CN105670998B - A kind of method of calcification cancer cell - Google Patents

A kind of method of calcification cancer cell Download PDF

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CN105670998B
CN105670998B CN201610027755.XA CN201610027755A CN105670998B CN 105670998 B CN105670998 B CN 105670998B CN 201610027755 A CN201610027755 A CN 201610027755A CN 105670998 B CN105670998 B CN 105670998B
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cancer cell
calcification
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folic acid
culture medium
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CN105670998A (en
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赵瑞波
唐睿康
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Zhejiang University ZJU
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins

Abstract

The invention discloses a kind of methods of calcification cancer cell, comprising the following steps: (1) by cancer cell inoculation in the culture medium containing folic acid, is incubated for, obtains the cancer cell of surface enrichment folic acid；(2) cancer cell of surface enrichment folic acid is placed in calcifying solution and is incubated for, the cancer cell of calcification is made.The invention also discloses folic acid to prepare the application in cancer cell calcification inducer.The present invention is based on cancer cell height expression folacin receptors and folacin-carboxyl to provide the characteristic of nucleation site for calcification, forms calcium phosphate calcified layer in cancer cell surfaces, activity capable of inhibiting cell destroys the structure of cell membrane and eventually leads to cancer cell death；Animal experiments show that calcification processing of the invention does not influence blood cell, there is preferable biological safety almost without damage without hepatotoxicity wind agitation, and to organ, therefore, the technical scheme is that oncotherapy provides feasible scheme.

Description

A kind of method of calcification cancer cell
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of method of calcification cancer cell.
Background technique
Cancer is one of highest disease of the death rate, and the high incidence that holding is lasting in the world.International Medical at present The method of the generally acknowledged treatment of cancer in boundary is still traditional chemotherapy and radiation, however Radiotherapy chemotherapy drug is killing the same of cancer cell When, body can be made by serious damage, such as kidney failure, alopecia, syntexis etc..In addition, metastases are that cancer is caused to be suffered from The major reason of person's death, patient occupies higher proportion because organ failure caused by its metastases is lethal, and radiotherapy and chemotherapy medicine Metastases cannot effectively be inhibited.According to statistics, the medical expense of annual cancer is very high, and in increased trend year by year, gives patient Family causes serious economic loss and psychological burden.
Although there are many emerging technologies to be used for treatment of cancer at present, for example, by using the mode of pharmaceutical carrier, carried by control The size and surface modification tumour-specific of body identify molecule, make drug-rich in tumor tissues, but its carrier has and exempts from Epidemic focus is identified and is removed by vivo immuning system quickly, to lose therapeutic effect after carrier enters blood.In recent years Come going deep into scientific research, constantly there is new drug to enter market, the anticancer drug (Nivolumab) of listing in 2015 is to be based on The small-molecule drug of apoptosis albumen PD-1 design obtains remarkable result in clinical test, and without obvious secondary Effect, however these drugs make the price of drug costly in the high-cost investment of development phase.According to US National health Research institute statistics obtain, by 2005, the treatment cost of american cancer was about 209,400,000,000 yuan, cancer disbursements in 2014 about The 20% of national product medical treatment total value is accounted for, it will thus be seen that patient will undertake high medical fee when using this drug.Tumour Transfer be major reason that it is difficult to cure, and inhibit the drug wretched insufficiency of metastases in many anticancer drugs at present, So that many patients after the treatment again occur it is helpless when metastatic tumor.Therefore, a kind of safe and effective and economic method is found Inhibit growth and the transfer of tumour extremely urgent.
In human body, calcification plays a significant role in the growth of bone and tooth, but the calcification meeting of the pathology of blood vessel Lead to the dysfunction of cell, finally causes a series of disease, such as chronic kidney disease becomes and cardiovascular pathological changes.If will Calcification, which is introduced into cancer cell surfaces then, may inhibit the activity of cancer cell or eventually lead to cancer cell death, can thus be cancer It treats and a kind of new approaches for not depending on drug is provided.In research in our prior, we are realized using the method for chemical modification The calcification of yeast cell wall, calcification can make yeast cells enter dormant period to stop division, this is because yeast cells have it is good Good anti-stress ability, and mammalian cell discovery is compared, mammalian cell cell membrane is more fragile and protects without cell wall, and And some researches show that after cell surface silication forms one layer of SiO2, the time of cell no more than 12h, illustrates in cell membrane outer layer One layer of mineral are formed, the activity of mammalian cell can be seriously affected.Different from normal tissue cell, there are many cancer cell surfaces Specific expressed molecular receptor, such as folacin receptor (FR), and FR in normal tissue cell almost without expression, and it matches Body folic acid (FA), just containing the carboxyl that can promote calcification.Therefore, it using the characteristic of cancer cell height expression folacin receptor, seeks The method of derivation carcinogenic cells calcification will be the effective means for inhibiting tumour growth transfer.
Summary of the invention
The present invention provides a kind of methods of calcification cancer cell, form layer mineral in cancer cell surfaces using this method, reach To the purpose for inhibiting tumor cell viability.
A kind of method of calcification cancer cell, comprising the following steps:
(1) it by cancer cell inoculation in the culture medium containing folic acid, is incubated for, obtains the cancer cell of surface enrichment folic acid；
(2) cancer cell of surface enrichment folic acid is placed in calcifying solution and is incubated for, the cancer cell of calcification is made.
The present invention is based on the characteristics of cancer cell surfaces height expression folacin receptor, by cancer cell inoculation to the culture containing folic acid In base, folic acid is made to be enriched in cancer cell surfaces, improves the carboxyl-content of cancer cell surfaces.Carboxyl provides nucleation and crystal for calcification The active site of growth leads to cancer cell calcification.
The calcification cancer cell obtained after technical method processing according to the invention is through scanning electron microscope and power spectrum elemental analysis table The ingredient for levying calcified layer is calcium phosphate, is to be crosslinked by many micro-, nanometer grade calcium phosphate clusters, and be adhered to cell membrane table Face.Calcium phosphate is covered in the surface of cancer cell, limits the physiological activity of cancer cell, inhibits tumor cell viability obviously, And eventually lead to the rupture of cell membrane.
Preferably, repetitive operation step (2) is for several times, the cancer cell of calcification is obtained.Fresh calcifying solution is added in repetition, makes Calcium ion, phosphate anion constantly react precipitating on carboxyl site, deposit more calcium phosphate in cancer cell surfaces.More Preferably, repetitive operation step (2) 2~5 times.
Preferably, the cancer cell is that human cervical carcinoma cell (HeLa), human breast cancer cell (MCF7) or people's ovary are thin Born of the same parents (SKVO-3).Research has shown that the folacin receptor of these three types of cells is overexpression, is conducive to the deposition of calcified layer.Uterine neck Cancer, breast cancer and oophoroma are the common malignant tumours of women, realize calcification for these three types of cancer cells, the treatment for tumour With directive significance.
In order to be enriched with folic acid to the maximum extent, cancer cell need to adequately be contacted with culture medium, therefore the inoculation of cancer cell is close Degree should not be too large, preferably, the inoculum density of cancer cell is the long 40-80% to culture hole plate base area.It is more highly preferred to , inoculum density accounts for the 60-70% of culture hole plate base area.Not only guaranteed that cancer cell was effectively contacted with culture medium in this way, but also energy Enough make full use of culture orifice plate.
Preferably, cancer cell is cleaned 2-3 times before inoculation using phosphate buffer.It is first carried out before cancer cell calcification processing Cleaning, avoids other substances from impacting calcification effect.
Folic acid provides the active site of nucleation and crystal growth for calcification, therefore, need to guarantee to contain sufficient amount in culture medium Folic acid, preferably, in step (1), the content of folic acid is 0.2-1.2mg/mL in culture medium.Culture medium is that conventional cancer is thin Born of the same parents' culture medium.Such as DMEM culture medium.More preferably, the content of folic acid is 0.5-1mg/mL in culture medium.
Preferably, the time of incubation is 5-30min in step (1).More preferably, the time of incubation is 10- 25min。
Preferably, the calcifying solution is the DMEM culture medium that calcium ion concentration is 5-20mM in step (2).
DMEM culture medium is as phosphoric acid root origin, by additional calcium ion, forms calcium phosphate (CaP) calcium in cell membrane surface Change layer.Calcium ion concentration is too low, is hardly formed precipitating, but being above 20mM can be to cell generation toxic side effect.More preferably, Calcium ion concentration is 10-15mM in DMEM culture medium.
Preferably, in step (2), incubation time 0.25-2h.Incubation time should not too long, as time went on, calcium The toxicity of ion pair cell also will increase.More preferably 0.5-1h.
Carbon dioxide accelerates the formation of calcium phosphate, therefore, carbon dioxide is passed through during incubation, carbon dioxide has few Amount is dissolved in liquid, the calcium binding with folic acid enrichment, and then precipitating is promoted to be formed.Preferably, incubation conditions include dioxy The concentration for changing carbon is 4-10%.
The present invention also provides folic acid to prepare the application in cancer cell calcification inducer.
It is that the present invention has the utility model has the advantages that (1) the present invention is based on cancer cell height expression folacin receptors and folacin-carboxyl The characteristic of nucleation site is provided for calcification, forms calcium phosphate calcified layer in cancer cell surfaces, activity capable of inhibiting cell destroys cell The structure of film simultaneously eventually leads to cancer cell death；(2) animal experiments show that calcification processing of the invention does not have shadow to blood cell It rings, there is preferable biological safety, therefore, technical solution of the present invention almost without damage without hepatotoxicity wind agitation, and to organ Feasible scheme is provided for oncotherapy.
Detailed description of the invention
Fig. 1 is optical microphotograph sem observation result after human normal cell line HEK293 and human cervical carcinoma cell HeLa calcification.
Fig. 2 is SEM observation (A) and HeLa calcigerous cell after human normal cell line HEK293 and human cervical carcinoma cell HeLa calcification Calcium, P elements imaging results (B).
The SEM that Fig. 3 is HeLa cell calcified layer CaP is magnified, and wherein A is 5000 times of amplification, and B is 50000 times of amplification.
Fig. 4 is the characterization result figure of calcigerous cell sample, and wherein A is that X-ray diffraction analyzes (XRD), and B is that Fourier is red Outer conversion spectrum analyzes (FTIR).
Fig. 5 is that the laser co-focusing of different time after HeLa cell calcification is observed as a result, wherein green fluorescence is calcified layer Calcium phosphate (calcein dyeing), red be cell membrane (PKH26 dyeing), blue be nucleus (Hoechest33342 contaminate Color).
Fig. 6 be the calcification for 24 hours of HeLa cell after life or death coloration result figure (A) and HEK293 and HeLa cell calcification after for 24 hours Survival rate MTT result (B), wherein in A upper figure be the non-calcification of HeLa cell coloration result, be living cells as the result is shown, under Figure is the coloration result after HeLa cell calcification for 24 hours, is dead cell as the result is shown.
Fig. 7 is the targeting calcification of mouse interior tumor tissue as a result, wherein A figure is the tumor tissues optics of different disposal group It takes pictures result；B figure is the micro-CT result of calcification tumor tissues；C figure is the tem observation knot of tumor tissue section after calcification Fruit, red four-headed arrow indicator cells gap, in iuntercellular, there are a large amount of calcium phosphate；D figure is tumor tissue section's fluorescent staining Laser co-focusing result figure afterwards, blue are the nucleus of Hoechst33342 dyeing.
Fig. 8 is targeting calcification to the safety evaluatio of mouse, and wherein A is mouse blood routine analysis of blood result；B is mouse Liver function biochemical studies；C is the HE coloration result of mouse major organs.
Fig. 9 is to target calcification for anti-tumor experiment in Mice Body as a result, wherein A is that the mouse tumor of different disposal group is raw Long volume change；B is mouse weight variation；C is the HE coloration result of mouse tumor tissue.
Specific embodiment
The present invention is further explained in the light of specific embodiments.These embodiments are for illustration purposes only, without For limiting use scope of the present invention.DMEM culture medium is commercial prod in experiment, and folic acid used (FA), has inorganic salts Machine small molecule and protein are the pure above import reagent of chemistry.
Embodiment 1
One, the preparation of calcification cancer cell-first is targeted
HeLa cell is laid in 24 orifice plates, every hole number of cells is about 2 × 105, orifice plate bottom area is grown to cell 70% or so, start calcification processing.The culture medium of cell is outwelled first, and PBS is cleaned 1-2 times, is then added and contains into orifice plate The DMEM culture medium of 300 μ g/mL FA, cell is placed in incubator, adjusts CO2Concentration is 5%, air 95%, temperature 37 DEG C, after being incubated for 15min, it is added into orifice plate and contains 11.8mM Ca2+(including the 10mM CaCl being newly added2) DMEM culture medium Calcifying solution, orifice plate is placed in calcification processing 30min in cell incubator, calcifying solution is sopped up fresh calcifying solution is added and continue with 60min.It is individually handled using FA and calcifying solution as control, solution is sopped up after the completion of processing, the cancer cell of calcification can be obtained.
Three, the preparation of calcification cancer cell-second is targeted
HeLa cell is laid in 24 orifice plates, every hole number of cells is about 2 × 105, orifice plate bottom area is grown to cell 70% or so, start calcification processing.The culture medium of cell is changed first, is cleaned 1-2 times with the phosphate buffer of pH7.2, Then the additional new DMEM culture medium that 200 μ g/mL FA are added is added into orifice plate, then by cell as adjusting in incubator CO2Concentration is 5%, air 95%, 37 DEG C of temperature, after being incubated for 20min in the incubator, sops up culture medium, is added into orifice plate Contain 21.8mM Ca2+(including the 20mM CaCl being newly added2) DMEM culture medium calcifying solution, orifice plate is placed in cell culture Calcification processing 30min in case.It is individually handled using FA and calcifying solution as control, solution is sopped up after the completion of processing, can be obtained The cancer cell of calcification.
Three, the preparation of calcification cancer cell-the third is targeted
HeLa cell is laid in 24 orifice plates, every hole number of cells is about 2 × 105, orifice plate bottom area is grown to cell 70% or so, start calcification processing.The culture medium of cell is changed first, is cleaned 1-2 times with the phosphate buffer of pH7.2, Then the additional new DMEM culture medium that 200 μ g/mL FA are added is added into orifice plate, then by cell as adjusting in incubator CO2Concentration is 5%, air 95%, 37 DEG C of temperature, after being incubated for 25min in the incubator, sops up culture medium, is added into orifice plate Contain 16.8mM Ca2+(including the 15mM CaCl being newly added2) DMEM culture medium calcifying solution, orifice plate is placed in cell culture Calcification processing 40min in case is reprocessed 1 time, is individually handled using FA and calcifying solution as control, is inhaled solution after the completion of processing Fall, the cancer cell of calcification can be obtained.
Four, the preparation of calcification cancer cell-fourth is targeted
HeLa cell is laid in 24 orifice plates, every hole number of cells is about 2 × 105, orifice plate bottom area is grown to cell 70% or so, start calcification processing.The culture medium of cell is changed first, is cleaned 1-2 times with the phosphate buffer of pH7.2, Then the additional new DMEM culture medium that 200 μ g/mL FA are added is added into orifice plate, then by cell as adjusting in incubator CO2Concentration is 5%, air 95%, 37 DEG C of temperature, after being incubated for 25min in the incubator, sops up culture medium, is added into orifice plate Contain 16.8mM Ca2+(including the 15mM CaCl being newly added2) DMEM culture medium calcifying solution, orifice plate is placed in cell culture Calcification processing 30min in case is reprocessed 2 times, is individually handled using FA and calcifying solution as control, is inhaled solution after the completion of processing Fall, the cancer cell of calcification can be obtained.
Five, the characterization of calcification cancer cell is targeted
After the completion of cell calcification, after the fixed 30min of 4% paraformaldehyde room temperature, PBS is cleaned 2-3 times, then uses gradient Washing dehydration is placed in scanning electron microscope after ethyl alcohol volatilization to ethyl alcohol (70%, 80%, 90%, 95% and 100%) respectively Lower observation, and elemental analysis is carried out to the calcified layer of the cell surface after calcification, while using using XRD and FTIR to calcification sample Product are analyzed.
Using human normal cell (HEK293) as control, make as above processing.
Without significant difference on the calcification cancer cell pattern of the first and second the third 4 kinds of methods of fourth preparations.The calcification cancer cell prepared with first For, as a result as shown in Figure 1,2,3, 4, HeLa cell is through calcification processing, and cell surface forms calcified layer, and Fig. 2 is using scanning electricity Mirror and power spectrum elemental analysis obtain calcified layer at containing calcium and phosphorus；Calcified layer amplifying observation display is formed by irregular cluster, Referring to Fig. 3；Fig. 4 mid infrared spectrum is as a result, be 1000cm in wave number-1And 500cm-1The phase transformation without now crystal is controlled, in conjunction with X- Ray diffraction results can illustrate that the calcified layer to be formed is unformed calcium phosphate.Human normal cell (HEK293) is by calcification After reason, cell surface does not form calcification calcium phosphate.The above result shows that using the method for the invention realizes the targets of cancer cell To calcification.
Embodiment 2
Using laser confocal microscope, dyeing and MTT experiment method are killed anyway to detect calcification processing to cancer cell Wound effect.Concrete operations are as follows:
One, the variation after cell calcification
Before calcification processing, live cell dye PKH26 and Hoechst33342 is respectively adopted to the cell membrane of cancer cell and thin Karyon carries out fluorescent staining.After the completion of calcification processing, fluorescent staining is carried out to ore bed with calcium specific fluorescence dye calcein, It changes with time respectively in confocal laser scanning microscope cellular morphology after the completion of dyeing, as a result referring to Fig. 5.
Two, cell dyes anyway
(1) HEK293, MCF7 and HeLa cell are with 1 × 104The density in a/hole is inoculated in respectively in 96 orifice plates, and culture is for 24 hours Afterwards, calcification processing is carried out to cell, calcification processing method is the same as embodiment 1.
(2) after the completion of calcification processing, DMEM culture medium is added, continues culture for 24 hours.
(3) PBS washs cell three times, and life or death staining reagent (Invitrogen) then is added, and is incubated in cell incubator 30min, under the microscope in fluorescence microscopy.
Three, MTT
(1) HEK293, MCF7 and HeLa cell are with 1 × 104The density in a/hole is inoculated in respectively in 96 orifice plates, and culture is for 24 hours Afterwards, calcification processing is carried out to cell, and is individually handled using FA and calcifying solution as control.
(2) after the completion of handling, culture medium is added and continues culture for 24 hours, 5mg/mL MTT (20 μ L) is then added and arrives reactant In system, 4h is cultivated, orifice plate solution is sopped up, 150 μ L DMSO are added and shake 5-10min, use microplate spectrophotometer (Bio-Tek) absorption value of 570nm, the absorption value and first a ceremonial jade-ladle, used in libation concentration (cell succinate dehydrogenase and MTT reaction product) are measured Directly proportional, which represent the activity of succinate dehydrogenase, and then indicate cell activity.
The results show that the calcium phosphate that calcification is formed is covered in the surface of cancer cell, the physiological activity of cell will limit, make thin Cytoactive is obviously inhibited, and eventually leads to the rupture of cell membrane, referring to Fig. 5.Cell life or death coloration result illustrates to target calcium Change can be used for killing cancer cell, referring to Fig. 6 A, and after calcification processing HeLa cell survival rate be suppressed 60% or more, and HEK29 cell is still able to maintain 80% or more survival rate, referring to Fig. 6 B, this is because the lower folic acid of HEK293 cell surface Caused by low calcium efficiency caused by expression of receptor amount, further illustrate that calcification can be specifically in the highly expressed cancer of folacin receptor Cell surface occurs.
Embodiment 3
Building HeLa animal model for tumour is used to detect the effect of targeting calcification in vivo.It operates as follows with testing process:
(1) HeLa cell strain is with 5 × 106A/amount only is inoculated into nude mice (20 nude mices, female) subcutaneously as kind of a mouse drug resistance Model grew to 80mm to gross tumor volume after 10 days3When, start to test.
(2) folic acid of 200 μ L, 1mg/mL is injected intraperitoneally, waits 15min or so, is enriched in tumor locus to folic acid, to swollen High calcium DMEM culture medium (50mM Ca is injected in tumor tissue2+), injection 1 time, continuous processing 15 days, observes the calcification of tumour daily Effect.Individually to inject calcifying solution and folic acid and blank group as control.
(3) using micro-CT technology to tumor scan, and it is scanned rear three-dimensional reconstruction, determines tumour calcification result； Calcification tumour is sliced, fluorescent staining is carried out to tumor biopsy using calcein, is seen after dyeing using laser co-focusing It examines；By tumor biopsy in the pattern of transmission electron microscope observation calcified layer and the form of cancer cell.
(4) in an experiment, the blood routine of mouse and biochemistry are analyzed, while HE dye is carried out to mouse major organs Color assesses the safety of this method.
The results show that calcification can effectively wrap up tumour cell, and there is necrosis ginseng in the Tumor Tissue Tumors after package See Fig. 7 A；For Micro-CT to the scanning result of tumour, arrow indicates tumor tissues and the calcium phosphate that calcification is formed respectively, referring to Fig. 7 B；The pattern of calcium phosphate and the form of tumour cell illustrate that calcification can be formed in tumor tissues in tem observation tumor biopsy Cancer cell surfaces, referring to Fig. 7 C；Tumor tissue section's laser co-focusing after Hoechst33342 staining cell core is taken pictures observation There is pyknosis in neoplastic cell nuclei as the result is shown, illustrates that calcification inhibits the activity of tumor tissues, referring to Fig. 7 D.In addition calcification processing The blood cell of mouse is not influenced, without hepatotoxicity wind agitation, and organ HE coloration result shows that calcification processing there almost is not organ It has damage, referring to Fig. 8.
Embodiment 4
Building HeLa mice with tumor animal model is used to detect the cancer resistant effect of targeting calcification.Operation and testing process are such as Under:
(1) HeLa cell strain is with 5 × 106A/amount only is inoculated into nude mice (40 nude mices, female) subcutaneously as nude mice mould Type grew to 80mm to gross tumor volume after 10 days3When, start to test.
(2) folic acid of 200 μ L 1mg/mL is injected intraperitoneally, then waits 10min or so, is enriched in tumor locus to folic acid, 100 μ L high calcium DMEM culture medium (50mM Ca are injected into tumor tissues2+), injection 1 time daily, continuous processing 15 days, observation was swollen The calcification effect of tumor.Individually to inject calcifying solution and folic acid and blank group as control.
(3) mouse sign is observed daily measure the longest diameter L and shortest diameter B of tumour every 2 day entry mouse weights, benefit With formula V=0.5 × L × B2Calculate mouse volume.
Mouse is put to death after (4) 30 days, is taken out tumour weighing, is photographed to record, and HE dyeing is carried out to tumour.
(5) mouse cancer cell lines 4T1/luc is chosen, with 5 × 106It is a/only amount be inoculated into mouse (40 nude mices, female) cream At gland, 80mm is grown to gross tumor volume after 10 days3Shi Zuowei model starts calcification processing, and processing method and step (2) are consistent.It adopts The transfer of mouse tumor is detected with small animal living body imaging technique, while recording the death rate of mouse.
The results show that tumor volume growth can be effectively suppressed in calcification, referring to Fig. 9 A；In addition the weight of mouse and control group phase Than not changing, further illustrate that this method does not have apparent side effect to mouse；HE coloration result shows that HE contaminates at calcification tumour Color can be deepened, and participate in Fig. 9 C FA/Ca processing group, the cell death of large area has been had already appeared around calcified tissue.Furtherly Bright this method can effectively inhibit tumour growth.In conjunction with Fig. 7, it can be deduced that, this method can realize cancer cell in tumour certain Calcification is targeted, and then inhibits the growth of tumour, to realize effective effect.It is said by mouse weight data (Fig. 9 B) in conjunction with Fig. 8 Bright this method has preferable biological safety.

Claims (6)

1. a kind of method of calcification cancer cell, which comprises the following steps:
(1) it by cancer cell inoculation in the culture medium containing folic acid, is incubated for, obtains the cancer cell of surface enrichment folic acid；
(2) cancer cell of surface enrichment folic acid is placed in calcifying solution and is incubated for, the cancer cell of calcification is made；
In step (1), the inoculum density of cancer cell is the long 40-80% to culture hole plate base area；Folic acid in culture medium Content is 0.2-1.2mg/mL；
In step (2), the calcifying solution is the DMEM culture medium that calcium ion concentration is 5-20mM, and incubation conditions include carbon dioxide Concentration be 4-10%.
2. the method as described in claim 1, which is characterized in that repetitive operation step (2) for several times, obtains the cancer cell of calcification.
3. the method as described in claim 1, which is characterized in that the cancer cell is people's cervical cancer cell HeLa, human breast carcinoma Cell MCF7 or people's gonad cell SKVO-3.
4. the method as described in claim 1, which is characterized in that in step (1), the time of incubation is 5-30min.
5. the method as described in claim 1, which is characterized in that in step (2), incubation time 0.25-2h.
6. folic acid is preparing the application in cancer cell calcification inducer.
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