CN101182491A - Vitro model capable of Dynamic modeling human cancer of liver displace - Google Patents
Vitro model capable of Dynamic modeling human cancer of liver displace Download PDFInfo
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- CN101182491A CN101182491A CNA200710113292XA CN200710113292A CN101182491A CN 101182491 A CN101182491 A CN 101182491A CN A200710113292X A CNA200710113292X A CN A200710113292XA CN 200710113292 A CN200710113292 A CN 200710113292A CN 101182491 A CN101182491 A CN 101182491A
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Abstract
The invention discloses a model in vitro which can simulate the metastasis of human liver cancer dynamically. The construction steps comprise the construction of an anti-cell adherence model by using human liver cancer cell line BEL7402 with high metastasis; the construction of metastasis model in vitro of liver cancer cell and the identification for the constructed in vitro metastasis model of liver cancer. The model of the invention can be used for the control research between the in situ cancer and the tumor in metastasis; the invention can further be used as a monitoring system for the biological behavior detection of the tumor cell in metastasis and the judgment of tumor prognosis. The model has important value for the research of clinical tumor metastasis mechanism and the screening of metastasis control drugs. The model can also be used as the screening technology platform for the preparation of anti-tumor transgenic vaccine and the construction of anti-tumor metastasis drugs.
Description
Technical field
The present invention relates to a kind of people's hepatoma Metastasis model, relate in particular to the external model of the anthropomorphic hepatoma Metastasis of a kind of dynamic analog.
Background technology
Liver cancer is as sickness rate and the high a kind of malignant tumour of case fatality rate at home, because of its course of disease progress fast, the grade malignancy height, rate of transform height, recurrence rate is high and become a great problem of long-term puzzlement clinicist and researcher.Though a few patients has obtained long-term survival because of diagnosis morning, early treatment, the transfer of liver cancer, recurrence but are a big obstacle that influences the liver cancer treatment effect all the time.Illustrating the mechanism of hepatoma Metastasis and it is carried out diagnosis early and prevents will be the key point that further improves the liver cancer treatment effect.
Liver cancer is the persistent ailment of serious harm human health, treatment means to liver cancer comprises operation, radio-frequency (RF) ablation, chemotherapy, get involved etc., but general curative effect is still undesirable, and a lot of patients course of disease when symptom takes place and makes a definite diagnosis liver cancer enters late period, behind the ocal resection tissue, transfer to liver cancer cell at a distance usually to radiotherapy, chemotherapy etc. are insensitive, thereby are difficult to be uprooted, and cause very high recurrence rate and mortality ratio.5 years recurrence rates of the postoperative of domestic report liver cancer patient have only about 30% up to 61.5%, 5 year survival rate.The cause of the death that we can say liver cancer patient is the transfer of liver cancer cell and non-liver cancer itself is extremely urgent to the hepatoma Metastasis Study on Mechanism.
In the process of hepatoma Metastasis, liver cancer cell has the general character of epitheliated type cell, i.e. the survival of cell needs the support of extracellular matrix (ECM) and the signal of surviving.If epitheliated type cell detachment ECM, cell then trends towards apoptosis, is called as anoikis (anoikis).Liver cancer cell is in entering the process of the recycle system, the support that has broken away from original position ECM is in and loses the nido attitude, and anoikis has taken place most liver cancer cells at this moment, and the minority liver cancer cell has obtained the ability of opposing anoikis, and can survive in lymphatic vessel or blood vessel and transfer at a distance.Therefore, the ability that obtains the opposing anoikis is the prerequisite that transfer takes place liver cancer cell, also is the focus of metastases research.
At present, mainly use the body inner model for the hepatoma Metastasis Study on Mechanism.But because normal mouse or rat, are difficult to real hepatoma Metastasis model normal mouse of immunologic function or rat construct in vitro people for the immunological rejection effect of people's hepatoma cell line.Therefore, the nude mice of immunologic hypofunction just becomes the first-selected model of research hepatoma Metastasis.But because nude mice price height, reasons such as difficult raising are greatly limited its application.With respect to the body inner model, it is few that external model has influence factor, and characteristics such as easy handling are significant for the mechanism of studying hepatoma Metastasis.
At present, in external research, mainly concentrate on the Mechanism Study of individual molecule or the detection of invasion by tumor cells transfer ability for hepatoma Metastasis.Analysis-by-synthesis domestic and international research present situation can find that the present research about metastases mainly concentrates on primary tumor, metastasis and the comparative study of the two.If the process of metastases is likened to a dumbbell, primary tumor and metastasis are the two ends of dumbbell, and vascular system plays the pivotal role that connects primary tumor and metastasis as the only way which must be passed of tumour distant metastasis.Therefore, can break away from primary tumor and survive by vascular system, be the prerequisite of tumour generation distant metastasis.And still do not consider this factor in the present applied hepatoma Metastasis model.By retrieval, paper and the patent of the biologic activity of the liver cancer cell that shifts being simulated dynamically with systematic research about the structure of the external model of the anthropomorphic hepatoma Metastasis of dynamic analog and the external model that effectively utilizes the anthropomorphic hepatoma Metastasis of dynamic analog yet there are no report.
Summary of the invention
At the deficiencies in the prior art, the problem to be solved in the present invention provides the external model of the anthropomorphic hepatoma Metastasis of a kind of dynamic analog.Utilize the high metastatic Bel7402 BEL7402 cell of external model of the present invention under suspended state, to resist anoikis, realization is judged to the drug screening research of the biological activity research of liver cancer under the research of hepatoma Metastasis process and mechanism, the transfering state, liver cancer treatment and for the prognosis of tumour, and can further be applied to the transfer process research of other tumour and the screening of related drugs.
The present invention must have from the liver cancer cell that can obtain transfer ability can break away from adhering to of extracellular matrix and hand is clicked and entered in this incision of tool survival ability, made up the hepatoma Metastasis model that does not rely on extracellular matrix and survive, and further separation has obtained to have the liver cancer cell subbreed of transfer ability, inquires into relevant metastasis for the biological activity research of metastatic carcinoma and lays a good foundation.
The selected research object BEL7402 clone of the present invention is the high metastatic Bel7402 of a strain tool, available from Shanghai cell institute.
The external model of the anthropomorphic hepatoma Metastasis of dynamic analog of the present invention, the modelling of being adhered to anti-cell by the metastatic Bel7402 BEL7402 of height cell, the foundation of the external metastasis model of liver cancer cell, the step that the external metastasis model of constructed liver cancer is identified make up and form, and it is characterized in that:
The establishment method that described anti-cell adheres to model is: with the dissolve with ethanol poly-HEMA[poly (2-Hydroxyethyl Methacrylate) of 95%-100%], be made into the solution that the poly-HEMA final concentration is 120mg/ml, 65 ℃ shook mixing 8-10 hour, got the poly-HEMA storing solution; With the alcohol dilution of 1: 10 by volume ratio of poly-HEMA storing solution with 95%-100%, making final concentration is the poly-HEMA working fluid of 12mg/ml then, and 4 ℃ of preservations are standby; Get the poly-HEMA working fluid and evenly be taped against on the bottom surface of Tissue Culture Plate or Tissue Culture Flask, the whole density of poly-HEMA working fluid is 1mg-3mg/cm during bed board
2, dry the poly-HEMA working fluid under the room temperature naturally, get anti-cell and adhere to model, 4 ℃ of preservations, standby;
The method that the external metastasis model of described liver cancer cell is set up is: choose the BEL-7402 cell that has high metastatic capacity, is in logarithmic phase, become single cell suspension with 0.25% trysinization after, the adjustment cell concn is 1-5 * 10
5Individual cell/ml is divided into two groups then, and a winding kind to above-mentioned anti-cell adheres in the model, be set at the suspension cell group, another group is seeded in the Tissue Culture Plate or Tissue Culture Flask that non-anti-cell adheres to model, is set at the attached cell group, treat even rearmounted 37 ℃ of cell shop, CO
2Cultivate 12h-48h in the incubator, the suspension cell group cell of acquisition is liver cancer cell and breaks away from the external metastasis model that adheres to, and the attached cell group cell of acquisition is the contrast model of liver cancer cell adherent growth;
The described index feature that the external metastasis model of constructed liver cancer is identified is: expect that at platform blue dyeing determines that suspension cell group and attached cell group cell survival rate are not less than under 97% the prerequisite, 1. suspension cell group and attached cell group apoptosis rate is lower than 5%; 2. suspension cell group and attached cell group cellular form contrast, the cell aggregation of suspension group is agglomerating, does not have projection to stretch out, cell still is a ball-shaped, assembling appears in cell 4h after suspension, and 36h cell aggregation is tighter, presents the fusion state, As time goes on, adherent group of cell begins to stretch gradually by stretching out projection, be paved into monolayer, and the cellular form of suspension group is rounded, and assemble agglomeratingly gradually, the cell mass that is gathered is As time goes on poly-more big more; 3. cell growth curve shows, adherent group of cell is vigorous growth curve, and suspension group cell remains static, propagation neither, and also apoptosis not is the curve that remains basically stable; 4. flow cytometer detects the cell distribution of each cell cycle and shows that suspension group cell is the cell that is in the stationary state of G0/G1 phase, and adherent group of cell is in the G2/M phase of molecular marker for increased proliferation mostly; 5. suspension group cell is running into when being suitable for adherent environment once more, can be under situation without any external stimulus, and adherent again, and reenter cell cycle and vigorous vegetative state.
In the method for the external model of the anthropomorphic hepatoma Metastasis of above-mentioned structure dynamic analog: it is 95% ethanol that described ethanol preferably uses concentration of volume percent.
In the method for the external model of the anthropomorphic hepatoma Metastasis of above-mentioned structure dynamic analog: the bed board density of described poly-HEMA is preferably 3mg/cm
2
In the method for the external model of the anthropomorphic hepatoma Metastasis of above-mentioned structure dynamic analog: the described anti-cell that is inoculated in adheres to the cell density that model or non-anti-cell adhere to model and is preferably 4 * 10
5Individual cell/ml.
In the method for the external model of the anthropomorphic hepatoma Metastasis of above-mentioned structure dynamic analog: in the index feature that the external metastasis model of described liver cancer is identified, when suspension group cell growth curve is drawn, because of the biological property of cell different with normal cell, the selection of used reagent also has difference, cell cultures is preferably used the reagent C CK8 reagent that changes the liquid operation without cell, to reduce operate miss.
In the method for the external model of the anthropomorphic hepatoma Metastasis of above-mentioned structure dynamic analog: in the index feature that the external metastasis model of described liver cancer is identified, when suspension group cell is carried out the flow cytometer detection, owing to assemble agglomerating can not directly the detection by flow cytometer, cell should be gone up machine testing again after 500 order copper mesh filter because of the cell of suspended state.
The external model of dynamic simulation hepatoma Metastasis process of the present invention has made up good platform to the correlative study of hepatoma Metastasis mechanism.Use this model, can realize the biological habit research of BEL7402 liver cancer cell in transfer process.Experimental result shows, uses this model and can obtain to simulate the BEL7402 liver cancer cell (can resist the liver cancer cell of anoikis) that keeps existence after the suspension of transfering state.The cell of surviving of uniting after the suspension is the cell of a group stationary state, and these cells are positioned at the G0/G1 phase of cell cycle mostly, by mutual gathering, provide the survival signal mutually, to survive, in case stop these cell aggregations by force, cell will no longer be survived, but start apoptosis pathway.The cell that is in this gathering survival form is all insensitive to radiotherapy, chemotherapy, is in the tumour patient body, can survive in vascular system, has metastatic potential, and dangerous, the most obstinate enemy of can escape body immunologic mechanism and various treatments.The suspension adherent growth again after the cell renewed vaccination of survival of uniting, this process dynamic simulation the tumour cell that shifts in the body run into suitable environment will adherent once more situation of growing.This be in stationary phase, in a single day the cell of surviving of uniting run into suitable environment, just can reenter the cell cycle, carries out vigorous propagation.This perhaps is the time of day of the cell of the inherent vascular system survival of body, transfer, and in a single day the resting cell of these survivals and the various kill mechanisms of escaping seeks suitable environment, just can breed again, grows up to new metastatic carcinoma piece.Experimental study also confirms: the suspension cell group is different with the transfer mode of the existence signal of attached cell group cell, attached cell group part or largely depend on AKT and ERK path, and as if the suspension cell group mainly do not rely on these two paths the existence signal be provided.
The external model of the anthropomorphic hepatoma Metastasis of dynamic analog provided by the invention can also further be applied to the relevant biological activity research of transfer of other tumours beyond the liver cancer.
The external model of the hepatoma Metastasis that the present invention set up is compared with metastasis model in the existing body, has easy and simple to handlely, and background is clear, characteristics such as favorable repeatability.
The external model outstanding feature and the advantage of dynamic simulation hepatoma Metastasis process of the present invention are: (1) this model can well analogue body in the dynamic transfer process of liver cancer cell.Wherein primary tumor one vascular system of tumour is unified the primary process of this transfer of metastasis and main incident can have in native system preferably and embody.(2) this model has remarkable advantages for the biologic activity of the liver cancer cell in the research transfer process, the biological characteristicses such as morphological change, propagation situation, cell cycle and apoptosis situation of the liver cancer cell of the transfer that can survive to suspension culture by this model are furtherd investigate, for the biological characteristics of illustrating transitional cell is had laid a good foundation.(3) method set up of this model, can compare research to the susceptibility that the cell that is in transfering state of the cell of adherent growth and suspension growth is treated measure to external world, and can pass through this system, carry out specificity clinically and screen at the medicine of metastatic tumor.
Description of drawings
Liver cancer cell is at the aspect graph of different time points under Fig. 1 transfering state
The BEL7402 liver cancer cell is inoculated in cultivation that does not add poly-HEMA and the culture plate that contains poly-HEMA, makes up the transitional cell model (F-J) of the opposing anoikis of the cell model (A-E) of adherent growth and suspension culture with this.The form of cell following time point carried out detection Oh (A, F), 12hrs (B, G), 24hrs (C, H), 36hrs (D, I), 48hrs (E, J).As seen from the figure, at each time point, the morphological differences of two groups of cells is huge, and As time goes on, adherent group of cell begins to stretch gradually, is paved into monolayer; And the cellular form of suspension group is rounded, and assembles agglomeratingly gradually, and the cell mass that is gathered is As time goes on poly-more big more.
The liver cancer cell that Fig. 2 dynamic simulation shifts runs into the suitable environment generating process figure of adherent growth once more
(A-C) the BEL7402 liver cancer cell makes up the suspension cultured cells model containing to cultivate with this on the culture plate of poly-HEMA.After 24 hours, once more move into the culture plate that do not contain poly-HEMA with assembling liver cancer cell agglomerating, that keep survival in the liver cancer cell suspension culture, cell is under the situation that is not subjected to any external stimulus, and is adherent once more.And As time goes on, assemble agglomerating cell stretch gradually, adherent, at typical morphological change stage 3hrs (A), 12hrs (B) and 24hrs (C) pair cell form are carried out Taking Pictures recording.(D) growth curve of adherent once more BEL7402 cell.As seen from the figure, cell adherent once more after the suspension culture begins propagation once more after having experienced a laundering period.This situation can be reacted the primary tumor cell well after having experienced vascular system with stationary state and shifting, can be when running into suitable living environment, and adherent once more, enter vigorous vegetative state, form metastatic tumor.
(E-H) the figure E-F BEL7402 that represent suspension culture is in the cell cycle of disengaging adhere to cultivation 12 hours and 24 hours, and the G1 phase cell of wherein adherent group and suspension group is respectively 55.5% and 74.6%.After 24 hours, cell is inoculated in the culture plate of no poly-HEMA once more in suspension culture, and spontaneous adherent and propagation situation has taken place cell, and in adherent once more 12 and 24 hours of cell, the cell cycle of attached cell was respectively 64.4% and 64.2% once more.
The apoptosis rate of liver cancer cell detects under Fig. 3 transfering state
To the detection of the apoptosis rate of the liver cancer cell of transfering state low suspension growth as shown in the figure, suspending to compare with adherent group with the apoptosis rate of the liver cancer cell of 36 cells in 24 hours does not all have notable difference (P＞0.05), i.e. the liver cancer cell that suspension group shifts does not have obvious apoptosis to take place.
The growth conditions of liver cancer cell research---the growth curve of the BEL7402 cell of adherent growth and suspension growth under Fig. 4 transfering state
As seen from the figure, in cell inoculation 48 hours, the cell of adherent growth is vigorous vegetative state, and the cell of suspension growth is not significantly bred phenomenon, and growth curve is the state of remaining basically stable.
The cell cycle of liver cancer cell changes under Fig. 5 transfering state
A refers to the detection of adherent 24 hours group BEL7402 cell cycles, and B refers to suspend and organized the detection of BEL7402 cell cycle in 24 hours, and suspension cell group and adherent group of cell are collected after cultivating 24 hours, and carry out the detection of cell cycle with flow cytometer.As shown in the figure, each cell cycle of adherent group and suspension group cell be distributed with notable difference, the cells ratio of the two G1 phase is respectively 64.3% and 85.4%, this shows that the retardance of cell cycle has taken place suspension cultured cells.
The mechanism of liver cancer cell survival is inquired under Fig. 6 transfering state
A refers to the growth inhibition ratio of Akt path retardance back liver cancer cell BEL7402, B refers to the growth inhibition ratio of ERK path retardance back liver cancer cell BEL7402, the specific inhibitor wortmannin that uses the specific inhibitor PD98059 of ERK path and AKT path respectively handles the cell of adherent group and suspension group, and detects the susceptibility of cell to inhibitor at each time point.As shown in the figure, inhibitor can make adherent group of cell proliferation be suppressed significantly, and suspension cultured cells is to the processing of inhibitor more insensitive (P＜0.05).
Fig. 7 chemotherapeutic is to the result of treatment of the liver cancer cell under the transfering state
As shown in the figure, compare with not medication group, chemotherapeutic 5-FU can make adherent group of cell growth be suppressed significantly, and suspension group cell is to this stimulation more insensitive (P＜0.05).
Fig. 8 inducer of apoptosis is to the fragmentation effect of liver cancer cell under the transfering state
As shown in the figure, inducer of apoptosis TRAIL can induce attached cell that apoptosis takes place significantly, and suspension cultured cells more insensitive (P＜0.05).
Embodiment
Below in conjunction with embodiment the external model and the application thereof of dynamic simulation hepatoma Metastasis process of the present invention are described further.
The structure of the external model of embodiment 1 dynamic simulation hepatoma Metastasis process
1. the anti-cell modelling of adhering to
Adherent growth is the prerequisite that the solid tissue cell can be survived.Poly-HEMA (available from Sigma company) is as a kind of anionic polymer, and the bottom that is layered on container can stop cell attachment, makes up the cell model of suspension growth behind the cell detachment with this.
Dissolve with ethanol poly-HEMA with 95% is made into the solution that the poly-HEMA final concentration is 120mg/ml (i.e. dissolving 2.4g poly-HEMA in the ethanol of 20ml95%), and 65 ℃ shook mixing 8 hours, got the poly-HEMA storing solution; Then with 1: 10 by volume ratio of poly-HEMA storing solution with 95% alcohol dilution, making final concentration is the poly-HEMA working fluid of 12mg/ml, 4 ℃ of preservations are standby; Get the poly-HEMA working fluid and evenly be taped against on the bottom surface of 6 porocyte culture plates, the whole density of poly-HEMA working fluid is 3mg/cm during bed board
2, dry the poly-HEMA working fluid under the room temperature naturally, get anti-cell and adhere to model, 4 ℃ of preservations, standby;
Use the preceding poly-HEMA plate is placed under the ultraviolet lamp to shine, to reach the aseptic requirement of cell cultures.
2. the foundation of the external metastasis model of liver cancer cell
Choose and have BEL7402 cell high metastatic capacity, that be in logarithmic phase (available from Shanghai cell institute), be digested to single cell suspension with 37 ℃ of 0.25% pancreatin after, adjust cell concn to 4 * 10
5Individual cell/ml contains the RPMI1640 substratum of 10% calf serum, described cell suspension inoculation to above-mentioned anti-cell is adhered to (6 porocyte culture plate) in the model, be set at the suspension cell group, simultaneously it is seeded in the Tissue Culture Plate or Tissue Culture Flask that non-anti-cell adheres to model, be set at the attached cell group, treat even rearmounted 37 ℃ of cell shop, CO
2Cultivate 24h in the incubator, be liver cancer cell with this suspension cell group cell that obtains and break away from the external metastasis model that adheres to, the attached cell group cell of acquisition is the contrast model of liver cancer cell adherent growth, and Nicon8700 cameras record morphological data.
Expect the activity of blue dyeing observation of cell by platform.
Get attached cell group and each 1ml of suspension cell group cell suspension, add 4% respectively and expect blue isotonic solution 3ul, left standstill in the room temperature 20 minutes, the centrifugal 3min of 1000rpm, blue supernatant liquor is removed in suction.Because dead cell can be expected that orchid dyes blueness by platform, and viable cell is refused to dye, and viable cell and dead cell can be separated thus.The judge existing state of cell of inverted microscope.The result shows, expects with platform and blue each group cell dyeed visible adherent group of cell survival; The suspension group cell survival of uniting, free cell indigo plant is dyed, and is dead cell.The cell survival rate of attached cell group and suspension cell group is all more than 97%, the two no significant difference.
3. with following index the external metastasis model of constructed liver cancer is identified
1. suspension cell group and attached cell group apoptosis rate is lower than 5%;
2. suspension cell group and attached cell group cellular form contrast, the cell aggregation of suspension group is agglomerating, does not have projection to stretch out, cell still is a ball-shaped, assembling appears in cell 4h after suspension, and 36h cell aggregation is tighter, presents the fusion state, As time goes on, adherent group of cell begins to stretch gradually by stretching out projection, be paved into monolayer, and the cellular form of suspension group is rounded, and assemble agglomeratingly gradually, the cell mass that is gathered is As time goes on poly-more big more;
3. cell growth curve shows, adherent group of cell is vigorous growth curve, and suspension group cell remains static, propagation neither, and also apoptosis not is the curve that remains basically stable;
4. flow cytometer detects the cell distribution of each cell cycle and shows that suspension group cell is the cell that is in the stationary state of G0/G1 phase, and adherent group of cell is in the G2/M phase of molecular marker for increased proliferation mostly;
5. suspension group cell is running into when being suitable for adherent environment once more, can be under situation without any external stimulus, and adherent again, and reenter cell cycle and vigorous vegetative state.
Arrive the dystopy dynamic simulation of adherent growth once more in the embodiment 2 cell transfer processes
Collect the anti-liver cancer cell that adheres to of simulation transfer of 24 hours of survival in vascular system, it is moved in the Tissue Culture Plate that does not contain poly-HEMA again,, contain 5% CO at 37 ℃
2Continue to cultivate 24h-48h in the incubator, every 12 hours with Nikon8700 cameras record-inferior morphological data.The collection when cultivating 12 hours and 24 hours of the cell of desiring once more adherent growth after will suspending simultaneously, and it is processed into single cell suspension, PBS washing, 70% ethanol fixedly spend the night the bromination third pyridine (PI, the Sigma product) dyeing, flow cytometer detects the cell distribution situation of each cell cycle.
Be collected in the cell of 96 orifice plate inner suspensions cultivation after 24 hours, its immigration is not contained in 96 orifice plates of poly-HEMA, got 3 every group multiple holes every 12 hours and add 10ul CCK8, behind the mixing, put 37 ℃, CO
2Continue to cultivate 90min in the incubator; Read at the 450nm place and record data with microplate reader, and draw the growth curve of adherent once more liver cancer cell according to data.
The result shows, by the detection of CCK8 test kit and flow cytometry to the propagation situation and the distribution situation of cell cycle of adherent once more liver cancer cell, spontaneous once more adherent liver cancer cell can enter the process of cell cycle and vigorous vegetative state once more after the stationary state of the suspension culture that has experienced 24 hours.
The detection of hepatoma cell apoptosis rate under embodiment 3 transfering states
6 porocyte culture plates of preparation poly-HEMA bag quilt are divided into suspension culture group and adherent culture group with 6 porocyte culture plates.Collect 24,36 hours liver cancer cell of suspension adherent growth respectively, the centrifugal 5min of 1000rpm; Per 10
6Individual cell adds 1ml cracking/sample buffer (0.2mM), places 30min on ice; Collect the cracking precipitated product of 24,36 hours liver cancer cell of suspension adherent growth respectively, the centrifugal 10min of 3000rpm; Preserve more than 18 hours for-20 ℃; Take out sample precipitation, 10 times of volume cracking/sample buffer dissolution precipitations from-20 ℃; The sample thief damping fluid adds in the enzyme plate, and the 100ul/ hole is with adhesive tape sealing, room temperature preservation 3 hours; Abandon supernatant, add 1 * lavation buffer solution washing three times, each every hole is filled 2min, pats subsequently, blots every hole lavation buffer solution; Get and detect in the antibody adding enzyme plate, the 100ul/ hole is with adhesive tape sealing, room temperature preservation 1 hour; Abandon supernatant, add 1 * lavation buffer solution washing three times, each every hole is filled 2min, pats subsequently, blots every hole lavation buffer solution; Get 1 * StreptavidinConjugate and add in the enzyme plate, the 100ul/ hole is with adhesive tape sealing, room temperature preservation 0.5 hour; Abandon supernatant, add 1 * lavation buffer solution washed twice, each every hole is filled 2min, pats subsequently, blots every hole lavation buffer solution; Abandon supernatant, add deionized water wash twice, each every hole is filled 2min, pats subsequently, blots every hole lavation buffer solution; Get 1 * substrate solution and add in the enzyme plate, the 100ul/ hole, with the adhesive tape sealing, room temperature kept in Dark Place 0.5 hour; Get 1 * Streptavidin Conjugate and add in the enzyme plate, the 100ul/ hole, with the adhesive tape sealing, room temperature kept in Dark Place 0.5 hour; Get 1 * stop buffer and add in the enzyme plate, read at the 450nm place and record data with microplate reader in 30min in the 100ul/ hole, according to the numerical value of typical curve calculating Nucleosome, calculates the apoptosis situation of suspension cell group and attached cell group.
The result shows that adherent growth group and suspension growth group all do not have tangible apoptosis that the apoptotic state there was no significant difference of the two takes place.
The variation of liver cancer cell growth curve under embodiment 4 transfering states
96 porocyte culture plates of preparation poly-HEMA bag quilt are divided into suspension culture group and adherent culture group with 96 porocyte culture plates; Liver cancer cell BEL7402 is inoculated into respectively in the suspension culture group and adherent culture group of 96 porocyte culture plates, the 100ul/ hole, behind the mixing to 37 ℃, CO
2Continue to cultivate 48h in the incubator, got 3 every group multiple holes every 12 hours and add 10ul CCK8, behind the mixing to 37 ℃, CO
2Continue to cultivate 90min in the incubator; Read at the 450nm place and record data with microplate reader, and draw the growth curve of liver cancer cell according to data.The result shows that adherent group is vigorous growth curve, and the suspension group is neither bred, and also apoptosis not is the curve that remains basically stable, and prompting suspension group cell is the cell that remains static.
The cell cycle of liver cancer cell changes under embodiment 5 transfering states
Preparation poly-HEMA wraps 6 orifice plates of quilt, prepares the liver cancer cell group of transfering state in 6 orifice plates, prepares the liver cancer cell group of adherent growth simultaneously.After cell is processed into single cell suspension with each group, collecting cell (comprising the cell that floats in the supernatant), washing, 70% ethanol fixedly spend the night, and bromination third pyridine (PI, Sigma product) is dyeed, and flow cytometer detects the cell distribution situation of each cell cycle.The result shows that compare with the control cells of adherent growth, the suspension cultured cells group has more cell to be arrested in the G0/G1 phase (P＜0.05).Flow cytometry detects the cell of adherent group and suspension group, and the 24h that chooses the difference maximum carries out the mensuration of cell cycle, confirms that suspension group cell is the cell that is in the stationary state of G0/G1 phase, and adherent group of G2/M phase that is in molecular marker for increased proliferation mostly.
The mechanism of liver cancer cell survival is inquired under embodiment 6 transfering states
PI-3K/AKT and ERK path are the main signal paths of keeping cell survival.For whether the liver cancer cell of judging the suspension growth that is in transfering state is to keep survival by these two paths, judge the survival mechanisms of cell by the specific inhibitor of using these two paths.
Mode prepares liver cancer cell and breaks away from the external metastasis model adhere to and the contrast model of adherent growth thereof as previously mentioned, and liver cancer cell BEL7402 is inoculated in 96 porocyte culture plates with preparation suspension culture group and adherent culture group.Using the specific inhibitor of PI-3K/AKT and ERK path handles each group cell.Wherein, the using dosage of the specific inhibitor wortmannin of PI-3K/AKT is 1 μ M, and the working concentration of the specific inhibitor PD98059 of ERK path is 50 μ M.12,24,36,48 hours application CCK8 detection kit pair cell survival conditions detect after each pathway inhibitor effect.
The growth curve result that CCK8 detects cell shows: the growth that the inhibitor of AKT and ERK can the obvious suppression attached cell, and for not obviously influence of suspension cell, the prompting existence signal that survival obtained of uniting behind the cell suspension may not rely on AKT or ERK path, and suspension cell may be kept survival by other the signal path that is different from the attached cell group.
Embodiment 7 chemotherapeutic are to the action effect research of the liver cancer cell under the transfering state
Whether the reactivity of chemotherapeutic is changed to some extent for exploring the cell that has transfer ability under the suspension culture state, the present invention chooses clinical chemotherapeutic 5-FU (SunRise commonly used, Shanghai, China) carry out drug effect research, and further compare the susceptibility of cell suspension front and back this chemotherapeutic.
Mode prepares liver cancer cell and breaks away from the external metastasis model adhere to and the contrast model of adherent growth thereof as described above, and promptly adherent culture groups of cells and suspended culture cell group when each group cell grows into 24 hours, add chemotherapeutic 5-FU.The concentration gradient of selected 5-FU is respectively 25ug/ul, 12.5ug/ul, 6.25ug/ul, 3.125ug/ul, 1.56ug/ul.After 12 hours, the growth existing state of pair cell detects in the 5-FU effect, whether can change the susceptibility of cell to chemotherapeutic to judge suspension culture.
The result shows that the liver cancer cell after the suspension obviously increases (P＜0.05) to the tolerance level of 5-FU.This explanation, the liver cancer cell that is in transfering state that the present invention the obtained lethal effect of chemotherapeutic of can escaping to liver cancer cell.
Embodiment 8 inducer of apoptosis are to the fragmentation effect of liver cancer cell under the transfering state
For whether the cell that has transfer ability under the research suspension culture state changes to some extent to the reactivity of inducer of apoptosis, the present invention chooses typical apoptosis induction molecule TRAIL and detects, and further relatively before and after the cell suspension to the susceptibility of inducer of apoptosis.
Mode prepares liver cancer cell and breaks away from the external metastasis model adhere to and the contrast model of adherent growth thereof as described above, and promptly adherent culture groups of cells and suspended culture cell group when each group cell grows into 24 hours, add inducer of apoptosis TRAIL and handle.The activity of TRAIL is respectively 0.2ng/ml, 2ng/ml, 20ng/ml.After 12 hours, the growth existing state of pair cell detects in the TRAL effect, whether can change the susceptibility of cell to the apoptosis induction material to judge suspension culture.
The result shows, the suspension cultured cells that is in transfering state is to the susceptibility of TRAIL obviously descend (P＜0.05).This explanation, the liver cancer cell that is in transfering state that the present invention the obtained lethal effect of inducer of apoptosis of can escaping to liver cancer cell.
Claims (5)
1. the external model of the anthropomorphic hepatoma Metastasis of a dynamic analog, the modelling of being adhered to anti-cell by the metastatic Bel7402 BEL7402 of height cell, the foundation of the external metastasis model of liver cancer cell, the step that the external metastasis model of constructed liver cancer is identified make up and form, and it is characterized in that:
The establishment method that described anti-cell adheres to model is: the dissolve with ethanol poly-HEMA with 95%-100%, be made into the solution that the poly-HEMA final concentration is 120mg/ml, and 65 ℃ shook mixing 8-10 hour, got the poly-HEMA storing solution; With the alcohol dilution of 1: 10 by volume ratio of poly-HEMA storing solution with 95%-100%, making final concentration is the poly-HEMA working fluid of 12mg/ml then, and 4 ℃ of preservations are standby; Get the poly-HEMA working fluid and evenly be taped against on the bottom surface of Tissue Culture Plate or Tissue Culture Flask, the whole density of poly-HEMA working fluid is 1mg-3mg/cm during bed board
2, dry the poly-HEMA working fluid under the room temperature naturally, get anti-cell and adhere to model, 4 ℃ of preservations, standby;
The method that the external metastasis model of described liver cancer cell is set up is: choose the BEL-7402 cell that has high metastatic capacity, is in logarithmic phase, become single cell suspension with 0.25% trysinization after, the adjustment cell concn is 1-5 * 10
5Individual cell/ml is divided into two groups then, and a winding kind to above-mentioned anti-cell adheres in the model, be set at the suspension cell group, another group is seeded in the Tissue Culture Plate or Tissue Culture Flask that non-anti-cell adheres to model, is set at the attached cell group, treat even rearmounted 37 ℃ of cell shop, CO
2Cultivate 12h-48h in the incubator, the suspension cell group cell of acquisition is liver cancer cell and breaks away from the external metastasis model that adheres to, and the attached cell group cell of acquisition is the contrast model of liver cancer cell adherent growth;
The described index feature that the external metastasis model of constructed liver cancer is identified is: expect that at platform blue dyeing determines that suspension cell group and attached cell group cell survival rate are not less than under 97% the prerequisite, 1. suspension cell group and attached cell group apoptosis rate is lower than 5%; 2. suspension cell group and attached cell group cellular form contrast, the cell aggregation of suspension group is agglomerating, does not have projection to stretch out, cell still is a ball-shaped, assembling appears in cell 4h after suspension, and 36h cell aggregation is tighter, presents the fusion state, As time goes on, adherent group of cell begins to stretch gradually by stretching out projection, be paved into monolayer, and the cellular form of suspension group is rounded, and assemble agglomeratingly gradually, the cell mass that is gathered is As time goes on poly-more big more; 3. cell growth curve shows, adherent group of cell is vigorous growth curve, and suspension group cell remains static, propagation neither, and also apoptosis not is the curve that remains basically stable; 4. flow cytometer detects the cell distribution of each cell cycle and shows that suspension group cell is the cell that is in the stationary state of G0/G1 phase, and adherent group of cell is in the G2/M phase of molecular marker for increased proliferation mostly; 5. suspension group cell is running into when being suitable for adherent environment once more, can be under situation without any external stimulus, and adherent again, and reenter cell cycle and vigorous vegetative state.
2. the external model of the anthropomorphic hepatoma Metastasis of dynamic analog as claimed in claim 1, it is characterized in that: the bed board density of described poly-HEMA is 3mg/cm
2
3. the external model of the anthropomorphic hepatoma Metastasis of dynamic analog as claimed in claim 1 is characterized in that: describedly be inoculated in anti-cell to adhere to the cell density that model or non-anti-cell adhere to model be 4 * 10
5Individual cell/ml.
4. the external model of the anthropomorphic hepatoma Metastasis of dynamic analog as claimed in claim 1 is characterized in that: in the index feature that the external metastasis model of described liver cancer is identified, and when suspension group cell growth curve is drawn, cell cultures CCK8 reagent.
5. the external model of the anthropomorphic hepatoma Metastasis of dynamic analog as claimed in claim 1, it is characterized in that: in the index feature that the external metastasis model of described liver cancer is identified, when suspension group cell was carried out the flow cytometer detection, cell should be gone up machine testing again after 500 order copper mesh filter.
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| CNA200710113292XA CN101182491A (en) | 2007-11-02 | 2007-11-02 | Vitro model capable of Dynamic modeling human cancer of liver displace |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102279186A (en) * | 2011-03-25 | 2011-12-14 | 山东省医学科学院基础医学研究所 | Method for detecting cell deformability by using poly-hydroxyethyl methacrylate (polyHEMA) |
| CN109112106A (en) * | 2018-09-07 | 2019-01-01 | 广州长峰生物技术有限公司 | The method for building up of the external model of the primary liver cancer tissue of people |
-
2007
- 2007-11-02 CN CNA200710113292XA patent/CN101182491A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102279186A (en) * | 2011-03-25 | 2011-12-14 | 山东省医学科学院基础医学研究所 | Method for detecting cell deformability by using poly-hydroxyethyl methacrylate (polyHEMA) |
| CN102279186B (en) * | 2011-03-25 | 2013-01-02 | 山东省医学科学院基础医学研究所 | Method for detecting cell deformability by using poly-hydroxyethyl methacrylate (polyHEMA) |
| CN109112106A (en) * | 2018-09-07 | 2019-01-01 | 广州长峰生物技术有限公司 | The method for building up of the external model of the primary liver cancer tissue of people |
| CN109112106B (en) * | 2018-09-07 | 2022-01-04 | 广州长峰生物技术有限公司 | Method for establishing in vitro model of human primary liver cancer tissue |
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