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Method for extracting, separating and identifying chilo suppressalis midgut stem cells

A technology of intestinal stem cells and identification methods, applied in the field of extraction, separation and identification of midgut stem cells of C. To achieve the effect of inhibiting bacterial growth, avoiding cell contamination, and regular cell shape

Active Publication Date: 2015-02-04
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the disadvantages of this method are: the amount of stem cells obtained is very small, the purity is not high, and there are many enterocytes mixed
The first step to carry out this research is to extract, isolate and identify midgut stem cells, and establish a pure line of midgut stem cells, but there is no report on the extraction, isolation and identification of midgut stem cells in C.

Method used

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  • Method for extracting, separating and identifying chilo suppressalis midgut stem cells
  • Method for extracting, separating and identifying chilo suppressalis midgut stem cells
  • Method for extracting, separating and identifying chilo suppressalis midgut stem cells

Examples

Experimental program
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Embodiment 1

[0044] In the absence of collagenase Ⅰ, Ficoll-Paque separation medium and density gradient centrifugation were used to extract, isolate and identify stem cells in the midgut of C. medinalis (taking 20 3rd instar larvae of C. medulla as an example);

[0045] 1.1 Under aseptic conditions, soak the 3rd instar larvae of Chilo borer in 2% sodium hypochlorite solution for 2 minutes, 75% ethanol for 2 minutes, and sterile dd H 2 In O for 2 minutes, carry out body surface disinfection;

[0046] 1.2 Put the worm body on a wax plate sterilized with 75% ethanol, and dissect the midgut of the larvae of Chilo suppressalis. Cut the ends of the thorax and abdomen separately, then cut along the worm body from the abdominal incision to the chest incision, and pull out the midgut side with tweezers;

[0047] 1.3 Take out the midgut, put it into a Petri dish filled with Ringer's solution, and use ophthalmic forceps to remove Malpighian tubes, peritrophic membranes and food residues, etc.;

[...

Embodiment 2

[0053] Collagenase I was added, and Ficoll-Paque separation solution and density gradient centrifugation were used to extract, separate and identify stem cells in the midgut of C. borer (taking the dissection of 40 third-instar larvae of C. borer as an example);

[0054] 2.1 Under sterile conditions, soak the 3rd instar larvae of Chilo borer in 2% sodium hypochlorite solution for 2 minutes, 75% ethanol for 5 minutes, and sterile dd H 2 In O for 2 minutes, carry out surface disinfection;

[0055] 2.2 Place the larvae in a wax plate sterilized with 75% ethanol, and dissect the midgut of the larvae of C. Cut the ends of the thorax and abdomen separately, then cut along the worm body from the abdominal incision to the chest incision, and pull out the midgut side with tweezers;

[0056] 2.3 Take out the midgut, put it into a petri dish filled with Ringer's solution, and use ophthalmic forceps to remove Malpighian tubes, peritrophic membranes and food residues, etc.;

[0057] 2.4 ...

Embodiment 3

[0065] Collagenase Ⅰ was added, and the midgut stem cells of C. borer were extracted, separated and identified by flow cytometry (take the dissection of 20 3rd instar larvae of C. borer as an example)

[0066] It is known from Example 2 that 0.1% collagenase I is beneficial to the extraction of midgut stem cells without damaging the cells, so in Example 3, 0.1% collagenase I was added to extract stem cells.

[0067] 3.1 Under aseptic conditions, soak the 3rd instar larvae of Chilo borer in 2% sodium hypochlorite solution for 2 minutes, 75% ethanol for 5 minutes, and sterile dd H 2 In O for 2 minutes, carry out surface disinfection;

[0068] 3.2 Put the worm body on a wax plate sterilized with 75% ethanol, and dissect the midgut of the larvae of Chilo suppressalis. Cut the ends of the thorax and abdomen separately, then cut along the worm body from the abdominal incision to the chest incision, and pull out the midgut side with tweezers;

[0069] 3.3 Take out the midgut, put i...

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Abstract

The invention relates to a method for extracting, separating and identifying chilo suppressalis midgut stem cells. The method comprises the following steps: carrying out enzymolysis on midgut cells by collagenase I so that the midgut cells fall off from the midgut wall, and then separating the stem cells and enteric cells by adopting a density gradient centrifugal process or a flow cytometry. By virtue of the method, high-purity chilo suppressalis midgut stem cells are efficiently obtained and can be applied to the subsequent stem cell function study.

Description

technical field [0001] The invention relates to the field of extraction, separation and identification of insect midgut cells, in particular to a method for extraction, separation and identification of midgut stem cells of Chilo suppressalis larvae. Background technique [0002] The insect midgut originates from the endoderm and is mainly responsible for the absorption of nutrients. It consists of four parts: peritrophic membrane, midgut epithelial cells, basement membrane and muscle layer. The midgut epithelial cells include: enteroblasts (columnar cells and goblet cells), stem cells and endocrine cells. Stem cells are located at the basement membrane of the epithelial cell layer and are a group of small cells sandwiched between goblet or columnar cells. They have a strong ability to renew and can continuously proliferate and differentiate to replace aging or damaged old cells. Produce the progeny cells necessary for the intestine, so that the intestine has a strong abilit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07G01N15/14
Inventor 韩兰芝周艳聪侯茂林彭于发
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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