CN1336431A - Establishment of immortal human ovary carcinoma cell strain - Google Patents
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Abstract
The present invention relates to the method of establishing human ovary cancer immortal cell strian, the immortal gene-SV40T antigen gene is introduced into human ovary cancer original generation cell, then screened, resistance clone enlarge culture to obtain immortal cell strain. The established ovary cancer cell strain is named as BVPH:OVSC-2 and is kept by CGMCC with keeping No.0606, its biological characteristics includes strong invading powder, cell is spindle sarcoma call shape, apparent heteromorphism, retains biological characters of original cell.
Description
Technical field:
The invention belongs to microorganism zooblast strain field, be specifically related to a kind of establishment method of immortal human ovary carcinoma cell strain and people's ovarian sarcoma sample cancer immortalized cell line of setting up according to this method.
Background technology:
In the female reproductive system malignant tumour, the sickness rate of ovarian cancer occupies the second, but mortality ratio occupy the first.Though operation, chemotherapy and radiation technology are updated, the five year survival rate of ovarian cancer is still paced up and down about 30% over past ten years.Major cause is that ovarian cancer histological type complexity, grade of malignancy height, individual difference are big, and the generation on its molecule and the cell levels, development mechanism are not clear.Owing to the fresh cancer cells life-span that obtains is too short, can't solve the whole process of the research of direction of medication usage and biotherapy etc. clinically in patient's body.Be to deepen understanding, need a large amount of models of dissimilar and individual ovarian cancer cell strain that derive from as in vitro study to ovarian cancer.The method of setting up the ovarian cancer cell strain at present has through the former foster spontaneous one-tenth of being commissioned to train to be or to plant to go down to posterity to be that the former becomes is that rate is very low, has only individual cells to become cell strain in nude mouse in a large amount of former foster samples of being commissioned to train; The bothersome effort of the latter, and the shortcoming that the cell that is exists human cell surface antigen to weaken or disappear of building.Therefore, the method of setting up the ovarian cancer cell strain at present can not satisfy the demand of in vitro study far away, so that limited on cell and molecular biology level, press for and set up a cover and can repeat to set up the method for ovarian cancer cell strain the understanding of ovarian cancer pathogeny, resistance mechanism, transfer correlative factor etc.
Cell immortalityization (Immortalization) also claim immortality, is the characteristic that cell obtains sustainable growth and multiplication capacity, and immortalized cells has the ability of indeterminate growth and propagation, and it is not dead to go down to posterity for a long time.The SV40T antigen gene imported set up the method that corresponding cell strain is the most frequently used cell immortalityization in the primary cell, reference is seen Jha KK, BangaS, Palejwala V, et al.SV40-mediated immortalization.Exp Cell Res, 1998,245:1-7.Utilize SV40 T antigen gene successively to make epithelial cell immortalizations such as segmental bronchus, stones in intrahepatic bile duct, uterine neck.It can improve the efficient of cell immortalityization, its immortalization effect is except that making the cell life cycle prolongation, the phenotypic differentiation that can also keep its initiating cell can reflect the biological characteristics of its initiating cell on cell levels, have that to become be rate height, characteristics repeatably, reference is seen Hopfer UJW, Jacobberger DC, Gruenert RL, et al.Immortalizationof epithelial cells.Am J Physiol, 1996,270 (Cell Physiol.39): C1-C11.But, still do not have the report that utilizes SV40T to set up immortal human ovary carcinoma cell strain at present both at home and abroad.
About nearly 119 kinds of the diagnosis names of the malignant tumour be made up of cancer and sarcoma, present unified title is a carcinoma sarcomatodes.Medical circle lacks understanding to this malignant tumour, causes confusion and arguement in the clinical diagnosis and treatment process easily.Carcinoma sarcomatodes belongs to epithelial cell source property, and essence is a kind of poor cancer of breaking up.Ovarian sarcoma sample cancer is very rare, easily causes mistaken diagnosis, and disease progression is very fast clinically, and growth is rapid, and invasiveness is strong, and survival rate was very low in 5 years, and majority is being diagnosed death in later six months.Oncologist and pathology doctor lack understanding to the clinical diagnosis and treatment of ovarian sarcoma sample cancer, and its biological characteristics is known little about it especially.Therefore, the cell strain donor that extremely needs to set up corresponding histological type is furtherd investigate outward, but the report that is is built in none strain still both at home and abroad at present.
Summary of the invention:
The purpose of this invention is to provide a kind of method of setting up immortal human ovary carcinoma cell strain, it is characterized in that immutalizing gene---SV40 T antigen gene imports the human ovarian cancer primary cell, through screening, the resistance clone enlarged culturing, reach the evaluation that SV40 T gene is expressed in transfectional cell, obtain immortalized cell line.
Another object of the present invention also is to utilize the method for above-mentioned immortalization that a kind of people's ovarian sarcoma sample cancer immortalized cell line is provided, its biological property is: invasiveness is strong, cell is rendered as fusiformis sarcoma cell form, be epithelial origin, atypia is obvious, and keeps the biological characteristics of its initiating cell.
People's ovarian sarcoma sample cancer immortalized cell line of the present invention, called after BUPH:OVSC-2, this cell strain are by China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, and preserving number is NO.0606.
One, the establishment method of immortal human ovary carcinoma cell strain of the present invention:
1.SV40 T antigen gene Construction of eukaryotic
PSV
3Neo contains SV40 T antigen (containing big T and little T antigen) gene fragment, and pcD2 is for efficiently expressing the eukaryotic vector plasmid.Utilize gene recombination technology that SV40 T antigen gene fragment cloning is gone in the pcD2 carrier, be built into pcD2-SV40.Recombinant plasmid is cut evaluation through enzyme.
2. former be commissioned to train foster:
The aseptic ovarian cancer fresh specimens of drawing materials of Operation theatre is supported the conventional processing tissue by former being commissioned to train.With final concentration 200U/ml collagenase I, 100U/ml hyaluronic acid enzymic digestion tissue.Digestion product through 45% and 20%Percoll solution carry out density gradient centrifugation, separate different cellular components, collect institute's isolated cells, be seeded in the plastic culture bottle.Substratum is that MCDB105: M199 adds 15% foetal calf serum, 0.2U/ml Regular Insulin, the cultivation of 2mmol/ml L-glutaminate, 37 ℃, 5%CO at 1: 1
2Cultivate in the incubator, 675U/ml collagenase I peptic cell goes down to posterity.
3. the foundation of immortalized cell line:
Select that growth conditions is good, the 80% early stage passage cell that converges is carrier transfection plasmid pcD2-SV40 with liposome Lipofectin2000.Cell screens through G418 after the transfection, the resistance clone enlarged culturing, and immortalized cell line is built up in the evaluation that SV40 T antigen gene is expressed in transfectional cell.After the transfection, cell changes the nutrient solution that contains 15% calf serum into and cultivates and 1: 1 had digestive transfer culture of 0.25% trypsinase: 0.02%ETDA at stable growth and after the certain period of going down to posterity.Become system's back cell to be undertaken frozen by the ordinary method of cell cultures and recovery.
Two, people's ovarian sarcoma sample cancer immortalized cell line (BUPH:0VSC-2)
Cell derives from gynaecology of The People's Hospital of Peking University patient with operation of being in hospital, and the people's ovarian sarcoma that has drawn from sample cancer stomach wall shifts tubercle, and it is identical with the ovary primary tumor that postoperative HE dyeing, immunohistochemical methods and Electronic Speculum confirm that stomach wall shifts tubercle character, is ovarian sarcoma sample cancer.
According to the people's ovarian sarcoma sample cancer immortalized cell line BUPH:OVSC-2 that sets up the method foundation of cell strain of the present invention, substratum is that MCDB105: M199 adds 15% foetal calf serum, 0.2U/ml Regular Insulin, the cultivation of 2mmol/ml L-glutaminate, 37 ℃, 5%CO at 1: 1
2Cultivate 1: 1 had digestive transfer culture of 0.25% trypsinase: 0.02%ETDA in the incubator.Undertaken frozen and recovery by the ordinary method of cell cultures, recovery back BUPH:OVSC-2 cell inoculation rate is about 80%.Generation surplus having reached 80 at present, can long term growth and stable going down to posterity.
Through experimental observation and checking, the BUPH:OVSC-2 of vitro culture has following feature:
Morphological observation: light microscopic observes down that viable cell is fusiformis and polygon in the culturing bottle, division stage cell become circle, the back cell monolayer that goes down to posterity is adherent to be dispersed in growth, density reaches 0.5 * 10
5/ cm
2The time, see packing phenomenon, lose contact inhibition (seeing accompanying drawing 1).HE dyeing visible cell differs in size, and form is irregular, and nucleus and tenuigenin ratio increase, and chromatin increases in the nuclear, and kernel increases, visible a plurality of kernels.Transmission electron microscope observing, iuntercellular mostly are general connection (seeing accompanying drawing 2), and there are secretory granules in intermediate filament and tension force silk under visible desmosome (seeing accompanying drawing 3), closely connection, fine hair, the after birth in the endochylema, and the confirmation cell is an epithelium genesis; The nucleus out-of-shape, the caryoplasm ratio is big, and heterochromatin is abundant in the nuclear, the big and limit collection of kernel, visible two above kernels; The endochylema inner cell organ is few.
Cell doubling time: about 42 hours.
Cloning efficiency: 11.8%.
Soft agar is cultivated: with the 30th generation cell carry out double-deck soft agar and cultivate, saw have cell colony to form in back 12 days in inoculation, illustrate that this cell has colony and forms ability.
Chromosome analysis: observe the 40th generation cell chromosome, count 50 mitotic figures at random, karyomit(e) is between the 43-46 bar, mode is 46.15 scattered G of random choose show the band sample, carry out karyotyping, all lack 1 X chromosome, many No. 20 karyomit(e), and each caryogram can both see 3 marker chromosomess, and wherein two is 1q and the mutual transposition of 12q, and another is 14p
+Its chromosome structure exists significantly unusual, but not normal diploid karyotype is pseudodiploid (seeing accompanying drawing 4).
Aggegation experiment: cell and con A have stronger agglutination reaction, and along with the increase of canavalin(e) concentration, agglutination reaction strengthens gradually, and it has cohesive force.
Friend's substance detects: cell is cultivated through the meat soup culture method, the detection of mycoplasma feminine gender.
Heterogenous animal inoculation: with 35 generations 1 * 10
7The cell inoculation nude mice is subcutaneous, sees the subcutaneous nodule joint in 7 days, and tumour was grown to about 1.5cm * 1.5cm size in 30 days, illustrated that it has tumorigenicity.Cut tumor tissues after 60 days, HE dyeing shows that the plantation tumour is consistent with former tumor tissues type and differentiation degree, cell presents the sarcoma sample form that the fusiformis bunchy is arranged, and does not see cancer nests and glandular epithelium (seeing accompanying drawing 5,6), illustrates that this cell strain still keeps its original biological characteristics.
Immunohistochemical methods: the immunohistochemical methods of monoclonal cell wax stone is Keratin sulfate and vimentin stained positive as a result, presents brown uneven grain in the endochylema, illustrates that it is an epithelium genesis.The AB-PAS stained positive, endochylema presents blue particle, shows that this cell has muciparous function.Above result is consistent with fundamental weave immunohistochemical methods result, confirms that it has kept the phenotypic differentiation of initiating cell.
The supernatant liquor markers tests: the ELISA method detects emiocytosis CEA, AFP, CA15-3, CA125, CA19-9 antigen situation, can detect CEA and CA19-9 antigen in the cells and supernatant.
The immortalization that the SV40 of utilization T antigen gene of the present invention carries out primary cell be a kind of feasible, repeatably set up human oophoroma cell line's method, this method can make histological types, different by stages, the Proliferation of Human Ovarian Cell of different differentiation degrees is immortalized, set up corresponding immortal human ovary carcinoma cell strain, one-tenth is that efficient is higher, can greatly enrich human ovarian cancer cell in vitro storehouse.
The cell strain BUPH:OVSC-2 that the present invention sets up, the grade of malignancy height, the biological characteristics that can reflect its initiating cell on cell levels is for the nosetiology of research people ovarian sarcoma sample cancer, metastasis, resistance mechanism, new medicament screen etc. provide the ideal experimental subjects.
Description of drawings:
Fig. 1 is BUPH:OVSC-2 cellular form (* a 200) photo under the inverted microscope.Visible cell is fusiformis and polygon, can overlappingly grow, and loses contact inhibition.
Fig. 2 is a BUPH:OVSC-2 cell nude mice plantation knurl transmission electron microscope observing photo.As seen the general connection between three epithelial cells.
Fig. 3 is a BUPH:OVSC-2 cell nude mice plantation knurl transmission electron microscope observing photo, and arrow is depicted as the desmosome structure.
Fig. 4 is a BUPH:OVSC-2 cell chromosome karyotyping photo.As seen lack 1 X chromosome, many No. 20 karyomit(e) can be seen 3 marker chromosomess, and wherein two is 1q and the mutual transposition of 12q, and another is 14p
+, be pseudodiploid.
Fig. 5 is the position tumor tissues HE dyeing photo of drawing materials.Visible cell presents the sarcoma sample form that the fusiformis bunchy is arranged, and atypia is obvious, does not see cancer nests and glandular epithelium.
Fig. 6 is a BUPH:OVSC-2 cell nude mice plantation knurl HE dyeing photo.As seen with the position tumor tissues form of drawing materials, the differentiation consistent.
Embodiment
One, the structure of SV40 gene eukaryotic expression vector
PSV
3Neo contains SV40 T antigen (containing big T and little T antigen) gene fragment, and the BamHI single endonuclease digestion obtains the SV40 T gene fragment of 3kb, and electrophoresis reclaims.PcD2 is for efficiently expressing the eukaryotic vector plasmid, and through the BamHI at multiple clone site place single endonuclease digestion, electrophoresis reclaims linear carrier, handles through calf intestine alkaline phosphatase again, and it can not connected certainly.It is connected under the effect of T4 dna ligase with the SV40 T gene fragment that reclaims, obtains recombinant plasmid pcD2-SV40.Recombinant plasmid is cut evaluation through enzyme.Through SmaI and HpaI double digestion, filter out pcD2-SV40 forward clone.
Two, people's ovarian sarcoma sample cancer cells former is commissioned to train foster
Cell derives from gynaecology of The People's Hospital of Peking University patient with operation of being in hospital, and the ovarian sarcoma that has drawn from sample cancer stomach wall shifts tubercle, and it is identical with the ovary primary tumor that postoperative HE dyeing, immunohistochemical methods and Electronic Speculum confirm that stomach wall shifts tubercle character, is ovarian sarcoma sample cancer.In the aseptic fresh specimens of drawing materials of Operation theatre, send the laboratory immediately back to.People's ovarian sarcoma sample cancerous tissue is cleaned in PBS, cut off necrotic tissue, fatty tissue and coating, be cut into 1cm
3Fritter is inserted in the penicillin bottle.Tissue block is cut into 1mm
3Fritter adds 5-6ml PBS piping and druming, makes its natural subsidence, the sucking-off supernatant.Add final concentration 200U/ml collagenase I, 100U/ml Unidasa and perfect medium in the penicillin bottle, 37 ℃ with 150 rev/mins of joltings 1 hour.Centrifugal collection digestion product is washed 2 times with PBS.Re-suspended cell by 400 purpose filter screens, is a single cell suspension with the assurance with it.In the 10ml plastic centrifuge tube, slowly add 45%Percoll solution, 20%Percoll solution and each 3ml of single cell suspension successively, centrifugal 30 minutes of 3000g.With the last confluent monolayer cells sucking-off of telling, to wash 2 times with PBS, inoculation goes in the 50ml plastic culture bottle to cultivate.Add 15% foetal calf serum, 0.2U/ml Regular Insulin, the cultivation of 2mmol/ml L-glutaminate at 1: 1 with substratum MCDB105: M199.After 5 days, see that cell is shuttle type or multiangular, the absence of vortices trend continues to cultivate.In the inoculation 11 days after with 675U/ml collagenase I had digestive transfer culture.When reaching for the 10th generation, select that growth conditions is good, 80% cell that converges is used for transfection.
Three, the transfection and the screening of people's ovarian sarcoma sample cancer cells
1. transfection is first day, get above-mentioned about 80% converge the 10th generation Proliferation of Human Ovarian Cell, change the perfect medium of no penicillin and streptomycin into, continue to cultivate 24 hours.
2. second day, following plasmid of preparation and liposome mixed solution in sterile glass tube were used for (in the 50ml plastic culture bottle) cultured cells in transfection 6 well culture plates.Solution A: 2 μ g (4 μ g) plasmid DNA is dissolved in 250 μ l (the 500 μ l) serum free medium.Solution B: 6 μ l (12 μ l) Lipofectin2000 reagent is dissolved in 250 μ l (the 500 μ l) serum free medium room temperature 5～30 minutes.Merge solution A and solution B, mix gently, put room temperature 20～50 minutes.Discard cell culture fluid.Add the no penicillin and streptomycin perfect medium of 2ml (4ml) to the Lipofectin2000-DNA mixed solution, add on the cell 37 ℃, 5%CO behind the mixing
2Incubator was cultivated 24 hours.The 3rd day, discard transfection liquid, continue to cultivate.
3. the screening of transfectional cell
(1) after the transfection 48～72 hours, treats that the cell growth goes down to posterity in 1: 4 ratio when converging.
(2) continue to cultivate, reach 50%～70% to cell density and converge.
(3) discard nutrient solution, being replaced with concentration is the G418 nutrient solution of 100 μ g/ml, simultaneously with not adding transfection
The cell of liquid compares.
(4) changed the G418 nutrient solution that once contains 100 μ g/ml in after this per 3～4 days, after 2～3 weeks, visible anti-
Sex clone formation and control cells are all dead, treat that the clone increases gradually, moves to 24 holes one by one with it
Plate continues to cultivate.
4. the transfer of clonal cell line and amplification
(1) clones in microscopically established, mark clone's position with marking pen.
(2) in super clean bench, the substratum that inclines is washed once with PBS.
(3) with the long handle tweezers, the aseptic filter paper sheet of a 0.2cm * 0.3cm of gripping, the libation at an ancient wedding ceremony is got Digestive system, is overlying on
On the cell clone, about about 10 seconds.
(4) filter paper is taken out (embrocating in the original place during taking-up), places 24 orifice plates that contain substratum immediately,
Continue to cultivate.
(5) treat that cell covers with after, be transferred to 6 orifice plates and continue amplification cultivation.
Four, the expression of SV40 T gene in the immortal human ovary carcinoma cell strain of transfection
1. the PCR of SV40T gene fragment
According to a pair of special primer of SV40 large T antigen cDNA gene order indoor design, amplified fragments should be the 372bp size.PcD2-SV40 makes positive control, and the primary cell of untransfected is made negative control, is template amplification with the genomic dna of immortal human ovary carcinoma cell strain.Through 30 circulations, the PCR product carries out 1% agarose gel electrophoresis analysis.As seen two samples have a specific amplified band respectively at the 370bp place, and are identical with the positive control band, and do not have amplified band in the primary cell genomic dna of untransfected.Illustrate in the successful transfered cell of SV40 T gene.
2.SV40T the RT-PCR of gene fragment
Utilize random primer to carry out the reverse transcription of the primary cell mRNA of transfectional cell and untransfected, make positive control with pcD2-SV40, the primary cell of untransfected is made negative control.A pair of special primer (with the PCR primer) with the indoor design of SV40 large T antigen cDNA gene order increases to these reverse transcription products, and product is the 372bp size.Pipe increases with confidential reference items primer GAPDH in addition simultaneously.The PCR product carries out 1% agarose gel electrophoresis analysis.The result confirms the reverse transcription success through the immortalized cells of transfection and the amplified band of the confidential reference items primer about all visible 300bp of negative control; The amplified band of the SV40 T gene specific primer of the visible 370bp of immortalized cells, identical with the positive control band, and the primary cell of untransfected is not seen band.There is the expression of SV40 T gene at transcriptional level in the immortalized cell line that transfection is described.
3.SV40 the Sothem Blot of T gene fragment PCR product
Cut the SV40 T gene fragment among the carrier pcD2-SV40, digoxin (DIG) is as probe on the employing random priming mark.With the amplified production of SV40 T special primer in the immortalized cells genomic dna and amplified production (positive control) electrophoresis in 1% sepharose of plasmid pcD2-SV40, through changeing film, prehybridization, hybridizing, wash film, colour developing, the colour developing band of brownish black all appears in result's two samples, and is identical with positive control.Illustrate the product that amplified can with the SV40 gene recombination, the gene of two cell inner expressions is a SV40 T gene.
Claims (2)
1. the foundation of immortal human ovary carcinoma cell strain is characterized in that step is as follows:
(1) utilize gene recombination technology with pSV
3SV40 T antigen among the neo (containing big T and little T antigen) gene fragment clone goes into to efficiently express among the gram nuclear vector plasmid pcD2, is built into pcD2-SV40, and recombinant plasmid is cut evaluation through enzyme;
(2) with final concentration 200U/ml collagenase I and 100U/ml hyaluronic acid enzymic digestion ovarian cancer tissue sample, digestion product through 45% and 20%Percoll solution carry out density gradient centrifugation, with substratum MCDB105: M1991: 1 adds 15% foetal calf serum, 0.2U/ml Regular Insulin, 2mmol/ml L-glutaminate cultivates, and 675U/ml collagenase I peptic cell goes down to posterity;
(3) select that growth conditions is good, the 80% early stage passage cell that converges is carrier transfection plasmid pcD2-SV40 with liposome Lipofectin2000, cell screens through G418 after the transfection, the resistance clone enlarged culturing, reach the evaluation that the SV40T gene is expressed in transfectional cell, obtaining thus can be in external growth steady in a long-term and the ovarian cancer cell strain of going down to posterity.
2. people's ovarian sarcoma sample cancer immortalized cell line of setting up according to the establishment method of immortal human ovary carcinoma cell strain according to claim 1, called after BUPH:OVSC-2, by China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, preserving number is NO.0606, can be external long term growth and stable going down to posterity, have following feature: 1. cellular form is based on fusiformis and polygon; 2. cell doubling time is about 42 hours; 3. karyomit(e) is pseudodiploid, and chromosome structure all lacks 1 X chromosome, many No. 20 karyomit(e), and each caryogram can both see 3 marker chromosomess, is 1q, 12q and 14P
+4. cell has muciparous function; 5. cell expressing Keratin sulfate and vimentin; 6. nude mice plantation tumor growth is fast, keeps former tumour form; 7. with in the SV40T antigen gene transfered cell and become immortalized cell line; The biological characteristics that 8. on cell levels, can reflect its initiating cell.
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