CN101186899A - Method applied for tumour cell and stem cell co-culture - Google Patents
Method applied for tumour cell and stem cell co-culture Download PDFInfo
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- CN101186899A CN101186899A CNA2007100599291A CN200710059929A CN101186899A CN 101186899 A CN101186899 A CN 101186899A CN A2007100599291 A CNA2007100599291 A CN A2007100599291A CN 200710059929 A CN200710059929 A CN 200710059929A CN 101186899 A CN101186899 A CN 101186899A
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Abstract
The invention relates to a novel process for co-culturing tumor cell and stem cell, which observes formal changes of mescenchymal stem cell in the co-culture, detects the chemical pigmentation result of two target immunologic tissues of VII factor and CD34, and analyzes chemical pigmentation difference of immunologic tissues mainly through non-contact co-culture of mescenchymal stem cell and malignant melanoma cell which is based on transwell. The experimental results show that parts of mescenchymal stem cell in the non-contact co-culture form polygon or short spindle, the statistic analysis shows that significant difference exists in the mescenchymal stem cell on the targets of VII factor and CD34 between the two culture processes by the comparison and observation of the chemical pigmentation result of two target immunologic tissues of VII factor and CD34 in the single culture and the non-contact co-culture. The invention makes that the non-contact co-culture can be applied to the study of the interaction between the mescenchymal stem cell and the malignant melanoma cell clear by comparing vitro single culture with the non-contact co-culture.
Description
Affiliated technical field
The present invention relates to the interactional experiment in vitro novel method of a kind of tumour cell and stem cell, especially relate to the contactless of tumour cell and stem cell and cultivate altogether.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSC) be meant the another kind of important stem cell of the non-hemopoietic tissue that is present in the bone marrow matrix, its the most significant biological property is the ability of existing self, has the potential to the histocyte differentiation in multiple mesoderm and neuroderm source again.It has source widely: comprise marrow, skeletal muscle, cartilage, bone, tendon etc., so have outstanding advantage in the stem-cell research field, have very important theoretical significance and clinical value.
We wish by based on the transwell model, set up the contactless co-culture model of MSC cell and malignant melanoma cell, observe the MSC form in the different culture models, detect multiple index immunohistochemical staining result, analyze difference, show tentatively with the contactless part MSC phenotype of cultivating altogether of malignant melanoma cell to change that clear and definite contactless cultivation altogether can be applied to Study of Interaction between MSC and the malignant melanoma cell.
The tight adherent growth of mescenchymal stem cell of cultivating in this experiment, form is than homogeneous, and its morphological specificity is essentially the inoblast shape of fusiformis, meets the mesenchymal stem cells MSCs form.Cell CD34 and VIII factor negative have been got rid of the possibility of hematopoietic cell and endotheliocyte.The analysis-by-synthesis The above results proves that cultured cells in this experiment contrast model meets the characteristics of mouse bone marrow cells mescenchymal stem cell, is not produce differentiation mouse bone marrow cells derived mesenchymal stem cell.
Blood vessel endothelium is meant one deck epithelial cell that blood vessel covers, have many important symbols, mainly comprise: VIII factor connection antigen (yon willebrand factor, vWF), can produce by all vascular endothelial cells of whole body, detect this factor and mainly use corresponding antibody.VWF has very high specificity, it has been generally acknowledged that it is the best index of reflection endotheliocyte, as people's vWF antibody the endotheliocyte of ox is not had cross reaction.
The contactless MSC light microscopic lower section cell of cultivating altogether is polygon or short fusiformis in this experiment; Immunohistochemistry and immunofluorescence result present the CD34 positive, the VIII factor positive, and all there were significant differences (P＜0.01) on CD34 and 2 indexs of the VIII factor with the contrast model to obtain co-culture model by statistical study.MSC part cell in the contactless cultivation altogether of above tentative confirmation as a result and B16 takes place to break up to the endotheliocyte direction.
Experiment this time by the transwell modelling contactless co-culture model, this kind culture model has been got rid of in the past the cell of contact co-culture model and has been mixed and be unfavorable for that the later stage observes and distinguish result's drawback, cell divide is clear and definite, changes to come into plain view.And when cell-cell interaction in the culture model need iuntercellular closely connects and/or when needing to adopt the mode of action of paracrine, can not cause that then target cell changes by setting up.According to contactless characteristics of cultivating altogether, can further infer malignant melanoma cell whether certain or some cytokine by secretion high level in the co-cultivation system MSC generation effect is caused the differentiation of MSC.
In sum, this experiment is by newly-built external contactless co-culture model, show tentatively with the contactless part MSC phenotype of cultivating altogether of malignant melanoma cell to change that clear and definite contactless cultivation altogether can be applied to the research of the two kinds of cell-cell interactions and the mode of action.Though, can't illustrate also that by present method B16 induces the concrete mode and the precise mechanism of MSC differentiation, but we have been familiar with the effect of MSC in tumor vessel forms, and contactless significance of cultivating altogether in the research cell-cell interaction and the mode of action.
Summary of the invention
By setting up the contactless co-culture model of MSC cell and malignant melanoma cell, observe the MSC form in the different culture models, detect multiple index immunohistochemical staining result, analyze difference, show tentatively with the contactless part MSC phenotype that is total in cultivating of malignant melanoma cell to change that clear and definite contactless cultivation altogether can be applied to Study of Interaction between MSC and the malignant melanoma cell.
The technical solution adopted for the present invention to solve the technical problems is: mainly utilize the Transwell culture plate in 0.4 μ m aperture to set up contactless co-culture model.
This modelling step is as follows: the trypsinase/EDTA liquid 5ml of adding 0.25% in MSC Tissue Culture Flask and B16 Tissue Culture Flask, and peptic cell 5～7 minutes, being mixed with MSC concentration is 3.0 * 10
4Individual/ml contactless cultivates the pre-training liquid of MSC altogether and B16 concentration is 7.5 * 10
4The contactless pre-training liquid of B16 of cultivating altogether of individual/ml.The contactless pre-training liquid of MSC of cultivating altogether of the 1ml of correspondence is added in the pre-culture hole of transwell, earlier with in the contactless common cultivation plug-in unit of cultivating altogether among the pre-training liquid adding of the B16 transwell (insert) of 0.1ml, again with the α-MEM perfect medium of normal 10% serum of 0.6ml (90% α-MEM+10% foetal calf serum+2% pair anti-), slowly inject along the sidewall of cultivating between plug-in unit (insert) and the lower floor's cell altogether, can reach the balance of the inside and outside liquid level of common cultivation plug-in unit (insert), finish pre-cultivation application of sample.Fully rock the mixing cell after application of sample is finished, cover tight transwell culture plate lid.Be placed on constant temperature CO
2Cultivate in the incubator, cultivate in advance and hatch 24 hours.The pre-cultivation after 24 hours taken out the transwell culture plate from incubator.Original nutrient solution suction among each hole and the insert is gone, the low serum perfect medium of α-MEM (5% foetal calf serum+about 95% α-MEM+2% is two anti-) that in the pre-culture hole of MSC, adds 5% serum of 0.6ml, with the insert that is mounted with the B16 cell one by one correspondence put into the pre-culture hole of MSC, the low serum perfect medium of α-MEM (5% foetal calf serum+about 95% α-MEM+2% is two anti-) that in each insert, slowly adds 5% serum of 0.1ml, the inside and outside liquid level of insert is balanced each other, promptly finish the foundation of the contactless co-culture model of transwell.Liquid feeding finishes, and the transwell culture plate is jiggled the mixing nutrient solution, covers tight culture plate lid.Be placed on constant temperature CO
2Cultivate in the incubator, begin to carry out contactless cultivation altogether in 72 hours.
The invention has the beneficial effects as follows, can set up the co-culture of cells system effectively, help sorting cells and sampling respectively in cultivating altogether, be convenient to the research of cell-cell interaction.
Description of drawings
The present invention is further described below in conjunction with drawings and Examples.
Fig. 1 is transwell of the present invention and plug-in unit thereof.
Fig. 2 is a MSC growth conditions in the contactless cultivation altogether.
Fig. 3 is a B16 growth conditions in the contactless cultivation altogether.
Fig. 4 is that B16 induces MSC to express the VIII factor in the contactless cultivation altogether.
Fig. 5 is that B16 induces MSC to express CD34 in the contactless cultivation altogether.
Embodiment
1 experiment material and method
1.1 laboratory animal
λ mouse source property mescenchymal stem cell, Medical University Of Tianjin's Pathological Staff Room
λ mouse source property malignant melanoma cell B16 clone, ring lake hospital Cytology Lab
1.2 main experiment reagent
λ CD34, the anti-mouse IgG of rabbit polyclonal antibody, Santa cruz company, article No. SC-9095
The λ VIII factor, the anti-mouse IgG of rabbit polyclonal antibody, genome company, article No. GA008202
1.3 main experiment equipment
λ is inverted and differs opticmicroscope, Olympus company
The λ image capturing system, Nikon/Dell company
λ controls constant incubator automatically, Jouan company
λ Transwell, 0.4um aperture, Costar company
1.4 the B16 cell is to the induction of differentiation of MSC cell in the contactless co-culture model
1.4.1 the contactless co-culture model of MSC cell and B16 cell sets up and contactless co-culture experiments is chosen MSC cell and B16 cell, carries out cell counting, being mixed with concentration is 3.0 * 10
4Pre-training liquid of the MSC of individual/ml and concentration are 7.5 * 10
4The pre-training liquid of the B16 of individual/ml.The pre-training liquid of 1ml MSC is added in the corresponding pre-culture hole of transwell; In the common cultivation plug-in unit among the pre-training liquid adding of the 0.1ml B16 transwell, again with the 0.6ml substratum, slowly inject lower floor's cell along sidewall, sample number into spectrum, the pre-cultivation after 24 hours, take out the transwell culture plate, with each hole and cultivate in the plug-in unit original nutrient solution altogether and inhale and go, the substratum (10% foetal calf serum+about 90% α-MEM+2% is two anti-) that in the pre-culture hole of MSC, adds 0.6ml, with the common cultivation plug-in unit that is mounted with the B16 cell one by one correspondence put into the pre-culture hole of MSC, to each substratum of cultivating 10% serum that slowly adds 0.1ml in the plug-in unit altogether, finish the foundation of the contactless co-culture model of transwell.Put into the constant temperature incubator, contactless cultivation altogether in 72 hours, routine observation cell growing state.
Contactless cultivate 72 hours altogether after, substratum among the exhaustion transwell is removed the common cultivation plug-in unit in each hole, and PBS cleans 2 times, the Paraformaldehyde 96 of adding 4% in each hole, fix 30 minutes, carry out mark, put into-20 ℃ and preserve (Medical University Of Tianjin, April 20 2007 shelf time, deposit number 0420 classification name BMSC), detects sample as the contactless co-culture model MSC cellular immunization histological chemistry that leaves and takes.
1.4.2 MSC cell contrast property single culture modelling and single culture experiment
Choose the MSC cell, carry out cell counting, being mixed with concentration is 3.0 * 10
4Individual/ml according to cultivating the MSC working fluid.The independent contrast culture MSC working fluid of 1ml is added in the pre-culture hole of corresponding transwell, sample number into spectrum, the pre-cultivation after 24 hours, taking-up is with the transwell culture plate, with each hole and cultivate in the plug-in unit original nutrient solution altogether and inhale and go, in each hole, add the 1ml substratum, finish the foundation of single culture model.Put into the constant temperature incubator and cultivate, 72 hours single culture, routine observation cell growing state.
After the single culture 72 hours, the substratum among the exhaustion transwell, PBS cleans 2 times, adds 4% Paraformaldehyde 96 in each hole, fixes 30 minutes, carries out mark, puts into-20 ℃ of preservations, detects sample as the single culture MSC immunohistochemistry of leaving and taking.
1.4.3 the immunohistochemistry of MSC surface molecular marker detects in two groups of experiments
The CD34 and the VIII level of factor immunohistochemistry of carrying out mouse mesenchymal stem cells MSCs in common cultivation and the single culture detect.Observe the colour developing situation in each hole, the optics inverted microscope is observed, and carries out IMAQ.Experimental data is represented with the average positive cell number and the average positive rate of every kind of index.Every group of numerical value is used x through after the stdn
2The method of inspection compares each group difference.
2 experimental results
As seen a large amount of closely cells of adherent growth are arranged in the single culture experiment, and the attached cell form mainly shows as spindle cell, and karyon is oval, and cellular form is consistent, is inoblast sample form, is typical MSC form.Contactless co-culture model part MSC cell light microscopic is inoblast sample form down; The part cell is polygon or short fusiformis, sharpness of border, and size is more even, and karyon is circular or oval, is positioned at cell central authorities.
In the induction of differentiation experiment of contactless cultivation group B16 cell altogether to MSC, cultivate 96 hole MSC immunohistochemistry and immunofluorescence results in (table 2) through paired observation single culture (table 1) and contactless being total to, present single culture group CD34 feminine gender (positive rate 4.8%), VIII factor negative (positive rate 5.5%); The contactless cultivation group CD34 positive (positive rate 50.9%) altogether, the VIII factor positive (positive rate 42.2%).And use the SPSS statistical software and carry out that the x2 check analysis is cultivated altogether and the difference (table 3) of single culture average positive cell number between CD34 and these 2 indexs of the VIII factor, the result shows common cultivation and single culture, and there were significant differences (P＜0.01) on CD34 and these 2 indexs of the VIII factor.
Table 1 single culture MSC detects index expression table
| The dyeing index | Expression | ||
| Average positive cell number | Average positive rate | The IHC staining power | |
| The CD34 VIII factor | 24 22 | 4.8％ 5.5％ | -～+ -～+ |
Annotate: the positive cell count of average positive rate accounts for the percentage of 400 observation of cell.
The contactless MSC that cultivates altogether of table 2 detects index expression table
| The dyeing index | Expression | ||
| Average positive cell number | Average positive rate | The IHC staining power | |
| The CD34 VIII factor | 204 169 | 50.9％ 42.2％ | ++～+++ +～++ |
Annotate: the positive cell count of average positive rate accounts for the percentage of 400 observation of cell.
Table 3 is cultivated altogether and single culture MSC detects index differential expression table
| The dyeing index | Be total to the cultivation group | Control group | Statistical study | |||
| Average positive cell number | Average positive rate | Average positive cell number | Average positive rate | x 2Value | The P value | |
| The CD34 VIII factor | 204 169 | 50.9％ 42.2％ | 24 22 | 4.8％ 5.5％ | 198.75 148.62 | ＜0.01 ＜0.01 |
Annotate: the positive cell count of average positive rate accounts for the percentage of 400 observation of cell.
Claims (2)
1. this novel method will change based on the phenotype that transwell (0.4 μ m aperture) set up the contactless co-culture model of tumour cell and mescenchymal stem cell and observed two kinds of cells, thereby clearly this contactless co-culture model can be applied to Study of Interaction between two kinds of cells.
2. according to claim 1, the phenotype of utilizing transwell (0.4 μ m aperture) to set up the contactless co-culture model of malignant melanoma cell and mescenchymal stem cell and observing two kinds of cells changes, and utilizes immunohistochemistry and immunofluorescence to detect its VIII factor and CD34 expression.Mescenchymal stem cell can be polygon or short fusiformis, V worker's II factor and CD34 up-regulated simultaneously by tumor cell induction in contactless co-culture model.Tentatively show and utilize tumour cell that transwell (0.4 μ m aperture) sets up and the part cell phenotype in the contactless co-culture model of mescenchymal stem cell to change, clearly this contactless co-culture model can be applied to Study of Interaction between mescenchymal stem cell and the pernicious melanocyte.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450858A (en) * | 2014-12-18 | 2015-03-25 | 中国农业科学院特产研究所 | Method and application for identifying cells participating in hair development |
| CN104623638A (en) * | 2015-01-20 | 2015-05-20 | 奥思达干细胞有限公司 | Melanoma-resistant stem cell patch and preparation method thereof |
| CN109266716A (en) * | 2018-09-13 | 2019-01-25 | 佛山科学技术学院 | A method of detection cell migration quantity |
| CN110651073A (en) * | 2017-03-22 | 2020-01-03 | 塞尔迈普有限责任公司 | Cell yields for synthetic tissue controls and synthetic tissue microarray controls |
-
2007
- 2007-10-18 CN CNA2007100599291A patent/CN101186899A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450858A (en) * | 2014-12-18 | 2015-03-25 | 中国农业科学院特产研究所 | Method and application for identifying cells participating in hair development |
| CN104623638A (en) * | 2015-01-20 | 2015-05-20 | 奥思达干细胞有限公司 | Melanoma-resistant stem cell patch and preparation method thereof |
| CN104623638B (en) * | 2015-01-20 | 2018-06-19 | 奥思达干细胞有限公司 | A kind of stem cell patch of melanoma and preparation method thereof |
| CN110651073A (en) * | 2017-03-22 | 2020-01-03 | 塞尔迈普有限责任公司 | Cell yields for synthetic tissue controls and synthetic tissue microarray controls |
| CN109266716A (en) * | 2018-09-13 | 2019-01-25 | 佛山科学技术学院 | A method of detection cell migration quantity |
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Open date: 20080528 |