CN111973635A - Method for researching lactation promoting effect of oysters - Google Patents
Method for researching lactation promoting effect of oysters Download PDFInfo
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Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/618—Molluscs, e.g. fresh-water molluscs, oysters, clams, squids, octopus, cuttlefish, snails or slugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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Abstract
The invention discloses a method for researching the lactation promoting effect of oysters, which comprises the following steps of S1, experimental materials, oysters, female rats and male rats. The experimental use is all SPF grade healthy SD rats, and the ratio of female rats to male rats is 2: 1, and both the female and male rats were 8 weeks old, unprimed rats, weight range of SD female: 180 g-200 g, weight range of SD male mouse: 200g to 250 g. S2, main experimental reagents, boric acid, anhydrous sodium carbonate, anhydrous ethanol, concentrated sulfuric acid, concentrated hydrochloric acid, potassium sulfate, glucose and n-butanol. According to the oyster lactation promoting effect research method, an overload lactation model is established, SD rats are taken as experimental objects, whether the oyster powder has the lactation promoting effect is researched, the comparison of the lactation amounts of the rats of an oyster powder group and a normal group shows that the oyster powder can improve the lactation amount of a lactating mother rat, compared with the visceral organ indexes of the mother rat of the normal group, the visceral organ indexes of the mother rat of the oyster powder group are higher than those of the normal group, and the normal healthy physiological level of the lactating mother rat is met.
Description
Technical Field
The invention relates to the technical field of application research of oysters, in particular to a method for researching the lactation promoting effect of oysters.
Background
The oyster is the first cultured shellfish in the world and one of the fourth cultured shellfish in China, the oyster has rich protein content, complete amino acid nutrition composition, unsaturated fatty acid, multiple vitamins and trace elements, is called as marine milk, and is a health-care food material which is approved by China and can be used as food and medicine，The cassia twig, dragon bone and oyster soup can treat various pediatric diseases and the like, and the medicinal use of the oysters is researched along with the application of the oystersThe kinds of actions are increasing.
With the improvement of life and the development of science and technology, people pay more and more attention to health care, wherein the research on oysters is gradually expanded, influence factors for treating postpartum hypogalactia are analyzed in the research on the synthesis and secretion of milk in modern medicine, and the research method for promoting lactation of oysters for treating postpartum hypogalactia is not provided for researching the effect of treating postpartum hypogalactia of oysters so far.
In order to solve the problems, an overload lactation rat model is urgently needed to be established to investigate whether the oysters can promote lactation.
Disclosure of Invention
The invention aims to provide a method for researching the lactation promoting effect of oysters, and aims to solve the problem that influence factors of treatment on postpartum hypogalactia are analyzed in modern medicine research on synthesis and secretion of milk in the background technology, and a comprehensive research method for the lactation promoting effect of oysters on treatment on postpartum hypogalactia is not available so far.
In order to achieve the purpose, the invention provides the following technical scheme: a method for researching lactation promotion effect of oysters comprises the following steps:
s1 test Material
(1) Oysters, mother rats and male rats.
(2) The experimental use is all SPF grade healthy SD rats, and the ratio of female rats to male rats is 2: 1, and both the female and male rats were 8 weeks old, unprimed rats, weight range of SD female: 180 g-200 g, weight range of SD male mouse: 200g to 250 g.
S2, main experimental reagent
Boric acid, anhydrous sodium carbonate, anhydrous ethanol, concentrated sulfuric acid, concentrated hydrochloric acid, potassium sulfate, glucose, n-butanol, copper sulfate pentahydrate potassium sulfate, trichloroacetic acid, barium chloride, sodium tartrate, sodium hydroxide, sodium chloride, xylene, hematoxylin eosin staining agent, chloral hydrate, zinc acetate, furin phenol and bovine serum albumin.
S3 preparation of oyster powder
(1) Cleaning fresh oysters by using a cleaning machine or a cleaning pool, filtering the cleaned oysters to dry, and preparing powder.
(2) Pre-freezing at 20 deg.C for 3 hr, deep-freezing in-80 deg.C refrigerator, and drying and storing.
S4 preparation of oyster protein
(1) 50g of oyster freeze-dried powder is dissolved in 1L of distilled water.
(2) Adjusting the pH value to 12-13 by 0.1mol/L sodium hydroxide solution.
(3) Stirring at 4 deg.C for 3 hr, centrifuging at 10000r/min and 4 deg.C for 15min, and extracting supernatant.
(4) Adjusting the pH of the supernatant to 4.5-5.1 by 0.1mol/L hydrochloric acid solution, centrifuging for 15min at 10000r/min and 4 ℃, and collecting the precipitate.
(5) Transferring the precipitate into a 12 ku-hole fiber dialysis bag, dialyzing for 48 hours, collecting the precipitate, freeze-drying, and storing in a dry manner.
S5 preparation of oyster enzymolysis powder
(1) Cleaning fresh Concha Ostreae, draining, homogenizing, adding distilled water at ratio of homogenate to water of 1: 3, adjusting pH to 7.0, and adding animal protein hydrolase.
(2) Keeping 1000U/g Carnis Ostreae in 53 deg.C water bath for 6 hr, centrifuging at 500r/min for 20min, extracting supernatant, removing water by rotary evaporation, freeze drying, and storing.
S6 establishment of overload lactation rat model
(1) The number of newborn mice fed by each litter of female mice is adjusted, the number of newborn mice is increased or reduced, so that each litter of female mice has the same number, the female mice feed the same number of newborn mice, the feeding requirements of a litter of newborn mice cannot be met, and the model is an overloaded rat model.
(2) The average is divided into a normal group and an oyster whole powder group, the normal group is filled with 1mL/100g of intragastric distilled water once a day for 20 consecutive days, and the oyster whole powder group is filled with 0.8mg/mL of oyster whole powder solution once a day for 20 consecutive days.
S7, serum collection and preservation
After the experiment is finished, all the female mice are anesthetized, the blood of the eyeballs is taken out of a centrifuge tube, the centrifuge tube is kept stand for 20min at 37 ℃, the centrifuge tube is placed in a high-speed centrifuge for centrifugation, the temperature is 4 ℃, 3000r/min is used for centrifugation for 15min, and supernate is collected and frozen in a refrigerator at the temperature of 20 ℃ below zero for storage.
S8, detecting the sample
(1) The serum sample is taken out and thawed, and the kit is taken out from the refrigerated environment and is used after being balanced for 15-30 minutes at room temperature.
(2) The method comprises the following operation steps:
and (3) diluting the standard:
sample adding: respectively arranging a blank hole (the blank control hole does not contain a sample and an enzyme-labeled reagent, and the rest steps are operated in the same way), a standard hole and a sample hole to be detected, accurately adding 50 mu L of the standard sample on an enzyme-labeled coating plate, adding 40 mu L of sample diluent in the sample hole to be detected, then adding 10 mu L of sample to be detected (the sample dilution is 5 times), adding the sample to the bottom of the hole of the enzyme-labeled plate, keeping the hole wall untouched as far as possible, and slightly shaking and mixing the sample and the sample hole;
and (3) incubation: incubating for 30 minutes at 37 ℃ after using a sealing plate membrane sealing plate;
preparing liquid: diluting 30 times of concentrated washing liquid with 3 times of distilled water for later use;
washing: carefully uncovering the sealing plate film, discarding liquid, patting to dry, filling washing liquid into each hole, standing for 30s, discarding, repeating the patting to dry for 5 times;
adding an enzyme: adding 50 mu L of enzyme labeling reagent into each hole except blank holes, incubating and washing;
color development: adding 50 μ L of color-developing agent A into each well, adding 50 μ L of color-developing agent B, gently shaking and mixing, and developing at 37 deg.C for 15min in dark;
and (4) terminating: stop solution 50. mu.L is added into each well to stop the reaction (blue color is immediately changed into yellow color);
and (3) determination: the blank wells were set to zero, and the absorbance (OD value) of each well was measured in order at a wavelength of 450nm, and the measurement was carried out within 15 minutes after the addition of the stop solution.
(3) Data processing
S9: observation of mammary gland tissue structure
Placing the anesthetized mother mouse on the back on an anatomical operating disk, cutting the skin of the chest and abdomen along the median line of the abdomen, separating the mammary tissue under the skin of the chest and abdomen, weighing and recording the weight of the mammary gland, cutting the same part of the mammary gland 1cm away from the top end of the mammary gland, placing the same part into 4% neutral paraformaldehyde with the volume about 10 times of the mammary gland for fixing for 24-72h, and standing overnight.
S10: tissue wax block production
(1) Fixed tissue trimming and Water washing
1. Firstly, placing the tissue block in a fixing solution for fixing for 24-72h, and then taking out the tissue block.
2. The tissue block is taken out, the shape of the tissue block is trimmed by a scalpel, the tissue block is placed in a dehydration frame for marking, and the tissue block is washed by running tap water for more than 2 hours to wash out the fixing liquid.
(2) Tissue dehydration
Placing the tissue block washed with the fixing solution into alcohol with gradient concentration for dehydration according to the following steps:
50% of ethanol: and (3) 30 min.
70% ethanol: and (3) 30 min.
80% of ethanol: and (3) 30 min.
95% ethanol: and (3) 30 min.
Anhydrous ethanol i: and (3) 30 min.
Anhydrous ethanol ii: and (4) 40 min.
(3) Is transparent
The dehydrated tissue blocks are operated according to the following steps:
1. placing in a xylene: alcohol 1:1 solution: and 15 min.
2. Xylene I: and 15 min.
3. Xylene II: and (4) 40 min.
(4) Wax dipping
Taking the completely transparent tissue block down from the dehydration basket, melting paraffin, and then soaking the tissue block in paraffin according to the following steps, wherein the temperature of the paraffin is about 64 ℃, and the temperature is adjusted according to the paraffin form.
1. Paraffin wax: xylene 1:1 mix: and (3) 30 min.
2. Paraffin wax I: and (4) 1 h.
3. And (3) paraffin II: and (4) 1 h.
4. Paraffin wax III: and (4) 1 h.
(5) Tissue embedding
And (3) placing the soaked tissue block in an embedding box, injecting the melted embedding paraffin into the embedding box, completely wrapping the tissue block by the paraffin, and simultaneously avoiding a gap between the solidified paraffin and the embedding box.
(6) Trimming wax block
Placing the embedding box in a ventilation place until the wax liquid is completely solidified, and freezing the embedding box in a refrigerator at-20 deg.C for 30min after the solidification.
After freezing, carefully taking out the wax block, making a clear tissue mark on one side of the wax block, then cutting off paraffin around the tissue block by using a blade, reserving paraffin of 1-2 mm around the tissue block, and trimming the wax block into a square.
S11: making tissue slices
(1) Slicing
The wax block is fixed on the holder, the wax block and the blade form an included angle of 5 degrees, the wax block is sliced, the tissue section in the wax block slice is complete and smooth, the blade is moved leftwards by a distance of about one wax block width, the slice thickness is adjusted to be 5 mu m, and then the slice is sliced, and the slice is continuously banded, complete and uniform.
(2) Exhibition piece
Preheating a baking machine at 46 ℃, dripping a small amount of egg white on a clean glass slide, uniformly smearing in the same direction, dripping a few drops of distilled water, carefully clamping a wax sheet on the glass slide, and finally placing the glass slide on the baking machine for drying.
(3) Deparaffinization and hydration of tissue sections
The completely dried tissue slices are placed on a slide rack and dewaxed according to the following steps:
1. xylene I: and 5 min.
2. Xylene II: and 5 min.
3. 1/2 xylene +1/2 absolute ethanol solution: and 5 min.
After dewaxing was complete, the tissue sections were washed to elute xylene as follows:
1. anhydrous ethanol: and 2 min.
2. 95% ethanol: and 2 min.
3. 90% ethanol: and 2 min.
4. 80% of ethanol: and 2 min.
5. 70% ethanol: and 2 min.
6. Washing with distilled water: and 2 min.
Sequentially placing dewaxed tissue slices into gradient ethanol, sequentially removing xylene according to the sequence of anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol and 70% ethanol, soaking in ethanol of each concentration for 2min, and finally soaking in distilled water for 2 min.
(4) Hematoxylin staining of tissue sections
Spin-drying the tissue slices with distilled water, soaking the tissue slices in hematoxylin staining solution for 4min, finally washing with distilled water for 2min, observing the staining result under a low power microscope, and repeating the previous steps until the staining is clear if the cell nucleus staining is not clear.
(5) Eosin staining of tissue sections
Decolorizing the tissue section after hematoxylin staining according to the following steps:
1. 70% ethanol: and 2 min.
2. 80% of ethanol: and 2 min.
3. 95% ethanol: and 20 min.
4. 100% ethanol i: and 2 min.
After the decolorization is finished, the tissue section is placed in a mixed solution of 95 percent ethanol and 0.5 percent eosin dye solution for soaking and dyeing for 12min, and then the elution is carried out according to the following steps.
1. 95% ethanol: and 2 min.
2. Anhydrous ethanol i: and 2 min.
3. Anhydrous ethanol ii: and 2 min.
4. 1/2 xylene +1/2 ethanol: and 2 min.
5. Xylene I: for 10 min.
6. Xylene II: for 10 min.
(6) Sealing piece of tissue slice
The stained slide glass is taken out from the xylene, and neutral gum is immediately dropped to cover the cover glass for mounting when the liquid on the surface of the slide glass is not dry.
(7) Observation of tissue sections
And (5) placing the slide in a microscopic imaging system for observation, and taking a picture for storage.
S12: sample analysis
S13: data processing and result determination
The experimental group and the control group have obvious difference, and the animal experiment result with the function of promoting the lactation function of the test sample can be judged to be positive.
Preferably, the collected raw material of the oysters is fresh material, and the oysters are cleaned to different degrees according to the degree of dirt attached to the surfaces of the oysters.
Preferably, the selected murines need to strictly select experimental mice with specific weight, sex and mouse age, and the specially selected experimental mice are used for establishing an overload mammal rat model.
Compared with the prior art, the invention has the beneficial effects that: the method for researching lactation promoting effect of Concha Ostreae is provided;
1. establishing an overload lactation model, taking an SD rat as an experimental object, researching whether the oyster powder has the function of promoting lactation, comparing the lactation amounts of rats in an oyster powder group and a normal group to show that the oyster powder can improve the lactation amount of a lactating mother rat, comparing the visceral organ indexes of the oyster powder group and the normal group mother rat, and ensuring that the visceral organ indexes of the oyster powder group and the normal group mother rat are all higher than those of the normal group, thereby conforming to the normal healthy physiological level of the lactating mother rat.
2. The PRL concentration of two groups of sera is compared, the oyster powder group is higher than the normal group, prolactin concentration can start and maintain lactation, thereby explaining that the oyster powder has the function of promoting lactation, the acinus of the oyster powder group is more and more intensive than the acinus of the normal group when observed under a microscope, explaining that the oyster powder promotes the development of mammary glands of a lactating mother mouse, and further explaining that the oyster powder can promote the lactation amount of the lactating mother mouse.
Drawings
FIG. 1 is a schematic diagram of the structure of the change of the degree of hydrolysis of oyster protein and oyster enzymolysis powder in the process of in vitro simulation of gastrointestinal digestion according to the present invention;
FIG. 2 is a schematic diagram showing the variation of the nitrogen release amount of the oyster protein and the oyster enzymolysis powder of the invention;
FIG. 3 is a schematic diagram of the structure of the average lactation volume data analysis of the present invention;
FIG. 4 is a schematic diagram of the analysis structure of mean value mouse body weight gain data according to the present invention;
FIG. 5 is a schematic diagram of the PRL content data analysis structure according to the present invention.
Detailed Description
Technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: a method for researching lactation promotion effect of oysters comprises the following steps:
s1 test Material
(1) Oysters, mother rats and male rats.
(2) The experimental use is all SPF grade healthy SD rats, and the ratio of female rats to male rats is 2: 1, and both the female and male rats were 8 weeks old, unprimed rats, weight range of SD female: 180 g-200 g, weight range of SD male mouse: 200g to 250 g.
S2, main experimental reagent
Boric acid, anhydrous sodium carbonate, anhydrous ethanol, concentrated sulfuric acid, concentrated hydrochloric acid, potassium sulfate, glucose, n-butanol, copper sulfate pentahydrate potassium sulfate, trichloroacetic acid, barium chloride, sodium tartrate, sodium hydroxide, sodium chloride, xylene, hematoxylin eosin staining agent, chloral hydrate, zinc acetate, furin phenol and bovine serum albumin.
S3 preparation of oyster powder
(1) Cleaning fresh oysters by using a cleaning machine or a cleaning pool, filtering the cleaned oysters to dry, and preparing powder.
(2) Pre-freezing at 20 deg.C for 3 hr, deep-freezing in-80 deg.C refrigerator, and drying and storing.
S4 preparation of oyster protein
(1) 50g of oyster freeze-dried powder is dissolved in 1L of distilled water.
(2) Adjusting the pH value to 12-13 by 0.1mol/L sodium hydroxide solution.
(3) Stirring at 4 deg.C for 3 hr, centrifuging at 10000r/min and 4 deg.C for 15min, and extracting supernatant.
(4) Adjusting the pH of the supernatant to 4.5-5.1 by 0.1mol/L hydrochloric acid solution, centrifuging for 15min at 10000r/min and 4 ℃, and collecting the precipitate.
(5) Transferring the precipitate into a 12 ku-hole fiber dialysis bag, dialyzing for 48 hours, collecting the precipitate, freeze-drying, and storing in a dry manner.
S5 preparation of oyster enzymolysis powder
(1) Cleaning fresh Concha Ostreae, draining, homogenizing, adding distilled water at ratio of homogenate to water of 1: 3, adjusting pH to 7.0, and adding animal protein hydrolase.
(2) Keeping 1000U/g Carnis Ostreae in 53 deg.C water bath for 6 hr, centrifuging at 500r/min for 20min, extracting supernatant, removing water by rotary evaporation, freeze drying, and storing.
S6 establishment of overload lactation rat model
(1) The number of newborn mice fed by each litter of female mice is adjusted, the number of newborn mice is increased or reduced, so that each litter of female mice has the same number, the female mice feed the same number of newborn mice, the feeding requirements of a litter of newborn mice cannot be met, and the model is an overloaded rat model.
(2) The average is divided into a normal group and an oyster whole powder group, the normal group is filled with 1mL/100g of intragastric distilled water once a day for 20 consecutive days, and the oyster whole powder group is filled with 0.8mg/mL of oyster whole powder solution once a day for 20 consecutive days.
(3) In vitro simulation of gastrointestinal digestion process oyster protein and oyster enzymolysis powder hydrolysis degree change (see figure 1).
(4) Variation in Nitrogen Release amount of oyster protein and oyster zymolyte powder (see FIG. 2)
S7, serum collection and preservation
After the experiment is finished, all the female mice are anesthetized, the blood of the eyeballs is taken out of a centrifuge tube, the centrifuge tube is kept stand for 20min at 37 ℃, the centrifuge tube is placed in a high-speed centrifuge for centrifugation, the temperature is 4 ℃, 3000r/min is used for centrifugation for 15min, and supernate is collected and frozen in a refrigerator at the temperature of 20 ℃ below zero for storage.
S8, detecting the sample
(1) The serum sample is taken out and thawed, and the kit is taken out from the refrigerated environment and is used after being balanced for 15-30 minutes at room temperature.
(2) The method comprises the following operation steps:
and (3) diluting the standard:
sample adding: respectively arranging a blank hole (the blank control hole does not contain a sample and an enzyme-labeled reagent, and the rest steps are operated in the same way), a standard hole and a sample hole to be detected, accurately adding 50 mu L of the standard sample on an enzyme-labeled coating plate, adding 40 mu L of sample diluent in the sample hole to be detected, then adding 10 mu L of sample to be detected (the sample dilution is 5 times), adding the sample to the bottom of the hole of the enzyme-labeled plate, keeping the hole wall untouched as far as possible, and slightly shaking and mixing the sample and the sample hole;
and (3) incubation: incubating for 30 minutes at 37 ℃ after using a sealing plate membrane sealing plate;
preparing liquid: diluting 30 times of concentrated washing liquid with 3 times of distilled water for later use;
washing: carefully uncovering the sealing plate film, discarding liquid, patting to dry, filling washing liquid into each hole, standing for 30s, discarding, repeating the patting to dry for 5 times;
adding an enzyme: adding 50 mu L of enzyme labeling reagent into each hole except blank holes, incubating and washing;
color development: adding 50 μ L of color-developing agent A into each well, adding 50 μ L of color-developing agent B, gently shaking and mixing, and developing at 37 deg.C for 15min in dark;
and (4) terminating: stop solution 50. mu.L is added into each well to stop the reaction (blue color is immediately changed into yellow color);
and (3) determination: the blank wells were set to zero, and the absorbance (OD value) of each well was measured in order at a wavelength of 450nm, and the measurement was carried out within 15 minutes after the addition of the stop solution.
(3) Data processing (see FIGS. 2, 4 and 5)
S9: observation of mammary gland tissue structure
Placing the anesthetized mother mouse on the back on an anatomical operating disk, cutting the skin of the chest and abdomen along the median line of the abdomen, separating the mammary tissue under the skin of the chest and abdomen, weighing and recording the weight of the mammary gland, cutting the same part of the mammary gland 1cm away from the top end of the mammary gland, placing the same part into 4% neutral paraformaldehyde with the volume about 10 times of the mammary gland for fixing for 24-72h, and standing overnight.
S10: tissue wax block production
(1) Fixed tissue trimming and Water washing
1. Firstly, placing the tissue block in a fixing solution for fixing for 24-72h, and then taking out the tissue block.
2. The tissue block is taken out, the shape of the tissue block is trimmed by a scalpel, the tissue block is placed in a dehydration frame for marking, and the tissue block is washed by running tap water for more than 2 hours to wash out the fixing liquid.
(2) Tissue dehydration
Placing the tissue block washed with the fixing solution into alcohol with gradient concentration for dehydration according to the following steps:
50% of ethanol: and (3) 30 min.
70% ethanol: and (3) 30 min.
80% of ethanol: and (3) 30 min.
95% ethanol: and (3) 30 min.
Anhydrous ethanol i: and (3) 30 min.
Anhydrous ethanol ii: and (4) 40 min.
(3) Is transparent
The dehydrated tissue blocks are operated according to the following steps:
1. placing in a xylene: alcohol 1:1 solution: and 15 min.
2. Xylene I: and 15 min.
3. Xylene II: and (4) 40 min.
(4) Wax dipping
Taking the completely transparent tissue block down from the dehydration basket, melting paraffin, and then soaking the tissue block in paraffin according to the following steps, wherein the temperature of the paraffin is about 64 ℃, and the temperature is adjusted according to the paraffin form.
1. Paraffin wax: xylene 1:1 mix: and (3) 30 min.
2. Paraffin wax I: and (4) 1 h.
3. And (3) paraffin II: and (4) 1 h.
4. Paraffin wax III: and (4) 1 h.
(5) Tissue embedding
And (3) placing the soaked tissue block in an embedding box, injecting the melted embedding paraffin into the embedding box, completely wrapping the tissue block by the paraffin, and simultaneously avoiding a gap between the solidified paraffin and the embedding box.
(6) Trimming wax block
Placing the embedding box in a ventilation place until the wax liquid is completely solidified, and freezing the embedding box in a refrigerator at-20 deg.C for 30min after the solidification.
After freezing, carefully taking out the wax block, making a clear tissue mark on one side of the wax block, then cutting off paraffin around the tissue block by using a blade, reserving paraffin of 1-2 mm around the tissue block, and trimming the wax block into a square.
S11: making tissue slices
(1) Slicing
The wax block is fixed on the holder, the wax block and the blade form an included angle of 5 degrees, the wax block is sliced, the tissue section in the wax block slice is complete and smooth, the blade is moved leftwards by a distance of about one wax block width, the slice thickness is adjusted to be 5 mu m, and then the slice is sliced, and the slice is continuously banded, complete and uniform.
(2) Exhibition piece
Preheating a baking machine at 46 ℃, dripping a small amount of egg white on a clean glass slide, uniformly smearing in the same direction, dripping a few drops of distilled water, carefully clamping a wax sheet on the glass slide, and finally placing the glass slide on the baking machine for drying.
(3) Deparaffinization and hydration of tissue sections
The completely dried tissue slices are placed on a slide rack and dewaxed according to the following steps:
1. xylene I: and 5 min.
2. Xylene II: and 5 min.
3. 1/2 xylene +1/2 absolute ethanol solution: and 5 min.
After dewaxing was complete, the tissue sections were washed to elute xylene as follows:
1. anhydrous ethanol: and 2 min.
2. 95% ethanol: and 2 min.
3. 90% ethanol: and 2 min.
4. 80% of ethanol: and 2 min.
5. 70% ethanol: and 2 min.
6. Washing with distilled water: and 2 min.
Sequentially placing dewaxed tissue slices into gradient ethanol, sequentially removing xylene according to the sequence of anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol and 70% ethanol, soaking in ethanol of each concentration for 2min, and finally soaking in distilled water for 2 min.
(4) Hematoxylin staining of tissue sections
Spin-drying the tissue slices with distilled water, soaking the tissue slices in hematoxylin staining solution for 4min, finally washing with distilled water for 2min, observing the staining result under a low power microscope, and repeating the previous steps until the staining is clear if the cell nucleus staining is not clear.
(5) Eosin staining of tissue sections
Decolorizing the tissue section after hematoxylin staining according to the following steps:
1. 70% ethanol: and 2 min.
2. 80% of ethanol: and 2 min.
3. 95% ethanol: and 20 min.
4. 100% ethanol i: and 2 min.
After the decolorization is finished, the tissue section is placed in a mixed solution of 95 percent ethanol and 0.5 percent eosin dye solution for soaking and dyeing for 12min, and then the elution is carried out according to the following steps.
1. 95% ethanol: and 2 min.
2. Anhydrous ethanol i: and 2 min.
3. Anhydrous ethanol ii: and 2 min.
4. 1/2 xylene +1/2 ethanol: and 2 min.
5. Xylene I: for 10 min.
6. Xylene II: for 10 min.
(6) Sealing piece of tissue slice
The stained slide glass is taken out from the xylene, and neutral gum is immediately dropped to cover the cover glass for mounting when the liquid on the surface of the slide glass is not dry.
(7) Observation of tissue sections
And (5) placing the slide in a microscopic imaging system for observation, and taking a picture for storage.
S12: sample analysis
S13: data processing and result determination
The experimental group and the control group have obvious difference, and the animal experiment result with the function of promoting the lactation function of the test sample can be judged to be positive.
The collected oyster raw materials are fresh materials, and oysters are cleaned to different degrees according to the degree of dirt attached to the surfaces of the oysters.
The selected rats need to be selected strictly with specific weight, sex and age, and the specially selected rats are used to establish an overload suckling rat model.
Those not described in detail in this specification are within the skill of the art.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (3)
1. A method for researching the lactation promoting effect of oysters is characterized by comprising the following steps: the method comprises the following steps:
s1 test Material
(1) Oysters, mother rats and male rats.
(2) The experimental use is all SPF grade healthy SD rats, and the ratio of female rats to male rats is 2: 1, and both the female and male rats were 8 weeks old, unprimed rats, weight range of SD female: 180 g-200 g, weight range of SD male mouse: 200g to 250 g.
S2, main experimental reagent
Boric acid, anhydrous sodium carbonate, anhydrous ethanol, concentrated sulfuric acid, concentrated hydrochloric acid, potassium sulfate, glucose, n-butanol, copper sulfate pentahydrate potassium sulfate, trichloroacetic acid, barium chloride, sodium tartrate, sodium hydroxide, sodium chloride, xylene, hematoxylin eosin staining agent, chloral hydrate, zinc acetate, furin phenol and bovine serum albumin.
S3 preparation of oyster powder
(1) Cleaning fresh oysters by using a cleaning machine or a cleaning pool, filtering the cleaned oysters to dry, and preparing powder.
(2) Pre-freezing at 20 deg.C for 3 hr, deep-freezing in-80 deg.C refrigerator, and drying and storing.
S4 preparation of oyster protein
(1) 50g of oyster freeze-dried powder is dissolved in 1L of distilled water.
(2) Adjusting the pH value to 12-13 by 0.1mol/L sodium hydroxide solution.
(3) Stirring at 4 deg.C for 3 hr, centrifuging at 10000r/min and 4 deg.C for 15min, and extracting supernatant.
(4) Adjusting the pH of the supernatant to 4.5-5.1 by 0.1mol/L hydrochloric acid solution, centrifuging for 15min at 10000r/min and 4 ℃, and collecting the precipitate.
(5) Transferring the precipitate into a 12 ku-hole fiber dialysis bag, dialyzing for 48 hours, collecting the precipitate, freeze-drying, and storing in a dry manner.
S5 preparation of oyster enzymolysis powder
(1) Cleaning fresh Concha Ostreae, draining, homogenizing, adding distilled water at ratio of homogenate to water of 1: 3, adjusting pH to 7.0, and adding animal protein hydrolase.
(2) Keeping 1000U/g Carnis Ostreae in 53 deg.C water bath for 6 hr, centrifuging at 500r/min for 20min, extracting supernatant, removing water by rotary evaporation, freeze drying, and storing.
S6 establishment of overload lactation rat model
(1) The number of newborn mice fed by each litter of female mice is adjusted, the number of newborn mice is increased or reduced, so that each litter of female mice has the same number, the female mice feed the same number of newborn mice, the feeding requirements of a litter of newborn mice cannot be met, and the model is an overloaded rat model.
(2) The average is divided into a normal group and an oyster whole powder group, the normal group is filled with 1mL/100g of intragastric distilled water once a day for 20 consecutive days, and the oyster whole powder group is filled with 0.8mg/mL of oyster whole powder solution once a day for 20 consecutive days.
S7, serum collection and preservation
After the experiment is finished, all the female mice are anesthetized, the blood of the eyeballs is taken out of a centrifuge tube, the centrifuge tube is kept stand for 20min at 37 ℃, the centrifuge tube is placed in a high-speed centrifuge for centrifugation, the temperature is 4 ℃, 3000r/min is used for centrifugation for 15min, and supernate is collected and frozen in a refrigerator at the temperature of 20 ℃ below zero for storage.
S8, detecting the sample
(1) The serum sample is taken out and thawed, and the kit is taken out from the refrigerated environment and is used after being balanced for 15-30 minutes at room temperature.
(2) The method comprises the following operation steps:
and (3) diluting the standard:
sample adding: respectively arranging a blank hole (the blank control hole does not contain a sample and an enzyme-labeled reagent, and the rest steps are operated in the same way), a standard hole and a sample hole to be detected, accurately adding 50 mu L of the standard sample on an enzyme-labeled coating plate, adding 40 mu L of sample diluent in the sample hole to be detected, then adding 10 mu L of sample to be detected (the sample dilution is 5 times), adding the sample to the bottom of the hole of the enzyme-labeled plate, keeping the hole wall untouched as far as possible, and slightly shaking and mixing the sample and the sample hole;
and (3) incubation: incubating for 30 minutes at 37 ℃ after using a sealing plate membrane sealing plate;
preparing liquid: diluting 30 times of concentrated washing liquid with 3 times of distilled water for later use;
washing: carefully uncovering the sealing plate film, discarding liquid, patting to dry, filling washing liquid into each hole, standing for 30s, discarding, repeating the patting to dry for 5 times;
adding an enzyme: adding 50 mu L of enzyme labeling reagent into each hole except blank holes, incubating and washing;
color development: adding 50 μ L of color-developing agent A into each well, adding 50 μ L of color-developing agent B, gently shaking and mixing, and developing at 37 deg.C for 15min in dark;
and (4) terminating: stop solution 50. mu.L is added into each well to stop the reaction (blue color is immediately changed into yellow color);
and (3) determination: the blank wells were set to zero, and the absorbance (OD value) of each well was measured in order at a wavelength of 450nm, and the measurement was carried out within 15 minutes after the addition of the stop solution.
(3) Data processing
S9: observation of mammary gland tissue structure
Placing the anesthetized mother mouse on the back on an anatomical operating disk, cutting the skin of the chest and abdomen along the median line of the abdomen, separating the mammary tissue under the skin of the chest and abdomen, weighing and recording the weight of the mammary gland, cutting the same part of the mammary gland 1cm away from the top end of the mammary gland, placing the same part into 4% neutral paraformaldehyde with the volume about 10 times of the mammary gland for fixing for 24-72h, and standing overnight.
S10: tissue wax block production
(1) Fixed tissue trimming and Water washing
1. Firstly, placing the tissue block in a fixing solution for fixing for 24-72h, and then taking out the tissue block.
2. The tissue block is taken out, the shape of the tissue block is trimmed by a scalpel, the tissue block is placed in a dehydration frame for marking, and the tissue block is washed by running tap water for more than 2 hours to wash out the fixing liquid.
(2) Tissue dehydration
Placing the tissue block washed with the fixing solution into alcohol with gradient concentration for dehydration according to the following steps:
50% of ethanol: and (3) 30 min.
70% ethanol: and (3) 30 min.
80% of ethanol: and (3) 30 min.
95% ethanol: and (3) 30 min.
Anhydrous ethanol i: and (3) 30 min.
Anhydrous ethanol ii: and (4) 40 min.
(3) Is transparent
The dehydrated tissue blocks are operated according to the following steps:
1. placing in a xylene: alcohol 1:1 solution: and 15 min.
2. Xylene I: and 15 min.
3. Xylene II: and (4) 40 min.
(4) Wax dipping
Taking the completely transparent tissue block down from the dehydration basket, melting paraffin, and then soaking the tissue block in paraffin according to the following steps, wherein the temperature of the paraffin is about 64 ℃, and the temperature is adjusted according to the paraffin form.
1. Paraffin wax: xylene 1:1 mix: and (3) 30 min.
2. Paraffin wax I: and (4) 1 h.
3. And (3) paraffin II: and (4) 1 h.
4. Paraffin wax III: and (4) 1 h.
(5) Tissue embedding
And (3) placing the soaked tissue block in an embedding box, injecting the melted embedding paraffin into the embedding box, completely wrapping the tissue block by the paraffin, and simultaneously avoiding a gap between the solidified paraffin and the embedding box.
(6) Trimming wax block
Placing the embedding box in a ventilation place until the wax liquid is completely solidified, and freezing the embedding box in a refrigerator at-20 deg.C for 30min after the solidification.
After freezing, carefully taking out the wax block, making a clear tissue mark on one side of the wax block, then cutting off paraffin around the tissue block by using a blade, reserving paraffin of 1-2 mm around the tissue block, and trimming the wax block into a square.
S11: making tissue slices
(1) Slicing
The wax block is fixed on the holder, the wax block and the blade form an included angle of 5 degrees, the wax block is sliced, the tissue section in the wax block slice is complete and smooth, the blade is moved leftwards by a distance of about one wax block width, the slice thickness is adjusted to be 5 mu m, and then the slice is sliced, and the slice is continuously banded, complete and uniform.
(2) Exhibition piece
Preheating a baking machine at 46 ℃, dripping a small amount of egg white on a clean glass slide, uniformly smearing in the same direction, dripping a few drops of distilled water, carefully clamping a wax sheet on the glass slide, and finally placing the glass slide on the baking machine for drying.
(3) Deparaffinization and hydration of tissue sections
The completely dried tissue slices are placed on a slide rack and dewaxed according to the following steps:
1. xylene I: and 5 min.
2. Xylene II: and 5 min.
3. 1/2 xylene +1/2 absolute ethanol solution: and 5 min.
After dewaxing was complete, the tissue sections were washed to elute xylene as follows:
1. anhydrous ethanol: and 2 min.
2. 95% ethanol: and 2 min.
3. 90% ethanol: and 2 min.
4. 80% of ethanol: and 2 min.
5. 70% ethanol: and 2 min.
6. Washing with distilled water: and 2 min.
Sequentially placing dewaxed tissue slices into gradient ethanol, sequentially removing xylene according to the sequence of anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol and 70% ethanol, soaking in ethanol of each concentration for 2min, and finally soaking in distilled water for 2 min.
(4) Hematoxylin staining of tissue sections
Spin-drying the tissue slices with distilled water, soaking the tissue slices in hematoxylin staining solution for 4min, finally washing with distilled water for 2min, observing the staining result under a low power microscope, and repeating the previous steps until the staining is clear if the cell nucleus staining is not clear.
(5) Eosin staining of tissue sections
Decolorizing the tissue section after hematoxylin staining according to the following steps:
1. 70% ethanol: and 2 min.
2. 80% of ethanol: and 2 min.
3. 95% ethanol: and 20 min.
4. 100% ethanol i: and 2 min.
After the decolorization is finished, the tissue section is placed in a mixed solution of 95 percent ethanol and 0.5 percent eosin dye solution for soaking and dyeing for 12min, and then the elution is carried out according to the following steps.
1. 95% ethanol: and 2 min.
2. Anhydrous ethanol i: and 2 min.
3. Anhydrous ethanol ii: and 2 min.
4. 1/2 xylene +1/2 ethanol: and 2 min.
5. Xylene I: for 10 min.
6. Xylene II: for 10 min.
(6) Sealing piece of tissue slice
The stained slide glass is taken out from the xylene, and neutral gum is immediately dropped to cover the cover glass for mounting when the liquid on the surface of the slide glass is not dry.
(7) Observation of tissue sections
And (5) placing the slide in a microscopic imaging system for observation, and taking a picture for storage.
S12: sample analysis
S13: data processing and result determination
The experimental group and the control group have obvious difference, and the animal experiment result with the function of promoting the lactation function of the test sample can be judged to be positive.
2. The method for researching lactation promotion effect of oysters according to claim 1, wherein the method comprises the following steps: the collected oyster raw materials are fresh materials, and oysters are cleaned to different degrees according to the dirt degree attached to the surfaces of the oysters.
3. The method for researching lactation promotion effect of oysters according to claim 1, wherein the method comprises the following steps: the selected rats need to be selected strictly with specific weight, sex and age, and the specially selected rats are used for establishing an overload lactation rat model.
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