CN111518746A - Preparation method and application of pellet in corneal micro-pocket surgery experiment - Google Patents
Preparation method and application of pellet in corneal micro-pocket surgery experiment Download PDFInfo
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- CN111518746A CN111518746A CN202010418577.XA CN202010418577A CN111518746A CN 111518746 A CN111518746 A CN 111518746A CN 202010418577 A CN202010418577 A CN 202010418577A CN 111518746 A CN111518746 A CN 111518746A
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Abstract
The invention discloses a preparation method of pellets in a corneal micro-pocket surgery experiment, which comprises the following steps: (1) the preparation of growth factors and the subpackaging process of Matrigel; (2) preparing Matrigel; (3) and a process for preparing the pellet; and scraping off the micro-pills by using a sterile blade when in operation. The invention also discloses application of the pellet in research on angiogenesis and lymphangiogenesis in corneal micro-pocket operation experiments, which is characterized in that in the experimental research on mice, the pellet is implanted into the cornea under an anatomical microscope in an anesthesia state of the mice, and the angiogenesis condition of the cornea of the mice can be observed by photographing with a slit lamp microscope on the 5 th to 7 th days after implantation. The pellet can be prepared as it is, the pellet preparation is simple, the operation is easy, the prepared pellets have the same size and strong uniformity, and the pellet can be prepared fresh according to the needs of each experiment.
Description
Technical Field
The invention belongs to the technical field of biological medicine, relates to a preparation method and application of a pellet in a corneal micro-pocket surgery experiment, and particularly relates to a novel pellet preparation method and application of the pellet in the research of angiogenesis and lymphangiogenesis in the technical process of a common mouse corneal micro-pocket experiment in the research field of angiogenesis and/or lymphangiogenesis.
Background
Tumor and cardiovascular diseases are two major diseases seriously harming human health, and Angiogenesis (angiogenisis) and Lymphangiogenesis (lymphangiogenisis) play important roles in tumor and cardiovascular diseases and are important research directions and treatment targets in the fields of medicine and life science. Clinical treatment with angiogenesis as a target, for example, anti-angiogenesis has achieved certain marked success in tumors and ophthalmic diseases, and angiogenesis promotion is an ideal strategy for treating cardiovascular diseases (such as myocardial ischemia and cardiac hypertrophy). Because the process of angiogenesis and lymphangiogenesis is complex and is a multi-stage process in which multiple cells and multiple factors participate in regulation, although an in vitro model also has certain advantages, an in vivo model is still a reliable choice for researching angiogenesis and lymphangiogenesis.
A mouse cornea micro-pocket model is a common model for researching pathological angiogenesis and lymphangiogenesis, and is characterized in that a cornea without blood vessel distribution is utilized for carrying out micromanipulation, a prepared micro-pill is implanted into the cornea to trigger the cornea to form a new blood vessel or a new lymphangiogenesis, and then the new blood vessels are visually evaluated under a slit lamp microscope, the angiogenesis condition is quantitatively evaluated according to the blood vessel area, or further cell level or molecular level analysis is carried out. Lymphatic vessels can also be visualized by specific molecular markers, thus enabling the study of lymphangiogenesis. In addition, the model can also be used for evaluating the influence of radiation on angiogenin, evaluating angiogenesis regulator drugs or treatment conditions, comparing the influence of different genetic backgrounds on angiogenesis and the like. Since new blood vessels grow in the native avascular tissue cornea, background blood vessels need not be measured, reducing background interference during the experiment. Therefore, the mouse cornea micro-pocket model is a visual, quantifiable, highly repeatable and flexible method for evaluating angiogenesis in vivo.
At present, the mouse cornea micro-pocket model is implanted into the mouse cornea by operation by using standardized sustained-release pellets containing specific growth factors, and the avascular cornea can be triggered to grow new blood vessels within 5 or 6 days. The preparation of pellets is the key of the model, and the pellets are required to be uniform, have no inflammatory activity, and have solubility and uniform distribution of molecules promoting angiogenesis or lymphangiogenesis. At present, the pellet is usually prepared by preparing a substrate from a vascular growth promoting factor such as basic fibroblast growth factor (bFGF) or Vascular Endothelial Growth Factor (VEGF) or a lymphatic growth promoting factor, mixing the substrate with sucralfate (sucralfate), fully mixing the substrate with hydrogen polymer (hydropolymer) added into alcohol to prepare a suspension, pouring the suspension onto a nylon mesh, and airing and tearing open meshes to obtain the pellet. However, the method is long in time consumption, complex in operation, many pellets are produced at one time, the obtained pellets are not strictly identical in size, and the pellets are easy to be unstable after being stored for a long time, so that the improvement of the pellet preparation method is urgently needed.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a preparation method and application of pellets in corneal micro-pocket surgery experiments which are simpler, easy to operate, economical and practical and can be prepared currently, so as to solve the problems that the method provided by the background art is long in time consumption and complex to operate, a lot of pellets are prepared at one time, the obtained pellets are not identical in size strictly and are easy to generate instability after long-time storage.
In order to solve the above technical problems, an embodiment of the present invention provides a method for preparing a pellet in a corneal micro-pocket surgery experiment, which is characterized by comprising the following steps:
(1) preparation of growth factor and subpackaging process of Matrigel
(1-1), VEGF configuration: dissolving VEGFA powder in sterile water, storing at 100ug/ml, and storing at-80 deg.C;
(1-2) dissolving the prepared Heparin in sterile water, storing the solution at the concentration of 25ku/ml, and storing the solution at the temperature of-20 ℃;
(1-3) subpackaging the Matrigel on an ice box by 1 ml/tube, and storing in a refrigerator at-80 ℃;
(2) preparation of Matrigel
(2-1) taking the Matrigel out of a refrigerator with the temperature of-80 ℃ according to the experimental requirement, and standing the Matrigel at the temperature of 4 ℃ overnight to fully dissolve the Matrigel into a liquid state;
(2-2) mixing the Matrigel and DMEM uniformly at a ratio of 1:1 the next day, then adding VEGFA and Heparin into the mixture, and mixing uniformly;
(2-3), the dose of VEGFA is 100 ng/pellet, and the concentration of Heparin is 30U/ml;
(3) process for preparing micropill
(3-1) preparing a sterile 10cm dish, placing the sterile 10cm dish on a sterile operating platform, adjusting a 10ul pipette to 8ul, sucking 8ul of mixed liquid prepared by Matrigel, and dripping the mixed liquid into a sterile culture dish to form a circular drop; then, the dish cover is covered and placed, the mark is made, and the dish cover is placed in a 37-degree incubator to be incubated for 1-2 hours;
(3-2), after incubation, standing at-20 ℃ overnight, and scraping off the pellets with a sterile blade when in operation.
Further, the pellets prepared in the step (3) can be stored in a refrigerator at-20 ℃ for 3 months, and the pellets in a 10cm dish can be scraped off when the pellet is used.
Further, the mixture in step (2) is kept on ice all the time and is operated aseptically.
The embodiment of the invention also provides application of the pellet in research on angiogenesis and lymphangiogenesis in a corneal micro-pocket operation experiment, which is characterized in that in the experimental research on mice, the pellet is implanted into the cornea under an anatomical microscope in an anesthesia state of the mice, and the angiogenesis condition of the cornea of the mice can be observed by photographing with a slit lamp microscope on the 5 th to 7 th days after implantation.
The technical scheme of the invention has the following beneficial effects:
(1) compared with the traditional method, the preparation method of the micro-pocket corneal surgery micro-pocket micro.
(2) The preparation method and the application of the pellet provided by the invention can enable a mouse cornea micro-pocket model to have better application prospect in the relevant research of tumor or ischemic diseases.
Drawings
Fig. 1 is a diagram showing the generation of blood vessels under a slit lamp microscope in a corneal micro-pocket surgery experiment by the pellet prepared by the preparation method in the corneal micro-pocket surgery experiment.
Detailed Description
In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.
Experimental Material
1、Matrigel（BD bioscience）；
2. VEGF (kyoto-yi-wangshu);
3、Heparin（Sigma）；
4、DMEM/HIGH GLUCOSE（HyClone）。
examples
First, preparation of growth factors such as VEGF, Heparin and packaging of Matrigel
Dissolving VEGFA powder in sterile water, storing at 100ug/ml, and storing at-80 deg.C; dissolving Heparin in sterile water, storing at 25ku/ml, and storing at-20 deg.C; the Matrigel was dispensed on an ice box at 1 ml/tube and stored in a freezer at-80 ℃.
Second, preparation of Matrigel
According to the experimental requirements, an appropriate amount of Matrigel was taken out of a-80 ℃ refrigerator and left to stand at 4 ℃ overnight to be sufficiently dissolved into a liquid state. The next day, Matrigel was mixed with DMEM at a 1:1 ratio, and then VEGFA and Heparin were added to the mixture and mixed well. VEGFA was used at 100 ng/pellet and Heparin at a concentration of 30U/ml (note: the mixture was kept on ice at all times and was handled aseptically).
Preparation of pellet
Preparing a sterile 10cm dish, putting the sterile 10cm dish on a sterile operating platform, adjusting a 10ul pipette to 8ul, sucking 8ul of mixed liquid prepared by Matrigel, dripping the mixed liquid into a sterile culture dish to form a circular drop, then covering the dish cover, putting the dish cover, marking, and placing the dish cover in a 37-degree incubator for incubation for 1-2 hours. Then, the mixture is placed at-20 ℃ overnight, and the pellets are scraped off by a sterile blade when the operation is performed.
Fourth, preservation of pellets
If more pellets are needed in the recent experiment, a plurality of pellets can be prepared at one time according to the experiment requirement, the pellets are stored in a refrigerator at the temperature of-20 ℃, and the pellets in 10cm dish can be scraped off when the pellet preparation is used, and the pellets can be stored for 3 months.
Fifth, the subsequent experimental procedures
Under the anesthesia state of the mouse, the pellet is implanted into the cornea under a dissecting microscope, and the angiogenesis condition is observed by taking a picture by a slit lamp microscope on the 5 th to 7 th days after the implantation.
The experimental results are as follows:as shown in fig. 1, the generation of blood vessels under a slit lamp microscope is clearly visible, and the generation of blood vessels of the cornea can be observed under the slit lamp microscope on the 6 th day after the operation by using the pellet prepared by the preparation method of the pellet in the corneal micro-pocket operation experiment. Compared with the pellets prepared by the traditional method, the preparation method of the pellets in the corneal micro-pocket operation experiment is simpler, is easy to operate, is economical and practical, can be prepared at present, and avoids the problem that a large amount of pellets are prepared at one time by the traditional method and need to be stored for a long time.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (4)
1. A preparation method of pellets in corneal micro-pocket surgery experiments is characterized by comprising the following steps:
(1) preparation of growth factor and subpackaging process of Matrigel
(1-1), VEGF configuration: dissolving VEGFA powder in sterile water, storing at 100ug/ml, and storing at-80 deg.C;
(1-2) dissolving the prepared Heparin in sterile water, storing the solution at the concentration of 25ku/ml, and storing the solution at the temperature of-20 ℃;
(1-3) subpackaging the Matrigel on an ice box by 1 ml/tube, and storing in a refrigerator at-80 ℃;
(2) preparation of Matrigel
(2-1) taking the Matrigel out of a refrigerator with the temperature of-80 ℃ according to the experimental requirement, and standing the Matrigel at the temperature of 4 ℃ overnight to fully dissolve the Matrigel into a liquid state;
(2-2) mixing the Matrigel and DMEM uniformly at a ratio of 1:1 the next day, then adding VEGFA and Heparin into the mixture, and mixing uniformly;
(2-3), the dose of VEGFA is 100 ng/pellet, and the concentration of Heparin is 30U/ml;
(3) process for preparing micropill
(3-1) preparing a sterile 10cm dish, placing the sterile 10cm dish on a sterile operating platform, adjusting a 10ul pipette to 8ul, sucking 8ul of mixed liquid prepared by Matrigel, and dripping the mixed liquid into a sterile culture dish to form a circular drop; then, the dish cover is covered and placed, the mark is made, and the dish cover is placed in a 37-degree incubator to be incubated for 1-2 hours;
(3-2), after incubation, standing at-20 ℃ overnight, and scraping off the pellets with a sterile blade when in operation.
2. The method for preparing pellets in corneal micro-pocket surgery experiments according to claim 1, wherein the pellets prepared in step (3) can be stored in a refrigerator at-20 ℃ for 3 months, and the pellets in 10cm dish can be scraped off when being used.
3. The method for preparing a micro pill for corneal micro-pocket surgery experiments as claimed in claim 1, wherein the mixture in step (2) is kept on ice all the time and is aseptically processed.
4. The application of the pellet in research on angiogenesis and lymphangiogenesis in corneal micro-pocket surgery experiments is characterized in that in the experimental research on mice, the pellet is implanted into the cornea under an anatomical microscope in an anesthetized state of the mice, and the angiogenesis condition of the cornea of the mice can be observed by photographing with a slit lamp microscope on the 5 th to 7 th days after implantation.
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| CN202010418577.XA CN111518746A (en) | 2020-05-18 | 2020-05-18 | Preparation method and application of pellet in corneal micro-pocket surgery experiment |
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| CN202010418577.XA CN111518746A (en) | 2020-05-18 | 2020-05-18 | Preparation method and application of pellet in corneal micro-pocket surgery experiment |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW394844B (en) * | 1994-03-04 | 2000-06-21 | Univ Australian | In-vitro angiogenesis assay |
| CN1741808A (en) * | 2002-11-25 | 2006-03-01 | 阿特努奥恩公司 | Peptides which inhibit angiogenesis, cell migration, cell invasion and cell proliferation, compositions and uses thereof |
| WO2008077236A1 (en) * | 2006-12-22 | 2008-07-03 | Bioaxone Therapeutique Inc. | Adp-ribosyl transferase fusion variant proteins |
| CN101583367A (en) * | 2006-11-07 | 2009-11-18 | 渗透治疗有限公司 | Materials and methods for treating and managing angiogenesis-mediated diseases |
| TW201532611A (en) * | 2014-02-26 | 2015-09-01 | Univ Nat Cheng Kung | Use of pharmaceutical composition in manufacturing drugs for treating cancer |
-
2020
- 2020-05-18 CN CN202010418577.XA patent/CN111518746A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW394844B (en) * | 1994-03-04 | 2000-06-21 | Univ Australian | In-vitro angiogenesis assay |
| CN1741808A (en) * | 2002-11-25 | 2006-03-01 | 阿特努奥恩公司 | Peptides which inhibit angiogenesis, cell migration, cell invasion and cell proliferation, compositions and uses thereof |
| CN101583367A (en) * | 2006-11-07 | 2009-11-18 | 渗透治疗有限公司 | Materials and methods for treating and managing angiogenesis-mediated diseases |
| WO2008077236A1 (en) * | 2006-12-22 | 2008-07-03 | Bioaxone Therapeutique Inc. | Adp-ribosyl transferase fusion variant proteins |
| TW201532611A (en) * | 2014-02-26 | 2015-09-01 | Univ Nat Cheng Kung | Use of pharmaceutical composition in manufacturing drugs for treating cancer |
Non-Patent Citations (3)
| Title |
|---|
| KUNIHIRO ASANUMA ET AL.,: "Protein C inhibitor inhibits breast cancer cell growth, metastasis and angiogenesis independently of its protease inhibitory activity", 《INT. J. CANCER》 * |
| ROBERT AUERBACH ET AL.,: "Angiogenesis assays: Problems and pitfalls", 《CANCER AND METASTASIS REVIEWS》 * |
| 陈 昕等: "定量研究血管新生的 Matrigel plug 体内模型建立", 《宁夏医科大学学报》 * |
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