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A method for screening and determining biomarkers of animal models of kidney yin deficiency

A technology of biomarkers and animal models, which is applied in the field of screening and determination of biomarkers of animal models of kidney yin deficiency, can solve difficulties in objectification and quantification, affecting the selection of treatment methods and medications, and evaluation of clinical curative effects. Medical research and development and other issues, to achieve the effect of easy carrier and scientific evaluation

Inactive Publication Date: 2018-09-28
FUJIAN UNIV OF TRADITIONAL CHINESE MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] "Syndrome" is the core of the theoretical system of traditional Chinese medicine. The characteristic of traditional Chinese medicine in treating diseases lies in syndrome differentiation and treatment, but syndrome differentiation is largely affected by the doctor himself (such as understanding of traditional Chinese medicine knowledge, clinical experience, etc.) Objectification and quantification lead to inconsistencies in the results of syndrome differentiation, affect the establishment of treatment methods, the selection of prescriptions and medications, and the evaluation of clinical efficacy, hindering the research and development of traditional Chinese medicine. The use of animal models in basic research of traditional Chinese medicine can simulate the reality of diseases to the greatest extent. The process is similar to the physiological and pathological state of "syndrome", so the use of animal models of kidney yin deficiency syndrome is the only way to study the essence of kidney deficiency syndrome
In this experiment, the symptoms, signs, serum cAMP, cGMP content detection and cAMP / cGMP ratio results of rats with kidney yin deficiency caused by gavage of thyroid tablet suspension were in line with the identification requirements of the commonly used kidney yin deficiency model. It shows that the animal model of kidney yin deficiency syndrome in this study was successfully constructed, 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 replication Whether the animal syndrome model is representative also involves the objectification and quantification of syndrome

Method used

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  • A method for screening and determining biomarkers of animal models of kidney yin deficiency
  • A method for screening and determining biomarkers of animal models of kidney yin deficiency
  • A method for screening and determining biomarkers of animal models of kidney yin deficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Establishment and identification of model rats with kidney yin deficiency:

[0038] 1 Experimental consumables

[0039] 1.1 Experimental animals

[0040] A total of 60 male 3-month-old healthy SPF grade WISTAR rats, weighing 200-240 g, were purchased by the Experimental Animal Center of Fujian University of Traditional Chinese Medicine from Shanghai Slack Experimental Animal Co., Ltd., experimental animal license number: SCXK (Shanghai) 2012 -0002, laboratory animal quality certificate number: 2007000583560. Experimental animal use license number of the Experimental Animal Center of Fujian University of Traditional Chinese Medicine: SYXK (Fujian) 2014-0001.

[0041] 1.2 Main reagents and drugs

[0042] Table 1 Main reagents and drugs

[0043]

[0044] 1.3 Main Instruments

[0045] Table 2 Main Instruments

[0046]

[0047] 2 Experimental methods

[0048] 2.1 Preparation of Thyroid Tablet Suspension

[0049] Put the thyroid slices in a grinder and grind the...

Embodiment 2

[0062] Screening of differential proteins, which includes the following steps:

[0063] 1. Extraction of total cortical bone protein ① Cut the frozen fresh femur stem with bone scissors, rinse the bone marrow with ultrapure water, and gently scrape off the cancellous bone of the femoral stem with a curette, then put it into a mortar (in advance liquid nitrogen precooling), cut into lmm with bone scissors 3 Add liquid nitrogen to the mortar and grind it quickly, repeat the grinding 3 to 5 times, take the powder and weigh it, and then add protein sample lysate according to the ratio of 1 g bone tissue to 1.5 mL lysate, Place in a refrigerator at 4°C to lyse and extract protein for 4 hours (mix well once every hour); ② Place the EP tube containing the bone tissue suspension in a low-temperature high-speed centrifuge, centrifuge at 14,000 rpm at 4°C for 1 hour, and draw Supernatant; ③Add nuclease: Add 2 ul DNase and 2 ulRnase per 1 ml of lysate, and place on ice for 15 minutes; ④...

Embodiment 3

[0088] Western Blot and RT-PCR technology double verification from protein and gene level, it includes the following steps:

[0089] 1 Western Blot detection of bone-related protein in femoral shaft cortex of rats in each group

[0090] 1.1 Extraction of total cortical bone protein. Randomly extract 8 rat femur dry cortical bones from each group in the -80°C freezer, place them on ice, quickly cut the bone tissue into pieces with a rongeur, and put them into a pre-cooled grinder Grind quickly to powder, add liquid nitrogen to cool at the right time during the grinding process, then transfer to an Eppendorf tube, weigh; add 1ml lysate to 1 g of bone tissue according to the proportion, oscillate with a micro-vortex for 5 s, and then place in 4 ℃ refrigerator, vortex shake once every 30 min, take out after 4 h; transfer to a low-temperature high-speed centrifuge, set the program 4 ℃, 14000 rpm, centrifuge for 20 min, absorb the supernatant.

[0091] 1.2 Determination of protein ...

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Abstract

The invention provides a kidney yin deficiency syndrome animal model biomarker screening and determining method. The method comprises the following steps: extracting a cortical bone total protein sample, measuring the protein concentration, carrying out bidirectional gel electrophoresis analysis, detecting the gel protein spot, displaying and analyzing differential protein spots, carrying out MALDI-TOF-MS of differential protein spots, searching the protein database to identify and determine differential proteins, identifying part of differential proteins through Western-Bolt and RT-PCR, and interactively verifying the identification results with ELISA detection results of differential proteins in the serum. The differential proteins, whose expression trends are consistent and are not in the normal value range, can be used as the biomarker for identifying a kidney yin deficiency syndrome animal model. The biomarker can be used as the discrimination index of kidney yin deficiency syndrome in animal experiments; and a base is provided for finding another kidney yin deficiency syndrome 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 yin deficiency. Background technique [0002] "Syndrome" is the core of the theoretical system of traditional Chinese medicine. The characteristic of traditional Chinese medicine in treating diseases lies in syndrome differentiation and treatment, but syndrome differentiation is largely affected by doctors themselves (such as understanding of traditional Chinese medicine knowledge, clinical experience, etc.) Objectification and quantification lead to inconsistencies in the results of syndrome differentiation, affect the establishment of treatment methods, the selection of prescriptions and medications, and the evaluation of clinical efficacy, hindering the research and development of traditional Chinese medicine. The use of animal models in basic research of traditional Chinese medicine can simul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447G01N33/68
CPCG01N27/447G01N33/6848
Inventor 苏友新詹正烜郑乃熙张英杰王旭张艺强蔡唐彦滕方舟
Owner FUJIAN UNIV OF TRADITIONAL CHINESE MEDICINE
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