CN110734950A - In vitro experimental method for correlation of NMN treatments with improved CMVA - Google Patents
In vitro experimental method for correlation of NMN treatments with improved CMVA Download PDFInfo
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Abstract
The invention discloses an in vitro experiment method for NMN treatments and correlation improvement of CMVA, which comprises the following steps of establishing two groups of HUVECs models which are divided into an NMN intervention group and a control group, wherein the NMN intervention group is used for incubating NMN, the control group is used for carrying out conventional culture on the HUVECs models, carrying out scratch experiment, Transwell experiment and tube forming experiment on the NMN intervention group and the control group, taking a mouse thoracic aorta ring, inoculating the mouse thoracic aorta ring into a serum-free culture dish with the thickness of 0.5mm, culturing the aorta ring into the NMN intervention group and the control group, carrying out aorta ring experiment on the NMN intervention group and the control group, detecting CITED2 protein and gene expression of cells and myocardial tissues by utilizing RT-qPCR and WB based on experiment, and detecting CITED2 expression and correlation of the CMVA, and further obtaining correlation of CICMD 2 expression and the VA.
Description
Technical Field
The invention relates to the technical field of medical treatment, in particular to an in vitro experimental method for treating and improving correlation of CMVA by NMN.
Background
With the development of the current society, people are constantly researching human vascular anti-aging by combining with the progress of scientific technology, the problem of the correlation between NMN treatment and CMVA improvement is researched in the research process of human vascular anti-aging, and no related in vitro experimental method exists in the prior art in the research process of the correlation between NMN treatment and CMVA improvement.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides an in vitro experimental method for improving the correlation of the NMN treatments and CMVA.
In order to achieve the purpose, the invention adopts the following technical scheme:
an in vitro assay for the correlation of NMN treatment with improved CMVA comprising the steps of:
s1, two groups of HUVECs models are created and divided into an NMN intervention group and a control group, the NMN intervention group is used for carrying out incubation on NMN, the replacement is carried out for times in days, and the control group is used for carrying out conventional culture on the HUVECs models;
s2: performing a scratch experiment, a Transwell experiment and a tube forming experiment on the NMN stem pre-group and the control group in the step S1, wherein in the scratch experiment, the time and the speed of cell proliferation of the NMN stem pre-group and the control group are observed, in the Transwell experiment, the number of HUVECs (human hematopoietic necrosis factor receptors) migrating in the NMN stem pre-group and the control group, namely the cell migration phenomenon, is observed, and in the tube forming experiment, the density of microtubules formed by the HUVECs in the NMN stem pre-group and the control group and the number of branch points, namely the tube density, are detected;
s3: inoculating chest aortic rings of a mouse into a serum-free culture dish in a thickness of 0.5mm, wherein the bottom of the plate contains 1mg/mL collagen matrix, each aortic ring is inoculated into a 96-well plate, VEGF is supplemented into a FBS-containing culture medium to stimulate blood vessels to germinate, the aortic rings are cultured into an NMN pre-treatment group and a control group, and the NMN pre-treatment group and the control group are incubated for 7 days and then observed;
s4: performing aortic annulus experiments on the NMN intervention group and the control group in the step S3, and observing the growth length, number and area of the microvascular buds, namely the tubulointing density of the cells in the aortic annulus experiments;
s5: establishing an aged mouse model, and dividing the aged mouse model into an NMN treatment group and a conventional feeding control group; the NMN treatment group is characterized in that the NMN is fed to the aged mice through drinking water, after 1 month, aiming at the aged mice of the NMN treatment group and the aged mice of the conventional feeding control group, the number of microtubules and the microtubule/myofiber ratio under each high-power visual field are detected by utilizing immunofluorescence and laser confocal microscopy, and the heart functions of the aged mice of the NMN treatment group and the aged mice of the conventional feeding control group are detected through NT-proBNP and heart color Doppler ultrasound;
and S6, based on the experimental, detection and observation data of the steps S2, S3 and S5, detecting the protein and gene expression of CITED2 of cells and myocardial tissues by RT-qPCR and WB in steps, and further acquiring the correlation between the expression of CITED2 and CMVA.
Preferably, in step S1, the NMN is at a concentration of 0.5 mM.
Preferably, in step S2, matrigel is applied during the tube forming experiment, the cell density is 40000/hole, and the formation of microvessels is observed in 12h, 24 h, 48h and 72h of the experiment respectively.
Preferably, in step S3, the content of FBS in the culture medium is 2.5%, and the concentration of VEGF is 30 ng/mL.
Preferably, in step S4, the observation process of the aortic ring experiment is photographing observation by a fluorescence microscope and a confocal laser microscope.
Preferably, the aged mice are mice older than 20 months, and the amount of NMN consumed by the aged mice is 400 mg/kg/d.
The in vitro experimental method for treating and improving the correlation of CMVA by NMN has the advantages that in the process of carrying out the method, a HUVECs model and a mouse thoracic aorta ring are divided into an NMN intervention group and a conventional control group, a scratch experiment, a Transwell experiment and a tube forming experiment are used for observing the proliferation, migration and tube forming conditions of HUVECs, the growth length, the number and the area of cross points of microvessels are observed through the aorta ring experiment, then a CITED2 gene and a protein expression level are detected, after the high-age mice are fed with NMN in the step, NT-proBNP, heart color Doppler ultrasound, immunofluorescence and a laser confocal microscope are used for observing the cardiac function of the mice and the density of microvessels in a myocardial section, then the CITED2 gene and the protein expression level are detected, after the CITED2 gene and the protein expression are finished, the correlation of the NMN treatment and the improvement of the CMVA can be obtained, the correlation of the NMN treatment and the improvement of the CMVA can be conveniently carried out after the CMVA is carried out on the CMVA experimental procedures, and the CMVA is easily improved.
Drawings
FIG. 1 is a schematic flow chart of the in vitro experimental method for the correlation of NMN treatments with CMVA improvement, which is proposed by the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments, not all embodiments, of the present invention .
Referring to fig. 1, an in vitro assay for the correlation of NMN treatments with improved CMVA, comprising the steps of:
s1, two groups of HUVECs models are created and divided into an NMN intervention group and a control group, the NMN intervention group is used for carrying out incubation on NMN, the replacement is carried out for times in days, and the control group is used for carrying out conventional culture on the HUVECs models;
s2: performing a scratch experiment, a Transwell experiment and a tube forming experiment on the NMN stem pre-group and the control group in the step S1, wherein in the scratch experiment, the time and the speed of cell proliferation of the NMN stem pre-group and the control group are observed, in the Transwell experiment, the number of HUVECs (human hematopoietic necrosis factor receptors) migrating in the NMN stem pre-group and the control group, namely the cell migration phenomenon, is observed, and in the tube forming experiment, the density of microtubules formed by the HUVECs in the NMN stem pre-group and the control group and the number of branch points, namely the tube density, are detected;
s3: inoculating chest aortic rings of a mouse into a serum-free culture dish in a thickness of 0.5mm, wherein the bottom of the plate contains 1mg/mL collagen matrix, each aortic ring is inoculated into a 96-well plate, VEGF is supplemented into a FBS-containing culture medium to stimulate blood vessels to germinate, the aortic rings are cultured into an NMN pre-treatment group and a control group, and the NMN pre-treatment group and the control group are incubated for 7 days and then observed;
s4: performing aortic annulus experiments on the NMN intervention group and the control group in the step S3, and observing the growth length, number and area of the microvascular buds, namely the tubulointing density of the cells in the aortic annulus experiments;
s5: establishing an aged mouse model, and dividing the aged mouse model into an NMN treatment group and a conventional feeding control group; the NMN treatment group is characterized in that the NMN is fed to the aged mice through drinking water, after 1 month, aiming at the aged mice of the NMN treatment group and the aged mice of the conventional feeding control group, the number of microtubules and the microtubule/myofiber ratio under each high-power visual field are detected by utilizing immunofluorescence and laser confocal microscopy, and the heart functions of the aged mice of the NMN treatment group and the aged mice of the conventional feeding control group are detected through NT-proBNP and heart color Doppler ultrasound;
and S6, based on the experimental, detection and observation data of the steps S2, S3 and S5, detecting the protein and gene expression of CITED2 of cells and myocardial tissues by RT-qPCR and WB in steps, and further acquiring the correlation between the expression of CITED2 and CMVA.
In step S1, the NMN concentration is 0.5 mM.
In step S2, matrigel was applied during the tube forming experiment, the cell density was 40000/well, and microvessel formation was observed at 12h, 24 h, 48h and 72h of the experiment, respectively.
In step S3, the content of FBS in the culture medium is 2.5%, and the concentration of VEGF is 30 ng/mL.
In step S4, the aortic annular experiment is observed by imaging with a fluorescence microscope and a confocal laser microscope.
The aged mice are mice more than 20 months old, and the amount of NMN eaten by the aged mice is 400 mg/kg/d.
In conclusion, in the process of carrying out the method, a HUVECs model and a mouse thoracic aorta ring are divided into an NMN intervention group and a conventional control group, a scratch experiment, a Transwell experiment and a tube forming experiment are utilized to observe the proliferation, migration and tube forming conditions of HUVECs, the growth length, the number of cross points and the area of microvascular buds are observed through the aortic loop experiment, then a CITED2 gene and a protein expression level are detected, after -step feeding of aged mice with NMN, NT-proBNP, heart color Doppler, immunofluorescence and a laser confocal microscope are utilized to observe the cardiac function of the mice and the change of microvascular density in myocardial slices, and then a CITED2 gene and a protein expression level are detected, after the CITED2 gene and the protein expression are completed, the correlation between NMN treatment and CMVA improvement can be obtained, so that the method realizes the experiment of the correlation between the NMN treatment and the CMVA improvement, the correlation between the NMN treatment and the VA improvement can be obtained after the experiment, the whole experiment procedure is convenient for carrying out the CMVA promotion, and the whole experiment is beneficial to carry out the simple operation process.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (6)
- An in vitro assay for the correlation of NMN treatment with improved CMVA, comprising the steps of:s1, two groups of HUVECs models are created and divided into an NMN intervention group and a control group, the NMN intervention group is used for carrying out incubation on NMN, the replacement is carried out for times in days, and the control group is used for carrying out conventional culture on the HUVECs models;s2: performing a scratch experiment, a Transwell experiment and a tube forming experiment on the NMN stem pre-group and the control group in the step S1, wherein in the scratch experiment, the time and the speed of cell proliferation of the NMN stem pre-group and the control group are observed, in the Transwell experiment, the number of HUVECs (human hematopoietic necrosis factor receptors) migrating in the NMN stem pre-group and the control group, namely the cell migration phenomenon, is observed, and in the tube forming experiment, the density of microtubules formed by the HUVECs in the NMN stem pre-group and the control group and the number of branch points, namely the tube density, are detected;s3: inoculating chest aortic rings of a mouse into a serum-free culture dish in a thickness of 0.5mm, wherein the bottom of the plate contains 1mg/mL collagen matrix, each aortic ring is inoculated into a 96-well plate, VEGF is supplemented into a FBS-containing culture medium to stimulate blood vessels to germinate, the aortic rings are cultured into an NMN pre-treatment group and a control group, and the NMN pre-treatment group and the control group are incubated for 7 days and then observed;s4: performing aortic annulus experiments on the NMN intervention group and the control group in the step S3, and observing the growth length, number and area of the microvascular buds, namely the tubulointing density of the cells in the aortic annulus experiments;s5: establishing an aged mouse model, and dividing the aged mouse model into an NMN treatment group and a conventional feeding control group; the NMN treatment group is characterized in that the NMN is fed to the aged mice through drinking water, after 1 month, aiming at the aged mice of the NMN treatment group and the aged mice of the conventional feeding control group, the number of microtubules and the microtubule/myofiber ratio under each high-power visual field are detected by utilizing immunofluorescence and laser confocal microscopy, and the heart functions of the aged mice of the NMN treatment group and the aged mice of the conventional feeding control group are detected through NT-proBNP and heart color Doppler ultrasound;and S6, based on the experimental, detection and observation data of the steps S2, S3 and S5, detecting the protein and gene expression of CITED2 of cells and myocardial tissues by RT-qPCR and WB in steps, and further acquiring the correlation between the expression of CITED2 and CMVA.
- 2. The in vitro assay of correlation of NMN treatment with improved CMVA of claim 1, wherein in step S1 the concentration of NMN is 0.5 mM.
- 3. The in vitro assay of NMN treatment-and-correlation improvement for CMVA according to claim 1, wherein in step S2, matrigel and cell density 40000/well were applied during the tube-forming assay and microvessel formation was observed at 12h, 24 h, 48h and 72h of the assay, respectively.
- 4. The in vitro experimental method for the treatment of NMN correlated with the improvement of CMVA of claim 1, wherein in step S3, the content of FBS in said culture medium is 2.5%, and the concentration of VEGF is 30 ng/mL.
- 5. The in vitro experimental method for NMN treatment and correlation of CMVA improvement according to claim 1, wherein in step S4, the observation process of the aortic annular experiment is photographing observation by fluorescence microscope and confocal laser microscope.
- 6. The in vitro experimental method of correlation of NMN treatment and improvement of CMVA according to claim 1, wherein said elderly mouse is a mouse older than 20 months, and said elderly mouse consumes NMN in an amount of 400 mg/kg/d.
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| CN107849534A (en) * | 2015-06-11 | 2018-03-27 | 阿尔伯特爱因斯坦医学院公司 | Tendon disease is intervened |
| US20190328761A1 (en) * | 2016-12-21 | 2019-10-31 | Newsouth Innovations Pty Limited | Methods for enhancing vascular density |
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| CN107849534A (en) * | 2015-06-11 | 2018-03-27 | 阿尔伯特爱因斯坦医学院公司 | Tendon disease is intervened |
| US20190328761A1 (en) * | 2016-12-21 | 2019-10-31 | Newsouth Innovations Pty Limited | Methods for enhancing vascular density |
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