CN102321568B

CN102321568B - New vascular tissue generation method

Info

Publication number: CN102321568B
Application number: CN201110231591XA
Authority: CN
Inventors: 张彦民; 贺浪冲; 代秉玲; 王楠; 郑蕾; 王嗣岑; 张�杰
Original assignee: Xian Jiaotong University
Current assignee: Xian Jiaotong University
Priority date: 2011-08-12
Filing date: 2011-08-12
Publication date: 2013-12-04
Anticipated expiration: 2031-08-12
Also published as: CN102321568A

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

A kind of New vascular tissue generation method

Technical field

The invention belongs to compound pharmacological action and field of molecular biotechnology, be specifically related to a kind of New vascular tissue generation method.

Background technology

Vasculogenesis (angiogenesis) refers to regeneration neovascularity on the basis of original vescular bed, is the process that is subject to multi factor control of a complexity, and it is different from the blood vessel generating process.Vasculogenesis be one by cytokine profiles 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 chamber subsequently, be subject to the minute adjustment of angiogenic stimulators and supressor.Physiological vasculogenesis is more common in wound healing, or the vasculogenesis of Cyclic Human Endometrium, but is a kind of pathologic phenomenon in some processes such as tumour, arteriosclerosis, psoriatic, diabetic retinopathy and endometriosis.Vasculogenesis is absolutely necessary in the generation of tumour and transfer process, the growth of most malignant tumours and to shift be all that blood vessel relies on, and tumor tissues can be induced the generation of neovascularity in its process of growth.The vasculogenesis phenomenon that this is induced in Tumor Growth, be referred to as tumor angiogenesis.The blocking-up vasculogenesis is a kind of means and approach of new treatment tumour.Destroy or suppress the new vessel generation of tumour, effectively stop the growth of tumour and the medicine of transfer to be referred to as tumor angiogenesis 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 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 simple, convenient and model that New vascular tissue that can be widely used generates.

Summary of the invention

The deficiency existed in order to overcome above-mentioned prior art, the object of the present invention is to provide a kind of New vascular tissue generation method, overcome the quantitative loaded down with trivial details and not high shortcoming of surviving rate, set up a kind of simple, convenient and model that New vascular tissue that can be widely used generates, the method can also be applied to the screening study of angiogenesis inhibitor.

In order to achieve the above object, the technical solution adopted in the present invention is:

A kind of New vascular tissue generation method, get mouse or rat chest aorta under aseptic condition, the heart, liver, spleen, lung, blood vessel, kidney and colon, the PBS liquid of putting into immediately precooling 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 cleaning with PBS liquid, under magnifying glass, the arterial ring that to cut into length in the DMEM of precooling substratum be 0.3mm or be cut into the artery piece that length is 0.3mm, and the heart, liver, spleen, lung, the treatment process of kidney and colon is for cutting with ophthalmologic operation and tweezers are removed after organizing peripheral adhesion organization and are cut into 1mm ³the tissue block of size, getting fibrinogen solution is laid in culture plate, every pore capacities of culture plate is 200 μ L, add zymoplasm 1 μ L in every hole of culture plate, shake up rear formation gel, the tissue block cut is planted on the gel of culture plate, add Fibrinogen solvent 200 μ L and zymoplasm 1 μ L again in every hole of culture plate, shake up rear formation gel, so just formed the sandwich structure of sandwich style, add the DMEM substratum 1mL containing mass concentration 3-10% foetal calf serum on this sandwich structure, put into the CO that volumetric concentration is 5% ₂with in the temperature incubator that is 37 ℃, cultivate, just can generate each self-corresponding New vascular tissue 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 vascular tissue of colon is followed successively by: the New vascular tissue of the New vascular tissue>blood vessel of the New vascular tissue >=colon of the New vascular tissue>kidney of New vascular tissue >=heart of the New vascular tissue>liver of the New vascular tissue >=spleen of lung, the process that its Midcolic New vascular tissue generates is divided into endotheliocyte and moves out and form two kinds of blood vessel and vasculogenesis.

Described DMEM substratum comprises VEGF or bFGF somatomedin.

The New vascular tissue of described lung adds thaspine after generating, can be inhibited for the New vascular tissue of lung, and the New vascular tissue of described colon adds 1822 pairs of colon's vasculogenesis of thaspine derivative inhibited after generating.

By a kind of New vascular tissue generation method, generate each self-corresponding New vascular tissue of aorta, the heart, liver, spleen, lung, kidney and colon by get mouse or rat chest aorta, the heart, liver, spleen, lung, kidney and colon under aseptic condition in conjunction with culture plate, overcome the quantitative loaded down with trivial details and not high shortcoming of surviving rate, set up a kind of simple, convenient and model that New vascular tissue that can be widely used generates, the method can also be applied to the screening study of angiogenesis inhibitor.

The accompanying drawing explanation

The angiogenic growth speed reduced co-ordinates figure of the various tissues of Fig. 1;

The affect coordinate diagram of Fig. 2 thaspine on the lung tissue vasculogenesis;

The affect coordinate diagram of Fig. 3 thaspine derivative 1822 on colon's vasculogenesis.

Embodiment

Below in conjunction with drawings and Examples, the present invention will be described in more detail.

New vascular tissue generation method, get mouse or rat chest aorta under aseptic condition, the heart, liver, spleen, lung, kidney and colon, the PBS liquid of putting into immediately precooling 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 cleaning with PBS liquid, under magnifying glass, the arterial ring that to cut into length in the DMEM of precooling substratum be 0.3mm or be cut into the artery piece that length is 0.3mm, and the heart, liver, spleen, lung, blood vessel, the treatment process of kidney and colon is for cutting with ophthalmologic operation and tweezers are removed after organizing peripheral adhesion organization and are cut into 1mm ³the tissue block of size, getting fibrinogen solution is laid in 48 well culture plates, 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 rear formation gel, the tissue block cut is planted on the gel of 48 hole culture hole, add Fibrinogen solvent 200 μ L and zymoplasm 1 μ L again in every hole of 48 hole culture hole, shake up rear formation gel, so just formed the sandwich structure of sandwich style, add the DMEM substratum 1mL containing the 3-10% foetal calf serum on this sandwich structure, put into the CO that concentration is 5% ₂with in the temperature incubator that is 37 ℃, cultivate, just can generate each self-corresponding New vascular tissue of aorta, the heart, liver, spleen, lung, kidney and colon with this.Described DMEM substratum comprises VEGF or bFGF somatomedin.After 3 days, the various tissue blood vessels of micro-Microscopic observation generate situation, 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 described generation aorta, the heart, liver, spleen, lung, the speed speed of kidney and each self-corresponding New vascular tissue of colon is followed successively by: the New vascular tissue of the New vascular tissue>blood vessel of the New vascular tissue >=colon of the New vascular tissue>kidney of New vascular tissue >=heart of the New vascular tissue>liver of the New vascular tissue >=spleen of lung, the process that its Midcolic New vascular tissue generates is divided into endotheliocyte and moves out and form two kinds of blood vessel and vasculogenesis.That at the New vascular tissue of lung, cultivates in addition adds thaspine on the 3rd day, the DMEM nutrient solution that contains mass concentration 3-10% foetal calf serum of take is the blank group, thaspine group low, in and high density be respectively 2.32, 4.63 and 9.25 μ mol/L, changed liquid every 3 days, establish 5 multiple holes for every group, carried out the tissue blood vessel counting at the 9th day, take the time as X-coordinate, the New vascular tissue number of lung is that ordinate zou is mapped respectively, comparative result is shown in Fig. 2, that at the New vascular tissue of colon, cultivates in addition adds thaspine derivative 1822 on the 3rd day, the DMEM nutrient solution that contains mass concentration 3-10% foetal calf serum of take is the blank group, thaspine derivative 1822 low, in and high density be respectively 4, 12 and 36 μ mol/L, changed liquid every 3 days, establish 5 multiple holes for every group, carried out the tissue blood vessel counting at the 9th day, take the time as X-coordinate, the New vascular tissue number of colon is that ordinate zou is mapped respectively, comparative result is shown in Fig. 3, the comparative result of synthesizing map 2 and Fig. 3 like this, the New vascular tissue of known described lung adds thaspine after generating, can be inhibited for the New vascular tissue of lung, and the New vascular tissue of described colon adds 1822 pairs of colon's vasculogenesis of thaspine derivative inhibited after generating.

Claims

1. a New vascular tissue generation method, it is characterized in that: get mouse or rat chest aorta under aseptic condition, the heart, liver, spleen, lung, kidney and colon, the PBS liquid of putting into immediately precooling 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 cleaning with PBS liquid, under magnifying glass, the arterial ring that to cut into length in the DMEM of precooling substratum be 0.3mm or be cut into the artery piece that length is 0.3mm, and the heart, liver, spleen, lung, the treatment process of kidney and colon is for cutting with ophthalmologic operation and tweezers are removed after organizing peripheral adhesion organization and are cut into 1mm ³the tissue block of size, getting fibrinogen solution is laid in culture plate, every pore capacities of culture plate is 200 μ L, add zymoplasm 1 μ L in every hole of culture plate, shake up rear formation gel, the tissue block cut is planted on the gel of culture plate, add Fibrinogen solvent 200 μ L and zymoplasm 1 μ L again in every hole of culture plate, shake up rear formation gel, so just formed the sandwich structure of sandwich style, add the DMEM substratum 1mL containing mass concentration 3-10% foetal calf serum on this sandwich structure, put into the CO that volumetric concentration is 5% ₂with in the temperature incubator that is 37 ℃, cultivate, just can generate each self-corresponding New vascular tissue 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 vascular tissue of colon is followed successively by: the New vascular tissue of the New vascular tissue >=spleen of lung > New vascular tissue of New vascular tissue >=heart of liver > New vascular tissue of New vascular tissue >=colon of kidney > New vascular tissue of blood vessel, the process that its Midcolic New vascular tissue generates is divided into endotheliocyte and moves out and form two kinds of blood vessel and vasculogenesis.

2. New vascular tissue generation method according to claim 1, it is characterized in that: described DMEM substratum also contains VEGF or bFGF somatomedin.