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Digestive juice preparation method

A technology of digestive juice and mother liquor, applied in the fields of biochemical equipment and methods, microorganisms, tumor/cancer cells, etc., can solve the problems of insufficient materials, affecting the patient's treatment plan, prognosis and outcome, long operation time, etc. Significant and accurate positive rate of lymph node metastasis and convenient material collection

Active Publication Date: 2018-07-31
SHAOXING PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the ex vivo tissue specimens of tumor lymph node dissection, thorough lymph node sampling is very important for tumor diagnosis and staging, formulation of treatment plans, and judgment of prognosis. However, neither conventional fixative solution nor existing specimen fixation methods can meet the requirements of rapid fixation while improving the quality of lymph nodes. The speed and detection rate of material collection make pathologists find lymph nodes in the long-term isolated adipose tissue with many disadvantages such as insufficient material collection and long operation time, which will ultimately affect the patient's treatment plan, prognosis and outcome

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: Digestive juice of different compositions constitutes

[0031] The core functional components of the digestive juice provided by this application are digestive enzymes, and the main components are proteinase K, trypsin and EDTA. Under different implementation environments, one of PBS or Tris-HCl can also be used as a buffer, and a CaCl 2 To stabilize the enzyme activity, the specific composition selection method can be found in Table 1.

[0032] Table 1 Digestive fluids with different compositions

[0033] serial number

Proteinase K

Trypsin

EDTA

PBS

Tris-HCl

CaCl 2

1

√

√

√

-

-

-

2

√

√

√

√

-

-

3

√

√

√

-

√

4

√

√

√

-

-

√

5

√

√

√

√

√

-

6

√

√

√

√

-

√

7

√

√

√

-

√

√

8

√

√

√

√

√

√

[0034] We tested the digestive juice of the core co...

Embodiment 2

[0037] At the same time, in different implementation environments, they were dissolved in different buffer solutions, and the role of the digestive solution will be described below by taking the solution in PBS solution and Tris-HCl as representatives respectively.

[0038] (1) PBS buffer: pH7.4, the main function is to buffer and maintain the pH value and salt ion concentration of the reaction system. In addition, 1-10mM CaCl can also be added 2 , to stabilize divalent cations required for enzyme activity.

[0039] (2) pH7.5 50mM Tris-HCl buffer solution, during the preparation process, 1-10mM CaCl can be added 2 , the preparation method is: add 6 grams of Tris-Base to 900ml of water, adjust the pH to 7.5 with HCl, and add CaCl 2 to a final concentration of 10 mM. The main function of Tris is to buffer and maintain the pH value and salt ion concentration of the reaction system, CaCl 2 Divalent cation required for stabilization of enzyme activity.

Embodiment 3

[0040] Embodiment 3: digestion effect contrast

[0041]In this example, six copies of fresh adipose tissue and lymph nodes resected by radical gastrectomy were taken and processed as follows: (1) The fixative was treated at room temperature for 3 hours, and the fixative was composed of components with the following concentration ratios: Formaldehyde 100ml / L, lecithin 50g / L, absolute ethanol 250ml / L, acetone 250ml / L, deoxycholic acid 47.5g / L, methanol 250ml / L, PBS solution 1×, methylene blue 0.1g / L;( 2) Treat with 0.1N HCl / PBS solution at room temperature for 1 hour; (3) Treat with 0.1N NaOH / PBS solution at room temperature for 1 hour; (4) Treat with digestive solution, 25mg trypsin-1%EDTA / ml+Protease K, 37℃ water bath 4 hours, and carry out parallel experiment to six samples with proteinase K of 0.5mg / ml, 0.25mg / ml, 0.1mg / ml, 0.5mg / ml, 0.25mg / ml, 0.1mg / ml respectively, the above treatment each Phase effects are observed.

[0042] Each tissue specimen processed in step (1) is...

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Abstract

The invention relates to a digestive juice preparation method and belongs to the technical field of preparation of samples for tests. The digestive juice preparation method comprises the following steps of weighing EDTA powder, dissolving the EDTA powder into PBS, regulating the pH value of the solution to be suitable during preparation, performing uniform stirring, and preparing an EDTA mother liquor; respectively weighing trypsin and protease K, dissolving the trypsin and the protease K into the PBS solution, dropwise adding the dissolved solution in the EDTA mother liquor according to a ratio, and supplementing the PBS with dropwise adding according to the needs to prepare a digestive juice; and the prepared digestive juice is prepared from the following components by concentration: 0.1-2mg / ml protease K, 1-100mg / ml trypsin and 0.1-2mg / ml EDTA. The digestive juice preparation method provided by the invention is applied to tumor lymph node tissue specimen handing, particularly is used to radically cure the specimen by combining with a stationary liquid and tumor lymph nodes subjected to acid and alkaline treatment, increases a hardness difference of fatty tissues and the lymph nodes, is convenient in lymph node material drawing, effectively increases a lymph node detection amount and the accuracy of the lymphatic metastasis positive rate.

Description

technical field [0001] The application relates to a method for preparing digestive juice, which belongs to the technical field of test sample preparation. Background technique [0002] Existing tumor lymph node dissected tissue specimens in vitro are usually soaked in formalin fixative solution, mainly because formaldehyde can preserve histomorphological and structural characteristics for a long time and has good economic utility at the same time. Although this type of specimen processing method can well fix the shape of the tissue, it also hardens the specimen containing adipose tissue, which greatly increases the difficulty in obtaining materials in pathology departments, especially lymph nodes. In the ex vivo tissue specimens of tumor lymph node dissection, thorough lymph node sampling is very important for tumor diagnosis and staging, formulation of treatment plans, and judgment of prognosis. However, neither conventional fixative solution nor existing specimen fixation ...

Claims

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Application Information

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IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2500/30C12N2501/734
Inventor 王诚陆晓青余美文侯传玲姚育英孙爱静
Owner SHAOXING PEOPLES HOSPITAL
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