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Separation and in vitro culture method of midgut cells of insects

A separation method and in vitro culture technology, applied in the field of insect cell research and culture, can solve the problems of separation effect and unsatisfactory cell growth state.

Inactive Publication Date: 2013-02-06
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, at home and abroad, the midgut tissue is mainly treated with enzymatic hydrolysis methods such as collagenase or trypsin to separate midgut cells, but the separation effect and cell growth status are not ideal.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: Isolation effect of dispase Ⅱ from midgut cells of silkworm (Bomyx mori) larvae

[0014] (1) Take healthy fourth-instar early larvae of silkworm, disinfect them with 10% sodium hypochlorite for 5 minutes, 75% alcohol for 5 minutes, and rinse with sterile water for 3 times in an ultra-clean workbench. Transfer the larvae into a Petri dish (33 mm in diameter) filled with 10 mL of sterilized LPS buffer, dissect the insects, and obtain the midgut tissue.

[0015] (2) Place the midgut tissue of the silkworm larvae in a sterilized petri dish, and use collagenase Collagenase (Roche Company), trypsin Trypsin (Thermo Company), and dispase II (Roche Company) at a concentration of 1 mg / mL, respectively, in a sterilized petri dish. ) and HyQTase enzyme (Thermo Company) and other 4 kinds of enzymes were treated, and the treatment was static at 28°C, and samples were taken at 15min, 30min, 60min, 90min and 120min respectively, and the midgut tissue and cells were gently bl...

Embodiment 2

[0019] Embodiment 2: In Vitro Culture of Silkworm Larval Midgut Cells

[0020] (1) Screening of the most suitable medium for midgut cells: take the midgut cells separated by Dispase Ⅱ enzymatic method, count them on a hemocytometer, add the cells to TNM-FH (Thermo Company) and TC-100 containing 15% FBS respectively (GIBCO Company), Grace (GIBCO Company) and IPL-41 (GIBCO Company) and other 4 different media, according to 1.0×10 per well 5 Inoculated into 24-well cell culture plates (BD Falcon Company), 1 mL per well, and cultured in a 28°C incubator. Samples were taken out every 7 days, the cell density was counted with a hemocytometer, and the viability of the cells was determined by trypan blue staining.

[0021] Bombyx mori midgut cells were cultured in 4 kinds of media including TNM-FH, TC-100, Grace and IPL-41. The initial cultured midgut cells were in good condition. With the prolongation of culture time, the cell density decreased and the cell survival rate also decrea...

Embodiment 3

[0026] Example 3: In vitro virus infection of silkworm larvae midgut cells

[0027] (1) Bombyx mori nuclear polyhedrosis virus (BmNPV) infected silkworm embryonic cell line Bm-Em-1 (Li Miaomiao et al., "Establishment and characterization of silkworm embryonic cell line Bm-Em-1", Acta Entomology, 2011, 54(12): 1341-1347) to propagate and make virus suspension.

[0028] (2) Take silkworm midgut cells cultured in vitro for 7 days, count on a hemocytometer, and measure 1.0×10 5 The amount of cells / well was inserted into a 24-well cell culture plate and repeated 3 times; cultured at 28°C for 2 hours, sucked off the medium, added BmNPV virus (MOI=10) to each well, cultured at 28°C and observed, and checked after 4 days Viral infection results.

[0029] (3) After inoculation with BmNPV, the cell morphology gradually changed, the number of round cells increased, the nucleus enlarged, the chromatin in the nucleus condensed into blocks, and the cells swelled. Polyhedrons began to appe...

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Abstract

The invention provides a separation and in vitro culture method of midgut cells of insects. The separated midgut tissues are subjected to enzymolysis by using a Dispase II enzyme, and the obtained midgu cells are subjected to in vitro culture by using a TNM-FH culture medium added with a conditioned medium and fetal bovine serum. The separation method of midgut cells of insects is simple, effective and suitable for the in vitro culture of midgut cells of insects, midgut physiology and pathology research. Enzyme liquid for dissociating the midgut cells, suitable culture media for maintaining the in vitro condition of cells, serum concentration and additives are included; the survival rate of the just separated midgut cells can reach above 80%, the survival rate of cells can reach above 20% after 60 days of the in vitro culture, the midgut cells can be infected by nuclear polyhedrosis virus under the in vitro condition, the secretion capacity such as alkaline phosphatase is provided, and the foundation is laid for research of the midgut physiological function of insects.

Description

technical field [0001] The invention belongs to the technical field of insect cell research and cultivation, in particular to a method for isolating and culturing midgut cells from the midgut tissue of silkworm (Bomyx mori) larvae. Background technique [0002] The midgut is the main part of the digestive tract of insects. The midgut cells are composed of columnar cells, goblet cells and stem cells, which play an important role in the life activities of insects. The midgut of insects is the main part of secreting digestive enzymes and other enzymes, which plays a role in digesting food and absorbing nutrients. At the same time, the midgut is also the target of various unfavorable factors including pathogenic microorganisms, pesticides, various toxins, and even unfavorable pH, such as nuclear polyhedrosis virus (Nuclear polyhedrosis virus, NPV) first proliferates in the midgut cells and then transfers When other tissues are infected, the insecticidal crystal protein of Bacil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07
Inventor 李长友郑桂玲周洪旭吴艳蕾童丹丹
Owner QINGDAO AGRI UNIV
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