CN107129962B - A kind of primary culture method of Hirudo japonica salivary gland cell - Google Patents
A kind of primary culture method of Hirudo japonica salivary gland cell Download PDFInfo
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- CN107129962B CN107129962B CN201710354224.6A CN201710354224A CN107129962B CN 107129962 B CN107129962 B CN 107129962B CN 201710354224 A CN201710354224 A CN 201710354224A CN 107129962 B CN107129962 B CN 107129962B
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- 241000237903 Hirudo Species 0.000 title claims abstract description 50
- 244000184734 Pyrus japonica Species 0.000 title claims abstract description 50
- 210000003079 salivary gland Anatomy 0.000 title claims abstract description 36
- 238000012136 culture method Methods 0.000 title claims abstract description 14
- 210000001519 tissue Anatomy 0.000 claims abstract description 25
- 241000238631 Hexapoda Species 0.000 claims abstract description 8
- 230000003068 static effect Effects 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 27
- 239000007853 buffer solution Substances 0.000 claims description 26
- 229930182555 Penicillin Natural products 0.000 claims description 12
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 12
- 230000001476 alcoholic effect Effects 0.000 claims description 12
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- 238000000034 method Methods 0.000 claims description 11
- 238000012545 processing Methods 0.000 claims description 10
- 238000004659 sterilization and disinfection Methods 0.000 claims description 10
- 230000028327 secretion Effects 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 8
- 210000003300 oropharynx Anatomy 0.000 claims description 4
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 2
- OQVYMXCRDHDTTH-UHFFFAOYSA-N 4-(diethoxyphosphorylmethyl)-2-[4-(diethoxyphosphorylmethyl)pyridin-2-yl]pyridine Chemical compound CCOP(=O)(OCC)CC1=CC=NC(C=2N=CC=C(CP(=O)(OCC)OCC)C=2)=C1 OQVYMXCRDHDTTH-UHFFFAOYSA-N 0.000 claims 1
- 241000545744 Hirudinea Species 0.000 abstract description 21
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- WQPDUTSPKFMPDP-OUMQNGNKSA-N hirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(OS(O)(=O)=O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]2CSSC[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N2)=O)CSSC1)C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)CSSC1)C(C)C)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 WQPDUTSPKFMPDP-OUMQNGNKSA-N 0.000 abstract description 16
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- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 4
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 4
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- 238000007654 immersion Methods 0.000 description 2
- 229960004408 lepirudin Drugs 0.000 description 2
- OTQCKZUSUGYWBD-BRHMIFOHSA-N lepirudin Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)C(C)C)[C@@H](C)O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)[C@@H](C)O)C1=CC=C(O)C=C1 OTQCKZUSUGYWBD-BRHMIFOHSA-N 0.000 description 2
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- 241000243818 Annelida Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 241000146385 Hirudo nipponia Species 0.000 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
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- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 230000000762 glandular Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 229960002773 hyaluronidase Drugs 0.000 description 1
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- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0601—Invertebrate cells or tissues, e.g. insect cells; Culture media therefor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
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- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
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- Biotechnology (AREA)
- Environmental Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
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Abstract
The invention belongs to technical field of bioengineering, disclose a kind of primary culture method of Hirudo japonica salivary gland cell, and the tissue block after shredding is inoculated in 25cm2Cell closing culture bottle in, be added 5mL SFX-INSCET Insect cellculture liquid, be placed in 29 DEG C of static gas wave refrigerators in biochemical cultivation case.The present invention will greatly improve the yield of natural hirudin；Minimal amount of wild Japanese doctor leech salivary gland cell can obtain a large amount of salivary gland cell after carrying out Cell culture invitro; separate and extract a large amount of natural hirudins; the protection for being conducive to wild Japanese doctor leech population reaches the target that not only deep development utilizes but also protects animal germ plasm resource energetically.The present invention is quickly, effective, repeatability is strong, is to provide reliable test data and basis to the perfect of leeches cell culture for the research of leeches cell primary culture.
Description
Technical field
The invention belongs to technical field of bioengineering more particularly to a kind of originally culture sides of Hirudo japonica salivary gland cell
Method.
Background technique
With the rapid development of life science, no matter for entire biotechnology, or the biology gram of one of them
For grand technology, cell culture is all an essential process, and pushes cell biology, molecular biology, biology
The development in the fields such as chemistry.Hirudo japonica (Hirudo nipponia) belongs to Annelida, there is annulus subphylum, Hirudinea, no kiss leech
Mesh, Yi Zhike cure leech category.According to statistics, it is distributed in global leeches animal and shares more than 600 kinds, it is close to be distributed in also having for China
100 kinds.They live in rivers, wet land and field, and some is with feeding fish, the frog, aquatic bird and ox, horse, people such one
The blood of a little mammals is made a living, also some by the invertebrate of feeding earthworm, shrimp, spiral shell, freshwater mussel etc body fluid, tissue so that
Entire body and existence.It is made a living with sucking mammalian, a variety of biologies such as hirudin is contained in salivary gland secretion
Active material and it is collectively referred to as medicine leeches in the world with the leeches animal of broad prospect of application in medicine.Research hair
Existing, various active substance is contained in medicine leeches salivary gland secretion: hirudin, leech hyaluronidase, peace replace Si Taxin, fiber crops
Liquor-saturated dose, vasodilator, prostaglandin etc..And important sources one of of the Hirudo japonica as natural hirudin, have reducing blood lipid,
Cardiovascular and cerebrovascular, anticoagulation and antitumor and other effects are protected, the title with " soft gold " even more has important research significance.Mesh
There are mainly two types of the preceding methods for obtaining hirudin upper both at home and abroad, and one is the salivary gland positions of direct career in medicine leech to separate and extract
Natural hirudin makes its glandular secretion that salivates with the stimulations such as arginine doctor leech, reuses special device and collects saliva, leads to
Cross that the natural hirudin yield that this method obtains is minimum, and Expenses Cost is higher；Another kind is closed by the method for genetic engineering
At lepirudin 023 ludon, although anticoagulating active is low, blood coagulation resisting function clinically with the difference of natural hirudin and little
Specificity will receive the influence of lepirudin 023 ludon concentration, and blood coagulation resisting function with high purity is also corresponding higher, therefore, clinically to natural
Hirudin demand is very big.
In conclusion problem of the existing technology is: the method for hirudin is obtained at present there are hirudin yield is minimum,
Expenses Cost is higher, the low disadvantage of anticoagulating active.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of originally culture sides of Hirudo japonica salivary gland cell
Method.
The invention is realized in this way a kind of primary culture method of Hirudo japonica salivary gland cell, the Hirudo japonica
The primary culture method of salivary gland cell includes that the tissue block after shredding is inoculated in 25cm2Cell closing culture bottle in, add
Enter 5mLSFX-INSCET insect cell medium, is placed in 29 DEG C of static gas wave refrigerators in biochemical cultivation case.
Further, it is needed before the inoculation:
Step 1, with the thick secretion of water removal Hirudo japonica body surface.After removing completely, first it is put in
It is impregnated in 0.3% liquor potassic permanganate, places into alcoholic solution and impregnate, carried out disinfection processing to Hirudo japonica；
Step 2 will be transferred to one equipped with PBS buffer solution in superclean bench by the Hirudo japonica of body surface sterilizing
In secondary property sterile petri dish, PBS buffer solution submerges Hirudo japonica, with the surgical scissors and tweezers by autoclave sterilization processing
Remove the leading head of Hirudo japonica；
Head is transferred in another disposable sterilized culture dish equipped with PBS buffer solution, PBS buffer solution by step 3
It is submerged, by the leading head of Hirudo japonica along being cut off in the middle part of oropharynx, removes pharynx wall endepidermis and external skin, be used in combination
PBS rinsing；
Salivary gland is placed in alcoholic solution after impregnating by step 4, transfers them to three rapidly respectively equipped with PBS buffer solution
It is respectively impregnated in the vessel of (penicillin/streptomycin containing 800U/mL/anphotericin mixed solution), and slow equipped with PBS in third
In the culture dish of fliud flushing (penicillin/streptomycin containing 800U/mL/anphotericin mixed solution), it is cut into tissue block.
Further, it is first put in 0.3% liquor potassic permanganate in the step 1 and impregnates 10min, place into 10% alcohol
10min is impregnated in solution, is carried out disinfection processing to Hirudo japonica.
Further, salivary gland is placed in 75% alcoholic solution in the step 4 after impregnating 5-7S, is shifted rapidly
It is each into three the respectively vessel equipped with 1mLPBS buffer (penicillin/streptomycin containing 800U/mL/anphotericin mixed solution)
Impregnate 5min.
Further, 0.5mm is cut into the step 43-1mm3The tissue block of size.
Another object of the present invention is to provide a kind of primary culture methods using the Hirudo japonica salivary gland cell
The Hirudo japonica of culture.
Advantages of the present invention and good effect are as follows: carry out Hirudo japonica salivary gland cell primary culture method, culture medium kind
The selection screening test of class, Antibiotics and concentration etc. establishes Hirudo japonica primitive cell culture system, realizes medicine leeches
The originally culture of salivary gland cell, to further realize secondary culture.If Hirudo japonica salivary gland cell in vitro culture can be real
It is existing, and secondary culture can be reached, natural hirudin acquisition modes --- from the Hirudo japonica salivary gland cell of in vitro culture
In directly extract separation, maintain the anticoagulating active of higher level always, and greatly improve the yield of natural hirudin, yield
It is apparently higher than the salivary gland position separation and Extraction natural hirudin of direct career in medicine leech and makes its point with the stimulations such as arginine doctor leech
Secrete the existing method that salivary gland secretion regathers saliva.Minimal amount of wild Japanese doctor leech salivary gland cell carries out cell in vitro training
A large amount of salivary gland cell can be obtained after supporting, separates and extracts a large amount of natural hirudins, is conducive to wild Japanese doctor leech population
Protection, reach not only deep development using the target of protection animal germ plasm resource energetically again.Using the present invention to Hirudo japonica saliva
Liquid gland cell has carried out originally culture, achieves preferable culture effect.
The present invention is quickly, effective, repeatability is strong, is to the perfect of leeches cell culture, is the culture of leeches cell primary
Research provides reliable test data and basis.
Detailed description of the invention
Fig. 1 is the primary culture method flow chart of Hirudo japonica salivary gland cell provided in an embodiment of the present invention.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Application principle of the invention is explained in detail with reference to the accompanying drawing.
The present invention is from RPMI1640 culture medium, SFX-INSCET insect cell medium, M199 culture medium this 3 kinds of culture mediums
In filter out most suitable culture medium SFX-INSCET insect cell medium；From kanamycins, neomycin, streptomysin, sulfuric acid
It is filtered out in 6 kinds of gentamicin, penicillin/streptomycin/amphotericin B mixed solution, ampicillin antibiotic most suitable
Antibiotics penicillin/streptomysin/amphotericin B mixed solution；Concentration gradient examination is carried out to penicillin/streptomycin/amphotericin B
It tests, filters out most suitable concentration 800U/mL；To which preliminary screening goes out to be most suitable for the culture body of Hirudo japonica cell primary culture
System.
As shown in Figure 1, the primary culture method of Hirudo japonica salivary gland cell provided in an embodiment of the present invention includes following
Step:
S101: with the thick secretion of water removal Hirudo japonica body surface.After removing completely, it is first put in 0.3%
10min is impregnated in liquor potassic permanganate, places into 10% alcoholic solution and impregnates 10min, is carried out disinfection processing to Hirudo japonica；
S102: it in superclean bench, will be transferred to by the Hirudo japonica of body surface sterilizing equipped with the primary of PBS buffer solution
Property sterile petri dish in, PBS buffer solution submerge Hirudo japonica, with by autoclave sterilization processing surgical scissors and tweezers take
The leading head of lower Hirudo japonica；
S103: head is transferred in another disposable sterilized culture dish equipped with PBS buffer solution, and PBS buffer solution will
It is submerged, and by the leading head of Hirudo japonica along cutting off in the middle part of oropharynx, removes pharynx wall endepidermis and external skin, and use PBS
Rinsing；
S104: salivary gland is placed in 75% alcoholic solution after impregnating 5-7S, transfers them to three rapidly and is respectively equipped with
5min is respectively impregnated in the vessel of 1mLPBS buffer (penicillin/streptomycin containing 800U/mL/anphotericin mixed solution), and
It, will in culture dish of the third equipped with PBS buffer solution (penicillin/streptomycin containing 800U/mL/anphotericin mixed solution)
It is cut into 0.5mm3-1mm3The tissue block of size or so；
S105: the tissue block after shredding is inoculated in 25cm2Cell closing culture bottle in, be added SFX-INSCET insect
Cell culture fluid is placed in 29 DEG C of static gas wave refrigerators in biochemical cultivation case.
Application effect of the invention is explained in detail below with reference to test.
Test specific steps are as follows:
Step 1, with the thick secretion of water removal Hirudo japonica body surface.After removing completely, first it is put in
10min is impregnated in 0.3% liquor potassic permanganate, places into 10% alcoholic solution and impregnates 10min, carry out disinfection to Hirudo japonica
Processing；
Step 2 will be transferred to one equipped with PBS buffer solution in superclean bench by the Hirudo japonica of body surface sterilizing
In secondary property sterile petri dish, PBS buffer solution submerges Hirudo japonica, with the surgical scissors and tweezers by autoclave sterilization processing
Remove the leading head of Hirudo japonica；
Head is transferred in another disposable sterilized culture dish equipped with PBS buffer solution, PBS buffer solution by step 3
It is submerged, by the leading head of Hirudo japonica along being cut off in the middle part of oropharynx, removes pharynx wall endepidermis and external skin, be used in combination
PBS rinsing；
Salivary gland is placed in 75% alcoholic solution after impregnating 5-7S, transfers them to three rapidly and be respectively equipped with by step 4
5min is respectively impregnated in the vessel of 1mLPBS buffer (penicillin/streptomycin containing 800U/mL/anphotericin mixed solution), and
It, will in culture dish of the third equipped with PBS buffer solution (penicillin/streptomycin containing 800U/mL/anphotericin mixed solution)
It is cut into 0.5mm3-1mm3The tissue block of size or so；
The screening of primary culture medium type: tissue block after shredding is inoculated in 25cm by step 52Cell closing
In culture bottle, divides three groups, be separately added into different types of culture medium (culture volume 5mL), be placed in biochemical cultivation case 29 DEG C
Static gas wave refrigerator replaces the culture solution of half after 7d, and adds 20% inactivation standard fetal calf serum；As a result it is cultivated in RPMI1640
The primitive cell culture carried out in base inoculated and cultured the 2nd day, is observed visually tissue block and is attached at wall bottom more, under the microscope
It can be seen that the tissue block shredded is not of uniform size, form of diverse, edge is rendered as the bright band more to become clear, and culture solution clarification does not have
Impurity；It inoculated and cultured the 3-4 days, is observed visually tissue block and is attached at wall bottom, culture solution clarification, microscopically observation to culture
There is a small amount of black impurity in liquid；Inoculated and cultured the 5th day, culture solution muddiness is visually observed, white clear culture medium slightly turns yellow, dirty
Contaminating object is in spot distribution, a large amount of black fine sand shape impurity pollutions is observed under inverted microscope, cell is without growth sign.In M199
The primitive cell culture carried out in culture medium inoculated and cultured the 2-3 days, visually observes that tissue block half is not adherent, and culture solution is clear
Clearly, observe that tissue is not of uniform size under inverted microscope, form of diverse, edge is rendered as the bright band more to become clear, and culture solution does not have
There is impurity；Inoculated and cultured 4-5 days, more than half tissue block was adherent not yet, culture solution clarification was observed under inverted microscope, cell is without growth
Sign；Inoculated and cultured the 6-7 days or so, the thin out muddiness of culture medium color, be observed visually float in culture solution one layer it is white
The pollutant of grenadine shape.Carry out primitive cell culture in SFX-INSCET insect cell medium, inoculated and cultured the 2-3 days,
It is observed visually tissue block and is attached at wall bottom more, observe that tissue is not of uniform size under inverted microscope, form of diverse, edge is presented
For more bright bright band, inoculated and cultured the 3-4 days, observation culture solution remained unchanged clarification under inverted microscope, and cell is temporarily without growth
Sign；Inoculated and cultured the 5-6 days, from the free a small amount of cell out of tissue block periphery, cellular morphology was circle, transparent.
The screening of originally culture Antibiotics: tissue block after shredding is inoculated in 25cm by step 62Cell closing
In culture bottle, after culture medium optimal in step S105 is added, it is divided into six groups, is separately added into variety classes same concentrations (200U/
ML antibiotic/PBS buffer solution mixed solution), is placed in 29 DEG C of static gas wave refrigerators in biochemical cultivation case；As a result compare same
In culture medium (i.e. SFX-INSCET insect cell medium) after the processing of same concentration 200U/mL difference Antibiotics
Primary cell growth survival and pollutional condition, hair now pass through at penicillin/streptomycin/amphotericin B mixed solution immersion
Time-to-live longest when tissue block originally culture after reason keeps culture solution not contaminated time also longest.
The screening of originally culture antibiotic concentration: tissue block after shredding is inoculated in 25cm by step 72Cell closing
In culture bottle, be added in step S105 after optimal culture medium, be divided into six groups, be separately added into without in concentration, step S106 most
Excellent antibiotic is placed in 29 DEG C of static gas wave refrigerators in biochemical cultivation case；As a result compare in same culture medium by same anti-
Still the different primary cell of antibiotic concentration grows survival and pollutional condition to immersion treatment to raw element solution (i.e. " three is anti-" solution),
Hair now passes through under the concentration that antibiotic concentration is 800U/mL (i.e. antibiotic: PBS buffer solution=800U:1mL), cell contamination
State is minimum, cell survival time longest, and the case where observed neoblast growth.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (4)
1. a kind of primary culture method of Hirudo japonica salivary gland cell, which is characterized in that the Hirudo japonica salivary gland cell
Primary culture method include that the tissue block after shredding is inoculated in 25cm2Cell closing culture bottle in, be added 5mL SFX-
INSCET insect cell medium is placed in 29 DEG C of static gas wave refrigerators in biochemical cultivation case；
It is needed before the inoculation:
It is high to be first put in 0.3% after removing completely with the thick secretion of water removal Hirudo japonica body surface for step 1
It is impregnated in potassium manganate solution, places into alcoholic solution and impregnate, carried out disinfection processing to Hirudo japonica；
Step 2 will be transferred to by the Hirudo japonica of body surface sterilizing equipped with the disposable of PBS buffer solution in superclean bench
In sterile petri dish, PBS buffer solution submerges Hirudo japonica, is removed with the surgical scissors and tweezers handled by autoclave sterilization
The leading head of Hirudo japonica；
Step 3, by head be transferred to another equipped with PBS buffer solution disposable sterilized culture dish in, PBS buffer solution by its
Submergence removes pharynx wall endepidermis and external skin, and floated with PBS by the leading head of Hirudo japonica along cutting off in the middle part of oropharynx
It washes；
Salivary gland is placed in alcoholic solution after impregnating by step 4, and all salivary gland are transferred to first equipped with containing antibiotic
PBS buffer solution vessel in, impregnate 5min；It is then transferred in second vessel equipped with antibiotic PBS buffer solution,
Impregnate 5min；It is finally transferred in a vessel equipped with antibiotic PBS buffer solution of third and impregnates 5min, while shredding tissue
Block；Wherein, antibiotic PBS buffer solution refers to the penicillin, streptomysin, anphotericin that joined final concentration respectively be 800U/mL
PBS buffer solution.
2. the primary culture method of Hirudo japonica salivary gland cell as described in claim 1, which is characterized in that the step 1
In carry out disinfection processing to Hirudo japonica method particularly includes: be first put in 0.3% liquor potassic permanganate and impregnate 10min, then put
Enter in 10% alcoholic solution and impregnates 10min.
3. the primary culture method of Hirudo japonica salivary gland cell as described in claim 1, which is characterized in that the step 4
The middle method impregnated in alcoholic solution that is placed in salivary gland is that salivary gland is placed in 75% alcoholic solution to impregnate 5-7s.
4. the primary culture method of Hirudo japonica salivary gland cell as described in claim 1, which is characterized in that the step 4
In shred tissue block and refer to and be cut into 0.5mm3-1mm3The tissue block of size.
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| CN111979174A (en) * | 2020-08-26 | 2020-11-24 | 中国计量大学 | Method for separating hirudo nipponica salivary gland cells |
| CN114657120A (en) * | 2022-04-20 | 2022-06-24 | 中国计量大学 | Method for obtaining hirudo nipponica salivary gland stem cell mass through enzymolysis and application |
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| US9433648B2 (en) * | 2012-09-17 | 2016-09-06 | Biopep Solutions, Inc. | Treating renal cancer with a whole, leech saliva extract |
| CN103820381A (en) * | 2014-01-23 | 2014-05-28 | 中国计量学院 | Primary culture method for chilo suppressalis cells |
| CN104611288A (en) * | 2014-12-30 | 2015-05-13 | 中国计量学院 | Primary culture method of ampullaria gigas heart tissue cell |
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Application publication date: 20170905 Assignee: Xinchang China Metrology University Enterprise Innovation Research Institute Co.,Ltd. Assignor: China Jiliang University Contract record no.: X2021330000071 Denomination of invention: A primary culture method of salivary gland cells of Japanese medical leech Granted publication date: 20190305 License type: Common License Record date: 20210816 |
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