediately cleans; The treatment process of thoracic aorta is for cutting with ophthalmologic operation and outer fiber and the fatty tissue of tweezers removal arterial, after the cleaning of PBS liquid, under reading lens; In the DMEM of precooling substratum, cut into length and be the arterial ring of 0.3mm or be cut into the artery piece that length is 0.3mm, and the treatment process of the heart, liver, spleen, lung, blood vessel, kidney and colon is for cutting with ophthalmologic operation and tweezers are cut into 1mm after removing the adhesion organization of tissue periphery
3The tissue block of size is got fibrinogen solution and is tiled in 48 well culture plates, and every pore capacities of 48 well culture plates is 200 μ L; Add zymoplasm 1 μ L in every hole of 48 hole culture hole; Shake up the back and form gel, the tissue block that cuts is planted on the gel of 48 hole culture hole, in every hole of 48 hole culture hole, add Fibrinogen solvent 200 μ L and zymoplasm 1 μ L again; Shake up the back and form gel; So just form the sandwich structure of sandwich style, on this sandwich structure, added the DMEM substratum 1mL that contains the 3-10% foetal calf serum, put into concentration and be 5% CO
2With temperature is to cultivate in 37 ℃ the incubator, just can generate each self-corresponding new organization blood vessel of aorta, the heart, liver, spleen, lung, kidney and colon with this.Comprise VEGF or bFGF growth factor in the described DMEM substratum.Microscopically is observed various tissue blood vessels and is generated situation after 3 days; Observe once every other day; Contrast the angiogenic growth speed of various tissues and form comparison diagram as shown in Figure 1 according to the vasculogenesis situation; Diagram shows that the speed speed of described generation aorta, the heart, liver, spleen, lung, kidney and each self-corresponding new organization blood vessel of colon is followed successively by: the new organization blood vessel of the new organization blood vessel＞blood vessel of the new organization blood vessel >=colon of the new organization blood vessel＞kidney of new organization blood vessel >=heart of the new organization blood vessel＞liver of the new organization blood vessel >=spleen of lung, the process of its Midcolic new organization vasculogenesis are divided into endotheliocyte and move out and form two kinds of blood vessel and vasculogenesis.Adding thaspine in the 3rd day that cultivates at the new organization blood vessel of lung in addition, is the blank group with the DMEM nutrient solution that contains mass concentration 3-10% foetal calf serum, the thaspine group low, in and high density be respectively 2.32; 4.63 with 9.25 μ mol/L, changed liquid at a distance from 3 days, establish 5 multiple holes for every group; Carrying out the tissue blood vessel counting at the 9th day, is X-coordinate with time, and the new organization vascular strip number of lung is that ordinate zou is mapped respectively; Comparative result is seen Fig. 2, and the 3rd day adding thaspine verivate of cultivating at the new organization blood vessel of colon in addition 1822 is the blank group with the DMEM nutrient solution that contains mass concentration 3-10% foetal calf serum; Thaspine verivate 1822 low, in and high density be respectively 4,12 and 36 μ mol/L, changed liquid at a distance from 3 days; Establishing 5 multiple holes for every group, carried out the tissue blood vessel counting at the 9th day, is X-coordinate with time; The new organization vascular strip number of colon is that ordinate zou is mapped respectively; Comparative result is seen Fig. 3, and the comparative result of synthesizing map 2 and Fig. 3 like this adds thaspine after can knowing the new organization vasculogenesis of described lung; Can be inhibited for the new organization blood vessel of lung, and 1822 pairs of colon's vasculogenesis of adding thaspine verivate are inhibited behind the new organization vasculogenesis of described colon.
Claims (4)
1. new organization vasculogenesis method; It is characterized in that: get mouse or rat chest aorta, the heart, liver, spleen, lung, kidney and colon under the aseptic condition; The PBS liquid of putting into precooling immediately cleans; The treatment process of thoracic aorta is for cutting with ophthalmologic operation and outer fiber and the fatty tissue of tweezers removal arterial, after the cleaning of PBS liquid, under reading lens; In the DMEM of precooling substratum, cut into length and be the arterial ring of 0.3mm or be cut into the artery piece that length is 0.3mm, and the treatment process of the heart, liver, spleen, lung, kidney and colon is for cutting with ophthalmologic operation and tweezers are cut into 1mm after removing the adhesion organization of tissue periphery
3The tissue block of size is got fibrinogen solution and is tiled in the culture plate, and every pore capacities of culture plate is 200 μ L; Add zymoplasm 1 μ L in every hole of culture plate; Shake up the back and form gel, the tissue block that cuts is planted on the gel of culture plate, in every hole of culture plate, add Fibrinogen solvent 200 μ L and zymoplasm 1 μ L again; Shake up the back and form gel; So just form the sandwich structure of sandwich style, on this sandwich structure, added the DMEM substratum 1mL that contains mass concentration 3-10% foetal calf serum, put into volumetric concentration and be 5% CO
2With temperature is to cultivate in 37 ℃ the incubator, just can generate each self-corresponding new organization blood vessel of aorta, the heart, liver, spleen, lung, kidney and colon with this.
2. new organization vasculogenesis method according to claim 1; It is characterized in that: the speed speed of described generation aorta, the heart, liver, spleen, lung, kidney and each self-corresponding new organization blood vessel of colon is followed successively by: the new organization blood vessel of the new organization blood vessel＞blood vessel of the new organization blood vessel >=colon of the new organization blood vessel＞kidney of new organization blood vessel >=heart of the new organization blood vessel＞liver of the new organization blood vessel >=spleen of lung, the process of its Midcolic new organization vasculogenesis are divided into endotheliocyte and move out and form two kinds of blood vessel and vasculogenesis.
3. according to claim 1 or the described new organization vasculogenesis of claim 2 method, it is characterized in that: can comprise VEGF or bFGF growth factor in the described DMEM substratum.
4. according to claim 1 or the described new organization vasculogenesis of claim 2 method; It is characterized in that: add thaspine behind the new organization vasculogenesis of described lung; Can be inhibited for the new organization blood vessel of lung, and 1822 pairs of colon's vasculogenesis of adding thaspine verivate are inhibited behind the new organization vasculogenesis of described colon.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021180024A1 (en) * | 2020-03-12 | 2021-09-16 | 中国医学科学院基础医学研究所 | Method for constructing mouse tumor model by using single tumor cell |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20020142459A1 (en) * | 2001-03-30 | 2002-10-03 | Williams Stuart K. | Prevascularized constructs for implantation to provide blood perfusion |
| CN101041080A (en) * | 2007-04-20 | 2007-09-26 | 东北师范大学遗传与细胞研究所 | Chicken embryo elk bursa film blood vessel generating model and the analyzing method and use |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020142459A1 (en) * | 2001-03-30 | 2002-10-03 | Williams Stuart K. | Prevascularized constructs for implantation to provide blood perfusion |
| CN101041080A (en) * | 2007-04-20 | 2007-09-26 | 东北师范大学遗传与细胞研究所 | Chicken embryo elk bursa film blood vessel generating model and the analyzing method and use |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021180024A1 (en) * | 2020-03-12 | 2021-09-16 | 中国医学科学院基础医学研究所 | Method for constructing mouse tumor model by using single tumor cell |
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