CN103695371A - Adult medulloblastoma cell system and application thereof - Google Patents
Adult medulloblastoma cell system and application thereof Download PDFInfo
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- CN103695371A CN103695371A CN201310722702.6A CN201310722702A CN103695371A CN 103695371 A CN103695371 A CN 103695371A CN 201310722702 A CN201310722702 A CN 201310722702A CN 103695371 A CN103695371 A CN 103695371A
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Abstract
The invention discloses an adult medulloblastoma cell system XQ625 with a collection number of CCTCC (China center for type culture collection)No:C2013171. The cell system is obtained by separation culture of a typical medulloblastoma specimen surgically removed from the adult cauda cerebelli and can be sub-cultured in vitro for a long term, and 1*104 cells can promote tumorigenesis in a nude mouse; the cell system can meet the need of an in-depth study on medulloblastomas, is a powerful tool for studying anti-medulloblastoma drugs, and has a good potential application prospect.
Description
Technical field
The invention belongs to cytobiology field, relate to a kind of tumor cell line.
Background technology
Medulloblastoma is the modal malignant brain tumors of children, and the onset peak age is 8 years old; Only 30% medulloblastoma betides adult.Because sickness rate is lower, the existing medulloblastoma cell in whole world system is very limited at present, only has Daoy, D283, D341Med, BO-1, MED-FU, ONS-76 and ONS-81 several, and all derives from children.In recent years, along with going deep into that medulloblastoma is studied, evidences show in a large number, and children and adult's medulloblastoma all has difference at aspects such as genetic background and clinical prognosis.For example, adult's medulloblastoma betides cerebellar hemisphere conventionally, and the multiple vermis of cerebellum of being born in of children's medulloblastoma; On adult's medulloblastoma histological type, mainly with short connective tissue proliferation type, be main, and poorer than children's medulloblastoma prognosis.Therefore, be necessary separation and set up a series of medulloblastoma cell systems with different biological characteristic, to meet the further investigation needs to medulloblastoma and medicine thereof.
Summary of the invention
In view of this, the object of the invention is to separated and set up the medulloblastoma cell system with different biological characteristic, to meet the further investigation needs to medulloblastoma and medicine thereof.
After deliberation, the invention provides following technical scheme:
Adult's medulloblastoma cell is XQ625, and preserving number is CCTCC No:C2013171.
Contriver has collected the 13 routine samples that 2011-2013 Nian Jian No.2 Hospital Attached to No.3 Military Medical College, PLA preoperative diagnosis is suspected to be medulloblastoma, draws materials and carry out the cultivation of primary cell in operation.Pathological diagnoses is the equal primary medulloblastoma cell of successful separation and Culture in vitro of 5 routine samples (MB1-MB5) of medulloblastoma, and rule goes down to posterity.In the culturing process that goes down to posterity, find, increase along with passage number, the multiplication capacity of cell weakens gradually, the differentiating phenomenon of 4 routine primary medulloblastoma cells cell after cultivating for tens generations is very obvious, Growth of Cells is stagnated, only 1 routine primary medulloblastoma cell (MB4) can continuous passage, and having set up thus stable medulloblastoma cell is XQ625.
Medulloblastoma cell is that XQ625 derives from adult, betides vermis of cerebellum, and its pathological diagnosis belongs to classic medulloblastoma according to WHO central nerve neuroma classification in 2007.
The biological characteristic research of XQ625 cell is found: the doubling time of cell is 42 hours; Cell cycle is G
1phase accounts for 53.93%, G
2phase accounts for 16.07%, the S phase and accounts for 30.00%; In nude mouse, 1 * 10
4individual XQ625 cell can initial tumour generation; Transplanted tumor cell is disperse and distributes, and cell is little and justify, and karyoplasmic ratio is large, and nuclear fission is easily shown in mutually, and endochylema is few, and endochylema inner cell organ is few, and rrna and plastosome, be more original undifferentiated state as seen; Immunohistochemical markers Vimentin is positive, and Ki67 approximately 95% cell is positive, and all the other immunohistochemical markers CD99, CgA, GFAP, Pan-CK, Syn, LCA, EMA, Desmin and S-100 are all negative.
Adult medulloblastoma clone XQ625 of the present invention can for the preparation of or screen the medicine of anti-medulloblastoma.For example, by experiment in vitro, filter out and there is the antibody that suppresses or kill and wound XQ625 cell, main component that can be using this antibody as treatment pith mother cells tumor medicine, or the antibody that filters out specific recognition XQ625 cell is as target instrument, prepares the targeted drug of anti-medulloblastoma.
Beneficial effect of the present invention is: the present invention is in the primary process of carrying out cell cultures to 5 routine medulloblastomas, successfully set up the medulloblastoma cell system that a strain betides adult, and its characteristics of cell biology is studied, this clone in vitro can Long Term Passages be cultivated, in nude mouse 1 * 10
4generation that can initial tumour, had both contributed to meet the further investigation needs to medulloblastoma, was again the powerful of the anti-pith mother cells tumor medicine of development, had potential good application prospect.
Biomaterial preservation
Adult's medulloblastoma cell is XQ625, on November 5th, 2013 be preserved in Chinese Typical Representative culture collection center (be called for short CCTCC, address: China. Wuhan. Wuhan University), preserving number is CCTCC No:C2013171.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearer, the invention provides following accompanying drawing and describe:
Fig. 1 is the HE dyeing of the primary sample of medulloblastoma, and wherein A-E is respectively MB1-MB5 tumor tissues HE dyeing, magnification * 200; F-J is respectively MB1-MB5 tumor tissues HE dyeing, magnification * 400.
Fig. 2 is that the inverted phase contrast microscope of medulloblastoma primary cell is observed, and wherein A-E is respectively the morphological specificity of MB1-MB5 medulloblastoma primary cell culture, magnification * 100.
Fig. 3 is that the inverted phase contrast microscope of the medulloblastoma cell XQ625 of different algebraically P1, P21 and P41 is observed, and wherein A-C is respectively the morphological specificity of XQ625 cell P1, P21 and P41, magnification * 100; D-F is respectively the morphological specificity of XQ625 cell P1, P21 and P41, magnification * 200.
Fig. 4 detects growth curve and the cell cycle of medulloblastoma cell XQ625, and wherein A is the growth curve that CCK8 detects XQ625 cell, and B is the cell cycle of cell cycle test kit flow cytometer detection XQ625 cell.
Fig. 5 is the karyotyping result of medulloblastoma cell XQ625.
Fig. 6 is that the nude mice by subcutaneous of medulloblastoma cell XQ625 becomes knurl, and wherein A is the size of different quantities level XQ625 knurl that cell becomes, and B is the weight of different quantities level XQ625 knurl that cell becomes.
Fig. 7 is XQ625 transplanted tumor HE dyeing and immunohistochemical staining, wherein A is HE dyeing, B-J is respectively vimentin, Ki67, CD99, CgA, GFAP, Pan-CK, Syn, LCA, EMA, Desmin, S-100 immunohistochemical staining, and magnification is * and 400.
Fig. 8 is XQ625 transplanted tumor electron microscopic observation, and wherein A shows that oncocyte is less, and size and form is more consistent, and karyoplasmic ratio is large, a visible nuclear fission phase; B shows that oncocyte nuclear membrane is more smooth, visible kernel, and endochylema is few, and endochylema inner cell organ is few, visible rrna and plastosome.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in preferred embodiment, conventionally according to normal condition, or the condition of advising according to reagent manufacturer is carried out.
The major equipment using in embodiment is as follows:
| Device name | Manufacturer (place of production) |
| Opticmicroscope, BX-40 type | Olympus company (Japan) |
| Opticmicroscope, band is adopted drawing system | Leica company (Germany) |
| Transmission electron microscope, TECNAI10 type | Philip company (Holland) |
| Inverted phase contrast microscope, CK40-F200 type | Olympus company (Japan) |
| FACS Calibur type flow cytometer | BD company (U.S.) |
Main agents is as follows:
The compound method of part reagent is as follows:
1. DMEM perfect medium: get 1 bag of DMEM culture medium dry powder (1L dress), dissolve with 800ml ultrapure water, add 2.38g HEPES and 3.5g NaHCO
3, then add penicillin-Streptomycin sulphate mixed solution (dual anti-, 100 *) 10ml(penicillin, Streptomycin sulphate final concentration to be respectively 100U/ml, 100 μ g/ml), with ultrapure water, be settled to 900ml, stirring makes to dissolve completely, adjusts pH to 7.2-7.4, obtains DMEM basic culture solution; Add again 100ml FBS, fully mix, vacuum filtration, 0.22 μ m filtering with microporous membrane degerming, obtains DMEM perfect medium, and 4 ℃ save backup.
2. trysinization liquid (containing 0.25% pancreatin and 0.02%EDTA): take 0.25g pancreatin and 0.02g EDTA, with 0.01M PBS (pH=7.4), dissolve and be settled to 100ml, 0.22 μ m filtering with microporous membrane degerming, 4 ℃ save backup.
3. I type and IV Collagenase Type mixed solution: take 100mg type i collagen enzyme and 100mg IV Collagenase Type, with DMEM/F12 substratum, dissolve and be settled to 100ml, regulate pH to 7.2-7.4,0.22 μ m filtering with microporous membrane degerming ,-20 ℃ are frozen standby.
4. Sodium Citrate antigen retrieval liquid (pH6.0): Sodium Citrate 14.7g is dissolved in and makes A liquid in 500ml deionized water; Citric Acid 10.5g is dissolved in and in 500ml deionized water, makes B liquid; Get 41ml A liquid and 9ml B liquid and mix, with deionized water, be settled to 500ml, obtain.
One, the primary sample clinical pathology of medulloblastoma information
Fresh medulloblastoma excision sample is taken from No.2 Hospital Attached to No.3 Military Medical College, PLA's Neurological Surgery.Case is diagnosed according to WHO central nerve neuroma classification in 2007, and every routine sample is provided diagnosis report by two pathologist.Clinical pathology information is as shown in table 1:
The primary sample clinical pathology of table 1 medulloblastoma information
| Numbering | Date of surgery | Sex | Age | Position | Pathological diagnosis |
| MB1 | 2011-7-26 | |
6 years old | Vermis of cerebellum | Short connective tissue proliferation type medulloblastoma |
| MB2 | 2011-9-13 | |
10 years old | Fourth ventricle | Classic medulloblastoma |
| MB3 | 2011-10-17 | Female | 9 years old | Fourth ventricle | Nodular type medulloblastoma |
| MB4 | 2012-06-25 | Man | 24 years old | Vermis of cerebellum | Classic medulloblastoma |
| MB5 | 2012-07-20 | |
6 years old | Vermis of cerebellum | Classic medulloblastoma |
As shown in Figure 1, visible oncocyte is little for the HE coloration result of the primary sample of above-mentioned 5 routine medulloblastoma, nuclear hyperchromatism, and endochylema is few, and karyoplasmic ratio is larger, the visible typical Homer-Wright chrysanthemum shape unity structure in subregion.
Two, primary medulloblastoma cell is cultivated
The primary sample of 5 routine medulloblastoma is placed in respectively to the DMEM perfect medium of precooling, with aseptic eye scissors, be cut into the fragment of about 1mm3 size, the centrifugal 5min of 800g, abandons supernatant, precipitation is resuspended by 10ml I type and IV Collagenase Type mixed solution, 37 ℃ of digestion 30min, the more centrifugal 5min of 800g, abandon supernatant, precipitation is resuspended with DMEM perfect medium, 37 ℃ of cultivations, 3 days visible tumour cell adherent growth afterwards, after growing to 60%-70% degrees of fusion by trysinization liquid had digestive transfer culture.
Result successful separation and Culture from the primary sample of 5 routine medulloblastoma obtains 5 routine primary medulloblastoma cells (MB1～MB5).Under inverted phase contrast microscope, observe, the form difference little (Fig. 2) of 5 routine primary medulloblastoma cells, in the situation that having serum, visible culturing cell endochylema is abundant, part district visible cell is the morphological specificity of spongiocyte, there is more elongated protrusion (Fig. 3), illustrate that the primary medulloblastoma cell of cultivating has occurred to the feature of spongiocyte direction differentiation.Along with the increase (approximately tens generations) of passage number, there are 4 routine primary medulloblastoma cells all to occur obvious differentiation and the stagnation of Growth of Cells in various degree, only 1 routine primary medulloblastoma cell (MB4) can continuous passage.
Three, foundation and the characteristic research thereof of medulloblastoma cell system
By the external continuous passage of primary medulloblastoma cell MB4, having set up stable medulloblastoma cell is XQ625.
1, cellular form is observed
Morphologic observation under the XQ625 cell mirror of different algebraically P1, P21 and P41, visible P1 cell has more elongated protrusion, is the feature of spongiocyte sample differentiation, and along with cultivating the increase of algebraically, the cell with spongiocyte sample differentiating characteristic is fewer and feweri; P21 cell minority is stellate cell sample form, has more endochylema projection, and all the other great majority are little circle, ellipse and polygonal morphological specificity, and caryoplasm is larger; P41 cell has loseed elongated protrusion, for compared with the cellular form of homogeneous (Fig. 1-3).
2, cell growth curve
Collect the P41XQ625 cell of logarithmic phase, adjust cell concn to 1 * 10
4individual/ml, the 96 every holes of orifice plate add cell suspension 0.1ml, put 37 ℃, 5%CO
2in incubator, cultivate 8 days, every 24 hours absorbances with CCK-8 kit measurement wavelength 450nm, draw cell growth curve.The results are shown in Figure 4A, the doubling time of cell is 42 hours.
3, cell cycle analysis
With trysinization P41XQ625 cell, become single cell suspension, the centrifugal 3-5min of 1000g, abandon supernatant, cell precipitation is also resuspended with the PBS washing of ice bath precooling, 70% ethanol that adds ice bath precooling, piping and druming mixes gently, fix 12 hours for 4 ℃, the more centrifugal 3-5min of 1000g, supernatant abandoned, cell precipitation is also resuspended with the PBS washing of ice bath precooling, add propidium iodide staining fluid (cell cycle detection kit), 37 ℃ of lucifuges are hatched 30min, with flow cytometer, detect, excitation wavelength is 488nm, with analysis software, carries out cell cycle analysis.The results are shown in Figure 4B, G
1phase cell accounts for 53.93%, G
2phase cell accounts for 16.07%, S phase cell and accounts for 30.00%.
4, karyotyping
The P41XQ625 cell of taking the logarithm vegetative period, before stop cultivating, within 2-4 hour, adding final concentration is that the colchicine of 0.2 μ g/ml makes cell fission stop at mid-term, after stopping cultivating, the centrifugal 10min of 1000g, abandon supernatant, precipitation adds the 0.075M KCl hypotonic medium that is preheated to 37 ℃, 37 ℃ of hypotonic 30min make cell expansion and Chromosome spread, adding stationary liquid is that volume ratio is that methyl alcohol-Glacial acetic acid mixed solution of 3:1 pre-fixes again, the centrifugal 10min of 1000g, abandon supernatant, precipitation adds stationary liquid again, room temperature is 30min fixedly, the centrifugal 10min of 1000g, abandon supernatant, after precipitation is resuspended with stationary liquid, dripping 2-3 drips on the slide glass soaking in frozen water, 80 ℃ of roasting sheet 15min, put the 8min that dyes in Giemsa dye liquor, washing removal floating color, dry, put micro-Microscopic observation chromosome specimen division phase number and deployment conditions.The results are shown in Figure 5, counting chromosome number is 60-62 bar.Karyotyping result is: 60, XX, der (1) t (1; 3) (p22; P11), der (1) t (1; 4) (q42; Q31,3) ,+1 ,+1, del (2) (q) der (2) (p), der (3) (q), der (5) t (3; 5) (q23; P15), del (6) (q11) ,+6 ,+7, der (9), der (10) t (2; 15), der (12) t (11; 12) (q23; Q13) ,+12, der (13) t (9; 13) (q12; P10) ,+14 ,-15 ,+16 ,-21 ,+der (19) t (19; 21) ,+i (11) (p10) ,+i (11) (p10) ,+i (11) (p10) ,+add (22) (q13) ,+mar ,+mar ,+der () ,+der ().
Four, medulloblastoma cell is the one-tenth knurl ability of XQ625
In order to detect XQ625 cell, whether have in body and become knurl ability, the P41XQ625 cell of different quantities level is got to nude mice oxter.Result is as shown in table 2, and 1 * 10
4individual XQ625 cell can initial tumour in nude mouse generation.Different quantities level XQ0625 cell becomes knurl size and weight to see Fig. 6.
Table 2 XQ625 cell becomes knurl experiment
| Cell quantity | Become knurl number of animals/ |
| 5×10 5 | 5/5 |
| 1×10 5 | 5/5 |
| 5×10 4 | 5/5 |
| 1×10 4 | 2/5 |
| 5×10 3 | 0/5 |
1, the HE of XQ625 transplanted tumor and immunohistochemical staining
Immunohistochemical staining method is as follows: organizes paraffin mass to be prepared into the tissue slice that thickness is 4-7 μ m transplanted tumor, puts under roasting sheet lamp and toast after 20min, and dewaxing, aquation, uses 3%H
2o
2solution soaks 30min blocking-up endogenous peroxydase in 37 ℃, 0.01M PBS rinsing 3 times, by Sodium Citrate antigen retrieval immersion, steep and carry out Microwave method (first 800W, 3min are heated to solution boiling, with 150W, maintain boiling state 17min again), room temperature naturally cooling after reparation, 0.01M PBS rinsing 3 times, add primary antibodie, 4 ℃ of overnight incubation, 0.01M PBS rinsing 3 times, then add HRP mark two anti-, hatch 30min for 37 ℃, 0.01M PBS rinsing 3 times, the colour developing of DAB nitrite ion, Hematorylin is redyed, dehydration, transparent, mounting, microscopy.Result as shown in Figure 7, learn the visible oncocyte disperse of form and distribute by XQ625 transplanted tumor HE dyeing mirror undertissue, and interstitial composition is few, and cell is little and justify, and endochylema is few, and nuclear fission is easily shown in mutually; Immunohistochemical staining shows that Vimentin is positive, Ki67 approximately 95% cell positive, and all the other immunohistochemical markers CD99, CgA, GFAP, Pan-CK, Syn, LCA, EMA, Desmin and S-100 are all negative.
2, the transmission electron microscope observing of XQ625 transplanted tumor
Get and be less than 1mm
3transplanted tumor tissue, by 2.5% glutaraldehyde, fix 24 hours, 0.1mol/L phosphoric acid rinsing liquid rinsing 3 times, 1% osmic acid stationary liquid is 2-3 hour fixedly, 0.1mol/L phosphoric acid rinsing liquid rinsing 3 times, in 4 ℃, use successively 50% ethanol again, 70% ethanol, 90% ethanol, 90% ethanol-90% acetone mixed solution (volume ratio 1:1), 90% acetone respectively soaks 15min, acetone room temperature rinsing 3 times, use successively again acetone+embedding liquid (volume ratio 2:1) room temperature embedding 3-4 hour, the embedding of acetone+embedding liquid (volume ratio 1:2) room temperature is spent the night, 37 ℃ of embedding 2-3 hour of embedding liquid, putting successively 37 ℃ of oven dryings spends the night again, 45 ℃ of oven dryings 12 hours, 60 ℃ of oven dryings 48 hours, finally section (thickness 70nm), the two dyeing of 3% acetic acid uranium-lead citrate, microscopy.As shown in Figure 8, the visible oncocyte of Electronic Speculum is less for result, and size and form is more consistent, and karyoplasmic ratio is large, and core is circular, oval, and nuclear membrane is smooth neat, also has nucleus circumference to be dentation complications, visible kernel, and nuclear fission is easily shown in mutually; It is neatly straight that cell circumference has, the complications that also have, and flanking cell plasma membrane is inlayed each other; Endochylema is few, and endochylema inner cell organ is few, and visible rrna and plastosome, have no microtubule or colloid microfilament bundle etc. to the differentiation of spongiocyte direction.
Finally explanation is, above preferred embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is described in detail by above preferred embodiment, but those skilled in the art are to be understood that, can to it, make various changes in the form and details, and not depart from the claims in the present invention book limited range.
Claims (2)
1. adult's medulloblastoma cell is XQ625, and preserving number is CCTCC No:C2013171.
2. described in claim 1, adult's medulloblastoma cell is XQ625 in preparation or screens the application in the medicine of anti-medulloblastoma.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108728533A (en) * | 2017-04-20 | 2018-11-02 | 常青 | The purposes of gene group and SNCA genes as the biomarker of 4 type medulloblastomas for medulloblastoma molecule parting |
| CN113866411A (en) * | 2021-08-19 | 2021-12-31 | 中国人民解放军陆军军医大学第一附属医院 | Medulloblastoma/cell marker and application thereof |
-
2013
- 2013-12-24 CN CN201310722702.6A patent/CN103695371B/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108728533A (en) * | 2017-04-20 | 2018-11-02 | 常青 | The purposes of gene group and SNCA genes as the biomarker of 4 type medulloblastomas for medulloblastoma molecule parting |
| CN108728533B (en) * | 2017-04-20 | 2022-06-14 | 常青 | Gene group for molecular typing of medulloblastoma and use of SNCA gene as biomarker of medulloblastoma type 4 |
| CN113866411A (en) * | 2021-08-19 | 2021-12-31 | 中国人民解放军陆军军医大学第一附属医院 | Medulloblastoma/cell marker and application thereof |
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