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Simple detection reagent for Warburg effect of human cells as well as preparation method and application of simple detection reagent

A technology for detecting reagents and human cells, applied in the field of medical testing, can solve the problems of no simple screening detection, human radiation hazards, and high operating costs of PET-CT, and achieve the effects of good specificity, good stability and high sensitivity

Active Publication Date: 2017-09-05
山东天倪生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problems of high operating cost of PET-CT, serious radiation hazards to the human body, and lack of reagents for simple screening and detection of early cancerous cells, the present invention provides a simple detection method for the Warburg effect of human cells quickly and accurately. Reagent

Method used

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  • Simple detection reagent for Warburg effect of human cells as well as preparation method and application of simple detection reagent
  • Simple detection reagent for Warburg effect of human cells as well as preparation method and application of simple detection reagent
  • Simple detection reagent for Warburg effect of human cells as well as preparation method and application of simple detection reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Reagent Preparation

[0051] (1) Preparation of Reagent A:

[0052] Add 5.00 g of subtilisin to 100 mL of 0.9 % NaCl isotonic solution and dissolve to obtain reagent A;

[0053] (2) Preparation of reagent B:

[0054] a) Dissolve 82g sodium acetate in 1000mL water to obtain 2 mol / L sodium acetate solution; dissolve 15g glacial acetic acid in 200mL water to obtain 2 mol / L acetic acid solution;

[0055] b) Mix the above solution at a ratio of 5:1 to obtain reagent B.

[0056] (3) Preparation of Reagent C:

[0057] a) Dissolve 0.15 g of 3,3’,5,5’-tetramethylbenzidine in 30 mL of absolute ethanol;

[0058] b) Add 0.25 mL of cumene peroxide and 0.125 mL of 6-methoxyquinoline to the above solution in a) in order to dissolve;

[0059] c) Dilute the purified water to 100mL to obtain reagent C;

[0060] (4) Preparation of reagent D:

[0061] a) Dissolve 0.05 g of toluidine blue O in 100 mL of purified water;

[0062] b) Add 0.5 mL of Triton X-100 to the above solu...

Embodiment 2

[0063] The usage method of embodiment 2 reagent

[0064] (1) Gently brush the epithelial exfoliated cells with a cell brush, then place the cell brush in 2mL of reagent A and gently stir to make a sample solution;

[0065] (2) Centrifuge the above sample solution at a high speed of 3000 rpm for 3 minutes;

[0066] (3) Take the supernatant of step (2), first add 1mL of reagent B and mix well, then add 1mL of reagent C, observe the color of the reaction solution in 3 minutes, and then measure the free prohemoglobin content of cells: if not Discoloration is negative, light blue is weak positive, blue is positive, dark blue is strong positive;

[0067] (4) Add 1mL of reagent D to the precipitate in step (2), and observe the color of the reaction solution at 3 minutes, which shows the content of reactive oxygen species in the cells: if it does not change color (blue), it is negative, and the blue color becomes Light blue is positive, and colorless is strong positive;

[0068] Re...

Embodiment 3

[0069] Example 3 Clinical verification

[0070] Collect cell samples from different parts of the human body, perform operations according to Example 2, and judge the results. Calculate the sensitivity and specificity of the reagent and the Youden index, and calculate the Youden index by the following formula:

[0071]

[0072] 3.1 Specimen source

[0073] Colonic mucosal cells: 118 samples of colonic mucosal epithelial exfoliated cells from colorectal cancer patients received by the Anorectal Department of a medical unit. Colorectal cancer was confirmed by pathological examination. 100 samples of colonic mucosal epithelial exfoliated cells from non-colorectal cancer patients admitted to the general surgery department of the same medical unit. Non-colorectal cancer was ruled out by cytology.

[0074] Body surface cells: 117 samples of body surface exfoliated cells from patients with surface malignant tumors received by the Department of Dermatology, Department of Gynecol...

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Abstract

In order to solve the problems that PET-CT operating cost is high, severe radiation hazards are caused on human bodies and a reagent capable of simply and conveniently screening and detecting early cancerous cells does not exist, the invention provides a reagent capable of rapidly and accurately performing simple detection on a Warburg effect of human cells as well as a preparation method and application of the reagent. The simple detection reagent is composed of a reagent A, a reagent B, a reagent C and a reagent D. The human cell collection parts comprise colonic mucosa, skin, vulva and mouth mucosa. The reagent disclosed by the invention is capable of detecting free heme and reactive oxygen intensity of cells in cast-off cell samples, and the detected cells do not have any Warburg effect.

Description

technical field [0001] The invention relates to a simple detection reagent for the Warburg effect of human cells, a preparation method and application thereof, and belongs to the field of medical examination. Background technique [0002] With the improvement of people's quality of life, the incidence of cancer is increasing year by year. In recent years, the number of new cases of malignant tumors in the world has increased year by year, and the number of deaths from malignant tumors in the world is nearly 10 million people every year. Colorectal cancer, malignant tumors on the body surface and oral cancer are several cancers that are more harmful, have a higher incidence rate and are more difficult to cure in recent years. [0003] There are as many as 930,000 new colorectal cancer patients in the world every year, and 130,000 to 160,000 new cases in my country every year. The incidence of colorectal cancer in digestive tract tumors is second only to gastric cancer; the c...

Claims

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Application Information

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IPC IPC(8): G01N21/78
CPCG01N21/78G01N2021/775
Inventor 郑德春
Owner 山东天倪生物技术有限公司
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