CN110420322B - Trichina ES complex inducer induced DC vaccine for preventing, treating and diagnosing trichinosis of pigs - Google Patents
Trichina ES complex inducer induced DC vaccine for preventing, treating and diagnosing trichinosis of pigs Download PDFInfo
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Abstract
The invention relates to a trichina ES complex inducer induced DC vaccine for preventing, treating and diagnosing trichinosis of pigs, belonging to the technical field of biology. Comprises the preparation of trichina ES, the respective addition of camptothecin and hypericum 0.61 mug and hypericum 0.25 mug, the construction of swine trichina DC vaccine, the separation of swine peripheral blood mononuclear cells, the acquisition of adherent cells and the acquisition of DC vaccine. The method has the advantages that the porcine peripheral blood mononuclear cells are utilized, the complete culture medium is adopted, and the trichina ES compound preparation is utilized to induce the maturation of the DC, so that the immunotherapy is carried out on the carnivorous pig infected with the trichina. The trichina ES compound inducer adopted is camptothecin and hypericin, can enhance the induction effect of trichina secretion on DC cells, can be used for preventing, treating and diagnosing the trichinosis of pigs, improves the quality of imports and exports of pork, ensures the food safety, reduces the economic loss of the trichinosis of pigs to the pig industry, and avoids the negative influence of the trichinosis of pigs on the food safety and the international trade of pork.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a DC vaccine for preventing, treating and diagnosing trichinosis in pigs.
Background
The trichinosis of pigs not only affects the health of people, but also causes huge economic loss to the pig industry, and directly brings negative reputation of international trade, and the current prevention and treatment situation provides new challenges for the prevention and treatment of the trichinosis.
With the development of international meat trade and tourism industry, and the improvement of ecological environments such as afforestation, returning to farming and the like, and the improvement of living standard of people, food diversification and excessive pursuit of consumption factors such as original ecological products and the like are stimulated together, trichinosis generally has new characteristics in recent years: 1. the incidence of diseases in the regions eating raw meat is reduced, while the incidence of diseases in the regions eating cooked meat is increased; 2. local outbreaks are the main causes, and the sources of infection are diversified. Current control situations present new challenges for the control of trichinosis. The use of albendazole as a pig feed additive has a good preventive effect on swine trichinosis, but the problem of drug residues in pork and the side effects thereof are not clear, so that vermin-proof feeding of pigs using vermin-proof drugs such as albendazole has been temporarily discouraged, and is also disapproved by the international trichinosis committee (ICT).
Antigen Presenting Cells (APCs) take, process and present antigens as a key link for starting adaptive immune response, dendritic Cells (DCs) are currently recognized as the full-time antigen presenting cells with the strongest functions in vivo, on one hand, specific humoral immune response of cellular immunity and T cell dependence can be started and regulated by antigen presenting and cell factor secretion, and on the other hand, regulatory T cells can be induced or corresponding T cell clone can be eliminated to achieve the effect of immunosuppression or immune tolerance induction. In recent years, with the development of DC vaccine application research, DC vaccine has become one of the most interesting focuses today. The types of the DC vaccine at present mainly comprise an antigen sensitized DC vaccine, an antigen polypeptide sensitized DC vaccine, a gene modified DC vaccine and the like. Compared with the first two DC vaccines, the DC vaccine with gene modification has more advantages, thus arousing the attention of more and more scholars. Although more than a dozen DC vaccines are in the phase III clinical research stage in recent years, the further development of the DC vaccines is greatly stimulated, and the DC vaccines are also shown to have wide application prospects. At present, no swine DC vaccine induced by using trichina ES complex preparation exists.
Disclosure of Invention
The invention provides a trichina ES complex inducer induced DC vaccine for preventing, treating and diagnosing trichinosis of pigs.
The technical scheme adopted by the invention is as follows: comprises the following steps:
preparation of trichina ES compound inducer
(1) Preparation of trichina ES
Orally infecting 300 trichina 10 Wistar rats to be killed by cervical dislocation, taking small intestines, removing adipose tissues and mesentery, longitudinally splitting the small intestines, flushing intestinal contents with flowing tap water, taking 300 g of the small intestines, washing the small intestines in 2000ml of physiological saline water with 200U/ml penicillin and 200U/ml streptomycin sulfate added at 37 ℃, collecting trichina adults by an artificial digestion method, and counting; pouring 50ml of liquid containing 5 ten thousand adult trichina into a clean glass plate with the diameter of 15cm, standing for 15min, removing the upper layer of liquid, cleaning the collected adult trichina, transferring the adult trichina into a 15ml centrifuge tube, centrifuging and washing for 2 times at the speed of 1000rpm/min by using RPMI-1640 culture solution containing double antibodies for 2min, and transferring the adult trichina which is cleaned and deposited at the bottom of the centrifuge tube to 25cm 3 The cell culture flask of (4), 15ml of the medium, 37 ℃,5% CO 2 Culturing in an incubator of 12h, centrifuging at 1000rpm/min for 2min, and collecting the supernatant; repeatedly centrifuging the supernatant at 9 deg.C and 6000rpm/min for 30min, concentrating until ES liquid becomes 8-10mL colorless liquid, i.e. Trichinella spiralis ES, transferring the colorless liquid into 1.5mL centrifuge tube, sealing, and storing at-20 deg.C;
(2) Preparation of trichina ES composite inducer
Adding camptothecin and fructus Rhodomyrti 0.61 μ g, and fructus Rhodomyrti 0.25 μ g per ml ES;
preparing camptothecin and hypericin, preparing the medicine into 200mg/mL according to storage concentration, and diluting to 0.1ug/mL when in use;
(II) construction of trichina swine DC vaccine
(1) Separation of peripheral blood mononuclear cells of pigs:
collecting 10ml of venous blood outside a front cavity of a pig under aseptic conditions, sucking upper serum for later use, adding PBS (phosphate buffer solution) with the same volume for diluting the blood, slowly adding 14ml of diluted peripheral blood into a centrifugal tube containing 7ml of NycoPrepTM 1.077A lymphocyte separation liquid along the tube wall by using a suction tube, centrifuging at 1800rpm for 20min, slightly sucking interface cells, namely mononuclear cells, in a liquid layer of the centrifugal tube separation liquid by using the suction tube, transferring to another aseptic centrifugal tube, lightly beating the PBS uniformly to fully dilute the cells, centrifuging at 1500rpm for 8min, washing twice, and collecting the mononuclear cells;
(2) Obtaining adherent cells
Suspending the obtained mononuclear cells with 10% fetal calf serum-containing RPMll640 liquid, blowing and beating uniformly, adding 90ul trypan blue into 10ul cell suspension, mixing uniformly, adding into a counting disc, counting living cells, and staining with trypan blue to prove that the cell activity is more than 95%,
calculating the formula: cell number/L =4 total number of viable cells/4 × 104 × dilution factor
Adjusting monocyte concentration to 2 × 10 6 Per ml, added to 6 well cell culture plates, 2ml per well, at 5% CO 2 Incubating at 37 ℃, washing off non-adherent cells after 90min, adding RPMI1640 without FCS for continuous incubation for 30min, washing off the non-adherent cells again after 30min, and obtaining the residual cells which are adherent mononuclear cells;
(3) Obtaining of DC vaccine
Adding 2ml of complete medium (recombinant colony stimulating factor (rmGSF) 160ng/ml + rmIL-4 50ng/ml +10% fetal bovine serum) per well, placing the culture plate in a sterile plastic bag, wrapping the bag, and adding 5% of CO into the cells 2 Culturing at 37 deg.C, washing with pipette after 7 daysCulturing the holes, sucking the cell suspension, adding the cell suspension into a centrifugal tube, centrifuging at 1500rpm for 8min, and collecting centrifugal precipitates; centrifuging the culture medium to obtain precipitate, changing culture medium on 3 days, adding trichina ES compound inducer 5ml on sixth day, adding TNF-alpha 100u/ml on seventh day, inducing to mature, blowing, mixing, collecting all suspension cells as trichina DC, and counting suspension cells to make the concentration of suspension cells 2 × 10 6 The number per ml is the trichina swine DC vaccine; the dosage of the pig per 100Kg of body weight is 1ml.
The method has the advantages that the porcine peripheral blood mononuclear cells are utilized, the complete culture medium is adopted, and the trichina ES compound preparation is utilized to induce the maturation of the DC, so that the immunotherapy is performed on the carnivorous pig infected with the trichina. The trichinosis ES compound inducer is obtained by screening 15 effective ingredients of traditional Chinese medicines, such as icariin, podophyllotoxin, kaempferol, chlorogenic acid, oleanolic acid, emodin, quercetin, forsythoside, camptothecin, chrysophanol, indirubin, indigo, hypericin, procyanidine, schizonepeta oil and the like, and proves that the camptothecin and the hypericin can enhance the induction effect of trichinosis secretion (ES) on DC cells.
Drawings
FIG. 1 is a graph of ES complex inducer-induced DCs and DCs not induced by ES complex inducer;
FIG. 2 is a graph of flow identification of ES complex inducer induced mature DCs;
FIG. 3 is a diagram of regulation of porcine blood cytokines by trichina ES complex inducer induced DCs;
FIG. 4 is a hypericin concentration screening graph of the present invention;
FIG. 5 is a screening graph of camptothecin concentration according to the invention.
Detailed Description
Comprises the following steps:
preparation of trichina ES compound inducer
(1) Preparation of trichina ES
Orally infecting 300 trichina 10 Wistar rats to be killed by cervical dislocation, taking small intestines, removing adipose tissues and mesentery, longitudinally splitting the small intestines, flushing intestinal contents with flowing tap water, taking 300 g of the small intestines, washing the small intestines in 2000ml of physiological saline water with 200U/ml penicillin and 200U/ml streptomycin sulfate added at 37 ℃, collecting trichina adults by an artificial digestion method, and counting; pouring 50ml of liquid containing 5 ten thousand adult trichinella into a clean glass plate with the diameter of 15cm, standing for 15min, removing the upper layer of liquid, cleaning the collected adult trichinella, transferring the adult trichinella into a 15ml centrifuge tube, centrifuging and washing for 2 times at 1000rpm/min by using RPMI-1640 culture solution containing double antibodies for 2min, and transferring the adult trichinella deposited at the bottom of the centrifuge tube after cleaning to 25cm 3 The cell culture flask of (4), 15ml of the medium, 37 ℃,5% CO 2 Culturing in an incubator of 12h, centrifuging at 1000rpm/min for 2min, and collecting the supernatant; repeatedly centrifuging the supernatant at 9 deg.C and 6000rpm/min for 30min, concentrating until the ES liquid becomes 8-10mL colorless liquid, i.e. Trichinella spiralis ES, transferring the colorless liquid into 1.5mL centrifuge tube, sealing, and storing at-20 deg.C;
(2) Preparation of trichina ES composite inducer
Adding camptothecin and fructus Rhodomyrti 0.61 μ g and 0.25 μ g per ml ES respectively;
preparing camptothecin and hypericin, preparing the medicine into 200mg/mL according to the storage concentration, and diluting to 0.1ug/mL when in use;
(II) construction of trichina swine DC vaccine
(1) Separation of peripheral blood mononuclear cells of pigs:
collecting 10ml of venous blood outside a front cavity of a pig under aseptic conditions, sucking upper serum for later use, adding PBS (phosphate buffer solution) with the same volume for diluting the blood, slowly adding 14ml of diluted peripheral blood into a centrifugal tube containing 7ml of NycoPrepTM 1.077A lymphocyte separation liquid along the tube wall by using a suction tube, centrifuging at 1800rpm for 20min, slightly sucking interface cells, namely mononuclear cells, in the separation liquid layer of the centrifugal tube by using the suction tube, transferring to another aseptic centrifugal tube, slightly blowing and beating the PBS uniformly to fully dilute the cells, centrifuging at 1500rpm for 8min, washing twice, and collecting the mononuclear cells;
(2) Obtaining adherent cells
Suspending the obtained mononuclear cells with 10% fetal calf serum-containing RPMll640 liquid, blowing and beating uniformly, adding 90ul trypan blue into 10ul cell suspension, mixing uniformly, adding into a counting disc, counting living cells, and staining with trypan blue to prove that the cell activity is more than 95%,
calculating the formula: cell number/L =4 total number of viable cells/4 × 104 × dilution factor
Adjusting monocyte concentration to 2 × 10 6 Per ml, added to 6 well cell culture plates, 2ml per well, at 5% CO 2 Incubating at 37 deg.C, washing off non-adherent cells after 90min, adding RPMI1640 without FCS, incubating for 30min, washing off non-adherent cells again after 30min, and collecting the residual cells as adherent mononuclear cells;
(3) Obtaining of DC vaccine
Adding 2ml of complete medium (recombinant colony stimulating factor (rmGSF) 160ng/ml + rmIL-4 50ng/ml + l0% fetal bovine serum) per well, placing the culture plate in a sterile plastic bag, wrapping the bag, and culturing the cells at 5% CO 2 Culturing at 37 deg.C, washing culture hole with pipette after 7 days, sucking cell suspension, adding into centrifuge tube, centrifuging at 1500rpm for 8min, and collecting centrifugal precipitate; completely culturing the obtained centrifugal precipitate in culture medium, changing culture medium on day 3, adding trichina ES compound inducer 5ml on day six, adding TNF-alpha 100u/ml on day seven, inducing, blowing, mixing, collecting all suspension cells, counting suspension cells to make suspension cell concentration be 2X 10 6 The number per ml is the trichina swine DC vaccine; the dosage of the pig per 100Kg of body weight is 1ml.
Experimental example 1 morphological examination
1. Phase contrast inverted microscope identification: the growth state and morphological changes of the cells were observed every day, and a photograph of the cells was taken on day 7.
2. And (3) identifying a scanning environment: centrifuging the collected suspension cells, removing 2/3 of supernatant, and resuspending the cells; sucking the cell suspension, carefully dropping the cell suspension on a glass slide coated with a thin film, flatly placing the glass slide in a culture dish and standing for 30min, then adding 2.5% glutaraldehyde into the culture dish to immerse the glass slide in the culture dish, fixing for 30min, fully rinsing with 0.1M buffer solution, fixing for 30min with 1% osmic acid, rinsing with the buffer solution, starting gradient dehydration with 30% alcohol, transiting for 5min with hexyl acetate, drying at a critical point, taking out the glass slide, sticking the glass slide on a specimen table by using a conductive adhesive, and performing ion sputtering coating; and scanning the environment to observe and take a picture.
And (3) transmission electric field identification: the cells were centrifuged to obtain cell pellets, fixed with 4% glutaraldehyde for 2h (pre-fixation), and rinsed with 0.1M phosphate buffer. Fixation after 1% osmic acid 2h,0.1M phosphate buffer rinse, 30% ethanol start gradient dehydration; replacing and soaking with acetone and resin, embedding and polymerizing, trimming and slicing, and dyeing with uranium acetate and lead citrate; the pictures are taken by transmission electronic observation. As shown in FIG. 1 (B, D), the ES complex inducer induces mature DC surface dendrites to be long and many, has a lot of folds, shows dendrites with different thicknesses, sizes, lengths and the like, has relatively straight protrusions and relatively curved protrusions, is distributed around the whole cell, and has unsmooth and uneven cell surface. Conforming to the morphological characteristics of a typical mature DC.
Immature DC cells which were not induced by an inducer are shown in FIG. 1 (A, C).
3. DC vaccine flow assay
The expression of CD86 was detected by flow cytometry on mature DC and immature DC cells (fig. 2e, b), CD11c being a characteristic surface molecule of myeloid-derived DCs, and CD11c + indicating that the extracted cells were myeloid-derived, not derived from the lymphatic system. Both immature and mature DCs highly expressed CD11c (FIG. 2A, D), whereas mature DCs expressed higher amounts of MHC-II (FIG. 2F, C) and CD86 than immature ones.
EXAMPLE 2 Immunotherapy of trichinosis in pigs
Induction of DC vaccine by ES Complex inducer to reduce insect ratio
Respectively infecting 40 healthy pigs according to 2000 trichina (trichina international serial number ISS 534)/head, and respectively treating 10 infected trichina pigs by inducing DC vaccine with normal saline, DC, ES and ES complex inducer; the usage amount of the pig per 100Kg of body weight is 1ml;
immunization strategy: after infection, immunizing all treated pigs with the single-dose DC vaccine on day 0 and day 2, double-dose immunotherapy pigs on day 7, and double-dose immunotherapy pigs on day 13 and day 17 after infection, respectively;
after 18 days, each group of pigs were killed, the muscle and intestinal trichina of each group of pigs were collected by digestion and counted, and the result of the reduction rate was calculated according to the following formula and is shown in table 1.
Reduction rate = (mean number of trichinella in normal saline treatment group-mean number of trichinella in treatment group)/mean number of trichinella in normal saline treatment group
TABLE 1 ES Re-inducer induced DC vaccine reduction
| Group of | Physiological saline | DC | ES | Induction of DC by ES re-inducer |
| Insect reduction (%) | 28.53 | 35.71 | 44.82 | 48.41 |
2. Regulation and control of trichina ES complex inducer induced DC on porcine blood cytokine
Respectively infecting 40 healthy pigs according to 2000 trichina (trichina international serial number ISS 534)/head, respectively inducing DC vaccine to treat 10 infected trichina pigs by using normal saline, DC, ES and ES complex inducer, wherein the using amount of each 100Kg of weight of the pig is 1ml; changes in cytokines in serum were detected by ELISA following the eBioscience kit instructions from the anterior vena cava bleeds at days 3, 6, 9, 12, 15, 18, 21, 24, and 27, respectively.
Evaluation of immune effect:
the changes in the various cytokines are shown in FIG. 4. The expression levels of IL-6 (FIG. 3B), IL-4 (FIG. 3C) and IL-10 (FIG. 3D) representing the immune response of trichina were down-regulated and IFN-. Gamma. (FIG. 3A) was up-regulated.
EXAMPLE 3 hypericin and camptothecin concentration screening
Hypericin and camptothecin acted on pig fetal fibroblasts (G418) according to six concentration groups of 8 mug/mL, 1.6 mug/mL, 0.32 mug/mL and 0.064 mug/mL, three time groups of 24h, 48h and 72h are set for each concentration, a solvent control hole and a blank hole are set, and 3 parallel holes are set for each group. The concentration of the drug that is 5% toxic to cells (i.e., the concentration of the drug at which the cell viability is 95%) is taken as the substantially non-toxic concentration of the drug to cells, and this concentration at 24h is used for subsequent experiments. Cell survival rate = (OD value experimental group-OD value control group)/(OD value control group-OD value blank group) × 100%. The results are shown in FIGS. 4 and 5.
Claims (4)
1. A preparation method of trichina ES complex inducer induced DC vaccine for preventing, treating and diagnosing trichinosis suis, which is characterized by comprising the following steps: comprises the following steps:
preparation of trichina ES compound inducer
(1) Preparation of trichina ES
Orally infecting 300 trichina 10 Wistar rats to be killed by cervical dislocation, taking small intestines, removing adipose tissues and mesentery, longitudinally splitting the small intestines, flushing intestinal contents under flowing tap water, taking 300 g of small intestines, washing the small intestines in 2000ml of physiological saline water at 37 ℃ and added with 200U/ml penicillin and 200U/ml streptomycin sulfate, collecting trichina imagoes by artificial digestion and counting; pouring 50ml of liquid containing 5 ten thousand adult trichinella into a clean glass plate with the diameter of 15cm, standing for 15min, removing the upper layer of liquid, cleaning the collected adult trichinella, transferring the adult trichinella into a 15ml centrifuge tube, centrifuging and washing for 2 times at 1000rpm/min by using RPMI-1640 culture solution containing double antibodies for 2min, and then cleaning and precipitatingTransferring the trichina imago settled at the bottom of the centrifuge tube to 25cm 3 The cell culture flask of (4), 15ml of the medium, 37 ℃,5% CO 2 Culturing in an incubator of 12h, centrifuging at 1000rpm/min for 2min, and collecting the supernatant; repeatedly centrifuging the supernatant at 9 deg.C and 6000rpm/min for 30min, concentrating until ES liquid becomes 8-10mL colorless liquid, i.e. Trichinella spiralis ES, transferring the colorless liquid into 1.5mL centrifuge tube, sealing, and storing at-20 deg.C;
(2) Preparation of trichina ES composite inducer
Camptothecin and hypericin are respectively added into each milliliter of ES by 0.61 mug and 0.25 mug for standby;
(II) construction of trichina swine DC vaccine
(1) Separation of peripheral blood mononuclear cells of pigs:
collecting 10ml of venous blood outside a front cavity of a pig under aseptic conditions, sucking upper serum for later use, adding PBS (phosphate buffer solution) with the same volume for diluting the blood, slowly adding 14ml of diluted peripheral blood into a centrifugal tube containing 7ml of NycoPrepTM 1.077A lymphocyte separation liquid along the tube wall by using a suction tube, centrifuging at 1800rpm for 20min, slightly sucking interface cells, namely mononuclear cells, in the separation liquid layer of the centrifugal tube by using the suction tube, transferring to another aseptic centrifugal tube, slightly blowing and beating the PBS uniformly to fully dilute the cells, centrifuging at 1500rpm for 8min, washing twice, and collecting the mononuclear cells;
(2) Obtention of adherent cells
Suspending the obtained mononuclear cells with 10% fetal calf serum-containing RPMll640 liquid, blowing and beating uniformly, adding 90ul trypan blue into 10ul cell suspension, mixing uniformly, adding into a counting disc, counting living cells, and staining with trypan blue to prove that the cell activity is more than 95%,
calculating the formula: cell number/L =4 big lattice total number of viable cells/4 × 104 × dilution factor
Adjusting monocyte concentration to 2 × 10 6 Per ml, added to 6 well cell culture plates, 2ml per well, at 5% CO 2 Incubating at 37 deg.C, washing off non-adherent cells after 90min, adding RPMI1640 without FCS, incubating for 30min, washing off non-adherent cells again after 30min, and collecting the residual cells as adherent mononuclear cells;
(3) Obtaining of DC vaccine
Adding 2ml of complete medium per well, placing the culture plate in a sterile plastic bag, wrapping the bag opening, and making the cells have a content of 5% CO 2 Culturing at 37 deg.C, washing culture hole with pipette after 7 days, sucking cell suspension, adding into centrifuge tube, centrifuging at 1500rpm for 8min, and collecting centrifugal precipitate; completely culturing the obtained centrifugal precipitate in culture medium, changing culture medium on day 3, adding trichina ES compound inducer 5ml on day six, adding TNF-alpha 100u/ml on day seven, inducing to mature, beating, mixing, collecting all suspension cells as swine trichina DC, counting suspension cells to make suspension cell concentration be 2X 10 6 And (4) each ml is the trichina swine DC vaccine.
2. The method for preparing trichinosis ES complex inducer induced DC vaccine for preventing, treating and diagnosing trichinosis of pigs according to claim 1, which is characterized in that: in the step (I), the trichina ES compound inducer is prepared, and in the step (2), the camptothecine and the hypericin are prepared, the medicine is prepared into 200mg/mL according to the storage concentration, and the medicine is diluted to 0.1ug/mL when in use.
3. The method for preparing trichinosis ES complex inducer induced DC vaccine for preventing, treating and diagnosing trichinosis of pigs according to claim 1, which is characterized in that: in the step (II) and the step (3) of obtaining the DC vaccine constructed by the swine trichinella DC vaccine, the complete culture medium added into each hole is as follows: recombinant colony stimulating factor (rmGSF) 160ng/ml + rmIL-4 50ng/ml +10% fetal bovine serum.
4. The method for preparing trichinosis ES complex inducer induced DC vaccine for preventing, treating and diagnosing trichinosis suis as claimed in claim 1, which is characterized in that: the dosage of the pig per 100Kg of body weight is 1ml.
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