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Method for screening and confirming kidney-yang deficiency animal model biomarker

A technology of biomarkers and animal models, applied in the field of screening and determination of biomarkers in animal models of kidney-yang deficiency syndrome, can solve the problems of lack of specificity and objectivity of evaluation indicators, and achieve the effect of promoting scientific evaluation and easy carrier.

Inactive Publication Date: 2017-05-10
FUJIAN UNIV OF TRADITIONAL CHINESE MEDICINE
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] In this experiment, the symptoms, signs, serum cAMP, cGMP content detection and cAMP / cGMP ratio results of the kidney-yang deficiency model rats simulated by intramuscular injection of hydrocortisone are in line with the identification requirements of the commonly used kidney-yang deficiency models. It shows that the kidney-yang deficiency model of this study is successful, but the above evaluation methods are mainly subjective evaluation, and the evaluation indicators lack specificity and objectivity. Can the research results scientifically clarify the essence of TCM "syndrome" and the replicated animal syndrome? Whether the phenotype model is representative also involves the problem of dialectical objectification and quantification

Method used

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  • Method for screening and confirming kidney-yang deficiency animal model biomarker
  • Method for screening and confirming kidney-yang deficiency animal model biomarker
  • Method for screening and confirming kidney-yang deficiency animal model biomarker

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Experimental program
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Effect test

Embodiment 1

[0038] Establishment and identification of model rats with kidney-yang deficiency syndrome:

[0039] 1 Experimental consumables

[0040] 1.1 Experimental animals

[0041] A total of 40 male 3-month-old healthy SPF (Specific Pathogen Free) grade WISTAR rats, weighing 200-240 g, were purchased from Shanghai Slack Experimental Animal Co., Ltd. by the Experimental Animal Center of Fujian University of Traditional Chinese Medicine. The experimental animal license number: SCXK (Shanghai) 2012-0002, laboratory animal quality certificate number: 2007000548015. Experimental animal use license number of the Experimental Animal Center of Fujian University of Traditional Chinese Medicine: SYXK (Fujian) 2014-0001.

[0042] 1.2 Main reagents and drugs

[0043] Table 1 Main reagents and drugs

[0044]

[0045] 1.3 Main Instruments

[0046] Table 2 Main Instruments

[0047]

[0048] 2 Experimental methods

[0049] 2.1 Animal grouping

[0050] Forty WISTAR rats were adaptively fe...

Embodiment 2

[0063] Screening of differential proteins, which includes the following steps:

[0064] 1. Extraction of cancellous bone total protein

[0065] ①Cut the frozen fresh femur stem with bone scissors, rinse the bone marrow with ultrapure water, and gently scrape the dry cancellous bone of the femur into the mortar (pre-cooled with liquid nitrogen) with a curette, and add Liquid nitrogen and rapid grinding, repeated grinding 3 to 5 times, the powder was taken and weighed, then protein sample lysate was added according to the ratio of 1 g bone tissue to 1.8 mL lysate, and the protein was lysed and extracted in a refrigerator at 4°C for 4 h (Mix thoroughly every 1 hour); ②Place the EP tube containing the bone tissue suspension in a low-temperature high-speed centrifuge, centrifuge at 100,000 rpm at 4°C for 1 hour, and absorb the supernatant; ③Add nuclease: Add 2 ulDNase and 2 ul RNase to 1 ml of the lysate, and place on ice for 15 min; ④ Ultrasound: use an ultrasonic cleaner to soni...

Embodiment 3

[0092] Western Blot and RT-PCR technology double verification from protein and gene level, it includes the following steps:

[0093] 1. Western Blot detection of cancellous bone-related protein in the femoral shaft of rats in each group

[0094] 1.1 Extraction of cancellous bone total protein

[0095] The cancellous femur bone of 8 rats in each group was randomly selected from the -80°C freezer, placed on ice, and the bone tissue was quickly cut into pieces with a rongeur, put into a pre-cooled grinder, and quickly ground to In powder form, liquid nitrogen was added to cool at the right time during the grinding process, then transferred to an Eppendorf tube, and weighed; 1 g of bone tissue was added to 1 ml of lysate according to the proportion, oscillated by a micro-vortexer for 5 s, and then placed in a refrigerator at 4 °C. Vortex once every 30 min, take it out after 4 h; transfer to a low-temperature high-speed centrifuge, set the program at 4 °C, 14000 rpm, centrifuge fo...

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Abstract

The invention provides a method for screening and confirming a kidney-yang deficiency animal model biomarker. The method comprises the following steps: extracting total protein of cancellous bone of a sample; determining the protein concentration; performing two-dimensional gel electrophoresis analysis; detecting gel protein spot and displaying and analyzing differential protein spot; performing MALDI-TOF-MS mass spectrometry for differential protein, retrieving and identifying a protein database; confirming the differential protein; identifying a part of differential protein through Western-Blot and RT-PCR and mutually verifying the detection result for differential protein ELISA in serum; using the differential protein in consistent expression tendency and beyond the normal value scope as a biomarker for identifying a kidney-yang deficiency animal model. The invention is beneficial to the supply of an identifying index to the kidney-yang deficiency in an animal experiment and provides a basis for further discovering the kidney-yang deficiency biomarker.

Description

technical field [0001] The invention belongs to the field of basic research, and in particular relates to a method for screening and determining biomarkers of animal models of kidney-yang deficiency. Background technique [0002] "Syndrome" is the core of the theoretical system of traditional Chinese medicine. The first priority of traditional Chinese medicine is syndrome differentiation and treatment. However, syndrome differentiation is largely affected by doctors themselves (such as understanding of traditional Chinese medicine knowledge, clinical experience, etc.) and subjective factors of patients, so it is difficult to objectively and Quantitative, resulting in inconsistent results of syndrome differentiation, affecting the establishment of treatment methods, prescription selection and evaluation of clinical efficacy, hindering the research and development of traditional Chinese medicine. The use of animal models in the basic research of TCM can simulate the real proce...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N27/447G01N27/62
CPCG01N27/447G01N27/62G01N33/6851
Inventor 苏友新郑乃熙郑良朴詹正烜赖兴泉黄露露朱亚菊
Owner FUJIAN UNIV OF TRADITIONAL CHINESE MEDICINE
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