阅读说明：本技术 一种rbp4作为肌少症治疗靶点的检测方法 (Detection method of RBP4 as sarcopenia treatment target ) 是由 高伟 鲁翔 王丽 赵璨 张康振 王玥 于 2020-05-14 设计创作，主要内容包括：本发明公开一种RBP4作为肌少症治疗靶点的检测方法,检测包括RBP4在肌少症患者的腓肠肌组织中表达变化,腺病毒沉默小鼠骨骼肌RBP4表达后小鼠运动耐量的改变,和RBP4在小鼠C2C12成肌细胞分化过程的表达变化。本发明检测方法通过肌少症患者腓肠肌组织中提取蛋白样品检测RBP4的表达水平；利用腺病毒沉默小鼠腓肠肌RBP4基因表达,测量小鼠运动耐量；以及从分化不同程度的C2C12成肌细胞中提取RNA检测RBP4的表达。操作简单、快速、稳定性好；为肌少症患者的临床诊治提供强有力的理论支持,具有临床意义和推广价值。(The invention discloses a detection method of RBP4 as a sarcopenia treatment target, which comprises the steps of detecting expression change of RBP4 in gastrocnemius tissues of sarcopenia patients, change of exercise tolerance of mice after adenovirus silences RBP4 expression of skeletal muscles of the mice, and expression change of RBP4 in the differentiation process of myoblasts of C2C12 of the mice. The detection method of the invention detects the expression level of RBP4 by extracting protein samples from gastrocnemius tissues of patients with sarcopenia; using adenovirus to silence the expression of mouse gastrocnemius RBP4 gene, measuring the exercise tolerance of mouse; and extracting RNA from the C2C12 myoblasts differentiated to different degrees to detect the expression of RBP 4. The operation is simple and rapid, and the stability is good; provides powerful theoretical support for clinical diagnosis and treatment of sarcopenia patients, and has clinical significance and popularization value.)

1. A detection method of RBP4 as a sarcopenia treatment target is characterized by comprising the steps of detecting expression change of RBP4 in gastrocnemius tissues of sarcopenia patients, change of exercise tolerance of mice after adenovirus silencing of RBP4 expression of skeletal muscles of the mice and expression change of RBP4 in a mouse C2C12 myoblast differentiation process.

2. The assay of claim 1, comprising RBP4 as a target for treatment of sarcopenia, wherein the change in expression of RBP4 in the gastrocnemius tissue of a sarcopenia patient comprises a Western Blot assay.

3. The method for detecting RBP4 as a target for sarcopenia treatment according to claim 2, wherein the Western Blot detection reagent comprises an antibody specific to RBP4 and an antibody specific to internal reference GAPDH.

4. The method for detecting the RBP4 as the target for sarcopenia therapy according to claim 3, wherein the RBP4 specific antibody is: ab 10919.

5. The method of claim 3, wherein the internal reference GAPDH-specific antibody 60004-1-Ig is used to detect RBP4 as a target for sarcopenia therapy.

6. The method for detecting RBP4 as a target for sarcopenia therapy according to claim 1, wherein the change of the exercise tolerance of the mice after the adenovirus silences the expression of the mouse skeletal muscle RBP4 comprises the measurement of the exercise distance and the exercise time of the mice.

7. The method for detecting RBP4 as a target for sarcopenia therapy according to claim 6, wherein the adenovirus silences mouse skeletal muscle RBP4 expression comprising RBP4 adenovirus sequences.

8. The method for detecting the RBP4 as the target point of sarcopenia therapy according to claim 1, wherein the expression change of the RBP4 in the mouse C2C12 myoblast differentiation process comprises a real-time fluorescent quantitative PCR detection reagent.

9. The method for detecting the target of RBP4 for sarcopenia treatment according to claim 8, wherein the real-time fluorescent quantitative PCR detection reagent comprises a specific primer for RBP4 and a specific primer for internal reference GAPDH;

the specific primers of the RBP4 comprise a specific primer forward primer of RBP4 and a specific primer reverse primer of RBP 4.

10. The method for detecting the target of RBP4 for sarcopenia treatment as claimed in claim 9, wherein the specific primers for the internal reference GAPDH comprise a GADH specific primer forward primer and a GAPDH specific primer reverse primer.

Technical Field

The invention relates to a detection method, in particular to a detection method of RBP4 as a sarcopenia treatment target.

Background

Skeletal muscle is one of the most viable and plastic tissues of the human body, accounting for approximately 40% of total body weight and containing 50-75% of all body proteins. Skeletal muscle atrophy occurs in a variety of conditions such as disease states (heart failure, cancer, diabetes, sepsis, etc.), denervation, fasting, aging, and drug therapy. Skeletal muscle atrophy is primarily due to excessive breakdown of muscle proteins, and protein breakdown is often accompanied by a decrease in protein synthesis, manifested by decreased skeletal muscle mass and dysfunction, leading to decreased quality of life for the patient, leading to increased morbidity and mortality. Therefore, the search for new effective muscle factors as intervention targets for the treatment of muscle atrophy is of great significance.

Retinol binding protein 4(RBP4), a transport protein for retinol (retinol) in circulating blood, is synthesized primarily in the liver and released to the external circulation, with a small proportion coming from adipocytes. Recent studies have shown that RBP4 induces insulin resistance and cardiovascular diseases through inflammatory pathways, and participates in the regulation of metabolism of multiple organs in the body. Recently we found that RBP4 expression was reduced in gastrocnemius tissue in patients with sarcopenia; after adenovirus silences RBP4 expression in mouse muscle tissue, the exercise tolerance of the mouse is reduced; further studies found that the expression level of RBP4 gradually increased as mouse C2C12 myoblasts differentiated.

Disclosure of Invention

The invention aims to provide a detection method of RBP4 as a target for treating sarcopenia, which detects the expression level of RBP4 by extracting a protein sample from gastrocnemius tissues of a sarcopenia patient; using adenovirus to silence the expression of mouse gastrocnemius RBP4 gene, measuring the exercise tolerance of mouse; and extracting RNA from the C2C12 myoblasts differentiated to different degrees to detect the expression of RBP 4. The operation is simple and rapid, and the stability is good; provides powerful theoretical support for clinical diagnosis and treatment of sarcopenia patients, and has clinical significance and popularization value.

The purpose of the invention can be realized by the following technical scheme:

a detection method of RBP4 as a sarcopenia treatment target comprises the steps of detecting expression change of RBP4 in gastrocnemius tissues of sarcopenia patients, change of exercise tolerance of mice after adenovirus silences RBP4 expression of skeletal muscles of the mice, and expression change of RBP4 in the differentiation process of myoblasts of C2C12 of the mice.

Further, the RBP4 expression changes in gastrocnemius tissue of a sarcopenia patient include a WesternBlot detection reagent.

Further, the WesternBlot detection reagent includes an RBP 4-specific antibody and an internal reference GAPDH-specific antibody.

Further, the RBP 4-specific antibody is: ab 10919.

Further, the internal reference GAPDH specific antibody is 60004-1-Ig.

Further, changes in mouse exercise tolerance following adenovirus silencing of mouse skeletal muscle RBP4 expression include measures of mouse distance and time of exercise.

Further, the adenovirus silences mouse skeletal muscle RBP4 expression and comprises RBP4 adenovirus sequences.

Further, the expression change of the RBP4 in the mouse C2C12 myoblast differentiation process comprises a real-time fluorescent quantitative PCR detection reagent.

Further, the real-time fluorescent quantitative PCR detection reagent comprises a specific primer of RBP4 and a specific primer of an internal reference GAPDH.

The specific primers of the RBP4 comprise a specific primer forward primer of RBP4 and a specific primer reverse primer of RBP 4.

Further, the primers specific to the internal reference GAPDH include a gaph specific primer forward primer and a GAPDH specific primer reverse primer.

The invention has the beneficial effects that:

1. the detection method of the invention detects the expression level of RBP4 by extracting protein samples from gastrocnemius tissues of patients with sarcopenia; using adenovirus to silence the expression of mouse gastrocnemius RBP4 gene, measuring the exercise tolerance of mouse; and extracting RNA from the C2C12 myoblasts differentiated to different degrees to detect the expression of RBP 4. The operation is simple and rapid, and the stability is good;

2. the detection method of the invention provides powerful theoretical support for clinical diagnosis and treatment of sarcopenia patients, and has clinical significance and popularization value.

Drawings

The invention will be further described with reference to the accompanying drawings.

FIG. 1 is a schematic representation of protein expression of RBP4 of the present invention in sarcopenia patients and normal human muscle tissue;

FIG. 2 is a schematic representation of protein expression of RBP4 of the present invention in sarcopenia patients and normal human muscle tissue;

FIG. 3 is a schematic diagram showing the measurement of the distance traveled by a mouse injected with RBP4 adenovirus, irrelevant control and physiological saline in gastrocnemius of the mouse according to the present invention;

FIG. 4 is a schematic diagram showing the measurement of the exercise time of mice injected with RBP4 adenovirus, irrelevant control and physiological saline in gastrocnemius of the mice according to the present invention;

FIG. 5 is a graph showing the change in expression of RBP4 at the mRNA level according to the change in the differentiation time of C2C12 cells of 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. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.