Construction and evaluation method of novel blood brain barrier model

Abstract

The invention provides a construction and evaluation method for establishing a novel blood brain barrier model. The method solves the following technical problems: (1) the influence of the change of binding protein related factors on the blood brain barrier is proved by a time gradient, and the optimal time for opening and closing the blood brain barrier is found to confirm whether the influence of illumination on the blood brain barrier is instant opening and instant closing; (2) advantages and disadvantages of a single cell culture model and a co-cell culture blood brain barrier model; (3) finding out the optimal illumination time and intensity according to the change and toxicity of the illumination time and intensity on binding proteins and related factors; (4) black phosphorus is added, and the influence on cells and related factors is tested through illumination to serve as a control group of pure illumination time intensity. And a research idea related to the blood-brain barrier aspect is provided for treating neurological degenerative diseases.

Description

Technical Field

[0001] This invention relates to the field of neurobiomedicine, and in particular to a novel method for constructing and evaluating a blood-brain barrier model. Background Technology

[0002] The future development trend of blood-brain barrier models will be to gradually evolve in vitro models that more closely resemble the brain's microenvironment, while ensuring ease of operation and cost-effectiveness, so that experimental data more accurately reflect reality. Initially, researchers simply cultured cerebral vascular endothelial cells, and subsequently introduced astrocytes and pericytes for co-culture. Currently, modeling methods have become more diversified. In vitro models play an irreplaceable role in studies of drug permeability and pathological changes in the blood-brain barrier.

[0003] Methods for assessing the integrity of the blood-brain barrier (BBB) ​​each have their advantages and disadvantages, and face numerous challenges in clinical application. Currently, the commonly used clinical assessment method involves invasively extracting cerebrospinal fluid (CSF). This method is not only invasive but can also lead to infection and other complications. Furthermore, CSF extraction only provides localized information and cannot comprehensively reflect the condition of the entire BBB. Non-invasive techniques are more in line with future development trends, but existing non-invasive detection methods still have many shortcomings. For example, the range of biomarkers is limited, making it impossible to comprehensively assess the functional status of the BBB; the established models are complex and expensive, increasing the cost of drug screening. Moreover, whether these models can accurately reflect the actual situation in the human brain remains an urgent problem to be solved. As research into the blood-brain barrier deepens, increasingly sophisticated in vitro models are expected to emerge. However, current models that closely approximate the BBB are typically complex and expensive, which not only increases research costs but also limits their widespread application in clinical practice and research. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a novel method for constructing and evaluating a blood-brain barrier model.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solution: (1) The time gradient test combined with the effect of changes in protein-related factors on the blood-brain barrier was used to find the optimal time for opening and closing the blood-brain barrier, in order to confirm whether the effect of light on the blood-brain barrier is instantaneous opening and instantaneous closing. (2) Advantages and disadvantages of single-cell culture model and co-cell culture blood-brain barrier model. (3) The effects of light duration and intensity on the changes and toxicity of binding proteins and related factors, and to find the optimal light duration and intensity. (4) The effects of black phosphorus and light on cells and related factors, serving as a control group for light intensity and duration alone.

[0006] In this invention, step (1) specifically includes: seeding immortalized mouse brain microvascular endothelial cells bEnd.3 onto a Transwell membrane, detecting their resistance value using a cell resistance meter, and achieving a resistance value of 200 Ω·cm approximately 8-9 days after cell seeding. 2 .

[0007] In this invention, step (2) specifically includes: setting 808nm near-infrared light to irradiate bEnd.3 cells seeded in a 96-well plate for different times, with the light irradiation time gradient set to 5min, 10min, 15min, 20min, 25min, and 30min.

[0008] In this invention, step (3) specifically includes: performing Western blotting experiments on the structural proteins ZO-1, Claudin-5, Occludin, as well as the transport proteins P-glycoprotein, ATP-binding cassette transporter, and advanced glycosylation receptor of the blood-brain barrier model exposed to light to test the changes in their protein content.

[0009] Secondly, the establishment of the blood-brain barrier and the determination of its impact after irradiation with 808nm near-infrared light.

[0010] Thirdly, a method for evaluating the effect of black phosphorus nanomaterials and near-infrared light irradiation on the blood-brain barrier structure.

[0011] The present invention has the following beneficial effects:

[0012] In order to effectively solve the technical problems mentioned in the background, the present invention provides a novel method for constructing and evaluating a blood-brain barrier model.

[0013] This invention establishes and utilizes a more rational and optimized in vitro BBB model, enabling a more comprehensive understanding of the actual BBB penetration of drugs.

[0014] This invention verifies the safety of black phosphorus nanomaterials using photothermal effects as an adjunct therapy within the time it takes for the blood-brain barrier to open, providing a new research approach for the application of near-infrared light to open the blood-brain barrier and deliver drugs.

[0015] Our research aims to simplify the testing procedure as much as possible and reduce costs to expand its application in medical research and advance the diagnosis and treatment of blood-brain barrier-related diseases. This invention has great potential for treating Alzheimer's disease. Detailed Implementation

[0016] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0017] Unless otherwise specified, the terms used in this specification have the same meaning as those commonly understood by those skilled in the art; however, in the event of any conflict, the definitions in this specification shall prevail.

[0018] The terms “comprising,” “including,” “containing,” “having,” “comprising,” or other variations thereof are intended to cover non-closed inclusion, and no distinction is made between these terms. The term “comprising” means that other steps and ingredients may be added without affecting the final result. The term “comprising” also includes the terms “consisting of” and “substantially consisting of”. The compositions and methods / processes of the present invention comprise, consist of, and substantially consist of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0019] All numerical values ​​or expressions relating to component amounts, process conditions, etc., used in the specification and claims are to be understood to be modified with “about” in all cases. All ranges relating to the same component or property include endpoints that can be independently combined. Because these ranges are continuous, they include every value between the minimum and maximum values. It should also be understood that any numerical range referenced in this application is intended to include all subranges within that range.

[0020] The present invention will now be described in detail with reference to the embodiments. These embodiments are merely preferred implementations of the present invention and are not intended to limit the present invention.

[0021] Example 1

[0022] This embodiment provides a method for constructing a blood-brain barrier model, wherein the specific method includes the following steps: (1) Immortalized mouse brain microvascular endothelial cells bEnd.3 were seeded onto Transwell membranes. (2) The resistance value was measured using a cell resistance meter, and the measurement was performed every 3 days. (3) The cell resistance value of bEnd.3 cells inoculated in (1) was measured. The resistance value reached 200 Ω·cm in about 8-9 days. 2 .

[0023] Example 2

[0024] This embodiment provides a novel method for evaluating a blood-brain barrier model, wherein the evaluation method specifically includes the following steps: (1) Construct a BBB model and explore the cell density of the model through parameters such as cell resistance value and intercellular space analysis. (2) The effects of NIR irradiation on cytotoxicity in cell models were investigated using CCK-8 and cell live / dead staining imaging methods.

[0025] Example 3 (1) The changes in the protein content of structural proteins ZO-1, Claudin-5 and Occludin in the blood-brain barrier model exposed to light were tested by Western blotting. (2) Starting from the physiological function of the blood-brain barrier, we explored the changes in protein content of transport proteins P-glycoprotein, ATP-binding cassette transporters, and late glycosylation advanced product receptors after NIR irradiation with different gradients. We used Western blotting experiments to test these changes. (3) By detecting oxidative stress and apoptosis-related cell metabolism in the blood-brain barrier model, we can confirm whether NIR irradiation has any effect on the cell model.

[0026] The above embodiments merely illustrate the implementation of the present invention, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the present invention. Any technical solutions obtained by adopting equivalent substitutions or equivalent transformations should fall within the protection scope of the present invention.

Claims

1. Using time gradients to demonstrate the effect of changes in protein-related factors on the blood-brain barrier, we aim to identify the optimal times for opening and closing the blood-brain barrier, in order to confirm whether the effect of light on the blood-brain barrier is instantaneous opening and closing.

2. To investigate the effects of light exposure time and intensity on the changes and toxicity of binding proteins and related factors, and to identify the optimal light exposure time and intensity.

3. Starting from the structure and function of the blood-brain barrier, we detected the changing trends of proteins related to adhesion and tight junctions of the blood-brain barrier after the blood-brain barrier model was irradiated with NIR at different gradients.

4. Starting from the physiological function of the blood-brain barrier, this study investigates the changes in transport proteins and regulatory factors of the blood-brain barrier model after irradiation with different gradients of NIR.

5. By detecting oxidative stress and apoptosis-related cellular metabolism in the blood-brain barrier model, we can confirm whether NIR irradiation has any effect on this cell model.

6. Effects of black phosphorus and light on cells and related factors, serving as a control group for light intensity and duration alone.

7. The application of the establishment and evaluation method as described in any one of claims 1-6 in a blood-brain barrier model.