CN102409021A

CN102409021A - Establishment and culture method of Jinhua pig fibroblast cell line

Info

Publication number: CN102409021A
Application number: CN2011104258521A
Authority: CN
Inventors: 郝柱; 王颖; 彭静; 沈一飞; 郭晓令; 徐宁迎
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2011-12-19
Filing date: 2011-12-19
Publication date: 2012-04-11

Abstract

The invention discloses a culture method of a Jinhua pig fibroblast cell line. Jinhua pig embryo muscle tissues are subjected to primary culture and secondary culture to finally obtain a high-purity and high-motility Jinhua pig fibroblast cell line, and the fibroblast cell line belongs to the field of biology. The fibroblast cell line obtained by culture does not contain epithelial cells and other parenchyma cells, thereby having high cell purity; the cryopreserved cells are stable in quality, and the motility of the cryopreserved cells can be up to 91.8-93.5% after anabiosis; and the cells subjected to secondary culture can stably grow, thereby being suitable for large-scale culture. The detection of cytologic characteristics shows that the cell line conforms to the cell line establishment requirements. The Jinhua pig fibroblast cells can be used for being manufactured into nucleus transplantation donors of transgenic pigs, can provide a raw material for the research of medicine and molecular biology, and can be used as the main raw material for vaccine production and a feeder layer for hepatic cell culture. Besides, the Jinhua pig fibroblast cell line can agriculturally enrich and improve indigenous pig breeds, thereby being a means for effectively protecting excellent indigenous pig breeds in China.

Description

The foundation and the cultural method thereof of Jinhua pig fibroblast

Technical field

The present invention relates to the cytobiology field, relate in particular to the foundation and the cultural method thereof of a kind of Jinhua pig fibroblast.

Background technology

Livestock and poultry genetic resources protection problem is early than proposing in the U.S. fifties.After the human environment meeting in Stockholm in 1972; Cause the attention of Food and Argriculture OrganizationFAO (FAO) and United Nations Environment Programme (UNEP) (UNEP); They have jointly carried out the protection action of domestic animal genetic resources in the whole world, the work of decades has been carried out in front and back so far, has obtained tangible result.China has just participated in a series of plans and the action of their tissue from beginning.But the situation of the livestock and poultry genetic resources of China protection at present is also pessimistic.In the generaI investigation of carrying out the eighties, estimate 600 of total kinds, wherein national local variety have 385.Have 10 local variety to become extinct, 8 kinds are endangered.The effective population size of 20 kinds sharply descends, account for the investigation kind several 364 13%.Again according to the Chinese Academy of Agricultural Sciences's herding be in " livestock and poultry genetic resources variety is replenished investigation " of accomplishing in 2000 with national livestock husbandry veterinarian general station kind livestock and poultry data claim; In 331 kinds of 17 provinces and regions investigating; Be in imminent danger and kind that will become extinct and have 59; Accounting for 18% of investigation sum has 7 kinds to become extinct in addition, the big general lucky pig in Yunnan for example, the Xiaoshan chicken in Zhejiang, mountain of papers goose, Simao goose, careless extra large goose etc.

China's livestock and poultry genetic resources is abundant, and they have brought into play important effect in herding production and new genetic resources forming process.Nearly 2O, for satisfying the demand of the people to livestock product such as meat, egg, milk, honey, China has introduced a large amount of external high-yield varieties in succession the low yield local variety has been improved, and the herding production level is significantly improved.But the aftermath of improvement makes some local variety cultivated kind or cross-fertilize seed gradually to be replaced, and causes the local variety colony quantity with rich and varied property to descend or disappearance.Research recently shows that colony's quantity of local livestock and poultry species is in decline in various degree more than 50%, and wherein some amount is in Critical Condition.For many years to protecting the practice and the experience of kind of work, telling us will effectively protect local good livestock and poultry resource key is to adopt the method for economical rationality, promptly makes the livestock and poultry resource well protected and practices thrift cost again both at home and abroad.Here the application cell biological method is set up the method that the clone of livestock and poultry is exactly an effective economy.In setting up the process of clone, simultaneously the basic biological characteristics of speech cell is studied, provided technology to support with theoretical for further studying later on.

The Jinhua pig is the famous local variety of China.Claim two crow again, originate in ground such as Dongyang, Zhejiang, Yiwu, Jinhua.Be unearthed according to Jinhua county ancient prescription the Western Jin Dynasty (265-316 in Christian era) pottery pig with the textual criticism of pottery pigsty, the quite prosperity of having raised pigs already of this band before 1600.The curing food of " local meat " is just arranged according to legend in ancient times, develop into ham thereafter.Along with ham is found a good sale in, the Jinhua pig is also become famous thereupon.Jinhua pig build is medium, and ear is sagging, and neck is short and thick, back of the body nick, and stern tilts, hard hooves.Whole body is by white in the middle of the hair, and neck, stern tail are deceived.With precocity be prone to fertile, the thin bone of skin is thin, meat is good, be suitable for cured ham is celebrated.When 70～75 kilograms of 7～8 monthly ages, body weight for butchering optimum period carcass lean meat percentage 40%～45%.At present, because the interference of a large amount of introductions of external pig kind and some social factors makes the population quantity continuous decrease of Jinhua pig, the combined material continuous decline of meat should be adopted an effective measure and strengthened guarantor's kind of Jinhua pig.

RESEARCH ON CELL-BIOLOGY all receives people's attention always very much; Along with development of scientific research; The research of people's pair cell is also more and more deep, and other development of life science that develops into of cytobiology provides the research basis, we can say that also cytobiology is the prerequisite of other life discipline development.This also reflects the importance that livestock and poultry resource cell is preserved from another side.In a single day these germ plasm resources just lose and can't study and use, so a lot of country all is devoted to the preservation of germ plasm resource at present.China protection species diversity aspect also is faced with many problems to be needed to solve.

The development of mammalian nuclear transfer technology also is gradually improved; The nuclear transplantation technology is that donorcells nuclear is moved into out in the non-nucleus egg mother cell; Making the latter is that vegetative propagation can be activated, divides and develop into new individuality without sexual processes such as sperm penetrate, makes that the gene of nuclear donor is duplicated fully.It has great function at aspects such as improvement of breed, preparation transgenic animal, gene therapy and organ transplantations, and inoblast is a kind of ideal donor cell in the nuclear transplantation.The Jinhua pig is as a good local pig breed, and it has great potential aspect breeding, and pig also is a kind of human organ's better provider, thereby the fibroblastic cording of building of Jinhua pig has important scientific research and productive value.

Summary of the invention

The objective of the invention is to overcome the deficiency of prior art, the foundation and the cultural method thereof of a kind of Jinhua pig fibroblast is provided.

For realizing above-mentioned purpose, the present invention takes following technical scheme:

The step of the cultural method of Jinhua pig fibroblast is following:

1) first culture: from the farrowing sow uterus, take out the embryo of 40 ages in days fast, clean 3 ~ 4 times with adding two anti-PBS; Separate longissimus dorsi muscle with eye scissors with the ophthalmology tweezer, clean 3 ~ 4 times with adding two anti-PBS again, be cut into 0.5 ~ 1.0mm then ³Tissue block; Tissue block is moved into culturing bottle, and also coating is even, is inverted culturing bottle, puts into 37 ~ 39 ℃, and percent by volume is 5%CO ₂Incubator in cultivate 3 ~ 4h; The complete adherent back of tissue block adds the nutrient solution of 10ml, and the nutrient solution composition: the DMEM+10% foetal calf serum continues to cultivate 8 ~ 12 hours;

2) cultivation of going down to posterity: pour out the nutrient solution in the step 1) process, after six usefulness PBS clean 2 ~ 3 times, stop digestion with the nutrient solution that adds 10 ~ 15ml behind the tryptic digestion; Postdigestive cell is divided into three bottles, puts into 37 ℃, 5%CO ₂Incubator in be cultured to frozen preceding 24 ~ 28h;

3) cell cryopreservation:

(1) the nutrient solution 10 ~ 15ml for preparing the original nutrient solution of frozen preceding 24 ~ 26h reject and more renew continues to cultivate 24 ~ 26h;

(2) pour out nutrient solution in the culturing bottle, clean 2 ~ 3 times with PBS after, stop digestion with the nutrient solution that adds 10 ~ 15ml behind the tryptic digestion;

(3) with the frozen preceding sum of red blood cell count(RBC) plate counting cells;

(4) 1000rpm eccentric visual cell 5min removes supernatant, adds the frozen storing liquid of 1 ~ 1.5ml, and piping and druming cell gently makes its suspension; The cells frozen storing liquid composition: percent by volume is that 50% foetal calf serum, percent by volume are that 40%DMEM and percent by volume are 10%DMSO;

(5) move on to cell in the frozen pipe, seal with sealing film;

The frozen pipe that (6) cell will be housed is transferred in 4 ℃ of refrigerators and is placed 1 ~ 2h, changes over to then in-20 ℃ of refrigerators and places 2 ~ 3h, is moved into-70 ℃ refrigerator overnight again, fast it is moved at last that liquid nitrogen is medium-term and long-term to be preserved, and carries out corresponding record.

The step of described trysinization is: the mass percent that in culturing bottle, adds 37 ~ 39 ℃ of preheatings is trypsinase 1.5 ~ 2ml of 2%; Examine under a microscope percent by volume and be and after 90 ~ 95% cell becomes circle trypsinase is outwelled; Knock Tissue Culture Flask with have gentle hands and make cell detachment, add the new nutrient solution of 10 ~ 15ml.

Described freeze-stored cell carries out recovery step: put into 37 ~ 39 ℃ of water-baths after frozen pipe is taken out from liquid nitrogen; After rocking 1 ~ 2min, be moved into cell and added in the full substratum centrifuge tube of 10 ~ 15ml, remove supernatant behind the centrifugal 5min of 1000r/min; The substratum that adds 2ml; After blowing and beating cell evenly gently cell is moved into the culturing bottle that nutrient solution is arranged, be positioned over 37 ℃, 5%CO ₂Incubator in cultivate.

The present invention is for the improvement adjustment of adherent culture method and culture medium culturing liquid composition, and the present invention cultivates the fibroblast that obtains and do not contain heteroproteose cells such as epithelial cell, and cell purity is high; The freeze-stored cell steady quality, cell cryopreservation recovery back motility rate can reach 91.8 ~ 97.5%, and the back cell growth of going down to posterity is stable, is fit to large scale culturing.Improvement for substratum and cultural method can significantly reduce the cultivation cost.The Jinhua pig inoblast that the present invention relates to can be used to make the nuclear transplantation donor of transgenic pig; Can starting material be provided for medical science and molecular biology research; Can be used as the main raw material of production of vaccine and the feeder layer of liver cell culture.Can enrich and improve local pig breed on the agricultural, be effective protective devices of the local good pig kind of China.

Because Jinhua pig germ plasm resource is deficient, making becomes blank for the research of Jinhua pig in every field.And high cell motility rate of the present invention and highly purified Jinhua pig fibroblast can provide material for later research, for later scientific research facilitates.

Description of drawings

Fig. 1 is Jinhua pig inoblast microscope of former generation figure below;

Fig. 2 for the Jinhua pig go down to posterity before microcytoscope figure below;

Fig. 3 is Jinhua pig subculture I microcytoscope figure below;

Fig. 4 is Jinhua pig subculture II microcytoscope figure below;

Fig. 5 (a) is by the cytological map of e. coli contamination;

Fig. 5 (b) is by the cytological map of mould contamination;

Fig. 6 (a) is a mycoplasma contamination positive cells map;

Fig. 6 (b) is the negative cell map of mycoplasma contamination;

Fig. 7 is a Jinhua pig growth of fibroblasts graphic representation;

Fig. 8 is the fibroblastic karyotyping figure of Jinhua pig;

Left side figure is malate dehydrogenase isozyme electrophoresis figure among Fig. 9 figure, and right figure is lactic dehydrogenase isozyme electrophoresis figure;

Figure 10 is that foreign gene 24h in the pig inoblast of Jinhua expresses figure (pEGFP-N3 GFP transfection plasmid);

Figure 11 is the Jinhua pig inoblast recovery apoptotic detection in back.

Embodiment

The step of the cultural method of Jinhua pig fibroblast is following:

1) first culture: from the farrowing sow uterus, take out the embryo of 40 ages in days fast, clean 3 ~ 4 times with adding two anti-PBS; Separate longissimus dorsi muscle with eye scissors with the ophthalmology tweezer, clean 3 ~ 4 times with adding two anti-PBS again, be cut into 0.5 ~ 1.0mm then ³Tissue block; Tissue block is moved into culturing bottle, and also coating is even, is inverted culturing bottle, puts into 37 ℃, and percent by volume is 5%CO ₂Incubator in cultivate 3 ~ 4h; The complete adherent back of tissue block adds the nutrient solution of 10ml, and the nutrient solution composition: the DMEM+10% foetal calf serum continues to cultivate 8 ~ 12 hours;

2) cultivation of going down to posterity: pour out the nutrient solution in the step 1) process, after six usefulness PBS clean 3 ~ 4 times, stop digestion with the nutrient solution that adds 10 ~ 15ml behind the tryptic digestion; Postdigestive cell is divided into three bottles, puts into 37 ℃, 5%CO ₂Incubator in be cultured to frozen preceding 24 ~ 28h;

3) cell cryopreservation:

(2) pour out nutrient solution in the culturing bottle, clean 3 ~ 4 times with PBS after, stop digestion with the nutrient solution that adds 10 ~ 15ml behind the tryptic digestion;

(5) move on to cell in the frozen pipe, seal with sealing film;

The step of described trysinization is: the mass percent that in culturing bottle, adds 37 ~ 39 ℃ of preheatings is 2% trypsinase 2ml; Examine under a microscope percent by volume and be and after 90 ~ 95% cell becomes circle trypsinase is outwelled; Knock Tissue Culture Flask with have gentle hands and make cell detachment, add the new nutrient solution of 10 ~ 15ml.

Below in conjunction with accompanying drawing and embodiment the present invention is further specified, so that the public has whole and sufficient understanding to summary of the invention.

Used key instrument and reagent source among the embodiment:

Jinhua pig embryonic origin: the pig farm is planted by the good China of Jinhua, Zhejiang

(1) key instrument equipment

37 ℃ of 5%CO2 incubators (German Heraeus)

PH030A type incubator (Shanghai)

DHG-9203A type electric heating constant temperature air dry oven (Shanghai)

DL-CJ-2N high-performance sterility test platform (Ha Donglian)

The vertical high-pressure sterilizing pot of YXO-LS-50SI (Shanghai)

IX-71 inverted phase contrast microscope (Japanese Olympus)

Laser scanning co-focusing microscope (Japanese Nikon)

CX31 biomicroscope (Japanese Olympus)

Electric-heated thermostatic water bath (Beijing)

TDL-40B horizontal centrifuge (Shanghai)

Water distillation apparatus (Shanghai)

Your Superpure water machine (German Pall) quite

Electronic pipettor (Germany)

-70 ℃ of cryogenic refrigerators (U.S.)

Liquid nitrogen container (Sichuan)

Blood counting chamber (Beijing)

96 well culture plates (Corning)

24 well culture plates (Corning)

DYY one 111 type voltage stabilization and current stabilization electrophoresis apparatuses (Liuyi Instruments Plant, Beijing)

Horizontal strip electrophoresis groove (Liuyi Instruments Plant, Beijing)

Vertical electrophoresis groove (Beijing 61 instruments)

(2) main agents

1, cell culture reagent

DMEM substratum (invitrogen), foetal calf serum (thermo), 0.20% trypsin solution (measure 0.2g trypsinase, be dissolved in the 100ml water, 0.22 μ m membrane filtration), PBS (take by weighing NaCl, 8.0g, Na ₂HPO ₄12H ₂0,2.89g, KCl, 0.2g, KH ₂PO ₄, 0.2g add an amount of ultrapure water make its dissolving after, with 1000ml volumetric flask constant volume.Regulate pH value about 7.2, autoclaving 30min)

2, chromosome specimen prepares reagent and preparation

0.20% trypsin solution (measure 0.2g trypsinase, be dissolved in the 100ml water, 0.22 μ m membrane filtration), DMEM perfect medium (invitrogen), PBS, NST-757 (SIGMA), Jim Sa (AMRESCO)

(1) NST-757 preparation: NST-757 10mg, PBS 10ml, suction filtration, this is a stoste.Working fluid concentration is 100 μ g/ml, promptly gets stoste l ml and is dissolved in the 9ml tri-distilled water, and stoste and working fluid all are stored in 4 ℃;

(2) preparation of Jim Sa stoste: measure glycerine 33ml; Methyl alcohol 33ml.0.5g puts into mortar with Jim Sa powder, adds a small amount of glycerine earlier, is ground to till the no particle; And then whole glycerine are poured into; Put in 56 ℃ of incubators behind the 2h, add methyl alcohol, the dye liquor sealing for preparing is preserved in the brown bottle (being better than 0～4 ℃ of preservation most);

The preparation of Jim Sa working fluid: Jim Sa stoste 1ml, phosphoric acid buffer 9ml (existing) with join at present;

(3) preparation of phosphoric acid buffer: 1/15 mol/L Na2HPO4 12H2O:2.39g is dissolved in the 100ml distilled water;

1/15 mol/L KH2PO4:0.907g is dissolved in the 100ml distilled water.Get 1/15 mol/L Na2HPO4 12H2O liquid 80ml, 1/15 mol/L KH2PO4 liquid 20ml mixes the phosphoric acid buffer that is pH7.38.

(4) stationary liquid: Glacial acetic acid min. 99.5 and methyl alcohol are with 1: 3 ratio (existing with join at present);

(5) hypotonic solution (0.5%KCl solution): 0.5gKC is dissolved in the 100ml tri-distilled water.

3, isozyme agents useful for same and preparation

EDTA (YD 30), Tris (PROMEGA), Acr (acrylic amide), glycocoll (Clycine)

Triton (Triton) X-100, TEMED (Tetramethyl Ethylene Diamine), PMS (azophenlyene sulfuric acid formicester; Phenazine Methosulfate), NAD (oxidisability coenzyme Ι), MTT (Thiazolyl blue; Thiazolyl Blue), Bis (methylene diacrylamide), ammonium persulphate (Ammonium Persulfate, AP), chlorination nitro blue tetrazolium (NBT), Sodium.alpha.-hydroxypropionate

(1) albumen extract: TritonX-100: 0.9%NaCl solution=1: 15 (v/v), pH7.1 contains 0.6mmol/L

EDTA, store in 4 ℃.

(2) separation gel liquid storage preparation:

1. 28%Acr～0.8% preparation: claim 14g Acr and 0.4g Bis, adding distil water is settled to 50ml, filters, and installs to brown bottle, and 4 ℃ keep in Dark Place.

2. 0.6M Tris – HCI (pH8.9)/TEMED preparation: claim 7.3g Tris-Base, be dissolved in the zero(ppm) water of 50ml, transfer pH value to 8.9 with 1N HCI, add 0.4ml TEMED, constant volume filters to 100ml, and 4 ℃ keep in Dark Place.

3. the preparation of 1%AP: claim 1.0g AP, be dissolved in the zero(ppm) water of 50ml that constant volume is to 100ml, filtration, install to brown with, 4 ℃ keep in Dark Place.

(3) concentrate the preparation of glue liquid storage:

1. 38%Acr～2%Bis preparation: claim 30g Acr and 2g Bis, adding distil water is settled to 100ml, filters,

Install to brown bottle, 4 ℃ keep in Dark Place;

2. 0.124M Tris – HCI (pH6.7) preparation: claim 1.5g Tris-Base, be dissolved in the zero(ppm) water of 50ml, transfer pH value to 6.7 with HCI, constant volume filters to 100ml, and 4 ℃ keep in Dark Place;

3. the preparation of 10%AP: claim 0.5g AP, constant volume is to 5ml, filtration, install to brown with, 4 ℃ keep in Dark Place.

4. TEMED original solution: a small amount of TEMED stoste of packing is in brown bottle, and 4 ℃ keep in Dark Place.

(4) electrode buffer preparation: claim 3.1g Tris-Base and 1.0g Gly, be dissolved in the water of 400ml, use 1N

HCI transfers pH value to 8.7, and constant volume is to 1000ml.

(5) glue

1. 7.5% separation gel (12ml)

PH8.9 Tris one HCl 5ml

30% acrylic amide 3ml

1%TEMED 0.9ml

Distilled water 6.51ml

10%AP 0.09ml

2. 3% concentrate glue (4ml)

PH6.7Tris one HCl 0.5ml

30% propylene phthalein amine 0.4ml

1%TEMED 0.3ml

Distilled water 2.74ml

10%AP 0.06ml

(6) bromjophenol blue sucrose solution

Bromjophenol blue 5mg

Sucrose 4g

Add distilled water to 10ml

(7) configuration of isozyme developer

0.05M PH8.0 Tris one HCl claim 0.61g Tris-Base, is dissolved in the zero(ppm) water of 50ml, transfers pH 8.0 with 1N HCI, constant volume filters to 100ml, and 4 ℃ keep in Dark Place.

1. MDH developer

NAD (NAD) 15mg

NBT 8mg

TMS 1mg

0.05M PH8.0 Tris one HCl 30ml

2. LDH developer

5M Sodium.alpha.-hydroxypropionate 6ml

NAD 20mg

NBT 15mg

PMS 2mg

0.05M PH8.0 Tris one HCl 40ml

4, draw cell proliferation curve agents useful for same

Trypan blue (sigma), PBS, DMEM cell culture medium (invitrogen)

5, cell transfecting agents useful for same

(Lipofectamine 2000, Invitrogen) and competent escherichia coli cell for pECFP-N1 (Invitrogen), cationic-liposome

6, cell bacterial contamination detects

Hoechst 33342 (sigma), 4% Paraformaldehyde 96 stationary liquid, PBS (take by weighing NaCl, after 8.0g, Na2HPO412H20,2.89g, KCl, 0.2g, KH2PO4,0.2g add an amount of ultrapure water and make its dissolving, with 1000ml volumetric flask constant volume.Regulate pH value about 7.2, autoclaving 30min)

7, apoptosis detects

Apoptosis test regent box (Wuhan Boster Biological Technology Co., Ltd.), 0.01M TBS, PH7.5 (joins method: add 8.5g sodium-chlor in the 1L tri-distilled water.1.2gTris with the pure acetate of 0.45-0.50ml), 0.01M PBS, PH7.4 (joins method: 7.9g sodium-chlor, 0.2g Repone K; 0.24g potassium primary phosphate; 1.8g potassium hydrogenphosphate is dissolved in the 800ml zero(ppm) water, transfers PH to 7.4 with hydrochloric acid, last adding distil water is settled to 1L)

The structure of embodiment 1 Jinhua pig fibroblast

(1) first culture: from the farrowing sow uterus, take out the embryo of 40 ages in days fast, clean 3 ~ 4 times with adding two anti-PBS; Separate longissimus dorsi muscle with eye scissors with the ophthalmology tweezer, clean 3 ~ 4 times with adding two anti-PBS again, be cut into 0.5 ~ 1.0mm then ³Tissue block; Tissue block is moved into culturing bottle, and also coating is even, is inverted culturing bottle, puts into 37 ℃, 5%CO ₂Incubator in cultivate 3 ~ 4h; The complete adherent back of tissue block adds the nutrient solution of 10ml, and the nutrient solution composition: the DMEM+10% foetal calf serum continues to cultivate 12 hours;

(2) cultivation of going down to posterity: pour out the nutrient solution in (1) process, clean 2 times with PBS after, stop digestion with the nutrient solution that adds 10ml behind the tryptic digestion; Postdigestive cell is divided into two bottles, puts into 37 ℃, 5%CO ₂Incubator in be cultured to frozen before 24h;

(3) cell cryopreservation:

1. the nutrient solution 10ml for preparing the frozen preceding original nutrient solution of 24h reject and more renew continues to cultivate 24h;

2. set by step (2) peptic cell adds the 10ml substratum and stops digestion;

3. the sum before using red blood cell count(RBC) plate counting cells frozen; 1000rpm eccentric visual cell 5min removes supernatant, adds the frozen storing liquid of 1ml, and piping and druming cell gently makes its suspension; The cells frozen storing liquid composition: 50% foetal calf serum, 40%DMEM and 10%DMSO, install to the cell branch in the frozen pipe, seal with sealing film;

The frozen pipe that 4. cell will be housed is transferred in 4 ℃ of refrigerators and is placed 1h, changes over to then in-20 ℃ of refrigerators and places 2 ~ 3h, is moved into-70 ℃ refrigerator overnight again, fast it is moved at last that liquid nitrogen is medium-term and long-term to be preserved, and carries out corresponding record.

(4) freeze-stored cell is recovered: put into 37 ℃ of water-baths after frozen pipe is taken out from liquid nitrogen; After rocking 1 ~ 2min, be moved into cell and added in the full substratum centrifuge tube of 10ml, remove supernatant behind the centrifugal 5min of 1000r/min; The substratum that adds 2ml; After blowing and beating cell evenly gently cell is moved into the culturing bottle that nutrient solution is arranged, be positioned over 37 ℃, 5%CO ₂Incubator in cultivate.

The detection of embodiment 2 Jinhua pig fibroblast biological characteristicses

The Jinhua pig fibroblast that embodiment 1 is cultivated carries out the inspection of biological characteristics, and method is following:

One, whether observation of cell growth conditions, nutrient solution colour-change situation and cell pollute.

Conclusion:

Like Fig. 1 is Jinhua pig inoblast of former generation; Fig. 2 for the Jinhua pig go down to posterity before microcytoscope figure below; Fig. 3 is Jinhua pig subculture I microcytoscope figure below; Fig. 4 is Jinhua pig subculture II microcytoscope figure below; Cell culture fluid is limpid transparent, and the tenuigenin projection is plentiful, and nucleus is high-visible, and whole cell is spindle shape, flame, and cell fission is rapid, and cellular form reaches unanimity, and is monokaryon fiber dress cell.Vigorous to the growth of whole cell colony observations proof cell, split speed is fast.

Two, microorganism detection

1, bacterium, fungi detect

Method: 1) get freeze-stored cell and fast frozen pipe put in 37 ℃ of water-baths, rock 1 ~ 2min after, be moved into cell and added in the full substratum centrifuge tube of 10ml, remove supernatant behind the centrifugal 5min of 1000r/min.

2) repeating step (1) adds the substratum of 2ml antibiotic-free, and cell is blown and beaten evenly gently.

3) cell is inoculated in 0.5ml in the culturing bottle that does not contain the microbiotic substratum, places the incubator of 37 ℃ and 26 ℃ to cultivate for two weeks respectively.

4) establish control group

(1) positive control: in rigidly connecting kind of the culturing bottle of recovery cell, place the incubator of 37 ℃ and 26 ℃ to cultivate for two weeks microbionation;

(2) negative control: the substratum of added with antibiotic is not put in two weeks in the incubator of 37 ℃ and 26 ℃.

Conclusion:

Visual inspection inoculation has the inoblast after the recovery and inoculates the substratum of inoblast and bacterium simultaneously, finds that only the fibroblastic substratum of inoculation recovery is as clear as crystal, and inoculates the substratum appearance muddiness of inoblast and bacterium simultaneously.Find only except inoblast, do not have other foreign material in the fibroblastic culturing bottle bottle of inoculation recovery with microscopic examination.

2, detection of mycoplasma

(1) get freeze-stored cell and fast frozen pipe put in 37 ℃ of water-baths, rock 1 ~ 2min after, be moved into cell and added in the full substratum centrifuge tube of 10ml, remove supernatant behind the centrifugal 5min of 1000r/min.

(2) repeating step (1) adds the substratum of 2ml antibiotic-free, and cell is blown and beaten evenly gently.Cell is inoculated in 0.5ml in the culturing bottle that does not contain the microbiotic substratum, places 37 ℃ incubator to cultivate 48h respectively;

(3) with PBS or suitable buffer preparation 10～50 μ M Hoechst33342 dyestuffs;

(4) in the Hoechst33342 dye solution adding cell culture with 1/10 cell culture medium volume (can use the Hoechst33342 dyestuff damping fluid of 1/10 concentration to replace substratum);

(5) 37 ℃ of culturing cells 10～20 minutes;

(6) wash cell twice with PBS or suitable damping fluid;

(7) directly add 4% Paraformaldehyde 96 stationary liquid, mix fixed cell 10min with nutrient solution by 1:3;

(8) with having the 350nm excitation wavelength, the fluorescence microscope cell of the spectral filter of 461nm emission wavelength.

Conclusion:

Hoechst33342 is a kind of blue fluorescent dyes that can permeates cell membranes, and the toxicity of pair cell is lower.Around fluorocyte, one deck tulle is arranged seemingly by the cell of mycoplasma contamination, do not form a sharp contrast with there being contaminated cell, as shown in Figure 6.From figure, can find this clone not by mycoplasma contamination, cell growth condition is good.

Three, the drafting of cell growth curve

Method:

(1) the preparation cell suspending liquid is got upgrowth situation good cell to be measured, treats cell length to 80 ~ 90% o'clock, with conventional method with cell dissociation;

(2) in 24 orifice plates, inoculate the cell of equivalent respectively, be preferably 2 * 10 ⁴Individual/ml, place 37 ℃, 5%CO ₂Incubator in cultivate;

(3) count from inoculation time, every separated 24h counts the cell density in 3 holes, with the sum of blood counting chamber difference counting cells, gets the MV in 3 holes, finishes up to the 8th day;

(4) be X-coordinate with the incubation time with, be ordinate zou with the cell density, the result is drawn on graph paper, obtain the growth curve of culturing cell.

Conclusion:

Through to the continuous 8 days counting of postvaccinal cell, obtain the growth curve of culturing cell.Can find that from curve cell inoculation growth in the 1st day is slower, this possibly be because the effect of pancreatin does not recover cell injury, is the cell adapted stage during this period of time; The 2nd day cell speed of growth in inoculation obviously accelerated, and the cell speed of growth reaches maximum in the time of the 3rd day; The cell speed of growth begins slack-offly after the 5th day, and this possibly be because the cell count quantitative change is many, causes nutritive substance consumption, the intensified competition between the cell.Cell density remained on maximum basically in 6 ~ 7 days, reduced to some extent to the 8th day cell quantity, and this possibly be that a spot of cell can not adapt to this aggravating circumstances and death because vying each other between the cell causes the living environment of cell further to worsen.

Four, the cell motility rate detects

Method: 1, before the cell cryopreservation, get the 1% trypan blue dye liquor that collection 10 μ l cell suspending liquids well add 10 μ l, mixing.(cell is transparent, and non-staining is viable cell for blood counting chamber living cell counting and dead cell; What cell was colored is dead cell).Count 1000 cells, the counting cells motility rate.

2, behind the cell recovery, get the 1% trypan blue dye liquor that 10 μ l cell suspending liquids add 10 μ l, mixing.Same step (1) counting cells motility rate.

Conclusion:

The cell cryopreservation front and back are that motility rate is measured to the Jinhua pig cell.The result shows: motility rate reaches 94.2 ~ 97.5% before the cell cryopreservation, and motility rate reaches 91.8 ~ 93.5% behind the cell recovery, can get cell cryopreservation thus after surviving rate still very high.After frozen, cell cultures is found that the form of cell meets fibroblastic form, and the BA of cell is not affected, the fibroblast quality of this Jinhua pig is more stable.

Five, isozyme separates

Method: discontinuous gradient polyacrylamide gel electrophoresis

1, the preparation of sample:

(1) gather Exponential growth expectation inspection cell, the centrifugal 5min of 1000g removes supernatant;

(2) add the PBS rinsing, centrifugal.Post rinse, centrifugal 2 times are abandoned supernatant at last;

(3) in cell precipitation, add extracting solution of protein, blow and beat repeatedly, cell is dissolved fully with suction pipe;

(4) change cytolysate in the 1.5ml centrifuge tube over to, blow and beat repeatedly with suction pipe again, cytolemma is thoroughly dissolved;

(5) insert in the microcentrifuge, high speed centrifugation (9000r/min, 2min) is collected supernatant, and break into portions stores in-70 ℃.

2, the preparation of gel and injection molding:

(1) according to desired concn and dosage, draw by gel component proportion speed ground with suction pipe earlier and respectively store liquid, in the centrifuge tube of 15ml, be mixed with the gel working fluid, each composition of uniform mixing leaves standstill 1min, takes out the air in the solution as far as possible.Pour into the gel working fluid that mixes wherein.Prevent alveolate generation in the encapsulating.After this slowly add zero(ppm) water and carry out water seal, height about the about 0.5cm of water layer;

(2) after room temperature left standstill about 45min, the water seal layer sharp interface occurred with separating between the glue-line.Pour out water layer, and draw residual moisture with inhaling paper.The concentrated glue working fluid that to prepare then injects above the separation glue in the gel chamber.Short on glass until distance along the 5mm place, immediately the sample comb is inserted the coagulant liquid top.This process also will avoid occurring bubble.Room temperature leaves standstill, and concentrates the glue polymerization behind the 1h and accomplishes.The careful sample comb that takes out is installed in the gel chamber on the electrophoresis frame, prepares to add sample;

(3) take out the protein extract that in-70 ℃, stores, thaw, add 40% half the sucrose solution of about protein extract and the tetrabromophenol sulfonphthalein indicator of 2.5 μ l fast, mixing, this moment, normal protein example can demonstrate mazarine.With micro sample adding appliance application of sample in sample cell, 10 μ l are advisable.On sample liquid, slowly add electrode buffer, each groove internal pore is filled fully.Groove on electrophoresis cartridge (negative pole) reaches down and pours electrode buffer respectively in the groove (positive pole).Electrophoresis chamber is inserted 4 ℃ of refrigerators, prepare electrophoresis;

(4) electrophoresis and dyeing: the about 30min of regulating voltage 60V electrophoresis, treat indicator behind the separation gel with VR for the about 6h of 120V electrophoresis, when the tetrabromophenol sulfonphthalein indicator migrates to the lower edge edge, stop electrophoresis, cut off the electricity supply; Take off the gel chamber, carefully remove sheet glass, the gel flat board is placed in the enamel pallet, inject staining fluid, 37 ℃ of lucifuge dyeing is till protein band occurs;

(5) take pictures and calculate: the gel after will dyeing with photographic camera is taken a picture, and measurement zone band swimming distance (d) and indicator swimming distance (D) are calculated relative mobility (Rf).

Conclusion: shown in Fig. 9 (left side); There are 2 bands to be cytosol type (s-MDH) faster in the zymogram of Jinhua pig inoblast malate dehydrogenase isozyme (MDH) near the anodic swimming; Near the swimming of negative electrode slower be plastosome type (m-MDH), the s-MDH of relative mobility and m-MDH are respectively 20.12% and 35%.Shown in Fig. 9 (right side); Have 4 bands from the negative electrode to the anode, to be followed successively by LDH1, LDH2, LDH3, LDH4 in the zymogram of Jinhua pig inoblast lactic dehydrogenase isozyme (LDH), relative mobility is respectively 15.87%, 33.33%, 47.62% and 63.49%.

Six, the preparation of caryogram

1, choose be in exponential phase of growth, with big flask culture, 80～90% confluent monolayer cultivate

Cell adds NST-757: from refrigerator, take out culturing bottle, add NST-757, making its ultimate density is 0.4 μ g/ml nutrient solution; Continue to cultivate 7h in the incubator;

2, gather somatoblast: the tryptic digestion collecting cell.Change in the centrifuge tube, with the centrifugal 5min of 1000r/min, collecting cell;

3, hypotonic: absorb supernatant, adding is preheated to 37 ℃, 0.5% KCl solution 2ml, and piping and druming evenly continues to add to 8ml gently, and piping and druming evenly.Incubation 30min in 37 ℃ of incubators;

4, pre-fix: add fresh stationary liquid 1ml in the suspension, beat even (beat to blow and beat gently when sparing and prevent cell rupture);

5, fixing: suspension with the centrifugal 5min of 1000r/min, is absorbed supernatant, add fresh stationary liquid 10ml, beat and spare, room temperature leaves standstill 20min;

6, heavily fixing: repeating step 6, the careful supernatant of absorbing in centrifugal back is looked suspension cell density size, residue stationary liquid 0.5～1.0ml, piping and druming is evenly;

7, film-making: take out the clean slide of precooling in refrigerator, when treating that the surface has one deck water smoke to occur, apart from slide about 50cm eminence with suction pipe rapidly droplet on 1～2 cell suspension.After dripping sheet, place natural drying at room temperature,, 2 ~ 3 times back and forth, sample is fixed on the slide glass dripping sheet fast through the flame envelope top of spirit lamp

8, Jim Sa working fluid dyeing 15min, tap water flushing, natural drying at room temperature;

9, microscopy karyotype selects the metacinesis that degree of scatter is good, nothing is lost, form is clear, background is clean to carry out photomicrography mutually.Whether the chromosome number to 100 cells is counted, be that whole diploid is analyzed to it, counts karyomit(e) 2n mode.

Conclusion: it is 85% that chromosome number statistics is obtained diploid number shared ratio in middle cell, and promptly 2n ≠ 38 proportions are 15%, and this has reached, and to build be the 75%-80% that normal diploid requires.

Seven, liposome-mediated DNA transfection cultured cell in vitro

Method:

1, the preparation of GFP reporter plasmid (pEGFP-N3): to specifications, extract purifying pEGFP-N ₃The GFP reporter plasmid; The plasmid concentration that uses ultraviolet spectrophotometer mensuration plasmid to obtain is 0.15 μ g/ml DNA; The OD260/OD280 value is between 1.75 ~ 1.83, and the result shows that the DNA purity that makes, concentration etc. are suitable for the transfection inoblast.

2, cell transfecting

(1) inoblast is contained in the DMEM nutrient solution of 10 % foetal calf serums with 96 porocyte culture plates.

(2) 37 ℃, 5 %CO ₂, the saturated humidity culturing cell grows to 50 %～80 % up to cell and converges.

(3) this is A liquid to get an amount of DMEM nutrient solution (antibiotic-free, serum) adding DNA mixing, leaves standstill.

(4) in DMEM nutrient solution (antibiotic-free, serum), add liposome (L; Ipofwctamine ^TM2000) mixing this be B liquid, leave standstill 5min.

(5) with A liquid and B liquid mixing slowly, room temperature leaves standstill 20min.

(6) before transfection, clean cell twice with PBS.

(7) in culture plate, add A liquid and B mixture also, be positioned over 37 ℃, 5 %CO2 are hatched 18～48h in the saturated humidity incubator, change to fresh 10 % foetal calf serums and the antibiotic DMEM nutrient solution of containing midway behind the 6h.

3, the observation of cell transfecting efficient: the transfection number of observation of cell under the 488nm exciting light, calculate transfection efficiency.

Conclusion: shown in figure 10, the highest at the transfection efficiency of 48h cell, transfection efficiency is 32.4%.Positive cell and transfection have the fluorescence intensity of the cell of fluorescence to begin to weaken behind the transfection 72h.

To compare transfection efficiency higher with general cell transfection rate 32.4% for the transfection efficiency of cell in this process, is suitable for later applied research.

Eight, apoptotic detection

Method: press apoptosis test regent box (the biological ltd of Wuhan doctor's moral, PIN MK1024) specification sheets and make an experiment.

Observations: the apoptosis test regent box that this process adopts can be dyed pale brown look with the nucleus of apoptotic cell, under simple microscope, observes the statistics apoptotic cell, calculates apoptosis rate.

Conclusion: shown in figure 11, detect to cultivate go down to posterity 50 generation cell apoptosis rate.The result shows that the apoptosis rate of cell is 2.0%, and this apoptosis rate possibly be because used method difference than the apoptosis rate height shown in other experiment, or adopts identical method and the criterion difference of the observation of cell apoptosis that adopts is caused.

Embodiment 2

1) first culture: from the farrowing sow uterus, take out the embryo of 40 ages in days fast, clean 3 times with adding two anti-PBS; Separate longissimus dorsi muscle with eye scissors with the ophthalmology tweezer, clean 3 times with adding two anti-PBS again, be cut into 1 ~ 0.5mm then ³Tissue block; Tissue block is moved into culturing bottle, and also coating is even, is inverted culturing bottle, puts into 37 ℃, and percent by volume is 5%CO ₂Incubator in cultivate 3h; The complete adherent back of tissue block adds the nutrient solution of 10ml, and the nutrient solution composition: the DMEM+10% foetal calf serum continues to cultivate 8 hours;

2) cultivation of going down to posterity: pour out the nutrient solution in the step 1) process, after six usefulness PBS clean 2 times, stop digestion with the nutrient solution that adds 10ml behind the tryptic digestion; Postdigestive cell is divided into three bottles, puts into 37 ℃, 5%CO ₂Incubator in be cultured to frozen before 24h;

All the other implementation methods are agent example 1 strictly according to the facts

3) cell cryopreservation:

(1) the nutrient solution 10ml for preparing the frozen preceding original nutrient solution of 24h reject and more renew continues to cultivate 24h;

(2) pour out nutrient solution in the culturing bottle, clean 2 times with PBS after, stop digestion with the nutrient solution that adds 10ml behind the tryptic digestion;

(4) 1000rpm eccentric visual cell 5min removes supernatant, adds the frozen storing liquid of 1ml, and piping and druming cell gently makes its suspension; The cells frozen storing liquid composition: percent by volume is that 50% foetal calf serum, percent by volume are that 40%DMEM and percent by volume are 10%DMSO;

(5) move on to cell in the frozen pipe, seal with sealing film;

The frozen pipe that (6) cell will be housed is transferred in 4 ℃ of refrigerators and is placed 1h, changes over to then in-20 ℃ of refrigerators and places 2h, is moved into-70 ℃ refrigerator overnight again, fast it is moved at last that liquid nitrogen is medium-term and long-term to be preserved, and carries out corresponding record.

The step of described trysinization is: the mass percent that in culturing bottle, adds 37 ℃ of preheatings is 2% trypsinase 1.5ml; Examine under a microscope percent by volume and be and after 90% cell becomes circle trypsinase is outwelled; Knock Tissue Culture Flask with have gentle hands and make cell detachment, add the new nutrient solution of 10ml.

Described freeze-stored cell carries out recovery step: put into 37 ℃ of water-baths after frozen pipe is taken out from liquid nitrogen; After rocking 1min, be moved into cell and added in the full substratum centrifuge tube of 10ml, remove supernatant behind the centrifugal 5min of 1000r/min; The substratum that adds 2ml; After blowing and beating cell evenly gently cell is moved into the culturing bottle that nutrient solution is arranged, be positioned over 37 ℃, 5%CO ₂Incubator in cultivate.

Embodiment 3

1) first culture: from the farrowing sow uterus, take out the embryo of 40 ages in days fast, clean 4 times with adding two anti-PBS; Separate longissimus dorsi muscle with eye scissors with the ophthalmology tweezer, clean 4 times with adding two anti-PBS again, be cut into 1 ~ 0.5mm then ³Tissue block; Tissue block is moved into culturing bottle, and also coating is even, is inverted culturing bottle, puts into 39 ℃, and percent by volume is 5%CO ₂Incubator in cultivate 4h; The complete adherent back of tissue block adds the nutrient solution of 10ml, and the nutrient solution composition: the DMEM+10% foetal calf serum continues to cultivate 12 hours;

2) cultivation of going down to posterity: pour out the nutrient solution in the step 1) process, after six usefulness PBS clean 3 times, stop digestion with the nutrient solution that adds 15ml behind the tryptic digestion; Postdigestive cell is divided into three bottles, puts into 37 ℃, 5%CO ₂Incubator in be cultured to frozen before 28h;

3) cell cryopreservation:

(1) the nutrient solution 15ml for preparing the frozen preceding original nutrient solution of 26h reject and more renew continues to cultivate 24 ~ 26h;

(2) pour out nutrient solution in the culturing bottle, clean 3 times with PBS after, stop digestion with the nutrient solution that adds 10 ~ 15ml behind the tryptic digestion;

(4) 1000rpm eccentric visual cell 5min removes supernatant, adds the frozen storing liquid of 1.5ml, and piping and druming cell gently makes its suspension; The cells frozen storing liquid composition: percent by volume is that 50% foetal calf serum, percent by volume are that 40%DMEM and percent by volume are 10%DMSO;

(5) move on to cell in the frozen pipe, seal with sealing film;

The frozen pipe that (6) cell will be housed is transferred in 4 ℃ of refrigerators and is placed 2h, changes over to then in-20 ℃ of refrigerators and places 3h, is moved into-70 ℃ refrigerator overnight again, fast it is moved at last that liquid nitrogen is medium-term and long-term to be preserved, and carries out corresponding record.

The step of described trysinization is: the mass percent that in culturing bottle, adds 39 ℃ of preheatings is 2% trypsinase 2ml; Examine under a microscope percent by volume and be and after 95% cell becomes circle trypsinase is outwelled; Knock Tissue Culture Flask with have gentle hands and make cell detachment, add the new nutrient solution of 15ml.

Described freeze-stored cell carries out recovery step: put into 39 ℃ of water-baths after frozen pipe is taken out from liquid nitrogen; After rocking 2min, be moved into cell and add in the full substratum centrifuge tube of  15ml, remove supernatant behind the centrifugal 5min of 1000r/min; The substratum that adds 2ml; After blowing and beating cell evenly gently cell is moved into the culturing bottle that nutrient solution is arranged, be positioned over 37 ℃, 5%CO ₂Incubator in cultivate, all the other steps are identical with embodiment 1.

Claims

1. the cultural method of a Jinhua pig fibroblast, its characteristic is that its step is following:

3) cell cryopreservation:

(5) move on to cell in the frozen pipe, seal with sealing film;

2. require the cultural method of described a kind of Jinhua pig fibroblast according to right 1; The step that it is characterized in that described trysinization is: the mass percent that in culturing bottle, adds 37 ~ 39 ℃ of preheatings is trypsinase 1.5 ~ 2ml of 2%; Examine under a microscope percent by volume and be and after 90 ~ 95% cell becomes circle trypsinase is outwelled; Knock Tissue Culture Flask with have gentle hands and make cell detachment, add the new nutrient solution of 10 ~ 15ml.

3. require the cultural method of described a kind of Jinhua pig fibroblast according to right 1, it is characterized in that described freeze-stored cell carries out recovery step and is: put into 37 ~ 39 ℃ of water-baths after frozen pipe is taken out from liquid nitrogen, rock 1 ~ 2min after; Be moved into cell and added in the full substratum centrifuge tube of 10 ~ 15ml; Remove supernatant behind the centrifugal 5min of 1000r/min, add the substratum of 2ml, cell is moved into the culturing bottle that nutrient solution is arranged after blowing and beating cell evenly gently; Be positioned over 37 ℃, 5%CO ₂Incubator in cultivate.