CN109792972B - In-vitro culture method for cysticercus cellulosae - Google Patents
In-vitro culture method for cysticercus cellulosae Download PDFInfo
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Abstract
本发明提供一种囊尾蚴体外培养方法，特别是一种豆状囊尾蚴离体培养方法。本发明的方法是：从宿主动物体内分离完整的囊尾蚴清洗干净后，转入其内加有培养液的培养容器内，培养容器置于二氧化碳细胞培养箱内，在温度37℃、CO2浓度5％、相对湿度70％条件下进行培养，虫体开始培养后的第6h及第12h各换液1次，每次换液时尽量除去陈旧培养液，加入新鲜的完全培养液8ml/瓶/次，以后每48h换液1次。本发明有效解决了豆状囊尾蚴离体长时间存活的问题，可以便捷地获得充足的研究材料，虫体体外存活时间长，可满足药物筛选及药理评价的筛选周期，同时可直接观察虫体‑药物作用。本发明的培养不依赖于宿主，实验背景易于控制，可对幼虫阶段具体实验做出较正确的评估。The invention provides a method for in vitro culture of cysticercosis, in particular a method for in vitro culture of cysticercoid bean. The method of the present invention is as follows: after the complete cysticercosis is separated from the host animal and cleaned, it is transferred into a culture container with a culture medium added therein, the culture container is placed in a carbon dioxide cell incubator, and the temperature is 37°C and the CO2 concentration Cultivate under the conditions of 5% and 70% relative humidity. Change the medium once at the 6th and 12th hour after the worm begins to cultivate. Try to remove the old culture medium every time the medium is changed, and add fresh complete culture medium 8ml/bottle/ times, and the medium was changed every 48 h thereafter. The invention effectively solves the problem of long-term in vitro survival of cysticercoid bean, can easily obtain sufficient research materials, has a long in vitro survival time of the parasite, can meet the screening cycle of drug screening and pharmacological evaluation, and can directly observe the parasite at the same time. ‑ Drug effect. The cultivation of the invention does not depend on the host, the experimental background is easy to control, and the specific experiments of the larval stage can be more accurately evaluated.
Description
Technical Field
The invention relates to an in vitro parasite culture method, which is an in vitro cysticercus cellulosae culture method, in particular to an in vitro cysticercus pisiformis culture method.
Background
Cysticercosis is a common parasitic disease, has wide distribution range and seriously threatens the development of animal husbandry and public health safety. The cysticercosis pisiformis is a common parasitic disease caused by the parasitosis of the middle-taeniasis pisiformis which is a stigmatic cysticercosis parasitic in rabbits. In China, the average infection rate of rabbits reaches 40%, the death rate is 4.0% -23.69%, and the development of rabbit breeding is seriously threatened. The cysticercus pisiformis is mainly parasitized on the liver, the omentum ventriculi, the mesenterium or the rectal posterior wall of the rabbit; the liver of a sick rabbit is damaged when the rabbit is infected by a large amount, and the rabbit clinically shows symptoms such as digestive disorder, inappetence, mental depression, emaciation, anemia and the like and dies suddenly when the rabbit is serious; in addition, pasteurellosis and the like can be generated secondarily, so that the feed return rate of the rabbits is reduced, and the economic benefit is reduced.
The tapeworm has a complex life history, and the complete life history can be completed by converting 2 hosts. Adults are usually parasitic in the small intestine of dogs, and eggs of the adults develop in the pregnancies of the adults and are released to the external environment along with the shedding of the pregnancies, thereby polluting food or water sources. After being swallowed by the rabbit, the eggs reach the liver along with the blood flow and move to the omentum majus, mesenterium and the like to develop into infectious cysticercus pisiformis. At present, China still faces a great problem in preventing and treating the disease, and screening and excavating specific drug targets and vaccine candidate molecules are urgently needed, so that a new means is provided for effective prevention, control and radical treatment of the disease. With the development of large-scale study of parasite functional genomes, gene function verification and novel drug analysis become bottlenecks in current parasite research. In the aspect of taenia pisiformis research, no effective stable cell line can be used, and a feasible in vitro evaluation model is not established yet.
In vitro culture is a valuable technical platform in the study of parasitology, and can carry out the related studies on parasite antigens, drug action, immune effect, parasite and host interaction and the like under intuitive and controllable conditions. In recent decades, with the continuous improvement and improvement of culture methods, the development of artificial culture technology research of parasites is promoted by establishing a suitable culture method by simulating certain factors and conditions of host organisms. However, the parasites are of various types and have complex life history, and no universal culture solution or uniform culture method is used for culturing the parasites in vitro.
The research on the taenia culture in vitro technology is relatively slow compared to protozoa, trematodes and nematodes. At present, live insect samples for researching bean-shaped cysticercus are mainly collected from naturally or experimentally infected rabbits, and the method has the following defects:
(1) the method for protecting the insects by means of the canine-rabbit circulating infection has high cost, large workload, great labor and more invested personnel; there are problems in safety and economy. In addition, the insect retention test period is long, and generally about 4 months are needed for completion.
(2) Depending on live cysticercus cellulosae samples in rabbits, the researches such as drug screening, drug effect evaluation and the like are not convenient to be carried out under intuitive or controllable conditions.
(3) Sufficient metabolic secretory antigens or exosomes cannot be collected at any time by depending on the cysticercus live insect sample in the rabbit.
Therefore, the feasible and stable method for culturing the lenticular cysticercus cellulosae in vitro is provided, the time and the cost for scientific research can be greatly shortened, and the method is beneficial to the research of antigen collection, drug screening, drug insecticidal effect analysis and the like in vitro; has important academic value and application significance for the research of tapeworm and the research of prevention and treatment technology thereof.
Disclosure of Invention
The invention provides an effective method for culturing cysticercus cellulosae in vitro, and particularly provides a method for culturing cysticercus cellulosae, which can realize the long-time survival of the cysticercus cellulosae in vitro. In addition, the invention also provides an in-vitro culture method of the phaeocysticercus cellulosae, which is convenient for preparing metabolic secretion antigens of the phaeocysticercus cellulosae, preparing exosomes, detecting the metabolism of the phaeocysticercus cellulosae, screening medicines and the like. Can also be used as a substitute model for researching other important tapeworm gene functions, such as cysticercosis cellulosae, cysticercosis bovis and the like. However, the in vitro culture process only keeps the larva morphology of the cysticercus, and does not relate to the development of the larva into imagoes through in vitro culture.
In order to achieve the purpose, the invention adopts the following technical scheme:
the method for culturing cysticercus in vitro comprises separating intact cysticercus from host animal, washing with PBS containing at least one antibiotic under aseptic condition, transferring into culture container containing culture solution, placing the culture container in carbon dioxide cell culture box, culturing at 37 deg.C and CO2Culturing under the conditions of 5% concentration and 70% relative humidity, changing the culture solution for 1 time in 6h and 12h after the start of culturing, removing old culture solution as much as possible during each liquid change, adding fresh complete culture solution 8 ml/bottle/time, and changing the culture solution for 1 time every 48 h.
The invention relates to a method for culturing coenuruses in vitro, which comprises the following steps: separating fresh Lepisciformis pisciformis from abdominal cavity of infected rabbit, washing with PBS containing at least one antibiotic under aseptic condition, transferring into culture container containing culture solution, placing the culture container in carbon dioxide cell culture box, and culturing at 37 deg.C under CO2Culturing under the conditions of 5% concentration and 70% relative humidity, changing the culture solution for 1 time in 6h and 12h after the start of culturing, removing old culture solution as much as possible during each liquid change, adding fresh complete culture solution 8 ml/bottle/time, and changing the culture solution for 1 time every 48 h.
Preferably, in the method for culturing the cysticercus pisiformis ex vivo, the PBS is added with 100U/ml penicillin and 100 mug/ml streptomycin, and the culture solution is RPMI1640 culture solution added with fetal bovine serum, penicillin, streptomycin, amphotericin B, L-glutamine and sodium bicarbonate, wherein: the fetal bovine serum content is 10% (v/v); the final concentration of the antibiotics is 100U/mL of penicillin, 100 mu g/mL of streptomycin and 1.25 mu g/mL of amphotericin B; the final concentration of the L-glutamine is 2.5 mM; and adding sodium bicarbonate to adjust the pH of the culture solution to 7.2-7.4.
Preferably, the addition amount of the initial culture solution of the method for culturing the cysticercus pisiformis in vitro of the invention is as follows: the adding amount of the 6-hole plate is 3 ml/hole, or the adding amount of the cell bottle is 8ml/(25 cm)2) And (7) a bottle.
The method for in vitro culture of the cysticercus fabarus used for collecting the exosomes of the cysticercus fabarus comprises the following steps: washing fresh and intact coenurosis pisiformis separated from abdominal cavity of infected rabbit under aseptic condition with PBS containing at least one antibiotic, transferring into culture container containing culture solution for treating with past exosome, placing the culture container in carbon dioxide cell culture box, and culturing at 37 deg.C under CO2Culturing under the conditions of 5% concentration and 70% relative humidity, changing the culture solution for 1 time in 6h and 12h after the start of culturing, removing old culture solution as much as possible during each liquid change, adding fresh complete culture solution 8 ml/bottle/time, and changing the culture solution for 1 time every 48 hours.
Preferably, the PBS used in the method for culturing the cysticercus cellulosae exosomes in vitro is added with 100U/ml of penicillin and 100. mu.g/ml of streptomycin, and the culture solution is RPMI1640 culture solution added with penicillin, streptomycin, amphotericin B, L-glutamine and sodium bicarbonate and fetal bovine serum treated by the exosomes in the past, wherein: the fetal bovine serum content is 10% (v/v); the final concentration of the antibiotics is 100U/mL of penicillin, 100 mu g/mL of streptomycin and 1.25 mu g/mL of amphotericin B; the final concentration of the L-glutamine is 2.5 mM; and adding sodium bicarbonate to adjust the pH of the culture solution to 7.2-7.4.
Preferably, the addition amount of the initial culture solution of the method for culturing the cysticercus pisiformis in vitro of the invention is as follows: the adding amount of the 6-hole plate is 3 ml/hole, or the adding amount of the cell bottle is 8ml/(25 cm)2) And (7) a bottle.
The method for collecting the metabolic secretion antigen and the exosome of the phacoenura pisiformis is to culture the phacoenura pisiformis according to the method, collect the culture solution of the phacoenura pisiformis once every 48 hours after the solution is changed for the second time, and further obtain the metabolic secretion antigen or the exosome by the methods of concentration, ultracentrifugation and the like.
The invention has the advantages that:
(1) provides an in vitro culture method for the cysticercus pisiformis, effectively solves the problem of long-time survival of the cysticercus pisiformis in vitro;
(2) sufficient research materials, such as metabolic secretion antigens and exosomes of the cysticercus pisiformis can be conveniently obtained;
(3) the survival time of the polypide in vitro is long, the screening period of drug screening and pharmacological evaluation can be met, and the polypide-drug action can be directly observed;
(4) the isolated culture is independent of a host, and the researches on metabolic antigen, immune effect, drug screening and the like of the cysticercus pisiformis are realized under controllable, intuitive and known conditions;
(5) the simple culture solution formula is adopted, and the experimental background is easy to control;
(6) the establishment of indexes is reasonable, the in vitro culture strictly stipulates that only the survival state of the cysticercus larvae is kept, and the concepts of the growth and development of the larvae and the reproduction are not involved, for example, the larvae grow into imagoes in vitro; more accurate assessments can be made of specific experiments on larval stages.
Drawings
FIG. 1 is a typical morphology observation picture of a light microscope for in vitro culture of the cysticercus faboidalis of the present invention. Where x 40 observes o: culturing the pre-polypide; a: RPMI-1640+ 6% rabbit bile culture group; b: a PBS culture group; c: RPMI-1640 culture group; d: complete broth 1 culture group. X 100 Observation e: the head form of the worm body of the a and b groups; f: and c and d groups of head node forms. Sc represents the head section; su denotes a suction cup; BW denotes a vesicle; ho denotes a small hook.
FIG. 2 is the result of western blot for metabolic secretion of antigen by cysticercus fabarus of the present invention. Wherein M: protein molecular weight marker; ESP: metabolically secreting an antigen; tpeno: the enolase of the cysticercus pisiformis has a natural protein molecular weight of about 48 kDa; tp 14-3-3: 14-3-3 of the cysticercus pisiformis, and the molecular weight of the natural protein is about 28 kDa.
FIG. 3 is the electron microscope identification picture of exosome of culture supernatant of phacoenuridae of the invention. In which the arrow indicates the positive-labeled exosome particles of CD 63.
Detailed Description
For a better understanding of the present invention, the contents of the present invention will be further explained with reference to the drawings and examples, but the contents of the present invention are not limited to the following examples.
Reagents and materials used in the following examples are commercially available unless otherwise specified. Fetal bovine serum, exosome-free fetal bovine serum, RPMI1640 medium, and 7.5% sodium bicarbonate solution were purchased from Gibco; L-Glutamine, penicillin, streptomycin and amphotericin B were all purchased from Sigma.
EXAMPLE 1 Long-term culture of Lemnaria pisiformis in vitro
1. Preparation of complete culture solution
The complete culture solution consists of mother solution and additives, and the preparation process is strictly aseptic.
1.1 complete broth 1
Taking RPMI1640 culture solution as mother solution; the mother liquor is a synthetic culture medium and contains various nutritional ingredients such as glucose, amino acids, balanced salts, vitamins, etc. The additives include fetal calf serum, penicillin, streptomycin, amphotericin B, L-glutamine and sodium bicarbonate. The content of the fetal calf serum in the complete culture medium is 10% (v/v); the final concentration of the antibiotics is 100U/mL of penicillin, 100 mu g/mL of streptomycin and 1.25 mu g/mL of amphotericin B; the final concentration of the L-glutamine is 2.5 mM; and adjusting the pH of the complete culture solution to 7.2-7.4 by using a proper amount of 7.5% sodium bicarbonate.
1.2 complete Medium 2
The serum additive in the complete culture solution 2 is non-exosome fetal calf serum, and the mother solution and other additives are the same as the complete culture solution 1.
2. Collection and pretreatment of phaeocysticercus pisiformis
The specific steps of collecting the cysticercus pisiformis and carrying out sterile treatment are as follows: parasitic lenticular cysticercus collected from abdominal cavity of infected rabbit, and tissues of parasitic parts such as omentum ventriculi are taken down simultaneously. Placing in a clean bench, cleaning with sterile PBS (containing double antibody) for several times, transferring the tissue to another clean plate, carefully separating polypide with ophthalmic scissors and forceps, carefully removing redundant host tissue, and taking care that the complete structure of polypide cannot be destroyed, i.e. ensuring the integrity of head segment and vesicle; the cells were washed repeatedly 5 times with sterile PBS (containing double antibody), 3 times with RPMI l640 medium, and transferred to a new clean plate for further use.
3. In vitro aseptic culture of cysticercus pisiformis
In a clean bench, whole coenuridae are picked and carefully transferred into a sterile cell bottle (25 cm)2) Adding 8ml of the complete culture solution 1, and culturing at a density of 100 per bottle; screwing down the bottle cap, and placing the bottle cap in a carbon dioxide cell incubator for culture.
Preferably, sugar, sterile and anaerobic environment are prerequisites to ensure survival of the insect in vitro. Therefore, in order to improve the culture efficiency of the phaeocystis piscifolia, the culture conditions are set as follows: constant temperature of 37 ℃ and 5% CO2Relative saturation humidity is about 70%. Continuously observing 1, 6, 12, 24, 36 and 48 hours after culturing and every day, taking the turning-out swing of the cysticercus cellulosae and the expansion of the wall under a microscope as the survival judgment standard of the cysticercus cellulosae, observing and recording the survival time and the form change of the lenticular cysticercus cellulosae in vitro, measuring the size of the cysticercus cellulosae and taking pictures.
Preferably, the liquid changing method comprises the following steps: the culture of the insect bodies is started in 6 th and 12 th hours, the old culture solution is removed by a sterile suction pipe as much as possible when the culture solution is changed, 8ml of fresh complete culture solution is added per bottle per time, the culture is continued under the preferable culture conditions, and the culture solution is changed 1 time every other day. During the period, the activity of the worm body can be observed in time, and the activity of the head section and the expansion state of the capsule wall can be observed under the microscope, so that the survival condition of the worm body can be known.
Preferably, when the metabolism secretion antigen and the exosome are collected, the polypide is cultured for 6h, the solution is changed, and the culture solution is discarded; adding fresh culture solution, culturing for 12 hr, discarding culture solution, replacing with new culture solution, and collecting 1 time every 48 hr.
Preferably, the culture process is strictly aseptic operation, so that no pollution is ensured; the concentration of the added antibiotics is maintained at a threshold value and is not too high, so that the disintegration and death of the polypide structure caused by toxic reaction are avoided.
Preferably, the culture method can provide physicochemical conditions, nutrients and sterility for the existence of the polypide. The worm survives for more than 3 months, and the requirements of test periods such as drug screening and the like are met.
Example 2 comparative study of three culture solutions and the culture solution provided by the invention for culturing cysticercus pisiformis
In an ultra-clean workbench, 40 pretreated worm bodies with consistent sizes and states are selected and randomly divided into 4 groups and 10 groups, and the groups are put into corresponding culture solutions for culture, wherein the culture solutions comprise a PBS (phosphate buffer solution) culture group, an RPMI-1640+ 10% rabbit bile culture group and a complete culture solution 1 culture group. The culture conditions were set as follows: 37 ℃ and 5% CO2Each group of culture medium contained a diabody (penicillin 100U/mL, streptomycin 100. mu.g/mL) and amphotericin B (1.25. mu.g/mL). Repeating the culture for 3 times; continuously observing 5min, 1, 2, 12, 24, 36 and 48h after culture and every day, taking the turning-out swing of the cysticercus cellulosae and the expansion of the cyst wall under a microscope as the survival judgment standard of the larva, observing and recording the turning-out time, the turning-out rate, the in-vitro survival time and the form change of the lenticular cysticercus cellulosae, measuring the size of the larva and taking a picture.
The general morphology of the cysticercosis in each culture group was changed as follows: the cysticercus cellulosae before culture is oval, the vesicle is transparent, the filling degree is good, the size is about 0.4cm multiplied by 0.7cm, the liquid in the cyst cavity is clear, and milky punctate head knots can be seen. Good telescopic movement of the head segment can be seen under a light microscope (x 40, fig. 1, o). The turning rate of the head of the polypide cultured by PBS is low, and is only 33% after 12 h; generally survived to 7d, and the worm peristalsis stopped (fig. 1, a); the head section under the microscope is obviously hooked and arranged orderly. The rabbit bile has obvious promotion effect on the turning-out of the soyabean cysticercus coenurus, and the turning-out speed of the worm coenurus is high. Culturing for 15min, wherein the head and neck segments of RPMI-1640+ 10% rabbit bile group cysticercus cellulosae can be completely and naturally turned out from the bursa, and the turning-out rate of the head and neck segments is 100%; the neck node part is thin and extends out by about 4-6 mm, and the neck node part can twist automatically and obviously; the vesicle becomes small, the mobility of the body wall and the head segment sucking discs is good (figure 1, b); under the observation of the mirror, the surface of the cervical vertebra is smooth, the folds are uniform and fine, the small spines are obvious in protrusion, the texture is thick and the cervical vertebra is in a sawtooth shape. After the group of polypide is cultured for 24 hours, the activity of polypide is weakened, polypide vesicles become small and partially disappear, and no liquid is full; the obvious sucking disc, the top projection and two circles of punctiform hooks (figure 1, e) can be seen by observing the head segment under the mirror, the capsule wall is thin, the structure is fuzzy, and the degree of contraction is weakened. As the culture time is prolonged, the activity of the larva stops, and the cysticercus pisiformis of the bile culture group can survive for 48h at most. The turning-out of the head section of the RPMI-1640 (figure 1, c) and complete culture solution 1 (figure 1, d) culture group worms is slow, the turning-out rate is low, and the culture time is only 20% after 12 h; however, as the culture time is prolonged, the turnover rate of the first node can be improved to 70%. The complete culture solution 1 group of insects has good physical activity; the suction cup of the knob is clearly visible under the scope of observation, but the small hook is not visible (fig. 1, f). The turning-out state of the head section of the worm body can be influenced by temperature and light; the cultured worms have different sizes and large differences, and the length of the worms ranges from 1.0 cm to 3.8cm, but most of the worms are 1.5 cm to 2 cm.
The most obvious difference between the individual culture groups is the change of vesicles with the increase of the culture time. The vesicles mainly undergo morphological changes, which are expressed as longer lengths, compared to those before culture; wherein the complete culture solution 1 group of polypide vesicles has good shape maintenance effect. Culturing in vitro for 3 months, wherein the survival rate of the insect body reaches more than 90 percent; as can be seen from Table 1, the length of the vesicle in the complete culture solution 1 group is measured to be 1.83 + -0.10 cm, the width is measured to be 0.85 + -0.05 cm, the vesicle filling degree is good, the vesicle liquid is clear, the edge of the vesicle wall is complete, and the vesicle can freely stretch and contract; the size difference of the wormhole vesicles with other culture groups is obvious (P is less than 0.05); the vesicle of the other cultured worm bodies is obviously reduced, the cyst fluid is reduced or disappeared, the cyst wall is thinned and loosened, and the edge is not integral.
The detection result shows that the insect body cultured by other culture methods has poor in-vitro culture state and short survival time, and the long-acting culture system of the complete culture solution 1 has good effects of maintaining the in-vitro survival activity of the insect body, prolonging the survival time, enhancing the adaptability of the insect body to the external environment and improving the survival rate. Can meet the requirements of different experimental purposes.
TABLE 1 Lenticercaria development status in different culture solutions
Note: different capital letters in the same column indicate significant differences (P < 0.05).
EXAMPLE 3 preparation of Metabolic secretory antigens of cysticercus pisiformis
3.1 culture of Bean-shaped cysticercus
The whole coenuridae are picked and carefully transferred into sterile cell flasks (25 cm)2) Adding 8ml of the complete culture solution 1, and culturing at a density of 100 per bottle; screwing down the bottle cap, and placing the bottle cap in a carbon dioxide cell incubator for culture. Continuously observing every day, and taking the turning-out swing of the cysticercus cellulosae and the expansion of the cyst wall under a microscope as the judgment standard of the survival of the larva.
Preferred culture conditions are: 37 ℃ and 5% CO2Relative saturation humidity is about 70%.
The preferable liquid changing method comprises the following steps: the culture solution is changed 1 time in 6 th and 12 th hours, the old culture solution is removed by a sterile pipette as much as possible during each change, fresh complete culture solution is added for 8 ml/bottle/time, the culture is continued under the preferable culture condition, and then 1 culture supernatant is collected every 48 hours.
3.2 separation, purification and identification of metabolic secretion antigen of Lepidoptera pisciformis
Centrifuging the collected culture supernatant of the worm body at 12000g for 10min at 4 ℃; sucking the supernatant, and filtering through a 0.45 mu m filter; transferring 15ml of filtrate into an Amicon Ultra-15 ultrafiltration tube (molecular weight cut-off of 10kDa) subjected to PBS balance treatment, centrifuging at 25 ℃ and 5000g for 30min, discarding liquid in a collection tube, adding PBS into an inner tube for buffer solution replacement, sucking and sampling samples gently for several times, centrifuging at 25 ℃ and 5000g for 30min, and sucking concentrated samples in the inner tube.
The metabolic secretion antigen in culture supernatant of the cysticercus pisiformis is separated by SDS-PAGE, anti-Tpeno monoclonal antibody 1C4 and anti-Tp 14-3-3 polyclonal antibody are taken as probes, and marker proteins enolase and 14-3-3 protein are subjected to immunoblotting detection by using a Western blot method. As a result: the metabolic secretion antigens in the culture supernatant of the phacoenura pisiformis are successfully concentrated, and the Westernblot result shows that the specific antibody can be used for detecting the parasite enolase and the 14-3-3 positive band, the sizes of the bands are respectively about 48kDa and 28kDa, and the sizes are consistent with the expected sizes.
Example 4 preparation of lenticular cysticercosis exosomes
4.1 culture of Bean-shaped cysticercus
The whole coenuridae are picked and carefully transferred into sterile cell flasks (25 cm)2) Adding 8ml of the complete culture solution 2, and culturing at a density of 100 per bottle; screwing down the bottle cap, and placing the bottle cap in a carbon dioxide cell incubator for culture. Continuously observing every day, and taking the turning-out swing of the cysticercus cellulosae and the expansion of the cyst wall under a microscope as the judgment standard of the survival of the larva.
Preferred culture conditions are: 37 ℃ and 5% CO2Relative saturation humidity is about 70%.
The preferable liquid changing method comprises the following steps: the culture solution is changed 1 time in 6 th and 12 th hours, the old culture solution is removed by a sterile pipette as much as possible during each change, fresh complete culture solution is added for 8 ml/bottle/time, the culture is continued under the preferable culture condition, and then 1 culture supernatant is collected every 48 hours.
4.2 separation and purification of lenticular cysticercosis exosomes
The method for extracting exosomes in culture supernatant of bean-shaped cysticercus cellulosae by ultracentrifugation comprises the following steps: collecting the culture supernatant of the worm, and centrifuging at 300g for 10min at 4 ℃; sucking the supernatant, and centrifuging at 2000g for 20min at 4 deg.C; collecting supernatant, centrifuging at 4 deg.C and 10000g for 40min, and collecting supernatant; filtering the supernatant with 0.22 μm filter, transferring to ultracentrifuge tube, centrifuging at 110000g for 120min at 4 deg.C with 12 ml/tube; washing the combined precipitate with PBS (0.22 μm filter), and centrifuging again at 110000g for 120min at 4 deg.C; the supernatant was discarded and the pellet was dissolved in 10. mu.l PBS (filtered through a 0.22 μm filter) and mixed well.
4.3 immunocolloidal gold electron microscope identification of Lepisma pisiformis
Adding 10 μ l of 2.5% paraformaldehyde into the exosome solution prepared in 4.2, mixing, contacting the droplets on the front surface of the electron microscope copper mesh, and incubating at room temperature for 30 min; washing the copper mesh with PBS and 50mM glycine, respectively; blocking the copper mesh with 5% BSA for 10min, and incubating with 20. mu.l of CD63 antibody (1:40) for 45 min; PBS 3 times washing, and 20 u l colloidal gold labeled goat anti mouse-IgG secondary antibody (1:20) incubation for 60 min; according to a conventional method, treating the copper mesh with 0.5% BSA, PBS, 2.5% paraformaldehyde and deionized water in sequence; adding 10 μ l phosphotungstic acid for negative dyeing for 1min, sucking off the dye solution by using filter paper, baking the copper mesh, and observing the exosome form by using a transmission electron microscope.
As a result: observing the lenticular cysticercus cellulosae exosomes as disc-shaped complete vesicles with the diameter of 50-150 nm under a transmission electron microscope; the result of an immunoelectron microscope shows that the marked transmembrane protein CD63 on the surface of the membrane is positive.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions within the technical scope of the present invention should be covered by the scope of the present invention. Therefore, the protection scope of the present application should be determined by the claims.
Claims (6)
1. An in vitro culture method of cysticercus cellulosae comprises separating the intact cysticercus cellulosae from the host animal, washing with PBS containing at least one antibiotic under aseptic condition, transferring into a culture container containing culture solution, placing the culture container in a carbon dioxide cell incubator at 37 deg.C and CO2Culturing under the conditions of 5% concentration and 70% relative humidity, changing the culture solution for 1 time in 6h and 12h after the start of culturing the polypide, removing old culture solution as much as possible during each liquid change, adding fresh complete culture solution for 8 ml/bottle/time, and changing the culture solution for 1 time in 48h, wherein the used PBS is added with 100U/ml penicillin and 100 mu g/ml streptomycin, and the culture solution is RPMI1640 culture solution added with fetal calf serum, penicillin, streptomycin, amphotericin B, L-glutamine and sodium bicarbonate, wherein: the fetal bovine serum content is 10% (v/v); the final concentration of the antibiotics is 100U/mL of penicillin, 100 mu g/mL of streptomycin and 1.25 mu g/mL of amphotericin B; the final concentration of the L-glutamine is 2.5 mM; and adding sodium bicarbonate to adjust the pH of the culture solution to 7.2-7.4.
2. An in vitro culture method of cysticercus pisiformis is characterized in that the cysticercus pisiformis freshly isolated and intact from abdominal cavity of infected rabbit is washed clean with PBS containing at least one antibiotic under aseptic condition, and then transferred into culture mediumCulturing in a carbon dioxide cell culture box at 37 deg.C and CO2Culturing under the conditions of 5% concentration and 70% relative humidity, changing the culture solution for 1 time in 6h and 12h after the start of culturing the polypide, removing old culture solution as much as possible during each liquid change, adding fresh complete culture solution for 8 ml/bottle/time, and changing the culture solution for 1 time in 48h, wherein the used PBS is added with 100U/ml penicillin and 100 mu g/ml streptomycin, and the culture solution is RPMI1640 culture solution added with fetal calf serum, penicillin, streptomycin, amphotericin B, L-glutamine and sodium bicarbonate, wherein: the fetal bovine serum content is 10% (v/v); the final concentration of the antibiotics is 100U/mL of penicillin, 100 mu g/mL of streptomycin and 1.25 mu g/mL of amphotericin B; the final concentration of the L-glutamine is 2.5 mM; and adding sodium bicarbonate to adjust the pH of the culture solution to 7.2-7.4.
3. The method for the ex vivo culture of cysticercus pisiformis according to claim 1 or 2, wherein the initial culture solution is added in an amount of: the adding amount of the 6-hole plate is 3 ml/hole, or the adding amount of the cell bottle is 8ml/(25 cm)2) And (7) a bottle.
4. An in vitro culture method of cysticercus pisiformis for collecting exosomes of cysticercus pisiformis is characterized in that the cysticercus pisiformis freshly separated and intact from abdominal cavity of infected rabbit is washed clean with PBS (phosphate buffered saline) added with at least one antibiotic under aseptic condition, and then transferred into a culture container added with culture solution for treating exosomes except before, the culture container is placed in a carbon dioxide cell culture box, and the temperature is 37 ℃ and CO2Culturing under the conditions of 5% concentration and 70% relative humidity, changing the culture solution for 1 time in 6h and 12h after the start of culturing the polypide, removing old culture solution as much as possible during each liquid change, adding fresh complete culture solution for 8 ml/bottle/time, and changing the culture solution for 1 time in 48h, wherein the used PBS is added with 100U/ml penicillin and 100 mug/ml streptomycin, and the culture solution is RPMI1640 culture solution added with penicillin, streptomycin, amphotericin B, L-glutamine and sodium bicarbonate and fetal calf serum treated by non-exosome in the past, wherein: the fetal bovine serum content is10% (v/v); the final concentration of the antibiotics is 100U/mL of penicillin, 100 mu g/mL of streptomycin and 1.25 mu g/mL of amphotericin B; the final concentration of the L-glutamine is 2.5 mM; and adding sodium bicarbonate to adjust the pH of the culture solution to 7.2-7.4.
5. The method for the ex vivo culture of cysticercus pisiformis as claimed in claim 4, wherein the amount of the initial culture solution added is: the adding amount of the 6-hole plate is 3 ml/hole, or the adding amount of the cell bottle is 8ml/(25 cm)2) And (7) a bottle.
6. A method for collecting metabolic secretion antigen and exosome of phacoenospora lenticidae, which is characterized in that phacoenospora lenticidae is cultured according to the method of claim 4 or 5, the culture solution of the phacoenospora lenticidae is collected once every 48h after the second liquid change, and the metabolic secretion antigen or exosome is further obtained by the methods of concentration or ultracentrifugation and the like.
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