CN103695371B - The medulloblastoma cell system of adult and application thereof - Google Patents
The medulloblastoma cell system of adult and application thereof Download PDFInfo
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- CN103695371B CN103695371B CN201310722702.6A CN201310722702A CN103695371B CN 103695371 B CN103695371 B CN 103695371B CN 201310722702 A CN201310722702 A CN 201310722702A CN 103695371 B CN103695371 B CN 103695371B
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Abstract
The invention discloses the medulloblastoma cell system XQ625 of a kind of adult, preserving number is CCTCC No:C2013171; This clone obtains from betiding separation and Culture the vermian classic medulloblastoma excision sample of adult, can cultivate, in nude mouse 1 × 10 by Long Term Passages in vitro
4individual cell can the generation of initial tumor; This clone had both contributed to meeting the further investigation needs to medulloblastoma, was again the powerful developing anti-pith mother cells tumor medicine, had potential good application prospect.
Description
Technical field
The invention belongs to cell biology, 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 the medulloblastoma of 30% betides adult.Because sickness rate is lower, the current whole world existing medulloblastoma cell system is very limited, 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 of studying medulloblastoma, a large amount of evidence shows, the medulloblastoma of children and adult all has difference in genetic background and clinical prognosis etc.Such as, the medulloblastoma of adult betides cerebellar hemisphere usually, and the multiple vermis of cerebellum of being born in of the medulloblastoma of children; The medulloblastoma histological type of adult is main mainly with short connective tissue proliferation type, and poorer than the medulloblastoma prognosis of children.Therefore, be necessary to be separated and set up a series of medulloblastoma cell system 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 be 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:
The medulloblastoma cell system XQ625 of adult, preserving number is CCTCC No:C2013171.
Contriver have collected 2011-2013 Nian Jian No.2 Hospital Attached to No.3 Military Medical College, PLA preoperative diagnosis and is suspected to be 13 of medulloblastoma routine samples, draws materials and carry out the cultivation of primary cell in operation.Pathological diagnoses is 5 routine samples (MB1-MB5) the primary medulloblastoma cell of successful separation and Culture all in vitro of medulloblastoma, and rule goes down to posterity.Find in Secondary Culture process, along with the increase of passage number, the multiplication capacity of cell weakens gradually, the differentiating phenomenon of 4 routine primary medulloblastoma cells cell after cultivation tens generation clearly, cell growth arrest, only 1 routine primary medulloblastoma cell (MB4) can continuous passage, thereby establish stable medulloblastoma cell system XQ625.
Medulloblastoma cell system 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 the generation of initial tumor; Transplanted tumor cell is Dispersed precipitate, and cell is little and justify, and karyoplasmic ratio is large, and mitosis figures is easily shown in, endochylema is few, and endochylema inner cell organ is few, visible rrna and plastosome, in more original undifferentiated state; Immunohistochemical markers Vimentin is positive, and Ki67 about 95% cell is positive, and all the other immunohistochemical markers CD99, CgA, GFAP, Pan-CK, Syn, LCA, EMA, Desmin and S-100 are feminine gender.
Adult medulloblastoma clone XQ625 of the present invention may be used for the medicine preparing or screen anti-medulloblastoma.Such as, the antibody having and suppress or kill and wound XQ625 cell is filtered out by experiment in vitro, can using the main component of this antibody as treatment pith mother cells tumor medicine, or the antibody filtering 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 carrying out in the process of cell cultures to 5 routine medulloblastomas are primary, successfully establish the medulloblastoma cell system that a strain betides adult, and its characteristics of cell biology is studied, this clone can be cultivated, in nude mouse 1 × 10 by Long Term Passages in vitro
4can the generation of initial tumor, both contributed to meeting the further investigation needs to medulloblastoma, and be again the powerful developing anti-pith mother cells tumor medicine, there is potential good application prospect.
Biomaterial preservation
Adult medulloblastoma cell system XQ625, on November 5th, 2013 be preserved in China typical 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 clearly, the invention provides following accompanying drawing and being described:
Fig. 1 is the HE dyeing of the primary sample of medulloblastoma, and wherein A-E is respectively MB1-MB5 tumor tissues HE and dyes, magnification × 200; F-J is respectively MB1-MB5 tumor tissues HE and dyes, 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 is growth curve and the cell cycle detection 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, wherein the size of A become to by different quantities level XQ625 cell knurl, and B becomes by different quantities level XQ625 cell the weight of knurl.
Fig. 7 is that XQ625 transplanted tumor HE dyes 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 display oncocyte is less, and size and form is relatively more consistent, and karyoplasmic ratio is large, a visible mitosis figures; It is more smooth that B shows oncocyte nuclear membrane, and visible kernel, 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, usually conveniently condition, or carry out according to the condition that reagent manufacturer advises.
The major equipment used in embodiment is as follows:
| Device name | Manufacturer (place of production) |
| Opticmicroscope, BX-40 type | Olympus company (Japan) |
| Opticmicroscope, drawing system adopted by band | 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 portion of reagent is as follows:
1. DMEM perfect medium: get 1 bag of DMEM culture medium dry powder (1L dress), dissolves with 800ml ultrapure water, adds 2.38g HEPES and 3.5g NaHCO
3, then add Pen .-Strep mixed solution (dual anti-, 100 ×) 10ml(penicillin, Streptomycin sulphate final concentration is respectively 100U/ml, 100 μ g/ml), 900ml is settled to ultrapure water, stirring makes to dissolve completely, and adjustment pH to 7.2-7.4, obtains DMEM basic culture solution; Add 100ml FBS again, fully mix, vacuum filtration, 0.22 μm of filtering with microporous membrane is degerming, obtains DMEM perfect medium, and 4 DEG C save backup.
2. trysinization liquid (containing 0.25% pancreatin and 0.02%EDTA): take 0.25g pancreatin and 0.02g EDTA, dissolve with 0.01M PBS (pH=7.4) and be settled to 100ml, 0.22 μm of filtering with microporous membrane is degerming, and 4 DEG C save backup.
3. I type and IV Collagenase Type mixed solution: take 100mg type i collagen enzyme and 100mg IV Collagenase Type, dissolve and be settled to 100ml with DMEM/F12 substratum, regulate pH to 7.2-7.4,0.22 μm of filtering with microporous membrane is degerming, and-20 DEG C frozen for subsequent use.
4. Sodium Citrate antigen retrieval buffers (pH6.0): Sodium Citrate 14.7g is dissolved in obtained A liquid in 500ml deionized water; Citric Acid 10.5g is dissolved in obtained B liquid in 500ml deionized water; Get 41ml A liquid and the mixing of 9ml B liquid, be settled to 500ml with deionized water, obtain final product.
One, medulloblastoma primary sample clinical pathology information
Fresh medulloblastoma excision sample takes 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 provides diagnosis report by two pathologist.Clinical pathology information is as shown in table 1:
Table 1 medulloblastoma primary sample clinical pathology information
| Numbering | Date of surgery | Sex | Age | Position | Pathological diagnosis |
| MB1 | 2011-7-26 | Man | 6 years old | Vermis of cerebellum | Short connective tissue proliferation type medulloblastoma |
| MB2 | 2011-9-13 | Man | 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 | Man | 6 years old | Vermis of cerebellum | Classic medulloblastoma |
As shown in Figure 1, visible oncocyte is little, nuclear hyperchromatism, and endochylema is few for the HE coloration result of the primary sample of above-mentioned 5 routine medulloblastoma, and karyoplasmic ratio is comparatively large, 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 the DMEM perfect medium of precooling, be cut into the fragment of about 1mm3 size with Sterile ophthalmic, the centrifugal 5min of 800g, abandons supernatant, precipitation 10ml I type and IV Collagenase Type mixed solution resuspended, 37 DEG C of digestion 30min, the more centrifugal 5min of 800g, abandon supernatant, precipitation DMEM perfect medium is resuspended, 37 DEG C of cultivations, visual tumors cell attachment growth after 3 days, by trysinization liquid had digestive transfer culture after growing to 60%-70% degrees of fusion.
Result successful separation and Culture from the primary sample of 5 routine medulloblastoma obtains 5 routine primary medulloblastoma cells (MB1 ~ MB5).Observe under inverted phase contrast microscope, the form difference little (Fig. 2) of 5 routine primary medulloblastoma cells, when there being serum, visible culturing cell endochylema enriches, part district visible cell is the morphological specificity of spongiocyte, there is more elongated protrusion (Fig. 3), illustrate that the feature to the differentiation of spongiocyte direction has appearred in the primary medulloblastoma cell cultivated.Along with the increase (about tens generations) of passage number, have 4 routine primary medulloblastoma cells all to occur obvious differentiation and cell growth arrest in various degree, only 1 routine primary medulloblastoma cell (MB4) can continuous passage.
Three, the foundation of medulloblastoma cell system and characteristic research thereof
Stable medulloblastoma cell system XQ625 is established by primary medulloblastoma cell MB4 serial passages in vitro.
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, the feature of the differentiation in spongiocyte sample, and along with the increase of cultivating 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 in little circle, ellipse and polygonal morphological specificity, and caryoplasm is larger; P41 cell loses elongated protrusion, is more homogeneous cellular form (Fig. 1-3).
2, cell growth curve
Collect the P41XQ625 cell of logarithmic phase, adjustment cell concn to 1 × 10
4individual/ml, the 96 every holes of orifice plate add cell suspension 0.1ml, put 37 DEG C, 5%CO
2cultivate 8 days in incubator, 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
Single cell suspension is become with trysinization P41XQ625 cell, the centrifugal 3-5min of 1000g, abandon supernatant, the precooling of cell precipitation ice bath PBS washing and resuspended, add 70% ethanol of ice bath precooling, blow and beat mixing gently, fix 12 hours for 4 DEG C, the more centrifugal 3-5min of 1000g, abandon supernatant, the precooling of cell precipitation ice bath PBS washing and resuspended, add propidium iodide stain liquid (cell cycle detection kit), 37 DEG C of lucifuges hatch 30min, with flow cytomery, excitation wavelength is 488nm, carries out cell cycle analysis with analysis software.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, within 2-4 hour, adding final concentration before stopping cultivating is that the colchicine of 0.2 μ g/ml makes cell fission stop at mid-term, after stopping cultivation, the centrifugal 10min of 1000g, abandon supernatant, precipitation adds the 0.075M KCl hypotonic medium being preheated to 37 DEG C, 37 DEG C of hypotonic 30min make cell expansion and Chromosome spread, adding stationary liquid and volume ratio is again that the methyl alcohol-Glacial acetic acid mixed solution of 3:1 pre-fixes, the centrifugal 10min of 1000g, abandon supernatant, precipitation adds stationary liquid again, room temperature fixes 30min, the centrifugal 10min of 1000g, abandon supernatant, after precipitation stationary liquid is resuspended, dripping 2-3 drips on the slide glass that soaked in frozen water, 80 DEG C of roasting sheet 15min, put in Giemsa dye liquor the 8min that dyes, washing removal floating color, dry, put number and the deployment conditions of basis of microscopic observation chromosome specimen split coil method.The results are shown in Figure 5, enumerating chromosomes 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, the one-tenth knurl ability of medulloblastoma cell system XQ625
Whether have in body become knurl ability to detect XQ625 cell, 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 the generation of initial tumor in nude mouse.Different quantities level XQ0625 cell becomes knurl size and weight to see Fig. 6.
Table 2 XQ625 cell becomes knurl to test
| Cell quantity | Become knurl number of animals/laboratory animal number |
| 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: transplanted tumor tissue paraffin block is prepared into the tissue slice that thickness is 4-7 μm, and after toasting 20min under putting roasting sheet lamp, dewaxing, aquation, uses 3%H
2o
2solution soaks 30min in 37 DEG C and blocks endogenous peroxydase, 0.01M PBS rinsing 3 times, soak with Sodium Citrate antigen retrieval buffers and carry out Microwave method (first 800W, 3min be heated to solution boiling, boiling state 17min is maintained again with 150W), room temperature naturally cooling after reparation, 0.01M PBS rinsing 3 times, add primary antibodie, 4 DEG C of overnight incubation, 0.01M PBS rinsing 3 times, then add HRP mark two resist, hatch 30min for 37 DEG C, 0.01M PBS rinsing 3 times, DAB nitrite ion develops the color, and Hematorylin is redyed, dehydration, transparent, mounting, microscopy.As shown in Figure 7, the visible oncocyte Dispersed precipitate of form is learned to result by XQ625 transplanted tumor HE dyeing mirror undertissue, and interstitial components is few, and cell is little and justify, and endochylema is few, and mitosis figures is easily shown in; Vimentin is positive in immunohistochemical staining display, and Ki67 about 95% cell positive, all the other immunohistochemical markers CD99, CgA, GFAP, Pan-CK, Syn, LCA, EMA, Desmin and S-100 are feminine gender.
2, the transmission electron microscope observing of XQ625 transplanted tumor
Get and be less than 1mm
3transplanted tumor tissue, 24 hours are fixed by 2.5% glutaraldehyde, 0.1mol/L phosphoric acid rinsing liquid rinsing 3 times, 2-3 hour fixed by 1% osmic acid stationary liquid, 0.1mol/L phosphoric acid rinsing liquid rinsing 3 times, 50% ethanol is used successively again in 4 DEG C, 70% ethanol, 90% ethanol, 90% ethanol-90% acetone mixture (volume ratio 1:1), 90% acetone respectively soaks 15min, acetone room temperature rinsing 3 times, again successively with acetone+embedding liquid (volume ratio 2:1) room temperature embedding 3-4 hour, acetone+embedding liquid (volume ratio 1:2) room temperature embedding is spent the night, embedding liquid 37 DEG C embedding 2-3 hour, put 37 DEG C of oven dried overnight more successively, 45 DEG C of oven dryings 12 hours, 60 DEG C of oven dryings 48 hours, finally cut into slices (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 relatively more consistent, and karyoplasmic ratio is large, and core is circular, oval, and nuclear membrane is neatly smooth, and also have nucleus circumference to be that dentation is tortuous, visible kernel, mitosis figures is easily shown in; It is neatly straight that cell circumference has, the complications also had, and flanking cell plasma membrane is inlayed each other; Endochylema is few, and endochylema inner cell organ is few, visible rrna and plastosome, has no the differentiation to spongiocyte direction such as microtubule or colloid microfilament bundle.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (2)
1. the medulloblastoma cell system XQ625 of adult, described medulloblastoma cell system XQ625 is confirmed by immunohistochemical staining method, and preserving number is CCTCC No:C2013171.
2. the application of the medulloblastoma cell system XQ625 of adult described in claim 1 in the medicine of the anti-medulloblastoma of screening.
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| TJ862髓母细胞瘤体外细胞系的建立及其特征;浦佩玉等;《中华神经外科杂志》;19881231;第4卷(第4期);第231页左栏第2段 * |
| wortmannin 对人髓母细胞瘤细胞系daoy增殖和凋亡的影响;杨晓非;《现代肿瘤医学》;20121130;第20卷(第11期);摘要 * |
| 小鼠髓母细胞瘤细胞系的建立与鉴定;王月圆等;《基础医学与临床》;20091231(第7期);摘要 * |
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