CN105219730A - Ovarian cancer cancerous tissue 3D cultural method and the application in evaluating drug effect thereof - Google Patents

Ovarian cancer cancerous tissue 3D cultural method and the application in evaluating drug effect thereof Download PDF

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CN105219730A
CN105219730A CN201510755769.9A CN201510755769A CN105219730A CN 105219730 A CN105219730 A CN 105219730A CN 201510755769 A CN201510755769 A CN 201510755769A CN 105219730 A CN105219730 A CN 105219730A
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nutrient solution
ovarian cancer
tissue
cultural method
tumor tissue
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李月娟
易月娥
顾阳
陆益彬
陈海宁
左从林
孙云霞
冯宇霞
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Joinn Suzhou Drug Research Center Co Ltd
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Abstract

The invention discloses a kind of 3D cultural method of ovarian cancer cancerous tissue, comprising step is: in orifice plate, drip Matrigel glue, room temperature or CO 2leave standstill in incubator and make gelling solid; The tumor tissue be dipped in nutrient solution moved on the described Matrigel glue after solidifying, then drip Matrigel glue in tumor tissue, same method makes gelling solid; Add nutrient solution and cultivate 1-3 days; Take out tumor tissue and fix.Can chemotherapeutics be added during cultivation, fixing organization blocks after cultivating, observation of cell shape after dyeing, calculate core abnormal rate.The cultural method that the present invention sets up and Testing index can evaluate effect and the action character of antitumor drug sooner, more accurately, the deficiency of cell 2D cultivation and experimentation on animals evaluation technique can be made up, be expected to promote the research and development of China's novelty cancer therapy drug and the process of precision treatment.

Description

Ovarian cancer cancerous tissue 3D cultural method and the application in evaluating drug effect thereof
Technical field
The present invention relates to field of biology, particularly relate to a kind of ovarian cancer cancerous tissue 3D cultural method and the application in evaluating drug effect thereof.
Background technology
Tumour is one of principal disease of serious harm people's health, the genesis mechanism of tumour is various and complicated, but chemotherapeutics conventional is at present nearly all the cell toxicity medicament for multiplication capacity rich cells, also produces toxic side effect to high proliferation cell normal in human body.Therefore, many medicament research and development mechanisms are all devoted to develop the targeting antineoplastic medicine thing of efficient, low toxicity and high specific, and the single screening anti-tumor medicine method of tradition has also been not enough to the demand dealing with the development of antitumor drug pharmacodynamics.
Along with the exploitation of deepening continuously of being familiar with tumor pathogenesis and tumor-targeting medicine, precision medical treatment becomes the trend of future tumors medical research and application gradually, and the key of precision treatment is that the diagnosis of oncogene and biomolecules mark, microenvironment are on the impact of tumour progression and curative effect and the individuation selection based on the cancer therapy drug kind under the former condition and dosage.
The 2D culture systems of the in vitro method evaluated of current screening anti-tumor medicine mainly tumour cell, in body, screening and evaluating system mainly uses animal model.
When 2D cultivates, tumour cell is in form, differentiation, amplification, very large different to all also existing from its state in vivo in the metabolic reaction stimulated and gene protein expression etc., many transgenations existed in tumor tissues, do not exist in tumor cell line.2D cultivates the microenvironment also lacking tumour cell existence, and the microenvironment of growth of tumour cell all has tremendous influence to its biological character and signal transduction path, can not true reappearance be this influences each other even the 2D of primary tumor cell cultivates, cannot provide with live tumor tissue closer to organoid and fresh tumor tissue 3D the powerful information that can provide is provided.In addition, the matrix components in microenvironment, vascular system, all kinds of somatomedin, immuno-stimulator and immunocyte etc. also have a great impact the biological character of cell and the susceptibility of antitumor drug and tolerance.And the Evaluation system used at present, owing to there is larger species variation with the mankind, the metabolic process of medicine in human body, drug action and toxic side effect can not be reflected completely.Therefore, many in pre-clinical assessment the potential medicine of tool, but have failed when clinical trial, demonstrate the Evaluation in Vivo and in Vitro system used at present and can not predict the validity of medicine in human body and potential safety hazard well.The 3D cultural method that foundation can simulate people's tumor tissues is better the active demand that antitumor drug develops.
The susceptibility of different tumour patients to different chemotherapeutics also exists difference, even same antitumor drug, also shows different results for the treatment of and prognosis with it at different tumour patients individuality.Occur that the major cause of this situation is relevant with the interaction of complicated Tumor Heterogeneity and tumour cell and microenvironment, therefore, provide the developing direction that precision diagnosis and treatment have targetedly become oncotherapy.But the main method that auxiliary precision diagnosis and treatment are implemented at present is the mark of gene sequencing and biomolecules; Though carried out the drug evaluation experiment of individuation clinically, but the method used mainly is cultivated and the appraisement system of people's tumor tissues in nude mouse (PDX) from the 2D of the tumour cell of specimens separation, not only there are the problems referred to above, and consuming time, effort, affect and remedy measures is targetedly taked in time to patient.
Summary of the invention
The technical problem that the present invention mainly solves is to provide a kind of ovarian cancer cancerous tissue 3D cultural method and the application in evaluating drug effect thereof, be expected to solve in the evaluation method of antitumor drug conventional at present also exist species variation on the impact of evaluation result, can not the problems such as accurate response drug effect, evaluation time be oversize.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is: provide a kind of ovarian cancer cancerous tissue 3D cultural method, comprising step is: (1) drips Matrigel glue in orifice plate, room temperature or CO 2leave standstill in incubator and make described Matrigel gelling solid;
(2) tumor tissue be dipped in the first nutrient solution is moved on the described Matrigel glue after solidifying, then in described tumor tissue, drip Matrigel glue, room temperature or CO 2leave standstill in incubator and make described Matrigel gelling solid;
(3) in the orifice plate of step (1), add the second nutrient solution to cultivate, take out the described tumor tissue after cultivating again and fix, the component that wherein said second nutrient solution comprises has DMEM in high glucose nutrient solution, F12 nutrient solution, epithelical cell growth factor EGF, fibroblast growth factor FGF and 10 × N 2.
In a preferred embodiment of the present invention, the DMEM dilution of Matrigel glue described in step (1) and step (2), the volume ratio of described DMEM and described Matrigel glue is 1:1-1:5.
In a preferred embodiment of the present invention, described in step (1) and step (2), each amount dripping the Matrigel glue after dilution is 10-50 μ L.
In a preferred embodiment of the present invention, described in step (2), the size of tumor tissue is 0.5-1mm 3, described tumor tissue is first with being dipped in DMEM nutrient solution again after buffer solution, the making processes of described tumor tissue is carried out on trash ice.
In a preferred embodiment of the present invention, the second nutrient solution described in step (3) is by described DMEM in high glucose nutrient solution, described F12 nutrient solution, described 10 × N 2by volume for 34:11:5 mixes, form mixed solution, described mixed solution is that 500:1:1 carries out being mixed to get described second nutrient solution with described epithelical cell growth factor EGF, described fibroblast growth factor FGF according to volume ratio again.
In a preferred embodiment of the present invention, described in step (3), incubation time is respectively 24h, 48h and 72h, stop cultivate after described tumor tissue is taken out from described second nutrient solution, be fixed on mass percent be 10% neutral formalin stationary liquid or mass percent be in the paraformaldehyde stationary liquid of 4%.
In a preferred embodiment of the present invention, also comprise in step (3) and paraffin embedding, section, HE dyeing and Ki67 immunohistochemical staining are carried out to fixing described tumor tissue, observe the change of ovarian cancer tissue's structure and cellular form.
In a preferred embodiment of the present invention, also comprise in step (3) and add chemotherapeutics in described second nutrient solution, continue to cultivate 24h, 48h and 72h, then described tumor tissue is taken out from described second nutrient solution, be fixed on mass percent be 10% neutral formalin stationary liquid or mass percent be in the paraformaldehyde stationary liquid of 4%, again paraffin embedding is carried out to fixing described tumor tissue, section, dyeing, observe the change of tumor tissues weave construction and cellular form, counting cells nuclear distortion number, calculate nuclear distortion rate, analyze the nuclear distortion rate feasibility as antitumor drug method of evaluating drug effect and index.
In a preferred embodiment of the present invention, described dyeing is HE dyeing or Ki67 immunohistochemical staining.
The invention has the beneficial effects as follows: the 3D cultural method of the human ovarian cancer tissue that the present invention sets up, the pharmacodynamic results of antitumor drug can be evaluated sooner, more accurately, the weak link of 2D cell cultures and experimentation on animals evaluation technique can be made up, be expected to promote the research and development of China's novelty cancer therapy drug and the process of precision treatment.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing used required in describing embodiment is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings, wherein:
Fig. 1 be in the present invention ovarian cancer tissue at cultivation 0 day (A), 1 day (B), 2 days (C), 3 days (D)
Time weave construction and cell shape, be HE coloration result shown in figure, magnification is 400 ×;
Fig. 2 is that in the present invention, ovarian cancer tissue adds docetaxel micella when cultivating simultaneously, continues cultivation 1
My god (A), 2 days (B), 3 days (C) time weave construction and the change of cell shape, be HE coloration result shown in figure, magnification is 400 ×;
Fig. 3 be in the present invention ovarian cancer tissue at cultivation 0 day (A), 1 day (B), 2 days (C), 3 days (D)
Time weave construction and cell shape, be Ki67 immunohistochemical staining result shown in figure, magnification is 400 ×;
Fig. 4 is that in the present invention, ovarian cancer tissue adds docetaxel micella when cultivating simultaneously, continues cultivation 1
My god (A), 2 days (B), 3 days (C) time weave construction and the change of cell shape, be Ki67 coloration result shown in figure, magnification is 400 ×;
Fig. 5 is that in the present invention, ovarian cancer tissue adds docetaxel micella when cultivating simultaneously, then cultivates 0
My god, 1 day, 2 days, 3 days time nucleus occur distortion ratio (per-cent), shown in figure be to Ki67 dyeing counting statistics result.
Embodiment
Be clearly and completely described to the technical scheme in the embodiment of the present invention below, obviously, described embodiment is only a part of embodiment of the present invention, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making other embodiments all obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment one:
There is provided a kind of ovarian cancer cancerous tissue 3D cultural method, comprising step is:
(1) making of tumor tissue
Get tumor tissues, adopt ophthalmic instruments to be cut into 0.5-1mm 3tissue block, with after phosphate buffered saline buffer (1 × PBS) washing, to be dipped in DMEM nutrient solution mid-for subsequent use on ice;
(2) tumor tissue cultivates
Get 6 orifice plates, 24 orifice plates or 96 orifice plates, the Matrigel glue of (be 1:1-1:5 with the Dilution ratio of DMEM) after diluting along base topical drops 10-50 μ L, room temperature or CO 2place 10-30min in incubator, carefully tissue block moved on this Matrigel solidified, then drip in tissue block 10-50 μ L dilute after the Matrigel glue of (be 1:1-1:5 with the Dilution ratio of DMEM), put CO 220-30min is left standstill in incubator.
Take out orifice plate, carefully add 0.4mL nutrient solution to 24 orifice plate, 0.1mL to 96 orifice plate or 2.5mL to 6 orifice plate and cultivate, the composition of described nutrient solution is as shown in the table, is the amount of various compositions contained in 100mL nutrient solution in table.
Nutrient solution composition Content
DMEM in high glucose nutrient solution 68 mL
F12 nutrient solution 22 mL
EGF 10-30 ng/mL
FGF 10-30 ng/mL
10×N 2 10 mL
Carefully take out tissue block respectively at after cultivation when 24h, 48h, 72h, put into rapidly mass percent be 10% neutral formalin stationary liquid or mass percent be 4% the fixing at least 72h of paraformaldehyde stationary liquid.
Carry out conventional paraffin embedding, section, HE dyeing and Ki67 immunohistochemical staining to fixing tumor tissue, wherein HE dyeing and Ki67 immunohistochemical staining are the dyeing processs as judging medicine effect.
Under the above-mentioned culture condition of Microscopic observation, the weave construction of tumor tissues and the change of cellular form, specifically refer to Fig. 1 and Fig. 3.Fig. 1 is the weave construction of ovarian cancer tissue when cultivating 0 day (A), 1 day (B), 2 days (C), 3 days (D) under above-mentioned culture condition and cell shape, is HE coloration result shown in figure, magnification is 400 ×.Fig. 3 is the weave construction of ovarian cancer tissue when cultivating 0 day (A), 1 day (B), 2 days (C), 3 days (D) under above-mentioned culture condition and cell shape, is Ki67 immunohistochemical staining result shown in figure, magnification 400 ×.As can be seen from Fig. 1 and Fig. 3, when ovarian cancer cultivates 24h, 48h under above-mentioned culture condition, weave construction does not have considerable change; When cultivating 72h, interstitial composition reduces, but nuclear shape and the dyeing depth change not obvious.
Embodiment two:
The tumor tissues that 3D cultivates is to the feasibility analysis of antitumor drug evaluating drug effect
When ovarian cancer tissue cultivates, different concns is added in nutrient solution, different types of chemotherapeutics, different time after cultivation, take out tissue, make frozen section or be fixed in above-mentioned stationary liquid, dye through HE, Ki67 immunohistochemical staining or accordingly other dyeing, Microscopic observation weave construction and cellular form, especially the change of nuclear shapes and the dyeing depth, the cell count that random counter nucleus occurs to distort accounts for the ratio (per-cent) of total cell count, compare the difference with the nuclear aberration rate of the sample not adding antitumor drug, evaluate the drug action of medicine, specifically consult Fig. 2 and Fig. 4.
Fig. 2 is that ovarian cancer adds docetaxel micella when cultivating under the culture condition of embodiment one simultaneously, weave construction when continuation cultivation 1 day (A), 2 days (B), 3 days (C) and the change of nuclear shapes, HE coloration result shown in figure, magnification is 400 ×.
Fig. 4 is that ovarian cancer adds docetaxel micella when cultivating under the culture condition of embodiment one simultaneously, weave construction when continuation cultivation 1 day (A), 2 days (B), 3 days (C) and the change of nuclear shapes, Ki67 immunohistochemical staining result shown in figure, magnification is 400 ×.As can be seen from Fig. 2, Fig. 4, to add after cytotoxic anti-tumor drug docetaxel micella 1 day, the nuclear distortion of ovarian cancer tissue and engrain and showed increased.Fig. 5 is counting to nuclear aberration and engrain rate and statistics.
Experimental result shows, the ovarian cancer 3D culture technique set up can as the index evaluating antitumor drug evaluating drug effect, and reproducible, result stable, can reflect pharmaceutically-active mechanism features, present short, that compare routine experiment in vitro more time-consuming than effect experiment in animal body and more can reflect the advantageous feature that tumor structure changes.Although tumor tissues after incubation 72h intercellular substance structure has comparatively considerable change, nuclear shape and dyeing change not obvious; And after medication during 24h the nucleus of tumor tissues there is obvious deformity, 3D cultivates the change of weave construction that 72h occurs does not affect the pharmacodynamic evaluation result of this method to antitumor drug.
Ovarian cancer is one of common malignant tumour of female sex organ, and sickness rate is only second to cervical cancer and carcinoma of uterine body and arranges the 3rd, residence, causes serious threat to women's life.
Docetaxel (docetaxel, DTX) is s-generation Japanese yew class high efficiency anti-tumor medicine, is that the precursor compound 10-DAB that extracts from Ramulus et folium taxi cuspidatae is through the semi-synthetic bearing taxanes obtained.Its mechanism of action strengthens tubulin polymerization effect and suppresses microtubule depolymerization effect, causes forming stable non-functional microtubule fasolculus, thus tumoricidal mitotic division.FDA approved is clinically for the treatment of ovarian cancer, mammary cancer, nonsmall-cell lung cancer and prostate cancer.The clinical research confirmation of I phase and II phase, docetaxel associating platinum medicine has good curative effect as first-line drug treatment ovarian cancer, and efficient is 58% ~ 89%.
The docetaxel formulation of current listing only has a kind of injection type, containing tween-80 in preparation, and tween-80 can cause serious anaphylaxis in vivo, and docetaxel can be disturbed to be combined with the form of concentration dependant with serum albumin, make it be that nonlinear pharmacokinetics changes.In order to increase its solubleness, reduce toxicity, there is multiple novel form if prodrug, emulsification, micella, liposome, nanoparticle etc. are all in research and development.
The present invention uses the Xenografts in nude mice experimental observation of the traditional evaluation antitumor drug antitumor drug effect of docetaxel micella, to find after administration 29 days, the knurl volume inhibiting rate of 5mg/kg, 10mg/kg, 20mg/kg docetaxel micella to the ovarian cancer of subcutaneous transplantation is respectively 45.79%, 82.44%, 80.40%; 50.04%, 83.41% and 83.04% is respectively to the tumor weight inhibiting rate of ovarian cancer.And using present method, after administration 1 day, the nuclear aberration rate that docetaxel micella (0.5 μ Lg/mL) causes ovarian cancer cell is 45.63%, has reacted action effect and the feature of medicine.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize description of the present invention to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other relevant technical field, be all in like manner included in scope of patent protection of the present invention.

Claims (9)

1. an ovarian cancer cancerous tissue 3D cultural method, is characterized in that, comprises step to be: (1) drips Matrigel glue in orifice plate, room temperature or CO 2leave standstill in incubator and make described Matrigel gelling solid;
(2) tumor tissue be dipped in the first nutrient solution is moved on the described Matrigel glue after solidifying, then in described tumor tissue, drip Matrigel glue, room temperature or CO 2leave standstill in incubator and make described Matrigel gelling solid;
(3) in the orifice plate of step (1), add the second nutrient solution to cultivate, take out the described tumor tissue after cultivating again and fix, the component that wherein said second nutrient solution comprises has DMEM in high glucose nutrient solution, F12 nutrient solution, epithelical cell growth factor EGF, fibroblast growth factor FGF and 10 × N 2.
2. ovarian cancer cancerous tissue 3D cultural method according to claim 1, is characterized in that, the DMEM dilution of Matrigel glue described in step (1) and step (2), the volume ratio of described DMEM and described Matrigel glue is 1:1-1:5.
3. ovarian cancer cancerous tissue 3D cultural method according to claim 2, is characterized in that, described in step (1) and step (2), each amount dripping the Matrigel glue after dilution is 10-50 μ L.
4. ovarian cancer cancerous tissue 3D cultural method according to claim 1, it is characterized in that, described in step (2), the size of tumor tissue is 0.5-1mm 3, described tumor tissue is first with being dipped in DMEM nutrient solution again after buffer solution, the making processes of described tumor tissue is carried out on trash ice.
5. ovarian cancer cancerous tissue 3D cultural method according to claim 1, is characterized in that, the second nutrient solution described in step (3) is by described DMEM in high glucose nutrient solution, described F12 nutrient solution, described 10 × N 2by volume for 34:11:5 mixes, form mixed solution, described mixed solution is that 500:1:1 carries out being mixed to get described second nutrient solution with described epithelical cell growth factor EGF, described fibroblast growth factor FGF according to volume ratio again.
6. ovarian cancer cancerous tissue 3D cultural method according to claim 1, it is characterized in that, described in step (3), incubation time is respectively 24h, 48h and 72h, stop cultivate after described tumor tissue is taken out from described second nutrient solution, be fixed on mass percent be 10% neutral formalin stationary liquid or mass percent be in the paraformaldehyde stationary liquid of 4%.
7. ovarian cancer cancerous tissue 3D cultural method according to claim 1, it is characterized in that, also comprise in step (3) and paraffin embedding, section, HE dyeing and Ki67 immunohistochemical staining are carried out to fixing described tumor tissue, observe the change of ovarian cancer tissue's structure and cellular form.
8. ovarian cancer cancerous tissue 3D cultural method according to claim 1, it is characterized in that, also comprise in step (3) and add chemotherapeutics in described second nutrient solution, continue to cultivate 24h, 48h and 72h, then described tumor tissue is taken out from described second nutrient solution, be fixed on mass percent be 10% neutral formalin stationary liquid or mass percent be in the paraformaldehyde stationary liquid of 4%, again paraffin embedding is carried out to fixing described tumor tissue, section, dyeing, observe the change of tumor tissues weave construction and cellular form, counting cells nuclear distortion number, calculate nuclear distortion rate, analyze the nuclear distortion rate feasibility as antitumor drug method of evaluating drug effect and index.
9. ovarian cancer cancerous tissue 3D cultural method according to claim 8, is characterized in that, described dyeing is HE dyeing or Ki67 immunohistochemical staining.
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CN109652376A (en) * 2019-01-08 2019-04-19 创芯国际生物科技(广州)有限公司 A kind of culture medium for ovarian cancer tissue 3D culture
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CN111040996A (en) * 2019-12-06 2020-04-21 北京科途医学科技有限公司 Method for preparing ovarian cancer organoid
CN111607568A (en) * 2020-05-06 2020-09-01 苏州济研生物医药科技有限公司 Culture method of primary ovarian cancer cells and application of primary ovarian cancer cells in drug screening
CN111876383A (en) * 2020-07-30 2020-11-03 中美冠科生物技术(太仓)有限公司 Quasi-organ lung cancer PDXO model, EGFR (epidermal growth factor receptor) engineering modification and application of PDXO model in tumor drug pharmacodynamic research

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Application publication date: 20160106