CN114426946B

CN114426946B - Preparation method of precious or endangered animal traditional Chinese medicinal material substitute based on stem cell/organoid technology

Info

Publication number: CN114426946B
Application number: CN202111564497.6A
Authority: CN
Inventors: 吴纯洁; 吴晓川; 叶洵; 刘子博; 彭伟; 申重阳
Original assignee: Chengdu University of Traditional Chinese Medicine
Current assignee: Chengdu University of Traditional Chinese Medicine
Priority date: 2020-12-18
Filing date: 2021-12-20
Publication date: 2022-10-11
Anticipated expiration: 2041-12-20
Also published as: CN114426946A

Abstract

The invention discloses a preparation method of a rare or endangered animal traditional Chinese medicinal material substitute based on a stem cell/organoid technology, which comprises the following steps: implanting dry cells or tissues of rare or endangered wild medicinal animals into a tissue engineering scaffold, adding growth factors, and continuously culturing. The invention also discloses application of the stem cells and/or the organoids in culturing animal traditional Chinese medicine substitutes. The method can prepare a substitute which is very close to the medicinal parts of the Chinese medicinal materials, is favorable for relieving the shortage of endangered Chinese medicinal materials, and promotes the harmonious development of high-quality sustainable development of the Chinese medicinal industry and the protection industry of wild animals.

Description

Preparation method of precious or endangered animal traditional Chinese medicinal material substitute based on stem cell/organoid technology

Technical Field

The invention belongs to the field of tissue cell culture, and particularly relates to a preparation method of a rare or endangered animal traditional Chinese medicinal material substitute based on a stem cell/organoid technology.

Background

Based on the clinical medicine requirement of traditional Chinese medicine, rare endangered animal medicines are an indispensable part all the time and play a key role in clinical curative effect, such as pangolin tablets, musk, tiger bones, bear gall powder, and cornu saigae tataricae. With the improvement of the acceptance of Chinese medicines and the emphasis on health, the demand of Chinese medicines is on the trend of rapid increase, and the clinical demand of rare or endangered animal medicinal materials is also continuously expanded.

At present, artificial breeding technologies of most medicinal rare or endangered animals are difficult to break through, wild resources are deficient, and negative effects are brought to the wild resources. The national protection force on rare or endangered animals is increasingly strengthened, and the shortage of rare or endangered animal medicinal materials causes the lack of completeness of part of classical prescriptions of the traditional Chinese medicine, so that the exertion of the clinical curative effect of the traditional Chinese medicine is restricted. The forbidden regulations of the national laws on the use of endangered medicinal materials have become the pain point and bottleneck of the inheritance and development of the cause of traditional Chinese medicine.

In traditional Chinese medicine and pharmacology, pangolin scales are very rare animal medicinal materials. The medicinal squama Manis is squama Manis of Manidae, and has effects of promoting blood circulation, resolving hard mass, dredging channels, promoting lactation, resolving carbuncle, and dissipating hard mass. It is used clinically to treat amenorrhea due to blood stasis, abdominal mass, rheumatic arthralgia, agalactia, carbuncle, scrofula, etc. But in recent years, the resource of pangolins has become extremely scarce due to over-killing and destruction of living environment. According to 2014 evaluation of the world conservation of nature alliance, 8 pangolins in the world are dangerous. Among them, chinese pangolin and Malaysia pangolin are classified as "extremely dangerous" species, which are rated "weak" two-stage higher than panda, and are second only to extinction and extinction in the field. The cornu saigae tataricae is originally recorded in Shen nong Ben Cao Jing (Shen nong's herbal), belongs to a famous and precious Chinese medicinal material, is salty in taste, enters liver and heart meridians, has the effects of calming liver wind, clearing liver and improving eyesight, dissipating blood and detoxifying and the like, and is mainly used for treating diseases such as liver wind stirring, convulsion and convulsion, eclampsia gravidarum, high-heat convulsion and syncope, epilepsy and mania, headache and dizziness, conjunctival congestion and nebula, toxic and heat macula, carbuncle and sore toxicity and the like. Because the Sijia antelope is mainly distributed in the middle Asia region, the quantity of the Sijia antelope is sharply reduced to less than 5 percent of the original quantity due to the excessive hunting and excessive hunting, and the Sijia antelope is listed in the red name list of endangered animals in the world by the world natural protection alliance (IUCN). Fel Ursi is gallbladder of black bear or brown bear of Ursidae. The traditional Chinese medicine considers that bear bile has the effects of clearing away heat and toxic materials, calming the liver, improving eyesight, killing parasites and stopping bleeding. Therefore, asian countries have the tradition of killing bear and taking gall for thousands of years. With the development of modern medicine and the arousal of animal protection consciousness, the action of the hunted bear to take the gall is gradually reduced, and the medicinal materials of the bear gall powder are in short supply.

In order to strengthen the protection of rare endangered wild medicinal animals, solve the resource limitation of the clinical use of medicinal materials and break the key bottleneck problem of hindering the development of the traditional Chinese medicine industry, the development of a new traditional Chinese medicine substitute is significant.

Disclosure of Invention

The invention aims to solve the problems that: provides a preparation method of a precious or endangered animal traditional Chinese medicinal material substitute based on a stem cell/organoid technology.

The technical scheme of the invention is as follows:

use of stem cells and/or organoids in culturing animal herbal substitutes.

The use as described hereinbefore, the animal is an endangered animal.

Further, the animal traditional Chinese medicinal materials are animal whole body, skin appendages (horns, scaly nails and the like), organ secretion products or bones; preferably, the animal Chinese medicinal materials are squama Manis, moschus, fel Ursi powder, cornu Saigae Tataricae, os Tigris, os pardi, cornu Rhinocerotis, corium Elephatis or cornu Cervi Pantotrichum.

A method for preparing an animal traditional Chinese medicinal material substitute based on stem cell/organoid technology, the method comprising:

taking corresponding basic animal cells and/or tissues of the traditional Chinese medicinal materials, implanting a tissue engineering scaffold, culturing, adding growth factors, and continuously culturing to obtain a substitute; or, taking the cell and/or tissue of the corresponding basic animal of the traditional Chinese medicinal material, purifying, differentiating or amplifying, differentiating and culturing to obtain a substitute;

the cells are dry cells and/or stem-free cells.

Further, the culture time before the addition of the growth factor is 4 hours to 14 days.

Further, the growth factor comprises EGF.

Further, the animal is an endangered animal.

Further, the animal traditional Chinese medicinal materials are animal whole body, skin appendages (squama, horn, etc.), organ secretion products or bones; preferably, the animal Chinese medicinal materials are squama Manis, moschus, fel Ursi powder, cornu Saigae Tataricae, os Tigris, os pardi, cornu Rhinocerotis, corium Elephatis or cornu Cervi Pantotrichum.

Further, when the animal traditional Chinese medicine is animal skin or skin appendages, the cells are epidermal stem cells and keratinocytes, and preferably, the number ratio of the epidermal stem cells to the keratinocytes is 3:1.

Further, the cells are inoculated on the tissue engineering scaffold according to the planting density of 100000-500000 cells/square centimeter.

Further, the material for preparing the tissue engineering scaffold comprises hydrogel microspheres and an artificial medical high polymer material wrapped outside the hydrogel microspheres;

the hydrogel microspheres are prepared from at least one of chitosan, collagen, sodium hyaluronate, chondroitin sulfate and the like;

the artificial medical high polymer material is prepared from at least one of polycaprolactone, polyamide, polylactic acid-glycolic acid copolymer, polyglycolic acid and the like.

Further, the hydrogel is made of at least one of collagen, sodium hyaluronate and chondroitin sulfate.

Further, the medical polymer material is polycaprolactone.

Further, when the animal traditional Chinese medicinal materials are organ secretion products, the cells are obtained by separating and digesting organ tissues; preferably, the Chinese medicinal material is bear gall powder, and the cells are obtained by separating and digesting liver tissues of the bear.

Furthermore, the cells are expanded and differentiated to obtain a substitute.

Further, the method comprises the following steps:

(1) Suspending the cells obtained by organ tissue separation and digestion in a substrate matrix, adding an amplification culture medium after solidification, culturing for 7-9 days, and replacing a fresh amplification culture medium every 2-3 days;

(2) Changing the culture medium into a differentiation culture medium for differentiation culture, culturing for 4-6 days, and changing a fresh differentiation culture medium every day;

(3) Supplementing a differentiation culture medium added with 30 mu M dexamethasone, replacing a fresh differentiation culture medium added with 30 mu M dexamethasone every day, and continuing differentiation culture for 5-7 days;

the amplification medium comprises the following components: basal medium, B27, amino acids, cytokines and hormones.

The differentiation medium for differentiation comprises the following components: basal medium, B27, amino acids, cytokines, small molecule inhibitors and hormones.

Further, the above-mentioned basal medium comprises ADMEM/F-12, penicillin and streptomycin, glutamine and HEPES;

the amino acid comprises at least one of N-acetylcysteine and nicotinamide;

the cell factor comprises at least one of Rspo1, EGF, FGF, HGF, FSK, noggin and Wnt 3A;

the small molecule inhibitor comprises at least one of a gamma-secretase inhibitor and an ALK4/5/7 inhibitor, the gamma-secretase inhibitor comprises DAPT, and the ALK4/5/7 inhibitor comprises A8301;

the hormone comprises gastrin.

Further, the basal medium is composed of the following components: 1% penicillin and streptomycin, 1% glutamine, 10mM HEPES, balance DMEM/F-12;

the amplification medium used for the amplification consists of the following components: 1-2% of B27, 1-1.25 mM N-acetylcysteine, 10mM nicotinamide, 100-250 ng/mL Rspo1, 50ng/mL EGF, 100ng/mL FGF, 25-50 ng/mL HGF, 10 μ M FSK, 10nM gastrin, and the balance of basal medium;

the differentiation medium comprises the following components: 1-2% of B27, 1-1.25 mM N-acetylcysteine, 50ng/mL of EGF, 100ng/mL of FGF, 50 nM-500nM of A8301, 10. Mu.M of DAPT, 10nM of gastrin, and the balance of basal medium.

Still further, the substrate matrix is a Matrigel substrate matrix.

Further, the culture conditions are 37 ℃,5% CO ₂ 。

Further, when the animal traditional Chinese medicinal material is a skin appendage, the cells are obtained by separating and digesting epidermal tissues; preferably, the Chinese medicinal material is sheep horn, and the cells are obtained by separating and digesting the epidermal tissues of the sheep horn protrusion part.

Further, the above cells are purified and differentiated to obtain a substitute.

Still further, the above method comprises the steps of:

(1) Carrying out differential adherent purification on cells obtained by separating and digesting epidermal tissues to obtain keratinocytes; digesting and purifying cell pancreatin obtained by separating and digesting epidermal tissues to obtain fibroblasts;

(2) Inoculating the fibroblasts and keratinocytes purified in the step (1) into an upper chamber of the collagen type I cell chamber, adding a KSFM culture medium into a lower chamber, culturing for 2 days, then replacing a fresh KSFM culture medium, and culturing for 2 days, wherein the amount of the culture medium exceeds the bottom of the cell chamber;

(3) Replacing the KSFM culture medium of the lower chamber with the KSFM culture medium added with 1.5mM calcium chloride, and continuing culturing for 2 days, wherein the amount of the KSFM culture medium exceeds the bottom of the cell chamber;

(4) The lower chamber was replaced with fresh KSFM medium supplemented with 1.5mM calcium chloride for 8 days, and every two days with fresh KSFM medium supplemented with 1.5mM calcium chloride in an amount just contacting the bottom of the cell chamber.

Further, the differential adherent purification of step (1) is at least 24h, and the pancreatin digestion purification does not exceed 2min.

Further, the ratio of the number of the seeded fibroblasts to the number of the seeded keratinocytes in the step (2) is 1:3, preferably, the density of the seeded fibroblasts is 4x10 ⁵ ～6x10 ⁵ Cells/cm ² 。

Further, the culture conditions were 37 ℃,5% CO ₂ 。

The animal Chinese medicinal material substitute prepared by the previous method.

The animal traditional Chinese medicine substitute is applied as a traditional Chinese medicine.

The term "sternness" refers to the ability of a cell to differentiate.

The term "stem cell" is a type of cell derived from an embryonic, fetal or adult human body which has the ability to self-renew and proliferate and differentiate without restriction under certain conditions, and is capable of producing daughter cells having the same phenotype and genotype as well as self-cells, and also capable of producing specialized cells constituting body tissues and organs, and also capable of differentiating into progenitor cells.

The term "organoid" is analogous to a tissue organ. In fact, it is a model that is highly similar to the source tissue or organ in vivo, based on a 3D in vitro cell culture system. Organoids are constructed mainly from two classes, one of which is differentiated by Induced Pluripotent Stem Cells (iPSCs) and the other of which is directly derived from tissues.

The Chinese medicinal materials related by the invention have the following names and animal-based sources:

pangolin scales: manis Manis, manis pendacactyla Linnaeus, lee.g., .

Musk: deer animals Moschus berezovski Flerov, musk Moschus sifanius Przewalski or original Musk Moschus moschefferus Linnaeus form dry secretions in mature male sachets.

Bear gall powder: bile obtained by draining Selenaretos thibetanus Cuvier of Ursidae animal is dried to obtain dried product.

Antelope horn: the horn of the bovine species antelope Saiga tatarica Linnaeus.

Tiger bone: bones of the feline tiger pandera tigris l.

Leopard bone: dried bones of the feline species panther a pardus Linnaeus, the ounce Panthera uncia Sch-reber, or the clouded animal of the genus clouded leopard Neofelis nebulosa Griffith.

Rhinoceros horn: the horn of the animal family Rhinoceros unicornis L.of the family Rhinoceros, rhinoceros R.Sondoides Desmarest, rhinoceros R.Sumatrensis Cuvier, rhinoceros R.bicornis L.and Rhinoceros R.simulans, etc.

Elephant skin: dried skin of the elephant animal asian elephant Elephas maximus l.

Pilose antler: cervus nippon Temminck or Cervus elaphus Linnaeus of Cervus elaphus L.

The method can prepare a substitute which is very close to the medicinal part of the traditional Chinese medicinal material, is beneficial to relieving the shortage of endangered traditional Chinese medicinal materials, breaks through the key bottleneck problem of hindering the development of the traditional Chinese medicine industry, innovatively drives the protection and utilization of rare endangered animals, and promotes the harmonious development of the high-quality sustainable development of the traditional Chinese medicine industry and the wild animal protection industry.

It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and common practice in the field.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows the growth of cells cultured in combination of different expansion media and differentiation media in example 1.

FIG. 2 shows a gene tree of the bear liver organoid and the bear gene prepared by the present invention.

FIG. 3 is a liquid mass spectrum of a lyophilized differentiation medium, an artificial bear gall powder containing the medium obtained by the method of the present invention, and a natural bear gall powder, wherein the lyophilized differentiation medium, the artificial bear gall powder containing the medium obtained by the method of the present invention, and the natural bear gall powder are sequentially arranged from top to bottom.

FIG. 4 is a structural diagram of staining of a horn-based tissue section of a young goat.

FIG. 5 is a microscopic image of purified goat fibroblasts, goat keratinocytes and differentiation-inducing goat horn organoid (histoid) cells of the present invention.

FIG. 6 shows the results of gel electrophoresis of a sample of goat horn and a sample of cultured goat horn organoid (tissue-like) of the present invention.

FIG. 7 is a mass spectrum of a goat horn organoid (tissue-like) sample and a native goat horn tissue sample.

FIG. 8 is an ion flow diagram of a goat horn organoid (tissue-like) sample and a native goat horn tissue sample.

FIG. 9 shows the results of immunofluorescent staining of cultured goat horn organoid (histioid) cells of the present invention.

Detailed Description

Example 1: preparation of artificial bear gall powder

1 preparation of artificial bear gall powder

1.1 isolation of liver tissue

a) The liver tissue of bear was taken and kept at low temperature (4 ℃) until processing.

b) Placing the tissue into a culture dish under aseptic condition, and cutting into pieces of 0.5-1mm ³ The block of (1).

c) The minced tissue was transferred to a centrifuge tube and washed by adding 10mL of ice-cold wash medium [ DMEM +1% Fetal Bovine Serum (FBS) +1% penicillin/streptomycin (4 ℃ C., 30 days old) ]. This washing step was repeated once.

d) The supernatant was taken and per gram of preheated digestive juice (fresh preparation: collagenase and dispase II were dissolved in washing medium at concentrations of 0.3 mg/mL) 4-5mL, and digestion was incubated at 37 ℃.

e) After incubation, the digestive juices were pipetted vigorously up and down and mixed, and the presence of single cells in the digestive juices was checked. If a small number of single cells were present, the solution was reduced to 37 ℃. Every 10 minutes for 90 minutes at the maximum. Digestion was stopped when the suspension contained 80-100% single cells.

f) After digestion was complete, cold wash medium was added. The digested material was passed through a 70 μm filter and ice cold wash medium was added.

g) Centrifuge at 1700rmp for 5min at 8 ℃. The supernatant was discarded and a further 15mL of cold wash medium was added to resuspend. The resuspended mixture was transferred to a centrifuge tube.

h) Centrifugation at 1700rmp for 5min at 8 ℃, supernatant was removed and discarded, the pellet was washed twice again with wash medium and once again with basal medium for 5min at 1700rmp at 8 ℃.

1.2 cell inoculation and expansion

a) The cells obtained in the previous step were taken and the desired number of cells (10000 cells per well in a 24-well plate) were resuspended in Matrigel-based matrix for seeding.

b) By adding a small drop of the base matrix in the center of each well, incubating at 37 ℃ for 5-10 minutes, or waiting for the base matrix to solidify.

c) The matrix droplets were covered with amplification medium (500. Mu.L per well in 24-well plates and 250. Mu.L per well in 48-well plates).

d)37℃，5％CO ₂ Culturing for 7-9 days under the condition, and changing the culture solution for 2-3 days.

1.3 differentiation of organoids

The medium was changed to differentiation medium. Replacing a fresh differentiation culture medium every day, supplementing a differentiation culture medium added with 30 mu M dexamethasone on the 14 th day, replacing the culture medium every day, culturing for 20 days to obtain organoids, and continuously culturing by adopting the differentiation culture medium (added with 30 mu M dexamethasone).

Collecting culture medium (containing secretion of organoid) containing culture medium of artificial bear bile, and lyophilizing to obtain artificial bear bile powder containing culture medium.

The artificial bear gall powder containing the culture medium can be obtained by removing the culture medium in a separation and purification mode.

The formulations of the amplification medium and the differentiation medium used for the culture were screened as follows:

combination 1 (medium formula No. 1):

amplification medium No. 1: basic medium +2% B27 (vitamin A not contained) +1% N2+1mM N-acetylcysteine +100ng/mL Rspo1+10mM nicotinamide +10nM gastrin +50ng/mL EGF +100ng/mL FGF10+25ng/mL HGF +10 μ M FSK +100ng/mL Noggin +150ng/mL Wnt 3a +10 μ M ROCK inhibitor + 500A8301 (4 ℃, capable of being stored for 14 days).

Differentiation medium No. 1: basal medium +1% B27 (without vitamin A) +1% N2+ 1.25mM N-acetylcysteine +10nM gastrin +50ng/mL EGF +100ng/mL FGF10+500nM A8301+10 μ M DAPT.

Combination 2 (medium formula No. 2):

amplification medium No. 2: basal medium +1% B27 (vitamin A-free) +1% N2+ 1.25mM N-acetylcysteine +250ng/mL Rspo1+10mM nicotinic amine +10nM gastrin +50ng/mL EGF +100ng/mL FGF10+50ng/mL HGF +10 μ M FSK +5 μ M A8301 (4 ℃ C., capable of being stored for 14 days).

Differentiation medium No. 2: basal medium +1% B27 (vitamin A-free) +1% N2+ 1.25mM N-acetylcysteine +10nM gastrin +50ng/mL EGF +100ng/mL FGF10+500nM A8301+ 10. Mu.M DAPT.

Combination 3 (formula of culture medium No. 3):

amplification medium No. 3: basal medium +2% b27 (vitamin a-free) +1mM N-acetylcysteine +100ng/mL Rspo1+10mM nicotinic amine +10nM gastrin +50ng/mL EGF +100ng/mL FGF10+50ng/mL HGF +10 μ M FSK (4 ℃, storable for 14 days).

Differentiation medium No. 3: basal medium +2% B27 (vitamin A not present) +1mM N-acetylcysteine +10nM gastrin +50ng/mL EGF +100ng/mL FGF10+50nM A8301+10 μ M DAPT (4 ℃, storable for 14 days).

The results of the different media are shown in FIG. 1. The growth vigor is better in the culture process of the No. 3 culture medium, the most preferable scheme is that the subsequent detection experiment adopts the combined culture of the two culture media of the combination 3.

2 detection

2.1 homology detection

Taking organoids obtained by the method of the invention, carrying out homology detection with separated bear liver tissues:

the specific primer fragments of bear designed by Primes 5.0 are shown in the following table.

The detection method comprises the following steps: and extracting the DNA of the sample to be detected by adopting an Ezup column type animal genome DNA extraction kit, and performing amplification sequencing by adopting the primer.

By performing blast (ref: hou WR, chen Y, wu X, hu JC, peng ZS, yang J, tang ZX, zhou CQ, li YM, yang SK, du YJ, kong LL, ren ZL, zhang HY, shuai SR. A complete mitochondal genome sequence of Asian Black bear subspace subspecies (Ursus thibetanus mupinensis). Int J Biol Sci.2006Dec 23 (2): 85-90.Doi 10.7150/ijbs.3.85.PMID: 051PMCID 1752222227), it was found that organoids cultured by the method of the invention were in-situ consistent with each other, with a similarity of 98.25% (FIG. 2).

2.2 secretion detection

Taking natural bear gall powder, a freeze-dried differentiation culture medium and the artificial bear gall powder containing the culture medium obtained by the method of the invention, metering the volume of methanol to 10mL, and filtering the solution through a 0.22 mu m microporous filter membrane.

(1) Chromatographic conditions

Mobile phase: acetonitrile is taken as a mobile phase A, and 0.1% formic acid is taken as a mobile phase B; gradient elution was performed according to the following table; flow rate 0.3mL/min, detector: DAD; detection wavelength: 203nm; column temperature: 30 ℃; the injection volume was 20. Mu.L.

(2) Conditions of Mass Spectrometry

Electrospray ion source (ESI); collecting positive and negative ions simultaneously; scanning range: 50-1200 Da; target molecular weight: 500Da; temperature of the drying gas: 350 ℃; flow rate of drying gas: 9.0L/min; the pressure of atomizing gas: 0.24MPa; capillary voltage: 4kV.

As shown in FIG. 3, FIG. 3 shows the results of the analysis of the lyophilized differentiation medium, the culture medium-containing artificial bear gall powder obtained by the method of the present invention, and the natural bear gall powder in the sequence from top to bottom. The corresponding peak-out times for the major components are shown in the following table:

therefore, the artificial bear gall powder containing the culture medium obtained by the method contains bezoar ursodesoxycholic acid, taurocholic acid, cholic acid and the like, and has similar components with natural bear gall powder.

According to the results, the bear liver organoid cultured by the method has the secretion function basically similar to that of individual bears, and can be used for preparing the artificial bear gall powder.

Example 2: preparation of artificial goat horn

In consideration of the protection of endangered animals, the antelope, the goat, the same animal, was used for research in the early stage of the design scheme.

1 preparation of Artificial goat horn

1.1 isolated culture of epidermal tissue cells at the horn-process site of young goat of Argy sheep

Intact epidermis, protease digestion for 24h, recovery of the remaining tissue, ophthalmic shearing to homogenate, addition of 4% pancreatin 2mL, digestion at 37 ℃ for 30min, digestion termination in 10% FBS in DMEM/F12 medium 5mL, centrifugation. The supernatant was discarded, 5mL of complete medium [ (DMEM/F12 +1% penicillin/streptomycin double antibody containing 10% FBS) ] was added thereto, the mixture was pipetted about 20 times, and the liquid was transferred to a petri dish, and 5mL of complete medium was further added thereto and cultured at 37 ℃. The culture medium is changed once after 48 h. Gently aspirate the medium with a pipette and discard, wash once with 3mL wash medium, and add 9mL complete medium.

1.2 purification of epidermal tissue cells

The fibroblast and keratinocyte are purified by a differential adherence method and a differential digestion method. Setting 5 time gradients of 2min,4min,6min,8min and 10min by a differential digestion method. The differential wall-pasting method is used for setting 5 time gradients of 10min,30min,1h,5h and 24h. It was found that adherent conditions were able to purify keratinocytes after 24 h. Digesting with pancreatin at 37 deg.C for 2min, and purifying to obtain fibroblast.

1.3 Induction differentiation of epidermal tissue cells to prepare organoids (tissue-like)

ALI gas-liquid two-phase culture dish induces keratinocyte keratinization, uses type I collagen to resuspend, digest and centrifugate fibroblasts, and has density of 5x10 ⁵ Cells/cm ² . Taking keratinocyte at 7.5x10 ⁵ Cells/cm ² The density of (a) was seeded in type I collagen cell chambers pre-coated with fibroblasts, the cell chambers were placed in a six-well plate, the lower chamber was seeded with 2.5mL KSFM medium, and the upper chamber was seeded with 1mL cell suspension. CO at 37 ℃ and 5% ₂ After 2 days of incubation under conditions, all media was aspirated from the wells and from the cell culture dish, creating a gas-liquid interface, and then 3mL of KSFM media was added to the lower chamber for 2 days of incubation. The medium was then cultured for 2 days by pumping from the lower chamber supplemented with fresh KSFM medium supplemented with 1.5mM calcium chloride.

The amount of KSFM medium added with 1.5mM calcium chloride in the lower cell chamber was reduced to 2mL by 6d to 14d so that the medium level just contacted the bottom of the cell chamber. The medium (KSFM medium supplemented with 1.5mM calcium chloride) was changed every two days to obtain organoids (organoids) of artificial goat horns.

2 detection

2.1 tissue staining and cell morphology analysis

Taking 1cm of skin at the base of the horn protrusion part of 12-day young sheep ³ The results obtained by staining paraffin-embedded sections after fixing with paraformaldehyde are shown in fig. 4, and it can be seen from the results of the sections that the skin at the position of the goat horn base bulge mainly contains two types of cells, namely goat fibroblasts and keratinocytes.

The purified goat fibroblasts and keratinocytes and organoid (histioid) cells of the artificial goat horn further induced to differentiate were again microscopically observed, and it was found that the organoid (histioid) cells of the artificial goat horn were successfully differentiated into keratinocytes (fig. 5).

2.2 gel electrophoresis analysis

(1) Total protein extraction

Pulverizing natural cornu Naemorhedi sample into powder (200 mesh), collecting 20mg of each sample, precisely weighing, placing in a centrifuge tube, adding 1.0mL protein extract (8 mol/L urea, 2% SDS,20mmol/L DTT, pH 7.4), mixing, placing the centrifuge tube in 65 deg.C water bath for 7h (mixing 1 time every 1 h), centrifuging at 12000r/min for 15min, collecting supernatant, and storing at-20 deg.C for use.

The organoids (tissue-like) of the artificial goat horns were washed three times with PBS, scraped off with a cell scraper, and centrifuged at 1000r/min for 5min to harvest the cells. Adding 500 μ L of the above protein extractive solution, mixing, performing the following steps to obtain protein supernatant, and storing at-20 deg.C.

(2) Protein content determination

The protein content of the extract was determined by Bradford method, using bovine serum albumin as standard protein.

(3) SDS-PAGE gel electrophoresis

Taking the prepared sample, adding a proper amount of ultrapure water and 5 times of loading buffer solution to prepare a loading solution with the protein mass concentration of 2g/L, carrying out water bath at 100 ℃ for 5min, and then centrifuging at 12000r/min for 5min for loading. SDS-PAGE vertical gel electrophoresis, the concentration of the separation gel is 12 percent, the concentration of the concentration gel is 5 percent, and the thickness of the gel is 1.5mm. Electrode buffer Tris-glycine buffer (25 mmol/L Tris,192mmol/L glycine, 0.1% SDS, pH 8.3) was used. The amount of protein loaded in each lane was 10. Mu.L, i.e., 20. Mu.g. Electrophoresis is carried out at 20 ℃, the electrophoresis conditions are 10mA of concentrated gel and 30mA of separation gel, and the electrophoresis is stopped when the tracer bromophenol blue migrates to the lower edge of the gel plate. The gel was stained with coomassie brilliant blue staining,

as shown in FIG. 5, the organoids (tissue-like) of the artificial goat horn cultured by the method of the present invention are similar to the protein components of the natural goat horn.

2.3 liquid Mass analysis

(1) Sample processing

Sampling 10mg of powder, adding 1mL of 8M urea, adding 10uL of 1M Dithiothreitol (DTT), heating in water bath at 60 deg.C for 30min, cooling, adding 30 μ L of 1M Iodoacetamide (IAA), standing in dark for 30min, and adding 1% NH ₄ HCO ₃ Diluting the solution to 10mL, adding trypsin solution 20 μ L (prepared with 1% NH4HCO3 solution to concentration of 1 μ g/uL), shaking, and performing enzymolysis at 37 deg.C for more than 12 h. Taking a proper amount of the solution, filtering the solution through a filter membrane of 0.22 mu m, and taking the subsequent filtrate to place in a sample bottle to obtain the product. Negative samples were prepared in the same manner.

(2) Detection conditions

Liquid phase conditions: the chromatographic column is Agilent SB-C18 (2.1x100mm, 1.8 μm); the flow rate is 0.3 mL/min; the column temperature is 40 ℃; the sample injection amount is 5 mu L; mobile phase a was 0.1% formic acid-water solution and B was 0.1% formic acid-acetonitrile solution, and gradient elution was performed as follows.

Mass spectrum conditions: the ionization mode is ESI +, the capillary voltage is 4kV, the taper hole voltage is 40V, the desolvation temperature is 450 ℃, the desolvation gas is 600L/h, and the ion source temperature is 120 ℃. Goat horn characteristic molecular ion peaks m/z673.3 → 764.0 and m/z673.3 → 920.0 were selected as detection ions.

(3) Analysis of results

After mass spectrometry of the cultured tissue sample of the in vitro cultured goat horn-like organ (tissue-like) can be matched with the molecular ion peaks m/z673.36 → 764.39 and m/z673.36 → 920.48 (FIG. 6), and the approximation of the characteristic molecular ion peaks of the goat horn described in the literature can be used to confirm that the cultured tissue sample of the in vitro cultured goat horn-like organ (tissue-like) has the characteristic peptide segment contained in the goat horn.

Further comparative analysis of organoids (tissue-like) from artificial goat horn and native goat horn tissue samples gave an approximate ion flowsheet (figure 7).

2.4 immunofluorescence

Goat KCs immunofluorescence

A permeabilization solution: 30. Mu.L of Triton X-100 stock solution was added to 10mL of PBS at a concentration of 0.3%.

PBST：50mLPBS+50μL Tween 20(0.1％)。

Sealing liquid: grouping skim milk:

(1) Soaking the cell-plated slide in PBS for 5min 3 times in the culture plate;

(2) Fixing the slide by 4% paraformaldehyde for 15min, and washing the slide by PBS for 3 times, 5min each time; the primary antibody solution is prepared in advance, and the diluted primary antibody needs to be thawed at 4 ℃ in advance.

(3) Allowing the permeabilization solution to permeate for 20min at a certain temperature;

(4) Soaking and washing the slide for 3 times by PBS (phosphate buffer solution), 5min each time, and sealing 200 mu L of skimmed milk for 30min at room temperature;

(5) The skim milk was aspirated off the absorbent paper, washed away, and primary antibody diluted with skim milk was added in groups and incubated overnight at 4 ℃.

The next day:

(6) Adding a fluorescent secondary antibody (in dark): PBST immersion-washing climbing film for 3 times, each time for 5min, PBST releasing fluorescent secondary antibody, incubation for 1h at 37 ℃ in a dark place, PBST immersion-washing for 3 times, each time for 5min; note that: all subsequent processing steps were performed as dark as possible from the addition of the fluorescent secondary antibody.

(7) Counterstaining the nucleus: dripping DAPI, incubating for 5min in dark place, staining the specimen for nucleus, washing PBST for 5min for 4 times, and washing off excessive DAPI;

(8) And (3) absorbing the liquid on the slide by using absorbent paper, sealing the slide by using sealing liquid containing an anti-fluorescence quenching agent, and observing and acquiring an image under a fluorescence microscope.

As shown in FIG. 8, it can be seen that the organoids (tissue-like) of the artificial goat horn of the present invention have keratin 14 expression in the cells, indicating that the organoids (tissue-like) of the cultured artificial goat horn of the present invention have the same composition as the goat horn.

As can be seen from the foregoing experiments, the artificial goat horn organoids (tissue-like) cultured by the method of the present invention have a composition similar to that of natural goat horns, and can be used for preparing artificial goat horns. The skin appendages such as other horns, scaly nails and the like, such as antelope horns of endangered animals, namely the Saiga antelope, can also be prepared by adopting the method.

In conclusion, the animal organs (tissue-like tissues) cultured by the method have the same or similar components and functions as the organs or the attachments of natural animals as the traditional Chinese medicinal materials, can be used as substitutes of the animal traditional Chinese medicinal materials, is favorable for relieving the shortage of endangered traditional Chinese medicinal materials, and promotes the harmonious development of high-quality sustainable development of the traditional Chinese medicine industry and the protection industry of wild animals.

SEQUENCE LISTING

<110> Chengdu university of traditional Chinese medicine

<120> preparation method of precious endangered animal traditional Chinese medicine substitute based on stem cell/organoid technology

<130> GY041-2021P0114241CCZ

<150> 2020115139031

<151> 2020-12-18

<160> 34

<170> PatentIn version 3.5

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gatcacacat aactgtggtg tcatg 25

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cggagactta catgtgtaat cttg 24

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taaaggtttg gtcctagcct tccc 24

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aagccctgtc tcttgggcag tattg 25

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agattaaaag aagtaaaagg aactcg 26

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atgtttataa ttaacactat ctcactag 28

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ttatatttgg gggggaatgc ttgct 25

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gtcctactaa tgaatggctc attcg 25

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ccacaacaac actatcactg tccc 24

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caattgactt ccaatcaatt agc 23

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aaactatatt tacagtaaat gggcccc 27

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cccgtaacca ttgactgaat agcccc 26

Claims

1. A preparation method of an animal traditional Chinese medicine substitute based on organoid technology is characterized by comprising the following steps: the traditional Chinese medicine is bear gall powder and comprises the following steps:

(a) Suspending the cells obtained by separating and digesting the liver tissue of the bear in a substrate matrix, adding an amplification culture medium after solidification for culturing for 7~9 days, and replacing fresh amplification culture medium every 2~3 days;

(b) Changing the culture medium into a differentiation culture medium for differentiation culture, culturing for 4~6 days, and changing a fresh differentiation culture medium every day;

(c) Supplementing a differentiation medium added with 30 mu M dexamethasone, replacing a fresh differentiation medium added with 30 mu M dexamethasone every day, and continuously culturing for 5~7 days;

the basic culture medium consists of the following components: 1% penicillin and streptomycin, 1% glutamine, 10mM HEPES, balance DMEM/F-12;

the amplification culture medium consists of the following components: 2% B27, 1mM N-acetylcysteine, 10mM nicotinamide, 100ng/mL Rspo1, 50ng/mL EGF, 100ng/mL FGF, 50ng/mL HGF, 10 μ M FSK, 10nM gastrin, and the balance basal medium;

the differentiation medium consists of the following components: 2% B27, 1mM N-acetylcysteine, 50ng/mL EGF, 100ng/mL FGF, 50nM A8301, 10. Mu.M DAPT, 10nM gastrin, balance basal medium.

2. A preparation method of an animal traditional Chinese medicine substitute based on organoid technology is characterized by comprising the following steps: the traditional Chinese medicinal materials are horns and scaly nails, and the method comprises the following steps:

(a') carrying out differential adherent purification on cells obtained by separating and digesting epidermal tissues at the horn process part to obtain keratinocytes; digesting and purifying cell pancreatin obtained by separating and digesting epidermal tissues to obtain fibroblasts; the differential adherent purification time is not less than 24h, and the pancreatin digestion purification time is not more than 2min;

(b ') the fibroblasts and keratinocytes purified in step (a') were seeded in the upper chamber of the type I collagen cell chamber, and the lower chamber was cultured for 2 days by adding KSFM medium, and then replaced with fresh KSFM medium for another 2 days, the amount of the KSFM medium exceeding the bottom of the cell chamber; the number ratio of the seeded fibroblasts to keratinocytes is 1:3; the density of the inoculated fibroblasts is 4x10 ⁵ ~6x10 ⁵ Cells/cm ² ；

(c') replacing the KSFM medium in the lower chamber with a KSFM medium supplemented with calcium chloride in an amount exceeding the bottom of the cell chamber, and continuing the culture for 2 days;

(d') the lower chamber medium was replaced with fresh calcium chloride supplemented KSFM medium for another 8 days, and every two days with fresh calcium chloride supplemented KSFM medium in an amount just contacting the bottom of the cell chamber.

3. The method of claim 2, wherein: squama Manis, cornu Saigae Tataricae, cornu Rhinocerotis or cornu Cervi Pantotrichum.

4. The method of claim 2, wherein: the KSFM medium added with calcium chloride is the KSFM medium added with 1.5mM calcium chloride.

5. The method of any of claims 1~4 wherein: the animal is an endangered animal.

6. An animal herbal replacement made by the method of any one of claims 1~5.

7. The animal herbal medicine substitute of claim 6, wherein: the Chinese medicinal materials include cornu Cervi Pantotrichum and squama Manis, wherein the squama Manis is squama Manis, and the cornu Saigae Tataricae, cornu Rhinocerotis or cornu Cervi Pantotrichum; or the traditional Chinese medicinal material is bear gall powder.

8. The animal herbal medicine substitute of claim 6, wherein: the animal is an endangered animal.