阅读说明：本技术 一种cited2异常打开与nmn治疗的相关性的体外实验法 (In vitro experimental method for correlation of CITED2 abnormal opening and NMN treatment ) 是由 张培东 吴畏 覃小桓 于 2019-11-14 设计创作，主要内容包括：本发明公开了一种CITED2异常打开与NMN治疗的相关性的体外实验法,包括如下步骤：构建CITED2基因过表达VECs,并分为NMN干预组和对照组,NMN干预组给予NMN进行孵育,对照组则进行常规培养；对NMN干预组和对照组均进行划痕实验、Transwell实验和成管实验；构建CITED2基因过表达小鼠模型,并分为NMN治疗组和常规喂养对照组,检测NMN治疗组和常规喂养对照组的高龄小鼠的心功能；基于实验、检测及观察数据,进一步的利用RT-qPCR和WB,检测细胞和心肌组织的CITED2蛋白和基因表达水平。本发明实现了CITED2异常打开与NMN治疗的相关性的实验,且整个实验过程程序简单,便于进行操作,进而有利于进行推广运用。(The invention discloses an in vitro experimental method for correlation between CITED2 abnormal opening and NMN treatment, which comprises the following steps: constructing CITED2 gene over-expression VECs, and dividing the CITED2 gene over-expression VECs into an NMN intervention group and a control group, wherein the NMN intervention group is used for incubation of NMN, and the control group is used for conventional culture; carrying out scratch experiments, Transwell experiments and tube forming experiments on the NMN intervention group and the control group; constructing a CITED2 gene overexpression mouse model, dividing the model into an NMN treatment group and a conventional feeding control group, and detecting the heart function of the aged mice of the NMN treatment group and the conventional feeding control group; based on experimental, detection and observation data, RT-qPCR and WB are further utilized to detect the CITED2 protein and gene expression level of cells and myocardial tissues. The invention realizes the experiment of the correlation between CITED2 abnormal opening and NMN treatment, and the whole experiment process has simple program and is convenient to operate, thereby being beneficial to popularization and application.)

1. An in vitro assay for the abnormal opening of CITED2 in relation to NMN treatment, comprising the steps of:

s1: constructing CITED2 gene over-expression VECs, and dividing the CITED2 gene over-expression VECs into an NMN intervention group and a control group, wherein the NMN intervention group is used for incubation by NMN and is replaced once a day, and the control group is used for conventional culture;

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: constructing a CITED2 gene overexpression mouse model, and dividing the model into an NMN treatment group and a conventional feeding control group, wherein the NMN treatment group is fed with NMN to feed the aged mice through drinking water, after the NMN treatment group lasts for 1 month, aiming at the aged mice of the NMN treatment group and the conventional feeding control group, the number of microtubules and the microtubule/myofiber ratio under each high-power visual field are detected by using immunofluorescence and laser confocal microscope technology, and the heart functions of the aged mice of the NMN treatment group and the conventional feeding control group are detected through NT-proBNP and heart color Doppler ultrasound;

s4: based on the experimental, detection and observation data of steps S2 and S3, RT-qPCR and WB are further utilized to detect the CITED2 protein and gene expression level of cells and myocardial tissues.

2. The in vitro assay of CITED2 for abnormal opening of correlation with NMN treatment of claim 1, wherein in step S1 the concentration of NMN is 0.5 mM.

3. The in vitro assay method of CITED2 for abnormal opening of correlation with NMN treatment according to claim 1, wherein in step S2, the scratch assay, the Transwell assay and the tube assay are all performed in step S1 for 12h, 24h, 48h and 72h of NMN intervention group incubation and control group culture.

4. The in vitro assay of CITED2 for abnormal opening of correlation with NMN treatment of claim 1, wherein in step S3 said aged mouse consumes 400mg/kg/d NMN.

Technical Field

The invention relates to the technical field of medical treatment, in particular to an in-vitro experimental method for correlation between CITED2 abnormal opening and NMN treatment.

Background

Along 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 abnormal opening of a CITED2 molecular switch and NMN treatment in the research process of human vascular anti-aging is always researched, and no related in-vitro experimental method exists in the prior art in the research process of the correlation between abnormal opening of the CITED2 molecular switch and NMN treatment. To this end, we propose an in vitro experimental method of CITED2 abnormal opening of association with NMN treatment.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provides an in vitro experimental method for abnormal opening of CITED2 and correlation of NMN treatment.

In order to achieve the purpose, the invention adopts the following technical scheme:

an in vitro assay for the abnormal opening of CITED2 in association with NMN treatment comprising the steps of:

s1: constructing CITED2 gene over-expression VECs, and dividing the CITED2 gene over-expression VECs into an NMN intervention group and a control group, wherein the NMN intervention group is used for incubation by NMN and is replaced once a day, and the control group is used for conventional culture;

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: constructing a CITED2 gene overexpression mouse model, and dividing the model into an NMN treatment group and a conventional feeding control group, wherein the NMN treatment group is fed with NMN to feed the aged mice through drinking water, after the NMN treatment group lasts for 1 month, aiming at the aged mice of the NMN treatment group and the conventional feeding control group, the number of microtubules and the microtubule/myofiber ratio under each high-power visual field are detected by using immunofluorescence and laser confocal microscope technology, and the heart functions of the aged mice of the NMN treatment group and the conventional feeding control group are detected through NT-proBNP and heart color Doppler ultrasound;

s4: based on the experimental, detection and observation data of steps S2 and S3, RT-qPCR and WB are further utilized to detect the CITED2 protein and gene expression level of cells and myocardial tissues.

Preferably, in step S1, the NMN is at a concentration of 0.5 mM.

Preferably, in step S2, the scratch test, the Transwell test and the tube test are all performed in the 12h, 24h, 48h and 72h sections of the NMN intervention group incubation and control group culture in step S1.

Preferably, in step S3, the amount of NMN consumed by the aging mouse is 400 mg/kg/d.

The in vitro experimental method for the correlation between CITED2 abnormal opening and NMN treatment provided by the invention has the beneficial effects that: in the process of the scheme, a CITED2 gene overexpression VECs model is constructed, NMN intervention is given, RT-qPCR and WB technologies are utilized to detect the expression change of CITED2, and the cell proliferation, migration and micro angiogenesis conditions are observed through scratch experiments, Transwell experiments and tube forming experiments; by constructing a CITED2 gene overexpression aged mouse model, adding NMN into drinking water for treatment, detecting the number of microtubules and microtubule/myofiber ratio under each high-power visual field by utilizing immunofluorescence and laser confocal microscope technology, and detecting the cardiac function of aged mice in an NMN treatment group and a conventionally fed control group by NT-proBNP and heart color Doppler ultrasound; the correlation between abnormal opening of the CITED2 molecular switch and NMN treatment can be obtained along with the completion of detection on CITED2 expression change and the completion of observation and detection on an elderly mouse, so that the experiment on the correlation between abnormal opening of the CITED2 molecular switch and NMN treatment is realized, the whole experiment process is simple in program, and the operation is convenient, so that the popularization and the application are facilitated.

Drawings

FIG. 1 is a schematic flow chart of an in vitro experimental method for the correlation of abnormal opening of CITED2 with NMN treatment according to 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 a part of the embodiments of the present invention, and not all of the embodiments.

Referring to fig. 1, an in vitro experimental method for CITED2 to abnormally open a correlation with NMN treatment, comprising the steps of:

s1: constructing CITED2 gene over-expression VECs, and dividing the CITED2 gene over-expression VECs into an NMN intervention group and a control group, wherein the NMN intervention group is used for incubation by NMN and is replaced once a day, and the control group is used for conventional culture;

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: constructing a CITED2 gene overexpression mouse model, and dividing the model into an NMN treatment group and a conventional feeding control group, wherein the NMN treatment group is fed with NMN to feed the aged mice through drinking water, after the NMN treatment group lasts for 1 month, aiming at the aged mice of the NMN treatment group and the conventional feeding control group, the number of microtubules and the microtubule/myofiber ratio under each high-power visual field are detected by using immunofluorescence and laser confocal microscope technology, and the heart functions of the aged mice of the NMN treatment group and the conventional feeding control group are detected through NT-proBNP and heart color Doppler ultrasound;

s4: based on the experimental, detection and observation data of steps S2 and S3, RT-qPCR and WB are further utilized to detect the CITED2 protein and gene expression level of cells and myocardial tissues.

In step S1, the NMN concentration is 0.5 mM.

In step S2, the scratch, Transwell and tube experiments were performed in steps S1 for 12h, 24h, 48h and 72h of NMN intervention and control incubations.

In step S3, the amount of NMN consumed by the aging mouse is 400 mg/kg/d.

In summary, the following steps: in the process of the invention, a CITED2 gene overexpression VECs model is constructed, NMN intervention is given, RT-qPCR and WB technologies are utilized to detect CITED2 expression change, and cell proliferation, migration and micro angiogenesis conditions are observed through scratch experiments, Transwell experiments and tube forming experiments; by constructing a CITED2 gene overexpression aged mouse model, adding NMN into drinking water for treatment, detecting the number of microtubules and microtubule/myofiber ratio under each high-power visual field by utilizing immunofluorescence and laser confocal microscope technology, and detecting the cardiac function of aged mice in an NMN treatment group and a conventionally fed control group by NT-proBNP and heart color Doppler ultrasound; the correlation between the abnormal opening of the CITED2 molecular switch and the NMN treatment can be obtained along with the completion of the detection of the expression change of the CITED2 and the observation and detection of the aged mice, so that the correlation experiment of the abnormal opening of the CITED2 molecular switch and the NMN treatment is realized, the whole experiment process is simple in program, and the operation is convenient, so that the popularization and the application are facilitated.

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.