TWI548873B - Method for quick evaluating neural function by establishing analysis module - Google Patents

Description

Establishing an analysis module to quickly evaluate neural function

The present invention relates to a method for establishing an analysis module for rapid assessment of neural function, in particular to a method for rapidly and representatively analyzing nerve growth function, including using an important marker sufficient for representing nerve growth, and optimizing with The image analysis process and program instructions; according to this, the method can not only quickly evaluate the early nerve growth and neural network formation, but also serve as a platform for screening drugs to quickly detect the effects of drugs on nerve function.

With the advancement of cancer treatment, the survival rate of patients has gradually increased. Due to the prolonged life of patients, the side effects of chemotherapy drugs have been paid attention to. Especially in the side effects of chemotherapy drugs, neuropathy (neuropathy) has the greatest impact on quality of life. Therefore, before receiving chemotherapy drugs, how to effectively test or screen drugs for neurotoxicity of nerve cells suffering from chemotherapy has become a research and development focus in related fields.

In methods for detecting whether a drug is cytotoxic, high-content analysis (HCA) is often used in conjunction with high-content analysis (HCA) to understand the effect of the drug on cell type. High-density analysis techniques have been widely used in biotechnology fields such as cancer, immunology, cardiovascular and neurological research, and with high-throughput screening to automate cellular fluorescence imaging and image analysis in porous discs. The experiment was carried out on a large scale and quickly, and a variety of quantitative information was available. However, there are still many problems in using molecular markers with high-throughput analysis to detect neural function; for example, Huang Y et al. revealed a high content neuron-based screening to explore neural cell types. State of computational architecture, which uses neuronal type III β-tubulin (TUJ1) as a molecular marker to study neuronal mechanisms (J Neurosci Methods. 2010 Jul 15;190(2):299-309); Harrill JA A high-intensity image analysis was used to detect the effect of chemicals on the growth of primary dendritic neurons in primary rat cortical neurons. The cells were stained with tubulin (β _III tubulin) (all) to deduct microtubule-associated protein 2 After (MAP2) staining area (dendritic), the remaining area represents the way of axon, analyzing the growth of nerve cells (Neurotoxicology. 2013 Jan; 34: 61-73); Nieland TJ et al. using MAP2, synapse Post-density protein 95-synaptic protein 1 (Psd95-Syn1), and postsynaptic protein-synaptic protein 1 (Gphn-Syn1) identify synaptic production (PLoS One.2014 Mar 14;9(3):e91744 ); the above method is only By a single way of neuronal axis outgrowth or synaptogenesis, it is impossible to comprehensively determine the nerve function; if you observe the axis, dendritic growth and synapse formation of nerve cells at the same time, you need to use a variety of Molecular markers, so the process is quite cumbersome and time consuming.

Furthermore, which kind of nerve cells are used for drug testing will also affect the analysis procedure and time; for example, although commercialized neural cell strains are better cultured, in order to stimulate nerve cell differentiation, additional drug stimulation is necessary, which may affect Test the role of the drug. In contrast, primary culture cells can be naturally differentiated, so the use of primary cultured cells can solve the problem of additional drugs, but compared with the use of cell lines, primary cell culture is not easy, and how to achieve the best differentiation time For drug testing, further investigation is needed.

Therefore, if you can screen out several representative molecular markers, and develop a set of The integrated functional assessment system, as a benchmark for assessing early nerve growth and neural network formation, provides a good balance between time-saving and complete assessment, and will quickly, massively and accurately assess neuronal survival and detect neurological function. Variety.

Nowadays, the inventor has made many improvements in the actual implementation of the method for assessing changes in neurological function. Therefore, it has been improved by its rich professional knowledge and years of practical experience, and has been developed accordingly. this invention.

The main object of the present invention is to provide a method for establishing an analysis module for rapid assessment of neural function, which is a rapid and representative method for analyzing nerve growth function, including using an important marker sufficient for representing nerve growth, and optimizing with The image analysis process and program; accordingly, the method can be further used as a platform for screening drugs to quickly detect whether the drug has protective or toxic effects on nerve cells.

In order to achieve the above-mentioned implementation object, the present invention provides a method for rapidly analyzing a nerve function by analyzing a module, which comprises the first step: using a fluorescence microscopic image system to capture a plurality of fluorescing cursors (including Hoechst dye to mark the nucleus) The cultured cells are subjected to image analysis, wherein the cultured cell line comprises nerve cells; Step 2: according to the size of the nucleus and the intensity of the fluorescence, the nerve cells of the expression axis and the dendritic cursor are selected, the non-neuronal cells are excluded, and the calculation is performed. The size of the nerve cell body, and the length of axons, dendrites and the number of branches, to confirm the axis of the nerve cells, dendritic growth, wherein the axis, dendritic cursor can be selected from microtubule-associated protein 2 (MAP2) Antibody or neuronal type III β-tubulin (TUJ1), while synaptic globose cursor is selected from Synaptophysin antibody or postsynaptic density protein 95 (PSD95); and step three: according to step two Axis, dendritic firefly cursor to determine the range, calculate the synapse on the nerve cells (synapse) The number is used to confirm the growth of nerve cells to evaluate nerve function.

In one embodiment of the present invention, the cultured cell line is prepared by the following steps: Step 1: Take a cerebral cortex tissue of a young mouse from 0 to 1 day after birth, and add a first medium (for example, serum-free DMEM) /F12 medium) to break up the cerebral cortex tissue to form a mixed solution; Step 2: Filter the mixture and obtain a cell pellet by centrifugation; and Step 3: Culture the cell pellet in a second medium 9~ 12 days (preferably 10 days) to prepare cultured cells; wherein the second medium contains Neurobasal ^® A medium (Neurobasal ^® A medium), L-glutamin, B-27 ^® Additive (B-27 ^® Supplement), and penicillin-streptomycin.

(S1)‧‧‧Step one

(S2)‧‧‧Step 2

(S3) ‧ ‧ Step 3

The first figure: the flow chart of the steps of the method for quickly analyzing the neural function of the analysis module of the present invention

Figure 2: Schematic diagram of nerve cells

Figure 3: Representative representation of nerve cells in different culture days

Figure 4: Effect of different culture days on the number of nerve cells

Figure 5: Effect of different culture days on the growth of nerve cells

Figure 6: Effect of different culture days on the number of synapses

Figure 7: Effect of different chemotherapy drugs on the number of nerve cells

Figure 8: Effect of different chemotherapy drugs on the number of synapses

Figure 9: Effect of different chemotherapy drugs on the length of axial dendrites

The object of the present invention and its structural and functional advantages will be explained in conjunction with the specific embodiments according to the structure shown in the following drawings, so that the reviewing committee can have a more in-depth and specific understanding of the present invention.

First, referring to the first figure, the method for establishing an analysis module for quickly evaluating nerve function includes the following steps: Step 1 (S1): using a fluorescence microscopic image system to capture a plurality of cursors The cultured cells (containing Hoechst stain to label the nucleus) for image analysis, wherein the cultured cell line contains nerve cells; Step 2 (S2): according to the size of the nucleus and the intensity of the fluorescence, select the expression axis, the dendritic cursor The nerve cells, exclude non-neuronal cells, and calculate the size of the nerve cell body, and the length of axons, dendrites and the number of branches to confirm the axis of the nerve cells, dendritic growth, where the axis, the dendritic It may be selected from a microtubule-associated protein 2 (MAP2) antibody or a neuronal type III β-tubulin (TUJ1), and the synaptophysin cursor is selected from a synaptophysin antibody or a post-synaptic density protein 95 ( PSD95); and step 3 (S3): According to the axis of step 2 and the range of the dendritic cursor, calculate the number of synapse cursors on the nerve cells to confirm the growth of the nerve cells. Assess nerve function. Among them, the schematic diagram of the nerve cells is shown in the second figure.

The "culturing cells" described in the above step 1 can be prepared, for example, but not limited to, by the following steps: Step 1: Take the cerebral cortex tissue of a young mouse (best mouse or rat 0 to 1 day after birth), and add A first medium (for example, serum-free DMEM/F12 medium) is used to break up cerebral cortical tissue to form a mixed solution; Step 2: filtering the mixture and obtaining a cell pellet by centrifugation; and Step 3: The cell pellet is cultured in a second medium (for example, Neurobasal ^® A medium (Neurobasal ^® A medium), L-glutamin, B-27 ^® Additive (B-27 ^® Supplement), And 9 to 12 days (preferably 10 days) in penicillin-streptomycin to prepare cultured cells.

In addition, the scope of the invention may be further exemplified by the following specific examples, which are not intended to limit the scope of the invention.

Example 1: Primary culture of cells and immunofluorescence staining

Compared with the method of inducing differentiation of the traditional cell line, the method of the primary culture of the nerve cell is closer to the state of the nerve cell, and the formation of the early neural network can be evaluated, and the experimental result is not affected by the differentiation drug, so the present invention is better. Primary cells were cultured using the original.

(1) Primary culture cells

The mouse cortex was taken from 0 to 1 day after birth (B6 mice pup). After removal of the meninges, the tissues were dispersed in serum-free DMEM/F12 medium to make up the volume of serum-free DMEM/F12. 5mL, add 0.5mL 1X trypsin (trypsin) to mix, water bath at 37 ° C for 5 minutes, use a 70μm filter membrane to infiltrate 1mL fetal bovine serum (FBS) to filter impurities, centrifuge 3,000rpm for 10 minutes, remove the supernatant To the solution, add the precipitated cells to the required amount of medium (medium preparation: Neurobasal ^® A medium 500 mL, L-glutamin 100X 1.3 mL, B-27 ^® Supplement 50 X 10 mL/vial, Penicillin/Streptomycin 100X 2.5 mL) for nerve Cell culture.

(2) The nerve cells were cultured using a transparent thin-bottom microporous black disk, and 70 μL of poly-D-lysine or poly-L-lysine was added to each well in the culture plate for coating. The cells were cultured in 2.8×10 ⁴ cells per well; after 7, 10, 14 and 21 days in vitro, the cells were fixed with 4% polyformaldehyde for 10 minutes, washed with PBS for 5 minutes twice; 1 M glycine was applied for 10 minutes, and then washed with PBS for 5 minutes twice; 0.05% Triton X-100 was added for 20 minutes, and washed with PBS for 5 minutes twice. Receiver, after blocking with 1% bovine serum albumin (BSA) for 1 hour, add primary antibody: (1) microtubule-associated protein 2 antibody (MAP2 antibody; MyBioSource; MBS502140) and (2) synaptic protein antibody (Anti-Synaptophysin antibody; Abcam; ab32127) for 12 to 16 hours; rinsed with PBST for 10 minutes twice, and added secondary antibody with a fluorescent cursor, respectively Alexa Fluor 488 (Jackson ImmunoResearch; 703-546- 155) and Alexa Fluor 647 (molecular probes; A31573), and adding Hurst dye (Hoechst 33258) for 1 hour, rinsing with PBST for 10 minutes 2 times, finally adding PBS, and performing fluorescence image extraction and analysis.

Embodiment 2: Image capture and image analysis

The image was taken using a fluorescence microscopic imaging system and image analysis of the above cultured cells was performed. Three kinds of fluorescent wavelengths DAPI, FITC, and Cy5 were used, and images were automatically taken for analysis.

For accurate analysis of neural function, use MetaXpress 3.1 software (website ftp://ftp.meta.moleculardevices.com/pub/uic/software/MX31R13/HelpDocs/MetaXpress/MetaXpress_3_1_Anaiysis_Guide.pdf) with the title "MetaXpress ^® Image Acquisition and Analysis It is found in the Software (Analysis Guide), which is hereby incorporated by reference in its entirety in its entirety herein in its entire entire entire entire entire entire entire entire entire entire entire portion Automated analysis was performed according to the following optimized image analysis indicators (a) to (c) in a total of 32 steps.

(a) defining nerve cells

Because cultured cells contain nerve cells and glial cells, This is based on the size of the nuclear area indicated by the Hoechst stain and the intensity of the fluorescence, with the MAP2 antibody to select the true nerve cells; for example, the built-in options in the MetaXpress 3.1 software can be set in 16 steps in the following order:

1: Configure Count Nuclei Data Log()

2: Configure Count Nuclei Summary Log()

3: Overwrite "Nuclear_All"=Count Nuclei(Src="DAPI")

4: Threshold Image ([3: Count Nuclei], 1, 65535, Inclusive)

5: Create Regions Around Objects([3:Count Nuclei])

6: Transfer Regions([3:Count Nuclei],"DAPI",ALLREGIONS and CLEARSOURCE and CLEARDEST)

7: Preferences()

8: Threshold Image["DAPI",1,65535,Inclusive]

9: Integrated Morphometry-Load State("NeuriteNuclear test")

10: Integrated Morphometry-Measure("DAPI")

11: Integrated Morphometry-Create Objects Mask()

12: Untitled=New(1392,1040,16,0)

13: Overwrite "Nuclear_IMA"=[12:New]+"IMA Objects Mask"

14: Configure Neurite Outgrowth Data Log()

15: Configure Neurite Outgrowth Summary Log()

16:[None]=Neurite Outgrowth(NeuriteSrc="FITC",NuclearSrc=[13:Arithmetic],NuclearDest=Overwrite"Nuclear_Neurite").

In this way, all the nuclei in the image are first counted, and then all the nuclei are distinguished from the fluorescence intensity by the size of the cell, and the nuclei of the cell and the neuronal cell marker MAP2 are overlapped to obtain true nerve cells.

(b) Analysis of the axis and dendritic growth of nerve cells

After selecting the nerve cells expressing microtubule-associated protein 2 (MAP2), calculate the size of the nerve cell body, and the length of axons, dendrites and the number of branches to confirm the axis and dendritic growth of the nerve cells; The built-in options in MetaXpress 3.1 software are set in 6 steps in the following order:

17: Threshold Image([16:Neurite Outgrowth], 1,65535, Inclusive)

18: Overwrite "NeuriteNuclear_Binary"=Clip Image(16,[16:Neurite Outgrowth])

19: Overwrite "Add"="FITC"+[18:Clip Image]

20: Configure Neurite Outgrowth Data Log()

21: Configure Neurite Outgrowth Summary Log()

22: Overwrite "Neurite"=Neurite Outgrowth(NeuriteSrc=[19:Arithmetic],NuclearSrc=[None],NuclearDest=[None])

Thereby, the range of the nerve cell body and the axis extending from the nerve cell can be analyzed, Density length and number of branches.

(c) Analysis of synaptic growth in nerve cells

Calculate the number of synapse that express Synaptophysin according to the above-mentioned range of axis and dendritic cursors to confirm the growth of nerve cells; for example, the options built into MetaXpress 3.1 software can be used in the following order. Set 10 steps:

23: Configure Cell Scoring Data Log()

24: Configure Cell Scoring Summary Log()

25: Overwrite "NeuronBinary"=Cell Scoring(All nuclei=[18:Clip Image],Positive marker="FITC")

26: Threshold Image ([25: Cell Scoring], 1, 65535, Inclusive)

27: Overwrite "Binary"=Binarize([25:Cell Scoring]),1,65535

28: Overwrite "AND"="Cy5" AND[27:Binary Operations]

29: Configure Transfluor Data Log()

30: Configure Transfluor Summary Log()

31: [None]=Transfluor(Pit/Vesicle Source=[28:Arithmetic],Nuclear Source=[18:Clip Image])

32: Close All (NOQUERY)

Thereby, the range of the nerve cell body and the microtubule-associated protein 2 (MAP2) is counted, and the number of synapse contained in the range of the nerve cell can be obtained.

Through the above 32 steps, you can understand the situation of nerve growth and neural network formation; for example, using this module for analysis, 1 hole and 3 kinds of fire cursors set 197 views with 591 images, only 2 hours Get the full result.

Please refer to the third figure for representative images of mouse cerebral cortical neurons cultured in vitro for 7, 10, 14 and 21 days (7~21 DIV). The green markers represent neuronal cell bodies and dendrites (anti- MAP2), the red mark represents the anti-synaptophysin, and the blue mark represents the nucleus (Hoechst); the scale length in the figure is 50 μm.

Further, using the above 32 steps for analysis, the experimental results of the primary cultured cells are as shown in the fourth to sixth figures. Looking at the fourth picture, the number of nerve cells measured by normal nerve cells in different culture days is best for 10 days; the fifth picture, the length of the main points, the length of the axis dendrites and the number of small branches are best in 14 days of culture. However, the sixth figure shows that the number of synapses is also the best in the 10th day of culture as shown in the fourth figure, and the number of synapses in the 14th day of culture is decreased; therefore, the number of culture days is 10 days after the rapid acquisition of nerve cells for subsequent analysis. For the best conditions.

Example 3: Detecting the effect of chemotherapeutic drugs on nerve cells

(1) Primary culture cells

The mouse cortex was taken from 0 to 1 day after birth (B6 mice pup). After removal of the meninges, the tissues were dispersed in serum-free DMEM/F12 medium to make up the volume of serum-free DMEM/F12. 5mL, add 0.5mL 1X trypsin (trypsin) to mix, water bath at 37 ° C for 5 minutes, use a 70μm filter membrane to infiltrate 1mL fetal bovine serum (FBS) to filter impurities, centrifuge 3,000rpm for 10 minutes, remove the supernatant To the solution, add the precipitated cells to the required amount of medium (medium preparation: Neurobasal ^® A medium 500 mL, L-glutamin 100X 1.3 mL, B-27 ^® Supplement 50 X 10 mL/vial, Penicillin/Streptomycin 100X 2.5 mL) for nerve Cell culture.

(2) The nerve cells were cultured using a transparent thin-bottom microporous black disk, and 70 μL of poly-D-lysine or poly-L-lysine was added to each well in the culture plate for coating. The cells were cultured in 2.8×10 ⁴ cells per well; after 10 days of in vitro culture, the chemotherapeutic drugs as shown in Table 1 were added to the cells for 24 hours, and the cells were fixed with 4% polyformaldehyde for 10 minutes, using PBS. Rinse twice for 5 minutes; add 1 M glycine for 10 minutes, rinse with PBS for 5 minutes twice; add 0.05% Triton X-100 for 20 minutes, rinse with PBS for 5 minutes twice. Primary antibody was blocked by blocking with 4% bovine serum albumin (BSA) for 1 hour: (1) microtubule-associated protein 2 antibody (MAP2 antibody; MyBioSource; MBS502140) and (2) synaptic protein antibody (Anti-Synaptophysin) Antibody; Abcam; ab32127) staining for 12-16 hours; rinsing with PBST for 10 minutes twice, and adding secondary antibodies with a fluorescent cursor, Alexa Fluor 488 (Jackson ImmunoResearch; 703-546-155) and Alexa Fluor 647 (molecular probes; A31573), and Hurst dye (Hoechst 33258) were added for 1 hour, washed with PBST for 10 minutes twice, finally added with PBS, and subjected to fluorescence image extraction and analysis.

(3) Using the fluorescence microscopic imaging system and method as in the second embodiment, capturing images for image analysis of the cultured cells, the image is magnified 200 times (200X), using fluorescent wavelengths DAPI, FITC, and Cy5 3 Kindly, the image is automatically accessed after the photo is taken for analysis.

Please refer to the seventh to ninth figures, respectively, for the effects of different chemotherapeutic drugs on the number of nerve cells, the number of synapses and the length of the axial dendrites, respectively, compared with the non-administered control group (relative control group%). From the results, vincristine has obvious neurotoxicity; compared with cisplatin Cisplatin, Carboplatin has lower neurocytotoxicity; and Topoisomerase inhibitor includes Ai The neurotoxicity of the doxorubicin and the liposome erythromycin (Lipo-Dox ^® ), as well as the nucleoside analogue gemcitabine, is lower.

After cell culture and image capture, a variety of analytical results can be quickly obtained by using a high-throughput analysis system and using the optimized module of the present invention (32 steps) to analyze nerve cell function. The method can be applied not only to the screening and detection of any nerve-related drugs, such as the toxicity or protection effect of drugs on the nervous system, but also the exploration of the molecules of the nervous system function and message transmission in the academic circle.

It can be seen from the above description that the present invention has the following advantages compared with the prior art:

1. The present invention has been repeatedly verified to screen out several representative molecular markers for assessing early nerve growth and neural network formation, as compared to current assessment of neuronal survival. The situation-based approach is more accurate in detecting changes in neurological function.

2. The image-and-type analysis method of the present invention designs neural cell growth and several important indicators into a complete functional evaluation module, and excludes non-discriminating indicators, in time-saving, large-scale and complete evaluation. A good balance between the two, so it can be applied to high-throughput screening and detection of any nerve-related drugs to quickly and accurately assess the effects of drugs on the nervous system.

In summary, the method for establishing a rapid analysis of neural function by the analysis module of the present invention can achieve the intended use efficiency by the above disclosed embodiments, and the present invention has not been disclosed before the application, and has been completely completed. Meet the requirements and requirements of the Patent Law.爰Issuing an application for a patent for invention in accordance with the law, and asking for a review, and granting a patent, is truly sensible.

The illustrations and descriptions of the present invention are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention; those skilled in the art, which are characterized by the scope of the present invention, Equivalent variations or modifications are considered to be within the scope of the design of the invention.

(S1)‧‧‧Step one

(S2)‧‧‧Step 2

(S3) ‧ ‧ Step 3

Claims (3)

A method for establishing an analysis module for rapid assessment of neural function includes the following steps: Step 1: Using a fluorescence microscopic imaging system to capture a cultured cell having a plurality of fluorescing cursors for image analysis of the cultured cells, wherein The fluorescence microscopic imaging system is internally provided with an analysis module including a plurality of instructions, which sequentially perform the steps 1 to 3. The culture cell line comprises nerve cells, and the plurality of fluorescent cursors comprise Hoechst dyeing. The agent is used to label the nucleus; Step 2: According to the size of the nucleus and the intensity of the fluorescein, the nerve cells of the expression axis and the dendritic fluorescein are selected, the non-neuronal cells are excluded, and the area of the nerve cell body is calculated, and the axon and the tree are calculated. The length of the protrusion and the number of branches to confirm the axis of the nerve cell and the growth of the dendrites, wherein the axis, the dendritic cursor is selected from the group consisting of microtubule-associated protein 2 (MAP2) antibody or neuron type III β-microtube Protein (TUJ1); and step three: according to the axis of the second step, the threshold of the dendritic cursor, calculate the number of synapse cursors on the nerve cells, to determine The case nerve cell growth, thereby neurological assessment, which is selected from the synapse fluorescence calibration synapsin (of Synaptophysin) antibody or synapse Post-density protein 95 (PSD95). The method for rapidly evaluating nerve function by establishing an analysis module as described in claim 1, wherein the cultured cell line is obtained by the following steps: Step 1: adding cells derived from the cerebral cortex tissue of a young rat to the first The medium is broken up to form a mixed solution, wherein the young mouse is a mouse or a rat of 0 to 1 day after birth, and the first medium is a serum-free DMEM/F12 medium; Step 2: Filtering the mixture and obtaining a cell pellet by centrifugation; and Step 3: culturing the cell pellet in a second medium for 9 to 12 days to prepare the cultured cells, wherein the second medium comprises Neurobasal ^® A medium (Neurobasal ^® A medium), L- Glutamic acid amide (L-glutamin), B- 27 ® additive (B-27 ^® Supplement), and penicillin - streptomycin (penicillin / streptomycin). A method for rapidly evaluating nerve function by establishing an analysis module as described in claim 2, wherein the cell pellet is cultured in the second medium for 10 days to prepare the cultured cell.