CN105670998A - Method for calcification of cancer cells - Google Patents
Method for calcification of cancer cells Download PDFInfo
- Publication number
- CN105670998A CN105670998A CN201610027755.XA CN201610027755A CN105670998A CN 105670998 A CN105670998 A CN 105670998A CN 201610027755 A CN201610027755 A CN 201610027755A CN 105670998 A CN105670998 A CN 105670998A
- Authority
- CN
- China
- Prior art keywords
- calcification
- cell
- cancer cell
- folic acid
- cancer cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Oncology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a method for calcification of cancer cells. The method comprises the following steps: (1) cancer cells are inoculated to a folic acid containing medium, incubation is carried out, and cancer cells with folic acid enriched on the surface are obtained; (2) the cancer cells with folic acid enriched on the surface are placed in calcification liquid for incubation, and calcified cancer cells are prepared. The invention also discloses an application of folic acid to preparation of a cancer cell calcification inducer. The method is based on the characteristic that cancer cells highly express folic acid receptors and folic acid carboxyl provides a nucleation site for calcification, calcium phosphate calcified layer is formed on the surface of the cancer cells, so that cell activity is inhibited, cell membrane structures are broken, and final cancer cell death is leaded to; animal experiments show that the calcification treatment does not influence blood cells and does not have hepatotoxicity, the calcification treatment almost does not damage organs and has good biosecurity, so that the technical scheme provides a feasible scheme for tumor treatment.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of method of calcification cancer cell.
Background technology
Cancer is one of disease that the death rate is the highest in the world, and keeps lasting high incidence. International medical community is generally acknowledged at presentThe method for the treatment of of cancer remain traditional chemotherapy and radiation, but Radiotherapy chemotherapy medicine is in killing cancer cell, can makeBody suffers serious damage, such as kidney exhaustion, alopecia, becomes thin etc. In addition, metastases is to cause cancer patient's deathMajor reason, the lethal higher proportion of occupying of organ failure that patient causes because of its metastases, and radiotherapy and chemotherapy medicine can not be effectiveSuppress metastases. According to statistics, the medical expense of annual cancer is very high, and is the trend increasing year by year, causes to patient familySerious economic loss and psychological burden.
Although have at present multiple emerging technology for treatment of cancer, for example, adopt the mode of pharmaceutical carrier, by controlling the chi of carrierVery little and finishing tumour-specific is identified molecule, medicine is enriched in tumor tissues, but its carrier has immunogenicity,When carrier enters after blood, the interior immune system recognition of very fast body is also eliminated, thus the result for the treatment of of losing. In recent years along with sectionLearn going deep into of research, constantly have new drug to come into the market, the cancer therapy drug (Nivolumab) of listing in 2015 is based on programmed cellThe small-molecule drug of dead albumen PD-1 design is obtained remarkable result, and be there is no apparent side effect in clinical testing, but thisA little medicines, in the expensive input of development, make the price of medicine comparatively expensive. Add up according to NIHGo out, by 2005, the treatment cost of american cancer was about 2,094 hundred million yuan, accounts for national product at the cancer disbursement of 201420% of medical treatment total value, can find out, patient will bear high medical fee in the time using this medicine thus. The transfer of tumour is itBe difficult to the major reason of curing, and in current many cancer therapy drugs, suppress the medicine wretched insufficiency of metastases, make many troubleWhen occurring metastatic tumor again after treatment, person feels simply helpless. Therefore, find the life that a kind of safe and effective and economic method suppresses tumourGrow and shift 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 causes carefullyBorn of the same parents' dysfunction, finally causes a series of disease, as CKD change and cardiovascular pathological changes etc. If calcification is drawnEnter to cancer cell surface may inhibition cancer cell active or finally cause cancer cell death, can thus be treatment of cancer providesA kind of new approaches that do not rely on medicine. In research before us, we adopt the method for chemical modification to realize yeast cellsThe calcification of wall, calcification meeting make yeast cells enter rest period stop division, this is because yeast cells has the good anti-energy of coercingPower, and contrast mammalian cell is found, mammalian cell cell membrane is more fragile and there is no cell membrane protection, and there are some researches showForm after one deck SiO2 in cell surface silication, the time of cell is no more than 12h, illustrate at the outer one deck mineral that form of cell membrane,Can have a strong impact on the activity of mammalian cell. Different from normal tissue cell, there are many specific expressed dividing on cancer cell surfaceSub-acceptor, as folacin receptor (FR), and FR does not almost express in normal tissue cell, and its part folic acid (FA), justContain well the carboxyl that can promote calcification. Therefore, utilize cancer cell height to express the characteristic of folacin receptor, seek to cause cancer cell calciumThe method of changing, suppresses by being the effective means that tumor growth shifts.
Summary of the invention
The invention provides a kind of method of calcification cancer cell, utilize the method to form layer mineral on cancer cell surface, reach inhibitionThe object of tumor cell viability.
A method for calcification cancer cell, comprises the following steps:
(1) cancer cell is inoculated in the culture medium that contains folic acid, hatches, obtain the cancer cell of surface enrichment folic acid;
(2) cancer cell of surface enrichment folic acid is placed in to calcifying solution and hatches, make the cancer cell of calcification.
The characteristic that the present invention is based on cancer cell surface high expressed folacin receptor, is inoculated into cancer cell in the culture medium that contains folic acid,Make folic acid be enriched in cancer cell surface, improve the carboxyl-content on cancer cell surface. Carboxyl is grown for calcification provides nucleation and crystalAvtive spot, causes cancer cell calcification.
The calcification cancer cell obtaining after processing according to technical method of the present invention is through ESEM and power spectrum elemental analysis calcification layerComposition be calcium phosphate, be to form by many micro-, nanoscale calcium phosphate clusters are crosslinked, and stick to surface of cell membrane. Calcium phosphateBe covered in the surface of cancer cell, limited the physiological activity of cancer cell, make tumor cell viability be subject to obvious inhibition, and finally causeBreaking of cell membrane.
As preferably, repeat step (2) for several times, obtain the cancer cell of calcification. Repeat to add fresh calcifying solution, make calcium fromSon, constantly reaction precipitation on carboxyl site of phosphate anion, at the more calcium phosphate of cancer cell surface deposition. More preferred, repeat step (2) 2～5 times.
As preferably, described cancer cell is human cervical carcinoma cell (HeLa), human breast cancer cell (MCF7) or people's gonad cell(SKVO-3). The folacin receptor that studies have shown that this three classes cell is overexpression, is conducive to the deposition of calcification layer. Cervical carcinoma,Breast cancer and oophoroma are the common malignant tumours of women, realize calcification for this three quasi-cancer cell, have for the treatment of tumourDirective significance.
For enrichment folic acid to greatest extent, cancer cell need contact fully with culture medium, and therefore the inoculum density of cancer cell is unsuitableExcessive, as preferably, the inoculum density of cancer cell is to grow to the 40-80% that cultivates orifice plate base area. More preferred, inoculationDensity accounts for the 60-70% that cultivates orifice plate base area. So both ensured that cancer cell effectively contacted with culture medium, and can make full use of againCultivate orifice plate.
As preferably, cancer cell uses phosphate buffer to clean 2-3 time before inoculation. Before cancer cell calcification processing, first clean,Avoid other materials to impact calcification effect.
Folic acid provides the avtive spot of nucleation and crystal growth for calcification, therefore, needs to ensure the folic acid that contains q.s in culture medium,As preferably, in step (1), the content of culture medium Folic Acid is 0.2-1.2mg/mL. Culture medium is conventional cancer cell culture medium.As DMEM culture medium. More preferred, the content of culture medium Folic Acid is 0.5-1mg/mL.
As preferably, in step (1), the time of hatching is 5-30min. More preferred, the time of hatching is 10-25min.
As preferably, in step (2), described calcifying solution is that calcium ion concentration is the DMEM culture medium of 5-20mM.
DMEM culture medium is originated as phosphate radical, by additional calcium ion, forms calcium phosphate (CaP) calcification at surface of cell membraneLayer. Calcium ion concentration is too low, is difficult to form precipitation, but can produces toxic and side effect to cell higher than 20mM. It is more preferred,In DMEM culture medium, calcium ion concentration is 10-15mM.
As preferably, in step (2), incubation time is 0.25-2h. Incubation time should not be too of a specified duration, along with time lengthening, calcium fromSon also can increase the toxicity of cell. More be preferably 0.5-1h.
Carbon dioxide accelerates the formation of calcium phosphate, therefore, passes into carbon dioxide in the process of hatching, and carbon dioxide has molten on a small quantityIn liquid, with the calcium binding of folic acid enrichment, and then promote precipitation to form. As preferably, incubation conditions comprises carbon dioxideConcentration be 4-10%.
The present invention also provides folic acid in the application of preparing in cancer cell calcification derivant.
The beneficial effect that the present invention possesses: (1) the present invention is based on cancer cell height expression folacin receptor and folacin-carboxyl is that calcification is carriedFor the characteristic in nucleation site, form calcium phosphate calcification layer on cancer cell surface, activity capable of inhibiting cell, the structure of destruction cell membraneAnd finally cause cancer cell death; (2) zoopery shows that calcification processing of the present invention does not affect blood cell, does not have liverToxicity, and organ is not almost damaged, there is good biological safety, therefore, technical scheme of the present invention is that tumour is controlledTreatment provides feasible scheme.
Brief description of the drawings
Fig. 1 is observation by light microscope result after human normal cell line HEK293 and human cervical carcinoma cell HeLa calcification.
Fig. 2 is that after human normal cell line HEK293 and human cervical carcinoma cell HeLa calcification, SEM observes (A) and HeLa calcigerous cellCalcium, P elements imaging results (B).
Fig. 3 is the SEM amplifying observation of HeLa cell calcification layer CaP, and wherein A is for amplifying 5000 times, and B is for amplifying 50000Doubly.
Fig. 4 is the characterization result figure of calcigerous cell sample, and wherein A is X-ray diffraction analysis (XRD), and B is that Fourier is infraredConversion spectrum is analyzed (FTIR).
Fig. 5 is the laser co-focusing observed result of different time after the calcification of HeLa cell, and wherein green fluorescence is the phosphorus of calcification layerAcid calcium (calcein dyeing), redness is cell membrane (PKH26 dyeing), blueness is nucleus (Hoechest33342 dyeing).
Fig. 6 is 24h after life or death coloration result figure (A) after HeLa cell calcification 24h and HEK293 and the calcification of HeLa cellSurvival rate MTT result (B), wherein in A, upper figure is the not coloration result of calcification of HeLa cell, result show be living cells,Figure below is the coloration result after HeLa cell calcification 24h, and it is dead cell that result shows.
Fig. 7 is the target calcification result of mouse interior tumor tissue, and wherein A figure is the tumor tissues optical photographing of different disposal groupResult; B figure is the micro-CT result of calcification tumor tissues; C figure is the tem observation result of tumor tissue section after calcification,, there is a large amount of calcium phosphate at iuntercellular in red four-headed arrow indicator cells gap; D figure is after tumor tissue section's fluorescent stainingLaser co-focusing result figure, the blue nucleus for Hoechst33342 dyeing.
Fig. 8 is the safety evaluatio of target calcification to mouse, and wherein A is mouse blood routine analysis of blood result; B is Mouse LiverFunctional biochemistry analysis result; C is the HE coloration result of mouse major organs.
Fig. 9 be target calcification for anti-tumor experiment result in Mice Body, wherein A is the mouse tumor growth of different disposal groupVolume Changes; B is that Mouse Weight changes; C is the HE coloration result of mouse tumor tissue.
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described. These embodiment are only for illustration purpose, and are not used in limitThe scope of application of the present invention processed. In experiment, DMEM culture medium is commercial prod, folic acid (FA), inorganic salts, You Ji little usedMolecule and protein are the above import reagent of chemical pure.
Embodiment 1
One, the preparation of target calcification cancer cell-first
HeLa cell is laid in 24 orifice plates, and every porocyte number is about 2 × 105, treat that cell grows to 70% of orifice plate bottom areaLeft and right, starts calcification processing. First outwell the culture medium of cell, PBS cleans 1-2 time, then in orifice plate, adds containing 300The DMEM culture medium of μ g/mLFA, is placed in incubator by cell, regulates CO2Concentration is 5%, air 95%, temperature37 DEG C, hatch after 15min, contain 11.8mMCa to adding in orifice plate2+(comprise the 10mMCaCl newly adding2) DMEMThe calcifying solution of culture medium, is placed in cell culture incubator calcification processing 30min by orifice plate, sops up calcifying solution and adds fresh calcifying solution to continueThe continuous 60min that processes. Process in contrast separately with FA and calcifying solution, after finishing dealing with, solution is sopped up, can obtain calcificationCancer cell.
Three, the preparation of target calcification cancer cell-second
HeLa cell is laid in 24 orifice plates, and every porocyte number is about 2 × 105, treat that cell grows to 70% of orifice plate bottom areaLeft and right, starts calcification processing. First the culture medium of cell is changed, used the phosphate buffer of pH7.2 to clean 1-2 time, thenTo adding the DMEM culture medium that additionally newly adds 200 μ g/mLFA in orifice plate, then by cell as for regulating CO in incubator2Concentration is 5%, air 95%, and 37 DEG C of temperature are hatched after 20min in incubator, sop up culture medium, in orifice plate, addContain 21.8mMCa2+(comprise the 20mMCaCl newly adding2) the calcifying solution of DMEM culture medium, orifice plate is placed in to cellCalcification processing 30min in incubator. Process in contrast separately with FA and calcifying solution, after finishing dealing with, solution is sopped up,Can obtain the cancer cell of calcification.
Three, the preparation of target calcification cancer cell-the third
HeLa cell is laid in 24 orifice plates, and every porocyte number is about 2 × 105, treat that cell grows to 70% of orifice plate bottom areaLeft and right, starts calcification processing. First the culture medium of cell is changed, used the phosphate buffer of pH7.2 to clean 1-2 time, thenTo adding the DMEM culture medium that additionally newly adds 200 μ g/mLFA in orifice plate, then by cell as for regulating CO in incubator2Concentration is 5%, air 95%, and 37 DEG C of temperature are hatched after 25min in incubator, sop up culture medium, in orifice plate, addContain 16.8mMCa2+(comprise the 15mMCaCl newly adding2) the calcifying solution of DMEM culture medium, orifice plate is placed in to cellCalcification processing 40min in incubator, reprocessing 1 time, processes in contrast separately with FA and calcifying solution, after finishing dealing withSolution is sopped up, can obtain the cancer cell of calcification.
Four, the preparation of target calcification cancer cell-fourth
HeLa cell is laid in 24 orifice plates, and every porocyte number is about 2 × 105, treat that cell grows to 70% of orifice plate bottom areaLeft and right, starts calcification processing. First the culture medium of cell is changed, used the phosphate buffer of pH7.2 to clean 1-2 time, thenTo adding the DMEM culture medium that additionally newly adds 200 μ g/mLFA in orifice plate, then by cell as for regulating CO in incubator2Concentration is 5%, air 95%, and 37 DEG C of temperature are hatched after 25min in incubator, sop up culture medium, in orifice plate, addContain 16.8mMCa2+(comprise the 15mMCaCl newly adding2) the calcifying solution of DMEM culture medium, orifice plate is placed in to cellCalcification processing 30min in incubator, reprocessing 2 times, processes in contrast separately with FA and calcifying solution, after finishing dealing withSolution is sopped up, can obtain the cancer cell of calcification.
Five, the sign of target calcification cancer cell
After cell calcification completes, adopt after the fixing 30min of 4% paraformaldehyde room temperature, PBS cleans 2-3 time, then uses gradientEthanol (70%, 80%, 90%, 95% and 100%) is washing dehydration respectively, after ethanol volatilization, is placed under SEMObserve, and the calcification layer of the cell surface after calcification is carried out to elementary analysis, adopt XRD and FTIR to calcification sample simultaneouslyProduct are analyzed.
With human normal cell (HEK293) in contrast, do as above to process.
On calcification cancer cell pattern prepared by the first and second the third 4 kinds of methods of fourth without significant difference. The calcification cancer cell of preparing taking first is example,As shown in Figure 1,2,3, 4, HeLa cell is through calcification processing for result, and cell surface forms calcification layer, and Fig. 2 adopts scanning electricityElementary analysis show that calcification layer becomes to contain calcium and phosphorus to mirror with power spectrum; The demonstration of calcification layer amplifying observation is formed by irregular cluster, ginsengSee Fig. 3; Fig. 4 mid infrared spectrum result is 1000cm in wave number-1And 500cm-1Left and right is the now phase transformation of crystal not, in conjunction withX-ray diffraction result can illustrate that the calcification layer of formation is unformed calcium phosphate. Human normal cell (HEK293) is through calcification placeAfter reason, cell surface does not form calcification calcium phosphate. Above result shows to adopt method of the present invention to realize the target of cancer cellCalcification.
Embodiment 2
Adopt laser confocal microscope, dyeing and MTT experimental technique detect the kill and wound work of calcification processing to cancer cell anywayWith. Concrete operations are as follows:
One, the variation after cell calcification
Before calcification processing, adopt respectively living cells dyestuff PKH26 and Hoechst33342 cell membrane and the nucleus to cancer cellCarry out fluorescent staining. After calcification processing completes, ore bed is carried out to fluorescent staining with calcium specificity fluorescent dyestuff calcein, dyeingAfter completing, respectively in confocal laser scanning microscope cellular morphology over time, result is referring to Fig. 5.
Two, cell dyeing anyway
(1) HEK293, MCF7 and HeLa cell are with 1 × 104The density in individual/hole is inoculated in respectively in 96 orifice plates, cultivates 24hAfter, cell is carried out to calcification processing, calcification processing method is with embodiment 1.
(2) after calcification processing completes, add DMEM culture medium, continue to cultivate 24h.
(3) PBS washed cell three times, then adds staining reagent (Invitrogen) anyway, hatches 30min in cell culture incubator,In fluorescence microscopy Microscopic observation.
Three, MTT
(1) HEK293, MCF7 and HeLa cell are with 1 × 104The density in individual/hole is inoculated in respectively in 96 orifice plates, cultivates 24hAfter, cell is carried out to calcification processing, and process in contrast separately with FA and calcifying solution.
(2) after finishing dealing with, add culture medium to continue to cultivate 24h, then add 5mg/mLMTT (20 μ L) to reaction systemIn, cultivate 4h, orifice plate solution is sopped up, add 150 μ LDMSO to jolt 5-10min, use microwell plate spectrophotometer(Bio-Tek) absorption value of measurement 570nm, this absorption value and first a ceremonial jade-ladle, used in libation concentration (cell succinate dehydrogenase and MTT product)Be directly proportional, it has represented the activity of succinate dehydrogenase, and then has indicated cytoactive.
Result demonstration, the calcium phosphate that calcification forms is covered in the surface of cancer cell, and the physiological activity of meeting restrictive cell is lived cellProperty is subject to obvious inhibition, and finally causes breaking of cell membrane, referring to Fig. 5. The calcification of coloration result explanation target can anyway for cellBe used for killing cancer cell, referring to Fig. 6 A, and after calcification processing, the survival rate of HeLa cell is suppressed more than 60%, and HEK29Cell still can keep more than 80% survival rate, and referring to Fig. 6 B, this is the folacin receptor lower due to HEK293 cell surfaceThe low calcification efficiency that expression causes causes, further illustrate calcification can be specifically at the cancer cell table of folacin receptor high expressedFace occurs.
Embodiment 3
Build the effect of HeLa animal model for tumour for detection of target calcification in body. Operation is as follows with testing process:
(1) HeLa cell line is with 5 × 106It is subcutaneous as planting mouse resistance model that individual/amount is only inoculated into nude mice (20 nude mices, female),After 10 days, treat that gross tumor volume grows to 80mm3Time, start test.
(2) folic acid of lumbar injection 200 μ L, 1mg/mL, waits for 15min left and right, treats that folic acid is enriched in tumor locus, toInjection high calcium DMEM culture medium (50mMCa in tumor tissues2+), inject every day 1 time, process continuously 15 days, observe swollenThe calcification effect of knurl. To inject separately calcifying solution and folic acid and blank group in contrast.
(3) adopt micro-CT technology to tumor scan, three-dimensional reconstruction after the line scanning of going forward side by side, determines tumour calcification result; By calciumChange tumour and cut into slices, adopt calcein to carry out fluorescent staining to tumor biopsy, after dyeing, adopt laser co-focusing to observe; WillTumor biopsy is in the pattern of transmission electron microscope observation calcification layer and the form of cancer cell.
(4) in experiment, blood routine and biochemistry to mouse are analyzed, and mouse major organs are carried out to HE dyeing simultaneously and commentEstimate the security of the method.
Result shows, calcification can be effectively by tumour cell parcel, and tumor tissues tumour after parcel occurs downright bad referring to Fig. 7 A;The scanning result of Micro-CT to tumour, arrow has been pointed out respectively the calcium phosphate that tumor tissues and calcification form, referring to Fig. 7 B;In tem observation tumor biopsy, the pattern of calcium phosphate and the form of tumour cell explanation calcification can be formed at tumor tissues cancer cellSurface, referring to Fig. 7 C; Tumor tissue section's laser co-focusing after Hoechst33342 staining cell core observed result of taking pictures is aobviousShow that pyknosis appears in neoplastic cell nuclei, illustrate that calcification has suppressed the activity of tumor tissues, referring to Fig. 7 D. Calcification processing is to little in additionThe blood cell of mouse does not affect, and there is no hepatotoxicity wind agitation, and organ HE coloration result demonstration calcification processing is not almost damaged organWound, referring to Fig. 8.
Embodiment 4
Build the cancer resistant effect of HeLa mice with tumor animal model for detection of target calcification. Operation is as follows with testing process:
(1) HeLa cell line is with 5 × 106It is subcutaneous as nude mice model that individual/amount is only inoculated into nude mice (40 nude mices, female), 10 daysAfter treat that gross tumor volume grows to 80mm3Time, start test.
(2) folic acid of lumbar injection 200 μ L1mg/mL, then waits for 10min left and right, treats that folic acid is enriched in tumor locus,In tumor tissues, inject 100 μ L high calcium DMEM culture medium (50mMCa2+), inject every day 1 time, process continuously 15 days,Observe the calcification effect of tumour. To inject separately calcifying solution and folic acid and blank group in contrast.
(3) observe mouse sign every day, every 2 day entry Mouse Weights, measure major diameter L and the minor axis B of tumour, profitWith formula V=0.5 × L × B2Calculate mouse volume.
After (4) 30 days, put to death mouse, take out tumour and weigh, Taking Pictures recording, and tumour is carried out to HE dyeing.
(5) choose mouse cancer cell lines 4T1/luc, with 5 × 106Individual/amount is only inoculated into mouse (40 nude mices, female) mammary gland place, 10After it, treat that gross tumor volume grows to 80mm3Shi Zuowei model starts calcification processing, and processing method is consistent with step (2). Adopt petty actionThing living imaging technology detects the transfer of mouse tumor, records the death rate of mouse simultaneously.
Result demonstration, calcification can effectively suppress gross tumor volume growth, referring to Fig. 9 A; The body weight of mouse is compared with control group in additionDo not change, further illustrating the method does not have apparent side effect to mouse; HE coloration result shows calcification tumor site HE dyeingCan deepen, participate in Fig. 9 CFA/Ca processed group, there is large-area cell death around in calcified tissue. FurtherlyBright the method can effectively suppress tumor growth. In conjunction with Fig. 7, can draw, this method can realize certain by cancer cell in tumourTarget calcification, and then the growth of inhibition tumour, thus realize effectively effect. Said in conjunction with Fig. 8 by Mouse Weight data (Fig. 9 B)Bright the method has good biological safety.
Claims (10)
1. a method for calcification cancer cell, is characterized in that, comprises the following steps:
(1) cancer cell is inoculated in the culture medium that contains folic acid, hatches, obtain the cancer cell of surface enrichment folic acid;
(2) cancer cell of surface enrichment folic acid is placed in to calcifying solution and hatches, make the cancer cell of calcification.
2. the method for claim 1, is characterized in that, repeats step (2) for several times, obtains the cancer cell of calcification.
3. the method for claim 1, is characterized in that, described cancer cell is human cervical carcinoma cell (HeLa), people's mammary glandCancer cell (MCF7) or people's gonad cell (SKVO-3).
4. the method for claim 1, is characterized in that, the inoculum density of cancer cell is cultivated orifice plate base area for growing to40-80%.
5. the method for claim 1, is characterized in that, in step (1), the content of culture medium Folic Acid is 0.2-1.2mg/mL。
6. the method for claim 1, is characterized in that, in step (1), the time of hatching is 5-30min.
7. the method for claim 1, is characterized in that, in step (2), described calcifying solution is that calcium ion concentration is 5-20The DMEM culture medium of mM.
8. the method for claim 1, is characterized in that, in step (2), incubation time is 0.25-2h.
9. the method for claim 1, is characterized in that, incubation conditions comprises that the concentration of carbon dioxide is 4-10%.
10. folic acid is in the application of preparing in cancer cell calcification derivant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610027755.XA CN105670998B (en) | 2016-01-15 | 2016-01-15 | A kind of method of calcification cancer cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610027755.XA CN105670998B (en) | 2016-01-15 | 2016-01-15 | A kind of method of calcification cancer cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105670998A true CN105670998A (en) | 2016-06-15 |
| CN105670998B CN105670998B (en) | 2019-01-25 |
Family
ID=56301025
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610027755.XA Active CN105670998B (en) | 2016-01-15 | 2016-01-15 | A kind of method of calcification cancer cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105670998B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111534488A (en) * | 2020-04-03 | 2020-08-14 | 浙江大学 | Chemically modified osteoclast, preparation method and application |
| CN114163508A (en) * | 2020-09-11 | 2022-03-11 | 王月志 | Amino acid sequences capable of destroying cells and related nucleotide sequences and related uses |
| CN114652852A (en) * | 2020-12-22 | 2022-06-24 | 浙江大学 | Molecule for inducing spontaneous calcification of tumor cells and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103417977A (en) * | 2013-07-30 | 2013-12-04 | 天津城建大学 | Efficient folate receptor alpha subtype-mediated target drug delivery system |
-
2016
- 2016-01-15 CN CN201610027755.XA patent/CN105670998B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103417977A (en) * | 2013-07-30 | 2013-12-04 | 天津城建大学 | Efficient folate receptor alpha subtype-mediated target drug delivery system |
Non-Patent Citations (2)
| Title |
|---|
| 赵杰等: "叶酸受体介导的抗肿瘤药物研究进展", 《药学学报》 * |
| 郭姗姗等: "模板法仿生合成磷酸钙纳米粒", 《中国科技论文在线精品论文》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111534488A (en) * | 2020-04-03 | 2020-08-14 | 浙江大学 | Chemically modified osteoclast, preparation method and application |
| CN111534488B (en) * | 2020-04-03 | 2021-12-21 | 浙江大学 | Chemically modified osteoclast, preparation method and application |
| CN114163508A (en) * | 2020-09-11 | 2022-03-11 | 王月志 | Amino acid sequences capable of destroying cells and related nucleotide sequences and related uses |
| WO2022053050A1 (en) * | 2020-09-11 | 2022-03-17 | 王月志 | Amino acid sequence that can destroy cells, and related nucleotide sequence and related uses thereof |
| CN114729021A (en) * | 2020-09-11 | 2022-07-08 | 王月志 | Amino acid sequences capable of destroying cells and related nucleotide sequences and related uses |
| CN114652852A (en) * | 2020-12-22 | 2022-06-24 | 浙江大学 | Molecule for inducing spontaneous calcification of tumor cells and application thereof |
| WO2022135381A1 (en) * | 2020-12-22 | 2022-06-30 | 浙江大学 | Molecule for inducing spontaneous calcification of tumor cells and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105670998B (en) | 2019-01-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108187072A (en) | The nano material of manganese dioxide and preparation method and application that albumin is stablized | |
| CN105062964B (en) | A kind of induction liquid for improving stem cell Osteoblast Differentiation efficiency and its application | |
| CN105106958B (en) | Copper-based human serum albumin nano-complex near infrared light fuel factor and its preparation method and application | |
| CN105670998A (en) | Method for calcification of cancer cells | |
| CN104223115B (en) | The new application of scale collagen polypeptide | |
| CN104971365B (en) | The new application of nano carbon mixed suspension injection | |
| CN103976803A (en) | Human colon cancer nude mouse skin subcutaneous transplantation tumor model establishing method | |
| CN113456836B (en) | Manganese-heme coordination polymer nanoparticle and preparation method and application thereof | |
| CN107952070A (en) | The photo-thermal therapy multifunctional nano hybrid and preparation method of dual imaging guiding | |
| EP3682899A1 (en) | Compound amphiphilic peptide nanomicelle, preparation and use thereof | |
| CN103073645B (en) | Biologically mineralized insulin protein nano particle and preparation method and application thereof | |
| CN103550282B (en) | A kind of method preparing water melon frost and its anti-infectious medical usage | |
| CN107715110A (en) | Biodegradable black phosphorus base X ray photosensitizer for photodynamic therapy and preparation method and application | |
| CN104127887B (en) | Fe5c2nano-particle application in preparing bio-imaging probe | |
| CN114432347B (en) | Application of nervonic acid-producing microalgae in preparation of product for treating spinal cord injury | |
| CN113318228A (en) | Photocatalytic/photothermal agent and application thereof in preparation of medicine for renal cancer photodynamic/photothermal therapy | |
| CN102716137B (en) | Preparation method and application of fluorescence nanometer diamond-doxycycline compound | |
| CN101831404B (en) | Lymph gland targeted metastatic human hepatoma cell strain and establishing method thereof | |
| BRAHMACHARI | The role of science in the recent progress of medicine | |
| CN107485714A (en) | A kind of smooth thermotherapeutic agent/photoacoustic contrast agent and its preparation method and application | |
| CN104312969B (en) | Brain and spinal cord acute ischemia/reperfusion injury cellular network model and its construction method and application | |
| CN113117078A (en) | Novel tumor treatment drug AuNCs @ GTTN and preparation method and application thereof | |
| CN102503892A (en) | Co-containing sandwich heteropolyacid as well as synthesis method and application thereof | |
| CN106176747B (en) | Tacrine is preparing the application in treating treating echinococcosis | |
| CN104825507A (en) | Radix actinidiae chinensis extract and application thereof in preparation of drugs for treating bile duct cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |