CN107904206A - A kind of purposes and method of lipopolysaccharide-induced tumour cell generation tumor stem cell - Google Patents
A kind of purposes and method of lipopolysaccharide-induced tumour cell generation tumor stem cell Download PDFInfo
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Abstract
The invention discloses a kind of purposes and method of lipopolysaccharide-induced tumour cell generation tumor stem cell, solve the problems, such as that the prior art is difficult to obtain the drug efficacy study requirement for meeting medicine, quantity enough and the tumor stem cell of controllable quantity.The single tumour cell to suspend is obtained the method for the present invention includes (1), progress monolayer adherence cell culture in culture dish is inoculated in, 4~10h of lipopolysaccharides culture is added in incubation；(2) Trypsin Induced is added, is washed out obtaining the single tumour cell after lipopolysaccharides processing；(3) the single tumour cell after lipopolysaccharides is handled is mixed with stem cell medium obtains cell suspension；(4) cell suspension obtains tumor stem cell after suspending and cultivating.The present invention can obtain the tumor stem cell for the drug efficacy study requirement for meeting medicine, and the tumor stem cell of inductive formation have the advantages that quantity dramatically increase, controllable quantity.
Description
Technical field
The present invention relates to a kind of inductive formation method of tumor stem cell, and in particular to a kind of lipopolysaccharide-induced tumour cell
Change into the purposes and method of tumor stem cell.
Background technology
The chemotherapy and targeted therapy of tumour are the main means of current oncotherapy.The former is in killing tumor cell
While, extremely strong toxic side effect is also produced to normal histocyte；The latter currently without handing over clearly to target also due to divide
Son and itself validity problem and cannot clinically extensive use.At the same time it is now recognized that above two therapeutic scheme master
If for the tumour cell with stronger multiplication capacity, and to the latent tumor stem cell in resting stage not kill and
Therapeutic effect, so that tumour cannot be cured fundamentally, leads recurrence and further deterioration after oncogenic treatment, ultimately results in
Tumor patient changes into cachexia and dead.Therefore, medicine of the research and development with killing tumor stem cell is prevention and controls
Treat the basic strategy of tumour.And in the prior art, the factor for restricting most important two aspects of realization of goal is：On the one hand
The medicine of the really effective killing tumor stem cell of research and development；On the other hand it is being useful for testing drug effect, quantity abundance
Tumor stem cell.Neither of the two can be dispensed.
Tumor stem cell is that a group has self-renewal capacity and multi-lineage potential, shows to start and rebuild tumour
Tissue phenotype ability and the tumour cell with high drug resistance.Since different types of tumor stem cell has different molecules
Marker, therefore define tumor stem cell and standard is still used as using above-mentioned cytology function.Tumor stem cell participates in turning for tumour
Move, recur and chemotherapy and radiation is resistant to.Therefore, the therapeutic strategy of targeting tumor stem cells carrys out the treatment zone being expected to as cancer
Wish.
At present, obtaining the method for tumor stem cell mainly has the serum-free cell suspension cultures method, the streaming of surface markers thin
Born of the same parents' instrument separating method, side population cell fluidic cell separating method and immunomagnetic beads fluidic cell separating method etc..But above-mentioned technical limit spacing swells
Knurl stem cell population is few, is simply possible to use in the research in laboratory and the identification of stem cell.Trained using serum-free cell Maitland culture
The technology for supporting stem-like cell tumour microballoon is by Brent A.Reynolds, Samuel Weiss (Developmental
Biology, 1996) invention.The technology is developed so far the technology for having become comparative maturity, and relatively generally acknowledges use the skill at present
The tumour bead cell that art is turned out be completely with above-mentioned tumor stem cell functional character knurl stem cell (Lawson,
D.A.et al,Proc Natl Acad Sci U S A,2007)。
Technical operation simplicity, the expense that serum free suspension cultivation sub-elects stem cell microballoon are low, therefore application is wide.
But this method is (can to obtain the higher tumour cell of grade malignancy) from cell line or tumor tissues, the cell through serum-free
Nutrient solution carries out the culture of at least two weeks in vitro, until forming stem-like cell tumour microballoon.The tumour microballoon includes cell
Through further identification, show as with the of self-replication capacity, multi-lineage potential, drug resistance biological property, is also expressed
Specific stem cell surface marker molecule.But the stem-like cell tumour microballoon quantity obtained through this technology is few, it is impossible to which repetition obtains
Take, be not used to antitumor drug efficacy study and the examination of medicine.
Thus, to obtain meet the requirements, quantity enough and controllable quantity tumor stem cell be used for medicine drug efficacy study,
Also it is difficult to reach with current existing technology.Therefore, develop generation sufficient amount and repeat the tumor stem cell of production
New technology becomes the task of top priority of antineoplaston medicament research and development.The technology one of the mass production tumor stem cell is set up, will
Greatly facilitate the progress of clinical drug therapy tumour.
The content of the invention
The technical problems to be solved by the invention are：The prior art is difficult to obtain drug efficacy study requirement, the number for meeting medicine
The problem of measuring the enough and tumor stem cell of controllable quantity, it is an object of the invention to provide the lipopolysaccharides to solve the above problems to lure
Tumour cell transformation is led into the purposes and method of tumor stem cell, by the optimization of this method, can effectively obtain and meet medicine
Drug efficacy study requires, quantity is enough and the tumor stem cell of controllable quantity.
The present invention is achieved through the following technical solutions：
A kind of lipopolysaccharides induces the purposes of tumour cell transformation formation tumor stem cell in vitro.
Further, the tumour cell is prostate tumor cells.
During lipopolysaccharides is applied to tumour cell transformation formation tumor stem cell by the present invention, conversion can be effectively increased
Efficiency, obtains the tumor stem cell for the drug efficacy study requirement for meeting medicine, and the increase of tumor stem cell quantity, the number of inductive formation
Measure it is controllable, while can duplication of production, effect is very notable.
A kind of method for inducing tumour cell transformation to form tumor stem cell in vitro using lipopolysaccharides, including：
(1) the single tumour cell to suspend is obtained, is inoculated in progress monolayer adherence cell culture culture in culture dish, culture
During add lipopolysaccharides culture 4~10h；
(2) Trypsin Induced is added, is washed out obtaining the single tumour cell after lipopolysaccharides processing；
(3) the single tumour cell after lipopolysaccharides is handled is mixed with stem cell medium obtains cell suspension；
(4) cell suspension obtains tumor stem cell after suspending and cultivating.
By the above method be applied to tumor stem cell induction production in, can effectively obtain meet medicine effect research will
The tumor stem cell asked, the repetition that can effectively realize tumor stem cell by this method are cultivated, the tumor stem cell cultivated
Quantity is more and controllable.
And the tumour cell induced by the method for the present invention has quick repair tissue, the ability of cellular damage, and
With significant clonality；EMT occurs during the inductive formation of tumor stem cell.And LPS induction outputs is swollen
Knurl microballoon stem cell expression stem cell markers molecule alpha-intergrin, CD44, Nestin, OCT-4, Nanog, BCRP
It is all remarkably higher than and does not add LPS control groups (* p<0.001)；Further prove that LPS has and induce and strengthen tumor stem cell formation
It is true.
Further, pass through in the suspension incubation more than once and change liquid culture, the process for changing liquid culture is：
Cell suspension is in 4%~8%CO2, after culture forms cell monolayer under conditions of 35~38 DEG C, disappeared with trypsase
Change, be washed out, be eventually adding stem cell medium.
The liquid number that changes cultivated that suspends is more than four times, and the incubation time for the culture that suspends is more than two weeks.
The tumour cell is prostate tumor cells, and the culture medium in culture dish is RPMI1640+10%FBS.It is described
The addition of lipopolysaccharides is 0.005~0.025ug/mL.The stem cell medium includes DMEM/F12 nutrient solutions, and final concentration
The hydrogen hydroxyl corticoid of insulin, 0.4~0.6mg/mL for 4~6mg/mL, 1~3% B27 cell culture addition
Agent, the epidermal growth factor of 18~22ng/mL.The concentration of cell is 3~5 × 10 in the cell suspension4Cells/ml.
In order to eliminate the non-specific adhesion power on surface in incubation, described change in liquid culture is used in advance through 1.2%
Poly-HEMA processing 6- holes culture hole in add 2~3 milliliters, or the Tissue Culture Dish of 100 mm dias in add 8~
12 milliliters are cultivated.
The present invention has the advantage that and beneficial effect：
1st, lipopolysaccharides converts the purposes to form tumor stem cell with induction prostate tumor cells in vitro in the present invention；
2nd, the tumor stem cell of inductive formation is more after the present invention is handled by lipopolysaccharides, is effectively applicable to medicine
Research and development and examination；
3rd, the generation quantity of tumor stem cell can effectively be adjusted by the method for the present invention, and can realizes that Tumor Stem is thin
The duplication of production of born of the same parents, is more applicable for industrialized production.
Brief description of the drawings
Attached drawing described herein is used for providing further understanding the embodiment of the present invention, forms one of the application
Point, do not form the restriction to the embodiment of the present invention.In the accompanying drawings：
The cell scratch experiment result figure for the tumour cell that Fig. 1 is generated by embodiment 1.
The cell scratch experiment result figure for the tumour cell that Fig. 2 is generated by embodiment 3.
Fig. 3 is the result relation schematic diagram of CK8/Vimentin and CK18/Vimentin ratios.
Fig. 4 is the microstructure schematic diagram of stem-like cell tumour microballoon.
Fig. 5 is the column schematic diagram of stem-like cell tumour microballoon quantity.
Fig. 6 is the microstructure schematic diagram of individual layer clonogenic tumor under regular growth culture.
Fig. 7 is the column schematic diagram of tumour cell quantity of formation under regular growth culture.
Fig. 8 is the column schematic diagram of the marker molecules expression of tumor stem cell.
Embodiment
For the object, technical solutions and advantages of the present invention are more clearly understood, with reference to embodiment and attached drawing, to this
Invention is described in further detail, and exemplary embodiment of the invention and its explanation are only used for explaining the present invention, do not make
For limitation of the invention.
Embodiment 1
A kind of method for inducing tumour cell transformation to form tumor stem cell in vitro using lipopolysaccharides, including：
First, the prostate tumor cells Trypsin Induced of individual layer will be grown into, the single DU145 for obtaining suspension swells
Oncocyte is inoculated in culture dish, and cellar culture is in the nutrient solution of RPMI1640+10%FBS.
Secondly, after when above-mentioned DU145 tumor cell cultures 12 are small, observed under inverted biologic microscope, it is ensured that growth
Cell in good condition carries out subsequent experimental.The culture supernatant of culture dish, adds where removing the good cell of growth conditions
The nutrient solution of lipopolysaccharides (LPS) containing 0.01ug/mL concentration continue culture 6 it is small when.
Then, with the above-mentioned cell handled through LPS of Trypsin Induced, the single DU145 tumour cells of suspension are obtained.
After DU145 tumour cells PBS washings three times, it is 40,000 cells/ml to be configured to concentration with the stem cell medium of serum-free
Cell suspension, and then be used for follow-up tumour microballoon culture.Wherein, stem cell medium includes DMEM/F12 nutrient solutions, pancreas
Island element, hydrogen hydroxyl corticoid, B27 cell culture additive, epidermal growth factor；The stem cell medium of the present embodiment
In, the final concentration of 5mg/mL of insulin, the final concentration of 0.5mg/mL of hydrogen hydroxyl corticoid, B27 cell culture addition
Final concentration of the 2% of agent, the final concentration of 20ng/mL of epidermal growth factor.
Finally, 2~3 milliliters of cell suspensions are added in the 6- holes culture hole handled in advance through 1.2%poly-HEMA,
It is placed in 6%CO2Cultivated with 37 DEG C of incubator.A not good liquor is changed within every four days, continues the culture that suspends as stated above.Used when changing liquid
Trypsase conventional digestion cell, centrifugation are obtained cell precipitation, stem cell medium are added after being washed using PBS and is configured to carefully
Born of the same parents' suspension.Co-culture and obtain tumor stem cell after being more than for four generations.
When carrying out conventional digestion using trypsase in the present embodiment, digestion time is 3 minutes, and digestion temperature is 37 DEG C.
Embodiment 2
The present embodiment is the control group of embodiment 1, and the present embodiment is differed only in embodiment 1, the present embodiment
LPS is not added in nutrient solution.
Embodiment 3
Difference lies in the final concentration of the LPS of addition is different, and specific setting is as follows with embodiment 1 for the present embodiment：
The present invention be respectively adopted final concentration of 0ug/mL, 0.0025ug/mL, 0.005ug/mL, 0.01ug/mL and
The LPS of 0.02ug/mL carries out tumour cell the tumour cell of processing acquisition.
The present invention is by experimental verification, and in the present embodiment, lipopolysaccharides can induce prostate tumor cells, as DU145 cells,
PC3 cells；Meanwhile theory is formed with tumor stem cell, it is believed that also can using this method according to malignant transformation of cells is theoretical
Other kinds of tumour cell generation tumor stem cell is induced, such as：Lung tumor cell, liver cancer cells, gastroenteric tumor, pancreas are disliked
Property tumour cell, head and neck neoplasm cell, etc. solid tumor cell, and leukaemia, etc. blood tumor cell.
The tumour cell (including control group and tumour cell through LPS processing) obtained to above-described embodiment carries out cell and draws
Trace, epithelial cell-mesenchyma conversion (EMT), the quantity of tumour microballoon formation, clonality, expression tumor stem cell sample
The experiment of molecule, experimental method and result are as follows：
First, cell scratch experiment
Using conventional cell scratch experiment method, the spacer of cut intermediate region under the conditions of different time sections is detected
From the tumour cell generated using embodiment 1 is tested, and experimental result is as shown in Figure 1.Meanwhile the present invention is in embodiment 3
The tumour cell obtained after the LPS processing of various concentrations carries out cell scratch experiment, and experimental result is as shown in Figure 2.
As seen in Figure 1, with the extension of time, damaged cell boundary border progressively narrows, can be seen by Fig. 2
Go out, with the increase of LPS concentration for the treatment of, damaged cell boundary border progressively narrows, and then can prove that LPS processing can accelerate
The migration of cell, promotes the reparation of tissue damage.
2nd, epithelial cell-mesenchyma conversion (EMT) experiment
Gather the gene expression of the tumour cell generated with enzyme chain reaction technology for detection embodiment 1 using reverse transcription, as a result show
Show gradually being reduced with the relation of time for CK8/Vimentin and CK18/Vimentin ratios, as shown in Figure 3.
By in the Fig. 3 the result shows that：The tumour cell of the LPS inductions used in the method for the present invention can be realized effectively
Conversion of the tumour cell from Epithelial to mesenchymal cell, meets the biological property of tumor stem cell formation.Further from point
The fact that the present invention can effectively induce and strengthen tumor stem cell formation is proved in handset reason.
3rd, the quantity experiment that tumour microballoon is formed
The tumor stem cell generated respectively to embodiment 1 and embodiment 2 under the identical cultivation time carries out micro- detection, observation
The quantity of stem-like cell tumour microballoon is formed in suspending nutrient solution, as shown in Figure 4 and Figure 5.
Pass through Fig. 4 and Fig. 5：The quantity of the stem-like cell tumour microballoon of embodiment 1 is significantly more than the dry thin of embodiment 2
The quantity of born of the same parents' sample tumour microballoon.Further prove：LPS can induce self-renewing and the propagation of tumor stem cell, and form tumour
The ability of microballoon, significantly improves the quantity of tumor stem cell.
4th, clonality is tested
By the tumor stem cell that embodiment 1 and embodiment 2 generate after Trypsin Induced forms individual cells, connect respectively
Kind carries out conventional single layer attached cell culture in culture dish.Respectively feelings are formed in first day and the 7th day observation tumour cell
Condition, as shown in Figure 6 and Figure 7.
Pass through the Fig. 6 and Fig. 7：The tumour cell quantity that embodiment 1 is turned out has bright for embodiment 2
Aobvious increase, and then prove that tumor stem cell that the embodiment of the present invention 1 generates can turn out tumour cell, thus the present invention generates
Tumor stem cell has self-renewing and differentiation, the ability for being proliferated into tumour cell.
5th, tumor stem cell sample molecule experiments are expressed
The related gene expression for the tumor stem cell that embodiment 1 and embodiment 2 generate is detected respectively, the results show that at LPS
Can be dramatically increased after reason induction stem-like cell tumour bead cell expression stem cell markers molecule alpha-intergrin,
CD44, Nestin, OCT-4, Nanog, BCRP, as shown in fig. 7, embodiment 1 is compared with the control group, * p<0.001.The result shows that
The tumour bead cell of LPS induced synthesis has more the characterization of molecules of stem cell.
Above-described embodiment, has carried out the purpose of the present invention, technical solution and beneficial effect further
Describe in detail, it should be understood that the foregoing is merely the embodiment of the present invention, be not intended to limit the present invention
Protection domain, within the spirit and principles of the invention, any modification, equivalent substitution, improvement and etc. done, should all include
Within protection scope of the present invention.
Claims (10)
1. a kind of lipopolysaccharides induces the purposes of tumour cell transformation formation tumor stem cell in vitro.
2. purposes according to claim 1, it is characterised in that the tumour cell is prostate tumor cells.
A kind of 3. method for inducing tumour cell transformation to form tumor stem cell in vitro using lipopolysaccharides, it is characterised in that bag
Include：
(1) the single tumour cell to suspend is obtained, progress monolayer adherence cell culture in culture dish is inoculated in, adds in incubation
Enter 4~10h of lipopolysaccharides culture；
(2) Trypsin Induced is added, is washed out obtaining the single tumour cell after lipopolysaccharides processing；
(3) the single tumour cell after lipopolysaccharides is handled is mixed with stem cell medium obtains cell suspension；
(4) cell suspension obtains tumor stem cell after suspending and cultivating.
4. a kind of tumour cell transformation is induced using lipopolysaccharides to form tumor stem cell in vitro according to claim 3
Method, it is characterised in that pass through in the suspension incubation more than once and change liquid culture, the process for changing liquid culture is：
Cell suspension is in 4%~8%CO2, after culture forms cell monolayer under conditions of 35~38 DEG C, with Trypsin Induced, so
After wash, be eventually adding stem cell medium culture.
5. a kind of tumour cell transformation is induced using lipopolysaccharides to form tumor stem cell in vitro according to claim 3
Method, it is characterised in that the culture that suspends changes liquid number as more than four times, the incubation time of the culture that suspends for two weeks with
On.
6. a kind of tumour cell transformation is induced using lipopolysaccharides to form tumor stem cell in vitro according to claim 3
Method, it is characterised in that the tumour cell is prostate tumor cells, and the culture medium of monolayer adherence cell is RPMI1640+
10%FBS.
7. a kind of tumour cell transformation is induced using lipopolysaccharides to form tumor stem cell in vitro according to claim 3
Method, it is characterised in that final concentration of 0.0025~0.02ug/mL after the lipopolysaccharides addition.
8. a kind of tumour cell transformation is induced using lipopolysaccharides to form tumor stem cell in vitro according to claim 3
Method, it is characterised in that the stem cell medium includes DMEM/F12 nutrient solutions, and the pancreas islet of final concentration of 4~6mg/mL
Element, the hydrogen hydroxyl corticoid of 0.4~0.6mg/mL, 1~3% B27 cell culture additive, 18~22ng/mL it is upper
Skin growth factor.
9. a kind of tumour cell transformation is induced using lipopolysaccharides to form tumor stem cell in vitro according to claim 3
Method, it is characterised in that the concentration of cell is 3~5 × 10 in the cell suspension cultures4Cells/ml.
10. one kind according to claim 3 induces tumour cell transformation to form tumor stem cell in vitro using lipopolysaccharides
Method, it is characterised in that it is described change in liquid culture using in advance through 1.2%poly-HEMA handle 6- holes culture hole in plus
Enter 8~12 milliliters of addition in 2~3 milliliters, or the Tissue Culture Dish of 100 mm dias to be cultivated.
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