CN104328082A - Mouse ovary surface epithelium in vitro malignant transformation culture method - Google Patents

Abstract

The invention relates to a mouse ovary surface epithelium in vitro malignant transformation culture method, which comprises the steps of ovary surface epithelium separation, early stage passage, middle stage passage and late period passage. A nutrient solution in a culture process employs a selectivity complete medium, a complete medium and a partial complete medium in order, the selectivity complete medium and the complete medium comprise fetal calf serum, mEGF, and a DMEM/F12 nutrient solution of insulin-transferrin-sodium selenite, the partial complete medium is the DMEM/F12 nutrient solution containing no mEGF; the fetal calf serum concentration in the selectivity complete medium, the complete medium and the partial complete medium can be increased in order, and mEGF concentration in the selectivity complete medium is higher than that in the complete medium. According to the mouse ovary surface epithelium in vitro malignant transformation culture method, carcinogenesis gene can not be transferred and no other induction factor is used, and the problem of normal wild type mouse ovary surface epithelium in vitro malignant transformation can be effectively solved.

Description

The external vicious transformation cultural method of mouse ovarian superficial epithelial cells

Technical field

The present invention relates to a kind of cell culture technology, particularly relate to the external vicious transformation cultural method of a kind of mouse ovarian superficial epithelial cells.

Background technology

In female reproductive system Incidence, the case fatality rate of ovarian cancer is the highest, and its survival rate is only 30%.Its major cause is that the early diagnostic rate of ovarian cancer is low, and grade malignancy is high, easily shifts, and easily produces resistance in therapeutic process.Although existing research shows that the ovarian cancer of about 80%-90% originates from ovarian surface epithelial cell, the cause of disease of ovarian surface epithelial cell vicious transformation and pathological process thereof be it be not immediately clear.In order to generation and the pathological development mechanism of more deep grasp epithelial cell ovarian cancer, need the process understanding ovarian surface epithelial cell vicious transformation, and set up the research model of an external vicious transformation of mouse ovarian superficial epithelial cells.

Although the people such as Yao Desheng are published in the paper " foundation of immortalization normal mouse ovarian epithelial cell system " of " Guangxi Medical University's journal " 04 phase in 2006, provide a kind of to be separated and cultivation has the mouse ovarian epithelial cell (MOSEC) of " immortalization " characteristic, but it also points out in paper, normal ovarian epithelial cell vitro culture growth is unstable, should not go down to posterity, easy aging stops division, therefore the method adopts the CD1 mouse proceeding to temperature sensitive antigen (TsAg) gene to carry out the foundation of MOSEC cell, also pertinent literature is had to point out, TsAg gene has conclusive impact (P S Jat for the cultivation of " immortalization " cell, C LCepko, R C Mulligan, and P A Sharp, Recombinant retroviruses encoding simian virus 40large T antigen and polyomavirus large and middle T antigens.Mol Cell Biol.Apr 1986, 6 (4): 1204 – 1217.), 2002, the people such as ARAKI Y utilized the same TsAg DNA murine that proceeds to turn out " the epididymis clone of immortalization " (ARAKI Y, et al.Journal of Andrology, 2002,23 (6): 854-869).Although TsAg gene does not have carinogenicity, the CD1 mouse proceeding to TsAg gene after all does not belong to the mouse of wild-type, has certain influence for the cause of disease of ovarian surface epithelial cell vicious transformation and the research of pathological process thereof.Lack effective means in prior art, solve this problem of the external vicious transformation of ovarian surface epithelial cell of normal wild type mouse.

In addition, for the epithelial separation of mouse ovarian in prior art, cultural method, also different with the mouse ovarian epithelial cell of wild-type, the raw MOSEC cell waiting people's separation and Culture of such as Yao De, it verifies as the CK-19 positive by immunohistochemical reaction, Vimentin is negative, and MOSEC is the two sexual cell of simple cuboidal-rectangular epithelial-mesenchymal on a class bag cystovarian surface under normal circumstances, it is periodicity ulceration propagation along with ovulation, to Ovarian surface reparation after Ovarian surface ulceration in ovulation process and ovulation, there is vital role (NELLY AUERSPERG et al.Endocrine Reviews 2001, 22 (2): 255 – 288, NUZHATAHMED et al.Cell.Physiol.2007 (213): 581 – 588), thus epithelial cell mark epithelial cell mark angling albumen (keratin) had both been expressed (as keratin7,8,18, and 19), continuous expression mesenchymal cell markers vimentin (Vimentin), N-cadherin and α-smooth muscle protein again, MOSEC Immunohistochemistry reaction assay is that Vimentin is positive in theory.In addition, DMBA (DMBA) is applied in rat ovary surface and sews up by the people such as Nishita in 1984, and the model for ovarian cancer is set up, after 36 weeks, there is ovarian tumor in nearly 50% animal, but DMBA is simultaneously induction of uterine tube, uterine endometrium and tumor of cervix.Thus, the Ovarian Cancer Model of DMBA induction does not have specificity, still can not determine that ovarian cancer is carcinoma in situ, there is many defects.

Because above-mentioned defect, the design people, actively in addition research and innovation, develop the external vicious transformation cultural method of a kind of mouse ovarian superficial epithelial cells.

Summary of the invention

For solving the problems of the technologies described above, the object of this invention is to provide a kind of do not proceed to oncogene, being immortalized characteristic TsAg gene or use other induction factor, effectively solve the external vicious transformation cultural method of mouse ovarian superficial epithelial cells of this problem of the external vicious transformation of normal wild type mouse ovarian superficial epithelial cells.

The external vicious transformation cultural method of mouse ovarian superficial epithelial cells of the present invention, comprises the following steps:

1) ovarian surface epithelial cell is separated: the ovary getting nonparous Healthy female C57BL/6 mouse in 6-8 age in week, rinse and peel off residual uterine tube, after fatty tissue and capsule of ovary, trysinization is utilized to be separated its ovarian surface epithelial cell, then perfect medium is added to stop trysinization process, centrifugal abandon supernatant liquor after again bringing Selection In property perfect medium moving in culture dish hatch in incubator, hatch and avoid mobile culture dish on the 2nd day, hatch and adopt half amount to change liquid method replacing nutrient solution according to cell growth status on the 3rd day, when ovarian surface epithelial cell density reaches 80%, use the expression of immuno-fluorescence assay epithelial cell mark angling albumen (Pan-keratin), when its positive rate reaches more than 95% and pebbles sample cuboidal epithelium reaches more than 80-85%, carry out next step,

2) go down to posterity in early days: inoculation algebraically is less than the ovarian surface epithelial cell in 15 generations, use half amount to change liquid method and go down to posterity in 2:3 to 3:2 ratio, generation time interval 5-7 days, nutrient solution adopts selectivity perfect medium;

3) go down to posterity mid-term: the ovarian surface epithelial cell of inoculation algebraically between 16 to 84 generations, in 16-60 generation, goes down to posterity in 1:2 to 1:4 ratio, and go down to posterity in 1:5 to 1:8 ratio after 60 generations, the generation time is spaced apart 3-5 days, and nutrient solution adopts perfect medium;

4) later passage: the ovarian surface epithelial cell that inoculation algebraically is greater than 85, go down to posterity in 1:8 to 1:10 ratio, the generation time is spaced apart 3-5 days, and nutrient solution adopts partial perfect medium.

Described selectivity perfect medium, perfect medium are the DMEM/F12 nutrient solution comprising foetal calf serum (FBS), mEGF and Insulin-Transferrin-Sodium Selenite (Insulin-Transferrin-Selenium), and described partial perfect medium is comprise foetal calf serum, Insulin-Transferrin-Sodium Selenite and not containing the DMEM/F12 nutrient solution of mEGF; In described selectivity perfect medium, perfect medium, partial perfect medium, the concentration of foetal calf serum increases successively, and in described selectivity perfect medium, the concentration of mEGF is higher than the concentration of mEGF in perfect medium.

Further, the ovarian surface epithelial cell of inoculation algebraically before 15 generations, the digestion time using trypsin solution when at every turn going down to posterity is 30-45s, and in described trypsin solution, pancreas enzyme concentration is 0.1% and does not contain EDTA.

Further, described selectivity perfect medium is the DMEM/F12 nutrient solution comprising 3-4% foetal calf serum (FBS), 1-1.2% Pen .-Strep (Penicillin-Streptomycin), 8-10ng/ml mEGF, 1-1.2% Insulin-Transferrin-Sodium Selenite (Insulin-Transferrin-Selenium).

Further, described perfect medium is the DMEM/F12 nutrient solution comprising 6-8% foetal calf serum (FBS), 1-1.2% Pen .-Strep (Penicillin-Streptomycin), 2-4ng/ml mEGF, 1-1.2% Insulin-Transferrin-Sodium Selenite (Insulin-Transferrin-Selenium).

Further, described partial perfect medium is the DMEM/F12 nutrient solution comprising 8-10% foetal calf serum (FBS), 1-1.2% Pen .-Strep (Penicillin-Streptomycin), 1-1.2% Insulin-Transferrin-Sodium Selenite (Insulin-Transferrin-Selenium).

Further, after described ovarian surface epithelial cell separating step completes, after carrying out identified by immunofluorescence and/or immunohistochemical methods qualification, when the step that goes down to posterity in early days after qualification result conformance with standard.

Further, described identified by immunofluorescence comprises the following steps:

1) hatch after the ovarian surface epithelial cell trysinization of 2nd generation, inoculation, when cell density reaches 80%, abandon substratum post rinsing, use stationary liquid fixed cell and confining liquid sealing treatment subsequently;

2) add the anti-angling albumen of primary antibodie (pan anti-cytokeratin) and process for some time post rinsing, add two anti-goat anti-mouse IgG process for some time post rinsings, add DAPI to dye for some time post rinsing, adopt with anti-quencher sealing subsequently;

3) basis of microscopic observation, calculates cell positive rate.

Further, described immunohistochemical methods qualification comprises the following steps:

1) with batch untreated intact mice ovary and being put in formaldehyde respectively with the mouse ovarian of trysinization spend the night;

2) being cut into tissue slice with after paraffin bag cystovarian, boiling process post rinsing with being placed in sodium citrate buffer solution;

3) surplus liquid is got rid of after dripping Normal Goat Serum confining liquid process for some time, drip the anti-angling albumen of primary antibodie (pan anti-cytokeratin) subsequently and process for some time post rinsing, drip two anti-goat anti-mouse IgG process for some time post rinsings;

4) after DAB color development treatment, haematoxylin redyeing, hydrochloride alcohol differentiation, PBS rinsing, dehydration, transparent, mounting process, identified under microscope.

By such scheme, the present invention at least has the following advantages: 1) by regulating MOSEC to grow the concentration of critical nutrients serum and mEGF and the primary MOSEC of purifying, realize propagation continuously to go down to posterity, solve " C57BL/6 healthy mice ovarian surface epithelial cell in vitro incubation growth is extremely slow; not easily cultivate, and growth is unstable, go down to posterity about 20 generation time; usually occur aging, stop division " this difficult problem; 2) the present invention does not adopt any mutagenesis or carcinogens, do not induce immortalization yet and proceeds to TsAg gene or oncogene establishes MOSEC vicious transformation model, be a kind of avirulent spontaneous vicious transformation model without foeign element induction, once experiment uses 5-6 mouse can obtain the MOSEC cell of higher degree and quantity; 3) for more understanding generation and the pathological development mechanism of epithelial cell ovarian cancer in depth, provide a kind of new ovarian cancer experimental research model, establish a mouse ovarian superficial epithelial cells longterm culture in vitro vicious transformation model, for the screening of oophoroma early diagnosis mark and preventing ovarian cancer generation development provide solution.

Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand technique means of the present invention, and can be implemented according to the content of specification sheets, coordinates accompanying drawing to be described in detail as follows below with preferred embodiment of the present invention.

Accompanying drawing explanation

The qualification schematic diagram that Fig. 1 is Primary mouse ovarian surface epithelial cell mark Pan-keratin, DAPI of the present invention and combines;

Fig. 2 is the impact of foetal calf serum and mEGF concentration versus cell purifying in the external vicious transformation cultural method of mouse ovarian superficial epithelial cells of the present invention;

Fig. 3 is the schematic diagram after the mouse ovarian superficial epithelial cells schematic diagram and amplification thereof gone down to posterity in early days in the present invention;

Fig. 4 be in the present invention mid-term the mouse ovarian superficial epithelial cells schematic diagram that goes down to posterity and the schematic diagram after amplifying thereof;

Fig. 5 is the mouse ovarian superficial epithelial cells schematic diagram that goes down to posterity of middle and advanced stage of the present invention and the schematic diagram after amplifying thereof;

Fig. 6 is the MOSEC growth curve schematic diagram of early stage in the present invention, mid-term, later passage;

Fig. 7 is the vicious transformation stove schematic diagram of the mouse ovarian superficial epithelial cells of later passage of the present invention.

Embodiment

Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.

Embodiment one

The external vicious transformation cultural method of a kind of mouse ovarian superficial epithelial cells described in a preferred embodiment of the present invention, comprises the following steps

1) ovarian surface epithelial cell is separated: under super clean bench, get the ovary of nonparous Healthy female C57BL/6 mouse in 4-6 6-8 all ages, be placed on damping fluid (PBS) (the 1000ml distilled water containing 400IU/ml Pen .-Strep, 1-2% foetal calf serum (FBS), NaCl 8.0g, KCl 0.2g, Na2HPO412H2O 3.48g, KH2PO40.2g, PH are 7.4) in damping fluid, then ovary is rinsed gently 3 times with the damping fluid of above-mentioned aseptic foetal calf serum (FBS), peel off residual uterine tube, fatty tissue and capsule of ovary, ovary after stripping is placed on pancreatin (0.25% pancreatin+EDTA is housed, 37 DEG C of preheatings) 1.5ml EP pipe in, digest in 37 DEG C of incubators containing 5% carbonic acid gas, for making Ovarian surface fully contact pancreatin, should ovary be made to separate as far as possible, after 30min, from incubator, take out 1.5ml EP manage, fluctuate about 10 times, take out ovary and transferred to another be equipped with in the 1.5ml EP pipe of pancreatin (0.25% pancreatin+EDTA) carry out second time digest, perfect medium (6-8% foetal calf serum (FBS) is added in former 1.5ml EP pipe, 1-1.2% Pen .-Strep (Penicillin-Streptomycin), 2-4ng/ml mEGF, 1-1.2% Insulin-Transferrin-Sodium Selenite (Insulin-Transferrin-Selenium)) stop digestion, epithelial centrifugal (1000r/min will be included, 5min), abandon upper liquid, add 1ml selectivity perfect medium and (comprise 3-4% foetal calf serum, 1-1.2% Pen .-Strep, 8-10ng/ml mEGF, the DMEM/F12 nutrient solution of 1-1.2% Insulin-Transferrin-Sodium Selenite) slowly blow and beat 6-10 time, collect the suspension containing mouse ovarian superficial epithelial cells, move into 35mm culture dish, hatch in 37 DEG C of incubators containing 5% carbonic acid gas.The step of second time digestion is identical with first time, ovarian surface epithelial cell is obtained by repeatedly digestion, the form of ovary can be kept on the one hand, prevent the ovary loose because losing tunicle chipping in digestive process, isolate the contaminating cell such as the interstitial of ovary inside, the difficulty simplified the operation on the other hand, makes MOSEC be separated relative ease.

Hatch and avoid mobile culture dish on the 2nd day, be beneficial to cell attachment growth, pericellular microenvironment can be maintained stablize simultaneously, hatch and adopt half amount to change liquid method replacing nutrient solution according to cell growth status on the 3rd day, when ovarian surface epithelial cell density reaches 80-85%, use the expression of immuno-fluorescence assay epithelial cell mark Pan-keratin, when its positive rate reaches more than 95% and pebbles sample cuboidal epithelium reaches more than 80%, carry out next step; Mouse ovarian superficial epithelial cells is that a class had both expressed epithelial cell mark Pan-keratin, expresses again the cell of mesenchymal cell markers Vimentin, the identified by immunofluorescence adopting Pan-keratin, DAPI and combine, and its result is see Fig. 1;

2) go down to posterity in early days: inoculation algebraically is less than the ovarian surface epithelial cell in 15 generations, use half amount to change liquid method to go down to posterity in 2:3 to 3:2 ratio, generation time interval 5-7 days, nutrient solution adopts selectivity perfect medium, go down to posterity and avoid mobile culture dish on the 2nd day all as far as possible, be beneficial to cell attachment growth, pericellular microenvironment can be maintained simultaneously and stablize; Now ovarian surface epithelial cell form mostly is a cube sample epithelial cell, and nucleus is regular ellipse, and the chromosome number of cell mostly is 40, and its effect of the ovarian surface epithelial cell after going down to posterity in early days as shown in Figure 3;

3) go down to posterity mid-term: the ovarian surface epithelial cell of inoculation algebraically between 16 to 84 generations, go down to posterity in 1:2 ratio before using mode 60 generation of all changing liquid, go down to posterity in 1:5 to 1:8 ratio after 60 generations, the generation time is spaced apart 3-5 days, and nutrient solution adopts perfect medium; Now, ovarian surface epithelial cell form is that cube and elongated spindle cell are mingled with existence, occurs more syncyte, kernel increases, the chromosome number of cell mostly is diploid or triploid, and plate clone Forming ability significantly improves, but does not also have soft-agar cloning Forming ability; Its effect of ovarian surface epithelial cell after going down to posterity mid-term as shown in Figure 4;

4) later passage: the ovarian surface epithelial cell that inoculation algebraically is greater than 85, the mode of all changing liquid is used to go down to posterity in 1:10 ratio, generation time is spaced apart 3-5 days, nutrient solution adopts partial perfect medium, now, ovarian surface epithelial cell form is many in elongated fusiformis, and the chromosome number of cell mostly is hypertetraploid, has soft-agar cloning Forming ability; Its effect of ovarian surface epithelial cell after later passage as shown in Figure 5.

Selectivity perfect medium is the DMEM/F12 nutrient solution comprising 3-4% foetal calf serum, 1-1.2% Pen .-Strep, 8-10ng/mlmEGF, 1-1.2% Insulin-Transferrin-Sodium Selenite; Perfect medium is the DMEM/F12 nutrient solution comprising 6-8% foetal calf serum, 1-1.2% Pen .-Strep, 2-4ng/ml mEGF, 1-1.2% Insulin-Transferrin-Sodium Selenite; Partial perfect medium is the DMEM/F12 nutrient solution comprising 8-10% foetal calf serum, 1-1.2% Pen .-Strep, 1-1.2% Insulin-Transferrin-Sodium Selenite; In selectivity perfect medium, perfect medium, partial perfect medium, the concentration of foetal calf serum increases successively, and in selectivity perfect medium, the concentration of mEGF is higher than the concentration of mEGF in perfect medium.

In above-mentioned steps, C57BL/6 mouse is selected to be because it is inbred mouse, the feature of inbred mouse is: the homozygosity of gene locus, the homology of genetic composition, phenotype consistence, long-term genetic stability, the resolvability of hereditary feature and the uniqueness etc. of genetic composition, namely have identical genetic composition and hereditary property; Be different from the CD1 mouse of prior art, be embodied in: 1) CD1 mouse is the mouse that two institutes of the U.S. cultivate from outbreeding system ICR mouse, the feature of outbred mice is: genetic composition has very high heterozygosity, carry a large amount of recessive deleterious alleles, there is stronger reproductivity and production cost low etc., namely there is genetic heterogeneity; 2) cell be separated from the CD1 mouse proceeding to TsAg gene has the feature of immortalized cells, can obtain and cultivate for a long time and go down to posterity.

In above-mentioned steps, nutrient solution selects DMEM/F12 but not normally used DMEM or a-MEM substratum, is because it contains specific amino acid, VITAMIN, inorganic salt and other composition.Contrast DMEM/F12, DMEM and α-MEM three kinds of medium components, add into altheine in its amino acid classes of result display DMEM/F12, L-Aspartic acid, Cys, L-PROLINE, add in VITAMIN into vitamin B12, add in inorganic salt into sulfate radical, copper, iron, the composition of zinc, and add xanthoglobulin, linolic acid, Thioctic Acid, un-added composition in DMEM and α such as putrescine and Thymine deoxyriboside-MEM, and the ratio of contained identical nutrient compositions is also different, as choline chloride 60, calcium pantothenate, niacinamide and vitamin B6 are no longer simple 1:1:1:1 ratios, carry out trickle adjustment, the interpolation of these compositions ensure that MOSEC can long term growth on the one hand, prevents epithelial cell aging on the other hand, if use containing 3-4%FBS, 8-10ng/ml mEGF, the DMEM selectivity perfect medium of 1-1.2% Insulin-Transferrin-Sodium Selenite-A, in long-term cultivation process, MOSEC cell does not survive, even if there is vicious transformation in MOSEC late, use partial perfect medium or the DMEM perfect medium of the preparation of DMEM substratum, cellular form can change, and can not keep vegetative state.

The vicious transformation mouse ovarian epithelial cell (MOSEC) obtained is cultivated through aforesaid method, it achieves continuous passage, and vicious transformation in vitro, its growth characteristics curve as shown in Figure 6, its late cell transforming focus is comparatively obvious, and see Fig. 7, wherein Fig. 7 A is early stage MOSEC cell, Fig. 7 B is MOSEC cell in mid-term, and Fig. 7 C is MOSEC cell in late period.It is early stage, mid-term and late period MOSEC chromosome karyotype analysis as shown in table 1.It should be pointed out that different from " immortalization " of prior art, " vicious transformation " not only has unlimited multiplication capacity, can also form clone's (anchor independent growth), also can become knurl at nude mice by subcutaneous on soft agar.Use early stage, mid-term, late period MOSEC cell in nude mouse, become knurl to detect, can find that early stage cell inoculation nude mice does not form lump, although it is dysplasia that but medium cell forms lump pathological diagnosis, tumour cell is not found in tumor tissue, and late cell inoculation nude mice forms larger lump, pathology detection finds many tumour cells, and late period, the tumor formation rate of MOSEC cell can reach 80%, and concrete correlation data refers to table 2.

Table 1 is early stage, mid-term and late period MOSEC chromosome karyotype analysis

Table 2 is early stage, mid-term, late period MOSEC cell nude mouse interior one-tenth knurl detects

It should be pointed out that the ovarian surface epithelial cell of inoculation algebraically before 15 generations, the digestion time using trypsin solution when at every turn going down to posterity is 30-45s, and in described trypsin solution, pancreas enzyme concentration is 0.1% and does not contain EDTA.Oval ovarian surface epithelial cell is responsive to pancreatin, short compared with other contaminating cell digestions time, utilizes this point, and collect the cell of trysinization 30s-45s, continuous passage is cultured to cell purification.

Embodiment two

Shown in Fig. 2 A, in ovarian surface epithelial cell separating step and in early days Secondary Culture, adopt and reduce serum-concentration and the selectivity perfect medium (the DMEM/F12 nutrient solution containing 3-4% foetal calf serum (FBS), 1-1.2% Pen .-Strep (Penicillin-Streptomycin), 8-10ng/ml mEGF, 1-1.2% Insulin-Transferrin-Sodium Selenite (Insulin-Transferrin-Selenium)) improving mEGF concentration, MOSEC obtains purifying, and namely rectangular-cube epithelial cell number is many.Shown in Fig. 2 B, in normal serum and lower concentration mEGF concentration cultures, inoblast competitive growth, namely volume is large, the fusiform of projection or the flats Constituent cell number of star many, MOSEC is difficult to purifying.

Comparative example one

In the early stage Secondary Culture of mouse ovarian superficial epithelial cells, use half amount to change liquid method to go down to posterity in 2:3 ratio, generation time interval 5-7 days, substratum adopts the DMEM substratum containing 3-4%FBS, 8-10ng/mL mEGF, use pancreatin (0.1% pancreatin, without EDTA) normal temperature digestion 30-45s, goes down to posterity after being no more than for 10 generations, necrocytosis.

Comparative example two

In the early stage Secondary Culture of mouse ovarian superficial epithelial cells, use half amount to change liquid method to go down to posterity in 2:3 ratio, generation time interval 5-7 days, substratum adopts containing the DMEM/F12 substratum being less than 3%FBS, 8-10ng/mL mEGF, use pancreatin (0.1% pancreatin, without EDTA) normal temperature digestion 30-45s, occur in early stage Secondary Culture process that early stage cytotrophy is not enough, grow slow problem.

Comparative example three

In the early stage Secondary Culture of mouse ovarian superficial epithelial cells, use half amount to change liquid method to go down to posterity in 2:3 ratio, generation time interval 5-7 days, substratum adopts containing the DMEM/F12 substratum being greater than 4%FBS, 8-10ng/mL mEGF, use pancreatin (0.1% pancreatin, without EDTA) normal temperature digestion 30-45s, due to foetal calf serum excessive concentration, cause occurring the problem that purifying rate is low, easily aging in early stage Secondary Culture process.

Comparative example four

In the early stage Secondary Culture of mouse ovarian superficial epithelial cells, use half amount to change liquid method to go down to posterity in 2:3 ratio, generation time interval 5-7 days, substratum adopts the DMEM/F12 substratum containing 3-4%FBS, 2-7ng/mL mEGF, use pancreatin (0.1% pancreatin, without EDTA) normal temperature digestion 30-45s, because somatomedin concentration is too low, cause occurring in early stage Secondary Culture process early stage vitro growth rates slowly, finally aging problem.

Comparative example five

In the early stage Secondary Culture of mouse ovarian superficial epithelial cells, use half amount to change liquid method to go down to posterity in 2:3 ratio, generation time interval 5-7 days, substratum adopts the DMEM/F12 substratum containing 3-4%FBS, 8-10ng/mL mEGF, use pancreatin (0.25% pancreatin+EDTA) normal temperature digestion 30-45s, occur going down to posterity in early stage Secondary Culture process not adherent problem.

Comparative example six

In the early stage Secondary Culture of mouse ovarian superficial epithelial cells, use half amount to change liquid method to go down to posterity in 2:3 ratio, generation time interval 5-7 days, substratum adopts the DMEM/F12 substratum containing 3-4%FBS, 8-10ng/mL mEGF, use pancreatin (0.1% pancreatin, without EDTA) normal temperature digestion lower than 30s, because pancreatin effect is not enough, there is the problem that cell dissociation amount is few, and under this pancreas enzyme concentration, extend digestion time, not only fail to increase the cell digested, and serious through the cell injury of digestion, easy aging death.

Comparative example seven

In the early stage Secondary Culture of mouse ovarian superficial epithelial cells, use half amount to change liquid method to go down to posterity in 2:3 ratio, generation time interval 5-7 days, substratum adopts the DMEM/F12 substratum containing 3-4%FBS, 8-10ng/mL mEGF, use pancreatin (0.1% pancreatin, without EDTA) normal temperature digestion higher than 45s, because digestion time is long, there is the problem that cell injury is serious, dead.

Comparative example eight

In the early stage Secondary Culture of mouse ovarian superficial epithelial cells, use half amount to change liquid method to go down to posterity in 2:3 ratio, generation time interval 5-7 days, substratum adopts the DMEM/F12 substratum containing 3-4%FBS, 8-10ng/mL mEGF, use pancreatin (0.1% pancreatin, without EDTA) normal temperature digestion 30-45s, Growth of Cells is good, purity is high, can cultivate by Long Term Passages.

Above comparative example one to comparative example eight, its Detailed Experimental result is as shown in table 3.

The early stage Secondary Culture Comparative result of table 3 mouse ovarian superficial epithelial cells

Embodiment three

The external vicious transformation cultural method of mouse ovarian superficial epithelial cells of the present invention, after ovarian surface epithelial cell separating step completes, after carrying out identified by immunofluorescence and/or immunohistochemical methods qualification, when the step that goes down to posterity in early days after qualification result conformance with standard.

A, identified by immunofluorescence comprise the following steps:

1) in aseptic super clean bench by the MOSEC pancreatin of 2nd generation (0.1% pancreatin+without EDTA) digestion, be inoculated on the creep plate in 24 orifice plates, hatch, when cell density reaches 80-85% in 37 DEG C of incubators containing 5% carbonic acid gas, abandon substratum, PBS rinsing 3 times; After the paraformaldehyde solution fixed cell 20-30min of 4%, penetrate 10-15min with 1-2%TritonX-100 and close 50-60min by 4.5-5% confining liquid room temperature again;

2) add the primary antibodie antikeratin antibody (pan anti-cytokeratin) of 1:500-1:800 dilution, spend the night in 4 DEG C of wet boxes, with PBS rinsing 3 × 3min; Add two anti-goat anti-mouse IgG of 1:1000-1:2000 dilution, 37 DEG C of lucifuge reaction 1-1.5h, then use PBS rinsing 3 × 3min; Add the DAPI that 8-10 μ g/ml dilutes, dyeing 15-20min, with PBS rinsing 3 × 3min, with anti-quencher sealing,

3) observe under confocal laser scanning microscope, CLSM, get 20-30 the visual field at random to detect in Xia Mei hole, 100 times of visuals field, count the positive cell number (N1) in each visual field and total cellular score (N) ratio, calculate cell positive rate, positive rate=N1/N × 100%.。

The qualification of b, immunohistochemical methods comprises the following steps:

1) same batch untreated intact mice ovary and to be put in 4% formaldehyde 4 DEG C respectively with the mouse ovarian of trysinization and to spend the night;

2) be cut into 6 μm of tissue slicies with after paraffin bag cystovarian, the ovary sample of de-paraffin be put in 0.01M sodium citrate buffer solution boil 10-15min, PBS rinsing subsequently;

3) drip Normal Goat Serum confining liquid, treat that 30min gets rid of surplus liquid; Drip the primary antibodie pan anti-cytokeratin 150 μ l of 1:100-1:200,4 DEG C are spent the night, PBS rinsing; Drip the two anti-goat anti-mouse IgG 150 μ l of 1:1000, be placed in 37 DEG C of 1h, PBS rinsings;

4) DAB colour developing 5-10min, PBS rinsing 10-12min, haematoxylin redyeing 2-2.5min, hydrochloride alcohol breaks up, PBS rinsing 10-15min, dehydration, transparent, mounting, microscopy.

Embodiment four

The present invention also provides a kind of mouse ovarian superficial epithelial cells external vicious transformation authentication method:

1) cell transformation stove: by intensive and the cell of overlap forms.When printing opacity checks, with the naked eye there is the white patch " transforming focus " differed in size all as seen; Basis of microscopic observation, after cell transformation, cell space increases, and become Polygons growth non-directional, lose contact inhibition, extend arrangement to three-dimensional space, cell is overlapped; At transforming focus edge, transformant and normal cell clear-cut, morphological differentiation is obvious;

2) chromosome karyotype analysis: normal mouse chromosome caryogram is 40XX (XY), uses routine chromosome tabletting technology Giemsa dyeing, observes the karyomit(e) that the ovarian surface epithelial cell of vicious transformation has occurred and mostly be hypertetraploid;

3) soft-agar cloning is formed:

Prepare bottom glue: make 1.2-1.4% low melting-point agarose liquid with distilled water, after autoclaving, be placed in 40 DEG C of water-bath equilibrium temperature 40-45min; By (2 × DMEM in high glucose+18-20% foetal calf serum+2-2.4%Insulin-Transferrin-Selenium) equalization temperature 40-45min in 40 DEG C of water-baths; By isopyknic above-mentioned two kinds of liquid blendings, to prepare containing 0.7% agaropectin; The above-mentioned mixed solution of 1.5ml is slowly injected 35mm culture dish, avoids bubble to produce, wait for that 30-40min solidifies completely to make glue;

Preparation upper strata glue: by above-mentioned 0.6-0.7% agaropectin equalization temperature 45min in 37 DEG C of water-baths; By the mouse ovarian superficial epithelial cells in early stage, mid-term and late period and positive control human oophoroma cell line SKOV-3 cell pancreatin (0.25% pancreatin+EDTA) digestion in aseptic super clean bench, trypan blue row dye, blood counting chamber counts, and makes cell count be 10 with substratum adjustment volume ⁵cell/ml; 1ml cell suspension is added in 15ml centrifuge tube, then add the 0.6-0.7% agaropectin of 3ml perfect medium and 3ml; Soft piping and druming mixing; On culture dish bottom glue, slowly inject the above-mentioned mixing liquid of 1.5ml, avoid bubble to produce, often organize setting 3 Duplicate Samples.Be placed in super clean bench (room temperature 25-27 DEG C) 80-90min, after upper strata gelling is solid, instill 500 μ L selectivity perfect mediums, perfect medium and partial perfect medium respectively gently on upper strata;

Culture dish is placed in 37 DEG C of incubators containing 5% carbonic acid gas to cultivate, period, every 3d, adds 500 μ l DMED/F12 perfect mediums; After cultivating 10d, under plate is placed in inverted microscope, clone's number of counting clone diameter >15 μm; Often organize setting 3 Duplicate Samples, independent experiment repeats 3 times; Soft-agar cloning rate of formation=Clone formation number/inoculating cell number × 100%;

4) external vicious transformation ability judges: compared with early and middle portion mouse ovarian superficial epithelial cells, the soft-agar cloning rate of formation of late cell obviously increases, compared with positive control human oophoroma cell line SKOV-3 cell, although cloning efficiency is low, but the volume of the clone formed is obviously bigger than normal, most diameter is the clone of 19-22 μm, and the mouse ovarian superficial epithelial cells outer vicious transformation of shedder in late period is described.

The above is only the preferred embodiment of the present invention; be not limited to the present invention; should be understood that; for those skilled in the art; under the prerequisite not departing from the technology of the present invention principle; can also make some improvement and modification, these improve and modification also should be considered as protection scope of the present invention.

Claims (8)

1. the external vicious transformation cultural method of mouse ovarian superficial epithelial cells, is characterized in that: comprise the following steps:

1) ovarian surface epithelial cell is separated: the ovary getting nonparous Healthy female C57BL/6 mouse in 6-8 age in week, rinse and peel off residual uterine tube, after fatty tissue and capsule of ovary, trysinization is utilized to be separated its ovarian surface epithelial cell, then perfect medium is added to stop trysinization process, centrifugal abandon supernatant liquor after again bringing Selection In property perfect medium moving in culture dish hatch in incubator, hatch and avoid mobile culture dish on the 2nd day, hatch and adopt half amount to change liquid method replacing nutrient solution according to cell growth status on the 3rd day, when ovarian surface epithelial cell density reaches 80%, use the expression of immuno-fluorescence assay epithelial cell mark angling albumen, when its positive rate reaches more than 95% and pebbles sample cuboidal epithelium reaches more than 80-85%, carry out next step,

2) go down to posterity in early days: inoculation algebraically is less than the ovarian surface epithelial cell in 15 generations, use half amount to change liquid method and go down to posterity in 2:3 to 3:2 ratio, generation time interval 5-7 days, nutrient solution adopts selectivity perfect medium;

3) go down to posterity mid-term: the ovarian surface epithelial cell of inoculation algebraically between 16 to 84 generations, in 16-60 generation, goes down to posterity in 1:2 to 1:4 ratio, and go down to posterity in 1:5 to 1:8 ratio after 60 generations, the generation time is spaced apart 3-5 days, and nutrient solution adopts perfect medium;

4) later passage: the ovarian surface epithelial cell that inoculation algebraically is greater than 85, go down to posterity in 1:8 to 1:10 ratio, the generation time is spaced apart 3-5 days, and nutrient solution adopts partial perfect medium.

Described selectivity perfect medium, perfect medium are the DMEM/F12 nutrient solution comprising foetal calf serum, mEGF and Insulin-Transferrin-Sodium Selenite, and described partial perfect medium is comprise foetal calf serum, Insulin-Transferrin-Sodium Selenite and not containing the DMEM/F12 nutrient solution of mEGF; In described selectivity perfect medium, perfect medium, partial perfect medium, the concentration of foetal calf serum increases successively, and in described selectivity perfect medium, the concentration of mEGF is higher than the concentration of mEGF in perfect medium.

2. the external vicious transformation cultural method of mouse ovarian superficial epithelial cells according to claim 1, it is characterized in that: the ovarian surface epithelial cell of inoculation algebraically before 15 generations, the digestion time using trypsin solution when at every turn going down to posterity is 30-45s, and in described trypsin solution, pancreas enzyme concentration is 0.1% and does not contain EDTA.

3. the external vicious transformation cultural method of mouse ovarian superficial epithelial cells according to claim 1, is characterized in that: described selectivity perfect medium is the DMEM/F12 nutrient solution comprising 3-4% foetal calf serum, 1-1.2% Pen .-Strep, 8-10ng/mlmEGF, 1-1.2% Insulin-Transferrin-Sodium Selenite.

4. the external vicious transformation cultural method of mouse ovarian superficial epithelial cells according to claim 1, is characterized in that: described perfect medium is the DMEM/F12 nutrient solution comprising 6-8% foetal calf serum, 1-1.2% Pen .-Strep, 2-4ng/mlmEGF, 1-1.2% Insulin-Transferrin-Sodium Selenite.

5. the external vicious transformation cultural method of mouse ovarian superficial epithelial cells according to claim 1, is characterized in that: described partial perfect medium is the DMEM/F12 nutrient solution comprising 8-10% foetal calf serum, 1-1.2% Pen .-Strep, 1-1.2% Insulin-Transferrin-Sodium Selenite.

6. the external vicious transformation cultural method of mouse ovarian superficial epithelial cells according to claim 1, it is characterized in that: after described ovarian surface epithelial cell separating step completes, after carrying out identified by immunofluorescence and/or immunohistochemical methods qualification, when the step that goes down to posterity in early days after qualification result conformance with standard.

7. the external vicious transformation cultural method of mouse ovarian superficial epithelial cells according to claim 6, is characterized in that: described identified by immunofluorescence comprises the following steps:

1) hatch after the ovarian surface epithelial cell trysinization of 2nd generation, inoculation, when cell density reaches 80%, abandon substratum post rinsing, use stationary liquid fixed cell and confining liquid sealing treatment subsequently;

2) add primary antibodie anti-angling albumen process for some time post rinsing, add two anti-goat anti-mouse IgG process for some time post rinsings, add DAPI and to dye for some time post rinsing, adopt with anti-quencher sealing subsequently;

3) basis of microscopic observation, calculates cell positive rate.

8. the external vicious transformation cultural method of mouse ovarian superficial epithelial cells according to claim 6, is characterized in that: described immunohistochemical methods qualification comprises the following steps:

1) with batch untreated intact mice ovary and being put in formaldehyde respectively with the mouse ovarian of trysinization spend the night;

2) being cut into tissue slice with after paraffin bag cystovarian, boiling process post rinsing with being placed in sodium citrate buffer solution;

3) get rid of surplus liquid after dripping Normal Goat Serum confining liquid process for some time, drip primary antibodie anti-angling albumen process for some time post rinsing subsequently, drip two anti-goat anti-mouse IgG process for some time post rinsings;

4) after DAB color development treatment, haematoxylin redyeing, hydrochloride alcohol differentiation, PBS rinsing, dehydration, transparent, mounting process, identified under microscope.