CN102911908B - Separation and in vitro culture method of midgut cells of insects - Google Patents
Separation and in vitro culture method of midgut cells of insects Download PDFInfo
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- CN102911908B CN102911908B CN201210475498.8A CN201210475498A CN102911908B CN 102911908 B CN102911908 B CN 102911908B CN 201210475498 A CN201210475498 A CN 201210475498A CN 102911908 B CN102911908 B CN 102911908B
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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
The invention belongs to insect cell research and culture technique field, be specifically related to separated midgut epithelial cells from the midgut tissue of silkworm (Bomyx mori) larva, and the method for cultivating.
Background technology
Middle intestines are the gastral chief components of insect, and midgut epithelial cells is comprised of mast cell, goblet cell and stem cell etc., in insect vital movement, plays an important role.Insect midgut, as the main position of secretion digestive ferment and other enzyme, plays a part digest food and absorbs nutritive substance.Simultaneously, intestines are also pathogenic micro-organism, agricultural chemicals, various toxin, even comprise the target of the multiple unfavorable factor effect of uncomfortable pH, as nuclear polyhedrosis virus (Nuclear polyhedrosis virus, NPV) first after midgut epithelial cells propagation, transfer to again other tissue infection, bacillus thuringiensis (Bacillus thuringiensis, Bt) the special receptors bind on insecticidal crystal protein and midgut epithelial cells film, the normal structure of intestines in destruction, reaches the effect of desinsection.Therefore, the structure of intestines and physiological function in research, significant for prevention that utilizes pathogenic microorganism prevention insect and beneficial insect disease etc.
At present, about the research of insect midgut physiological function, mainly in living insects, carry out both at home and abroad, owing to interacting widely between the complicacy of intestines structure in vivo and midgut epithelial cells, make middle intestines physiology, pathology and the toxicologic research difficulty that becomes, and the function that isolated culture is research midgut epithelial cells provides simply, means and desirable external model fast and accurately.From eighties of last century nineties, more external laboratories are attempted and report the separation of insect midgut cell and former culture, comprise lepidopterous maduca sexta (Manduca sexta), Heliothis virescens (Heliothis virescens), gypsymoth (Lymantria dispar), choristoneura fumigerana (Choristoneura fumiferana) etc., the insects such as the red turpentine beetle of dipterous Aedes aegypti (Aedes aegypti) and Coleoptera (Dendroc tonus valens), but all existing cell obviously weakens in vitro vigor, the problems such as the survival time is not long.Wherein, the isolation technique of midgut epithelial cells is the principal element that causes cell to be difficult to survive, and when separated midgut epithelial cells, enzyme and mechanical effect can cause damage in various degree to midgut epithelial cells, selects the less treatment process of midgut epithelial cells injury particularly important.At present, be mainly to adopt the enzymolysis processs such as collagenase or trypsinase to process the separated midgut epithelial cells of midgut tissue both at home and abroad, separating effect and cell growth state are unsatisfactory.Therefore, how reducing damage, the raising in vitro survival rate of cell of midgut epithelial cells and extend the in vitro time of surviving of cell is problem demanding prompt solution.
Summary of the invention
Shortcoming for prior art, technical problem to be solved by this invention is to provide a kind of simple and effective and to the lightest cell isolation method of insect midgut cell damage, and separated insect midgut cell can be used for the isolated culture of cell, middle intestines physiology and pathological research etc.
First the present invention provides a kind of separation method of silkworm larva midgut epithelial cells, that separated midgut tissue is carried out to enzymolysis with Dispase II enzyme, after enzymolysis finishes, midgut tissue after piping and druming enzymolysis, and enzymolysis solution is filtered to remove middle intestines counterdie tissue, by the centrifugal supernatant discarded of cell filtrate of collecting, obtain the midgut epithelial cells of separated silkworm larva.
Above-mentioned enzymatic hydrolysis condition is as follows: the concentration of Dispase II enzyme in LPS damping fluid is 1mg/mL, and under 28 ℃ of conditions, standing processing 30min completes enzymolysis.
The formula of above-mentioned LPS damping fluid is as follows: the CaCl of the NaCl of 178mmol/L, the KCl of 4.3mmol/L, 4.3mmol/L
2, 3.8mmol/L NaHCO
3, the gentamicin of 100mg/L is, the amphotericin B of 2.5mg/L; PH is 6.5.
Described filtration is to filter with the filtering net that aperture is 100 μ m.
The isolated culture method of the midgut epithelial cells of separated silkworm larva is as follows: separated silkworm larva midgut epithelial cells is suspended in the TNM-FH substratum that is added with conditioned medium and foetal calf serum, cultivates and keep active at 25～30 ℃; The preparation method of described conditioned medium is as follows: Silkworm Embryo Cell Lines Bm-Em-1 is cultured to logarithmic growth after date in being added with the TNM-FH substratum of foetal calf serum, and the filtrate by culture supernatant after membrane filtration is conditioned medium.
The interpolation volume by volume concentration of above-mentioned conditioned medium in TNM-FH substratum is 20%.
The invention provides a kind of simple insect midgut cell isolated culture that is effectively applicable to, the insect midgut cell isolation method of middle intestines physiology and pathological research, comprise the enzyme liquid that the midgut epithelial cells that dissociates is used, maintain applicable substratum and serum-concentration and the additive of the in vitro state of cell, midgut epithelial cells survival rate under just separation is reached more than 80%, cell survival rate after isolated culture 60d is more than 20%, in vitro midgut epithelial cells can be infected by nuclear polyhedrosis virus, and there is the secretion capacity of alkaline phosphatase etc., for being engaged in the research of insect midgut physiological function, lay the foundation.
Embodiment
Below in conjunction with specific embodiment, method of the present invention is described in detail.
Embodiment 1: the separating effect of silkworm (Bomyx mori) larva midgut epithelial cells Dispase Dispase II
(1) get healthy silkworm primary larva in four ages, in Bechtop, through 10% clorox sterilization 5min, 75% alcohol disinfecting 5min, uses aseptic water washing 3 times.Larva is moved into and is equipped with in the culture dish (diameter 33mm) of 10mL sterilizing LPS damping fluid, and dissecting insects, obtains midgut tissue.
(2) by the midgut tissue of the silkworm larva taking out in sterilizing culture dish, the collagenase Collagenase(Roche company that is 1mg/mL by concentration respectively), trypsinase Trypsin(Thermo company), 4 kinds of enzymes such as Dispase Dispase II (Roche company) and HyQTase enzyme (Thermo company) are processed, standing processing under 28 ℃ of conditions, respectively at 15min, 30min, 60min, 90min and 120min sampling, with suction pipe, blow and beat gently midgut tissue and cell, being added to the filtering net that aperture is 100 μ m (BD Falcon company) filters, collecting cell is considered liquid, the centrifugal 5min of 1000r/min, suck supernatant, cell precipitation is suspended in the TNM-FH substratum of 15%FBS, obtain separated midgut epithelial cells.
(3) midgut epithelial cells being suspended in TNM-FH substratum is moved in 24 porocyte culture plates (BD Falcon company), every hole 1mL, being placed in 28 ℃ of incubators cultivates, in inverted phase contrast microscope IX71(Olympus company) lower observation of cell state, density by blood counting chamber computational solution from midgut epithelial cells, and by the survival rate of determination of trypan blue staining cell.
(4) by cell counting, cellular form are observed and Trypan Blue etc., analyze, result shows, silkworm larva midgut epithelial cells with the separation of Dispase II enzyme process, state in relatively disperseing in TNM-FH substratum, mostly be unicellular, only a few cell is agglomerating, and cell diopter is good, is uniform suspended state; The type of midgut epithelial cells mainly contains three kinds of goblet cell, mast cell and regenerative cells, and wherein goblet cell and cylindrocellular ratio are about 1:5; By Trypan Blue, the cell survival rate that enzyme is processed 30min is 82.1%.From aspects such as cell state and survival rates, Dispase II enzyme process is compared with the good separating effect of other several enzymes, after HyQTase enzyme, collagenase Collagenase and trypsinase Trypsin process, cell has into reunion heap phenomenon, dispersion effect is bad, and there is cell debris, the cell survival rate that enzyme is processed 30min is respectively 78.8%, 77.6% and 62.6%.
Midgut epithelial cells with the separation of Dispase II enzyme process, prolongation along with enzyme processing time, midgut epithelial cells quantity under dissociating is on the increase, processing 120min midgut epithelial cells can all disintegrate down from intestinal tissue, process 15min, 30min, the cell proportion under 60min and 90min dissociate is respectively 38.3%, 71.2%, 83.9% and 92.4%; Along with the prolongation of enzyme processing time, the shared ratio of viable cell declines gradually, processes 15min, 30min, and 60min, after 90min and 120min, the survival rate of midgut epithelial cells is respectively 86.6%, 82.1%, 68.8%, 62.2% and 62.0%.Comprehensively think, with Dispase II enzyme, under 28 ℃ of conditions, process midgut epithelial cells state and separating effect the best that midgut tissue 30min obtains.
Embodiment 2: the isolated culture of silkworm larva midgut epithelial cells
(1) screening of midgut epithelial cells optimum medium: the midgut epithelial cells of getting the separation of Dispase II enzyme process, after blood counting chamber counting, cell is added respectively to the TNM-FH(Thermo company containing 15%FBS), TC-100(GIBCO company), Grace(GIBCO company) and IPL-41(GIBCO company) etc. in 4 kinds of different substratum, according to every hole 1.0 * 10
5the concentration of individual cell, is inoculated in 24 porocyte culture plates (BD Falcon company), and every hole 1mL, is placed in 28 ℃ of incubators and cultivates.Every 7d, take out sample, by blood counting chamber counting cells density, and by the survival rate of determination of trypan blue staining cell.
Midgut of Silkworm, Bombyx Mori cell is in 4 kinds of substratum such as TNM-FH, TC-100, Grace and IPL-41, and the initial midgut epithelial cells epidemic situation comparison of cultivating is good, and along with the prolongation of incubation time, cell density reduces, and cell survival rate is also on a declining curve.From being inoculated into, cultivate three weeks, the cell density of midgut epithelial cells in TNM-FH, TC-100, Grace and IPL-41 is from 1.0 * 10
5cells/mL drops to respectively 8.9 * 10
4cells/mL, 6.3 * 10
4cells/mL, 5.3 * 10
4cells/mL and 3.9 * 10
4cells/mL, the cell density of the midgut epithelial cells of cultivating in TNM-FH is the highest, is secondly TC-100, Grace, IPL-41.From being inoculated into, cultivate three weeks, the cell survival rate of midgut epithelial cells in TNM-FH, TC-100, Grace and IPL-41 drops to 26.1%, 19.8%, 17.6% and 14.3% from initial 83.7% respectively, the cell survival rate of the midgut epithelial cells of cultivating in TNM-FH is the highest, is secondly TC-100, Grace, IPL-41.By above result, can be drawn, growth conditions, cell density and the cell survival rate of the Midgut of Silkworm, Bombyx Mori cell in TNM-FH is all good compared with other three kinds of substratum.
(2) the midgut epithelial cells substratum screening of suitable serum-concentration: the midgut epithelial cells of getting the separation of Dispase II enzyme process, after blood counting chamber counting, by cell add respectively containing 0%, 5%, 10%, 15% and the TNM-FH substratum (Thermo company) of 20%FBS in, according to every hole 1.0 * 10
5the concentration of individual cell, is inoculated in 24 porocyte culture plates (Becton Dickinson company), and every hole 1mL, is placed in 28 ℃ of incubators and cultivates.Every 7d, take out sample, by blood counting chamber counting cells density, and by the survival rate of determination of trypan blue staining cell.
Midgut of Silkworm, Bombyx Mori cell isolated culture is after three weeks, and the cell density in the TNM-FH that contains 0%, 5%, 10%, 15%, 20% serum is from initial 1.0 * 10
5cells/mL drops to respectively 3.6 * 10
4cells/mL, 5.1 * 10
4cells/mL, 5.6 * 10
4cells/mL, 7.1 * 10
4cells/mL and 3.8 * 10
4cells/mL.Same, the cell survival rate of midgut epithelial cells in the TNM-FH that contains 0%, 5%, 10%, 15%, 20% serum drops to respectively 11.5%, 13.8%, 16.8%, 22.3% and 12.6% from initial 81.6%.In a word, in the best state containing cell in the TNM-FH of 15% serum, the also isolated culture of applicable midgut epithelial cells.
(3) impact of conditioned medium on midgut epithelial cells isolated culture: get Silkworm Embryo Cell Lines Bm-Em-1(Li Miao seedling that growth conditions is good etc., " foundation of Silkworm Embryo Cell Lines Bm-Em-1 and characteristic research ", < < insect journal > >, 2011, 54(12): 1341-1347) be inoculated in the TNM-FH substratum that is added with 15% foetal calf serum, under 28 ℃ of conditions, cultivate 5d to logarithmic growth after date, after the centrifugal 10min of 4000r/min, by supernatant liquor via hole diameter, be that filtrate after the membrane filtration of 0.22 μ m is conditioned medium.According to the ratio of 1:4 (v/v), conditioned medium is joined in the TNM-FH substratum containing 15%FBS again, make the TNM-FH substratum containing 20% conditioned medium and 15%FBS.Get the midgut epithelial cells of Dispase II enzyme process separation, after blood counting chamber counting, cell is added respectively containing conditioned medium and not containing in the TNM-FH substratum of conditioned medium, according to every hole 1.0 * 10
5the concentration of individual cell, is inoculated in 24 porocyte culture plates (BDFalcon company), and every hole 1mL, is placed in 28 ℃ of incubators and cultivates.Every 7d, take out sample, by blood counting chamber counting cells density, and by the survival rate of determination of trypan blue staining cell.Test-results shows, the midgut epithelial cells of cultivating after adding conditional substratum no matter aspect cell density or cell survival rate all higher than adding conditional substratum not.
(4) isolated culture of silkworm larva midgut epithelial cells: the midgut epithelial cells of getting the separation of Dispase II enzyme process, after blood counting chamber counting, inoculating cell is in the TNM-FH substratum containing 20% conditioned medium and 15%FBS, be sub-packed in 24 porocyte culture plates (BD Falcon company), being placed in 28 ℃ of incubators cultivates, every 15d, change 1/2 fresh culture, in inverted phase contrast microscope IX71(Olympus company) lower observation of cell state, and by the survival rate of determination of trypan blue staining cell.Isolated culture 60d, the survival rate of cell still has 25.6%.Above-mentioned research and result prove, the complex medium of the present invention's configuration is for keeping the activity of silkworm larva midgut epithelial cells to have outstanding effect.
Embodiment 3: the in vitro virus infection of silkworm larva midgut epithelial cells
(1) Bombyx mori nuclear polyhydrosis virus (BmNPV) infected silkworm embryo cell line Bm-Em-1(Li Miao seedling etc., " foundation of Silkworm Embryo Cell Lines Bm-Em-1 and characteristic research ", < < insect journal > >, 2011,54(12): 1341-1347), breed, make viral suspension.
(2) get the Midgut of Silkworm, Bombyx Mori cell of isolated culture 7d, after blood counting chamber counting, by 1.0 * 10
5the amount of cells/well accesses in 24 porocyte culture plates, repeats for 3 times; Cultivate 2h for 28 ℃, suck substratum, every hole adds BmNPV virus (infection multiplicity MOI=10), cultivates and observes for 28 ℃, checks virus infection result after 4d.
(3) after inoculation BmNPV, cellular form changes gradually, round cell quantity increases, nucleus increases, and in core, chromatin condensation becomes piece, cell expansion, after virus infection 4d, in cell, start to occur polyhedron, during virus infection 6d, in cell, polyhedrosis quantity increases, and occurs typical virus infection symptom.As can be seen here, the Midgut of Silkworm, Bombyx Mori cell of isolated culture can be infected by BmNPV.
Embodiment 4: Midgut of Silkworm, Bombyx Mori isolated cells alkaline phosphatase (alkaline phosphatase, AKP) determination of activity
(1) get the Midgut of Silkworm, Bombyx Mori cell of isolated culture 7d, the centrifugal 5min collecting cell of 1000r/min, be suspended in damping fluid (100mM N.F,USP MANNITOL, 10mM Hepes-Tris, pH 7.2) in, after ultrasonic treatment cell, the concentration determination of total protein of cell adopts Bradford method, the OD value of spectrophotometry 595nm, calculates cell protein concentration by typical curve.
(2) activity of alkaline phosphatase is to measure the p-nitrophenol concentration that the hydrolysis of p-p-nitrophenyl phosphoric acid ester produces, use cell alkaline phosphatase activities colorimetric determination detection kit (Genmed company), with reference to product description, in the OD of spectrophotometric determination 400nm value, according to formula: protein content (mg) in AKP specific activity (U/mg)=every mL cell ZhongAKP activity unit's number (U)/every mL cell, calculates enzymic activity.
(3) after measured, cultivate in the in vitro Midgut of Silkworm, Bombyx Mori cell of 7d, the activity of alkaline phosphatase can be detected, its activity is 143 ± 16mU/mg.
Above-mentioned result shows that the inventive method can effectively maintain the activity of midgut epithelial cells in vitro, thereby the Midgut of Silkworm, Bombyx Mori isolated cells of separation and Culture can be infected by nuclear polyhedrosis virus, and there is the secretion capacity of alkaline phosphatase etc., for being engaged in the research of insect midgut physiological function, lay the foundation.
Claims (3)
1. the separation method of a silkworm larva midgut epithelial cells, it is characterized in that, described separation method is that separated midgut tissue is carried out to enzymolysis with Dispase II enzyme, after enzymolysis finishes, midgut tissue after piping and druming enzymolysis, and enzymolysis solution is filtered to remove middle intestines counterdie tissue, by the centrifugal supernatant discarded of cell filtrate of collecting, obtain the midgut epithelial cells of separated silkworm larva; Described enzymatic hydrolysis condition is as follows: the concentration of Dispase II enzyme in LPS damping fluid is 1mg/mL, and under 28 ℃ of conditions, standing processing 30min completes enzymolysis; Described LPS damping fluid composed as follows: the CaCl of the NaCl of 178mmol/L, the KCl of 4.3mmol/L, 4.3mmol/L
2, 3.8mmol/L NaHCO
3, the gentamicin of 100mg/L is, the amphotericin B of 2.5mg/L; PH is 6.5;
Separated silkworm larva midgut epithelial cells is suspended in the TNM-FH substratum that is added with conditioned medium and foetal calf serum, at 25～30 ℃, cultivates and keep active; The preparation method of described conditioned medium is as follows: Silkworm Embryo Cell Lines Bm-Em-1 is cultured to logarithmic growth after date in being added with the TNM-FH substratum of foetal calf serum, and the filtrate by culture supernatant after membrane filtration is conditioned medium.
2. separation method as claimed in claim 1, is characterized in that described enzymolysis solution filters, and is to filter with the filtering net that aperture is 100 μ m.
3. separation method as claimed in claim 1, is characterized in that the volume by volume concentration that described conditioned medium adds in TNM-FH substratum is 20%.
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| CN201310314719.8A CN103352024B (en) | 2012-11-19 | 2012-11-19 | In vitro cultivation method for insect mesenteron cells |
| CN201210475498.8A CN102911908B (en) | 2012-11-19 | 2012-11-19 | Separation and in vitro culture method of midgut cells of insects |
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| CN201210475498.8A CN102911908B (en) | 2012-11-19 | 2012-11-19 | Separation and in vitro culture method of midgut cells of insects |
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| US5298418A (en) * | 1991-09-16 | 1994-03-29 | Boyce Thompson Institute For Plant Research, Inc. | Cell line isolated from larval midgut tissue of Trichoplusia ni |
| CN102093979A (en) * | 2009-12-14 | 2011-06-15 | 中国人民解放军军事医学科学院野战输血研究所 | Method for separating cells from different tissues |
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