CN106367392B - One kind is derived from advanced stage signet ring cell gastric carcinoma cell line and its method for building up and application - Google Patents
One kind is derived from advanced stage signet ring cell gastric carcinoma cell line and its method for building up and application Download PDFInfo
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Abstract
The present invention relates to a kind of new derived from advanced stage signet ring cell gastric carcinoma cell line gc-006-03 and its method for building up and application, and the cell strain is deposited in China typical culture collection center, and deposit number is CCTCC NO:C2016152.Signet ring cell gastric carcinoma cell line gc-006-03 of the invention can stablize passage, and to study the generation of stomach signet ring cell cancer from now on, development is shifted, and then the pathogenesis for disclosing stomach signet ring cell cancer provides certain research clue and experimental model.Meanwhile establishing gc-006-03 cell strain not is only that the researchs such as gastric cancer molecular biology, immunology and Comprehensive Clinical diagnosis and treatment provide advantage, and the platform provided convenience for screening prevention and treatment gastric cancer medicament.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of cell strain derived from people's advanced gastric signet ring cell cancer and its
Method for building up and application.
Background technique
Gastric cancer is to seriously endanger the malignant tumor of digestive tract of people's health.According to the literature, in worldwide, stomach
989,600 people of cancer new cases accounts for the 8% of whole world cancer neopathy number；The associated death number 738 as caused by gastric cancer,
000 people accounts for the 10% of whole world number of cancer deaths.New cases and death more than 70% occur in development China
Family.East Asia Region incidence gastric cancer rate ranks first in the world.China is the hotspot of gastric cancer, and annual new patients with gastric cancer of sending out is up to 30~40
Ten thousand people, death toll account for the 23% of mortality of malignant tumors number up to 300,000 people.In recent years, the age of onset of gastric cancer has youth
Change trend, the past is in the majority with 40 years old~60 years old age group, with 35 years old~55 years old age group is then more now.
Tumor cell line/strain foundation, for further research tumour generation, infiltration metastasis Ji ﹑ screening anticancer medicine etc.
Provide convenience the cell model with cheaper.The gastric carcinoma cell lines reported in world wide at present are many, such as: day
9 plants of this MKN-1, MKN-28, MKN-45, MKN-74 etc.；16 plants of SNU-1, SNU-5, SNU-16 of South Korea etc.；U.S. ATCC is
Compare authoritative cell bank, the stomach cancer cell line in constant demand sold there are 6 plants, wherein the asian ancestry crowd of 4 plants of source Korea Ss, Japan.China is
The High Risk For Gastric Cancer country, but only three plants of stomach cancer cells are widely used as the further investigation of elaboration of tumour mechanism at present, respectively
It is BGC823, SGC-7901 and MGC803, and they have been passed on 20-30.Therefore, it is special more to establish new China
The stomach cancer cell line of color, and it is particularly important using the research that it carries out China's incidence gastric cancer mechanism.
Stomach signet ring cell cancer is one of gastric cancer specific type, with the characteristics of cell mucilage secretion, with other histologies
Type gastric cancer is compared, and signet ring cell cancer is in diffuse infiltrative growth more, is apt to occur in that young woman, invasiveness be strong, poor prognosis.Due to
Stomach signet ring cell cancer ultra microstructure, pathological manifestations, function differentiation phenotype, etc. have its spy in terms of related neoplasms molecular biology
Different property.At present in limited stomach cancer cell line, signet ring cell gastric carcinoma cell line is actually rare, therefore there is an urgent need to find typical case
Stomach signet ring cell cancer cell, for carrying out the further investigation of gastric cancer generation, infiltration metastasis mechanism.
Since tumour has the characteristics that heterogeneous, complexity and by not agnate and region bring otherness, even if
It is the cell line/strain set up with identical tumour originally culture, because of its Background sources difference, the correlative study obtained may also
It cannot reflect whole features of corresponding disease completely.Moreover, many tumor cell line/strains can be with the extension or culture of incubation time
The variation of environment and occurrence features change, or even lose its original characteristic completely, especially most of gastric carcinoma cell lines/strains are limited
It is low or the problems such as homogeneity is poor in tumor formation rate, it is difficult to the drug screening applied to extensive xenograft tumor models.Therefore, constantly
The research of tumour cell originally culture is carried out, establishing more gastric carcinoma cell lines/strains is very important.
Summary of the invention
It is low for above-mentioned China's human gastric cancer cell line/strain bio-diversity, differed with the biological character of clinical gastric cancer compared with
The deficiencies of big, it is an object of that present invention to provide a kind of new to be derived from advanced stage signet ring cell gastric carcinoma cell line gc-006-03.The cell
Strain is deposited in China typical culture collection center (address: Wuhan University, wuchang, wuhan area, Hubei China province collection),
Deposit number is CCTCC NO:C2016152, and the deposit date is on August 9th, 2016, classification naming: people's stomach signet ring cell cancer was thin
Born of the same parents' strain.
Its character of gc-006-03 cell strain of the present invention is stablized, and can stablize passage > 100 generations, and tumor formation rate is high, and incubation period is short,
One property is good, provides a kind of experimental material of new clinical tumor biological characteristics for gastric cancer research.
Cell strain of the invention is derived from the ascites of 55 years old women stomach signet ring cell cancer patient, using percoll solution
Isolation technics establishes stomach signet ring cell cancerous cell line gc-006 through vitro cell expansion secondary culture.When separating cell strain,
It is grown in big plate using first low concentration cell, after culture 7-10 days, cell growth is vigorous, and at the monoclonal of dispersion
Shape, then row select monoclonal cell.The clone being selected first cultivates in coated 24 orifice plate of poly-L-sine, holds cell
It is easily adherent to survive.And then the cell strain of high-purity is obtained, it is named as gc-006-03.
The cell strain has following biological characteristics:
1, cell adherent growth, contactless inhibition, in superposition growth；
2, cell doubling time was close to 36 hours；
3, cell strain has very strong clonality, and agar colony formation rate is 70%；Kind plate efficiency 88%；
4, cellular immunity groupization is as the result is shown: this cell is in keratin (+++), vimentin (-), ck7 (2+), ck20
(-), ttf1 (-), Li-cad (+), cdx2 (2+), CEA (3+), Ki-67 stained positive rate about 45%；
5 ﹑ flow cytometer detection CD326+> 98%: gc-006-03 is epithelium source cell as the result is shown for this, and other height are equal
One property.
6, chromosome analysis prompts: this plant of cell is stable hypo-triploid cell strain, and chromosome is distributed in 38-54, sub-
Triploid accounts for 82.9%, wherein the cell of 50,51,52,53 chromosomes accounts for 58.5%, has dicentromere, dystopy and derivative
Phenomenon；
7, tumor formation rate is high: whole tumor formations after nude inoculation 5, this cell；
8 ﹑ short tandem repeat (short tandem repeat, STR) result:
The result is compared with progress cell STR in ATCC ﹑ DSMZ ﹑ JCRB and National Laboratory resource cell shared platform,
The homologous cell of gc-006-03 is not found.Illustrate that the cell is unique.
Another object of the present invention is to provide a kind of method of unicellular strain for separating adherent growth, and the method includes such as
Lower step: the digestion of 1. pancreatin: the digestion of New cell line pancreatin need to grope experimental condition, such as pancreas enzyme concentration and digestion time.Because of mistake
Degree digestion then influences cell activity, and not enough then cell is agglomerating for digestion；2. terminating digestion: to cell rounding, patting culture bottle, make thin
Born of the same parents fall off.The serum-containing media or HBSS liquid (Hank ' the s liquid of calcium-magnesium-containing) for being rapidly added 10-20 times terminate digestion；3. inoculation
Cell: according to the cloning efficiency of prediction, candidate cell being serially diluted, and is inoculated with respectively with the range of 100-2000/ware
In several 10cm, gently piping and druming dispersion；4. culture: cell sets 2-10%CO2, 95 humidity, 37 DEG C cultivate 7-15 days, generally not
Need to change liquid, cell is in the monoclonal shape dispersed at this time, and grows dynamic；5. selecting monoclonal: Colony forming process
In periodically with micro- sem observation, determine its Clonal Origin, more dispersed region is cloned in selection, carries out mark, and removal part is trained
Nutrient solution is not necessarily to add pancreatin with the direct picked clones of 1ml pipette tips, to reduce the damage to cell；6. amplification cultivation: picking purpose
Clone, which is first inoculated in coated 24 orifice plate of poly-L-sine, to be cultivated, this like cell easy sticker wall survives.Every 3-5 days into
Half amount of row changes liquid, and the 80-90% that cell covers with bottom of bottle is passed in time, then gradually amplification cultivation, forms the monoclonal of sufficient amount
Cell strain.
Conventional separation cell strain is all made of microtiter culture plate and is cloned, however microtiter culture plate operation is numerous
It is miscellaneous, and cell is not easy to survive in single individual.Method of the invention, which is taken, first does series for the cell of gc-006 cell line
Dilution is seeded in 5 10cm respectively with the range of 100-2000/ware, is carried out with 1640 culture mediums of 10% fetal calf serum
Culture, sets 37 DEG C, 5%CO2It is cultivated 7-10 days in incubator.Cell is in the monoclonal shape dispersed at this time, and grows vigorous.This
When carry out accurate monoclonal cell again and select, when picked clones without plus pancreatin, reduction cellular damage as far as possible.The clone chosen
Cell is first cultivated in coated 24 orifice plate of poly-L-sine, makes cell is easily adherent to survive.Then gradually amplification cultivation.Pass through
The above method has been successfully set up the signet ring cell gastric carcinoma cell line gc-006- of the high-purity for capableing of continuous passage of the invention
03。
It is also an object of the present invention to provide the applications of gc-006-03 signet ring cell gastric carcinoma cell line.The application
Including but not limited to: (animal) screening prevention and treatment gastric cancer medicament in vitro or in vivo；Study the genesis mechanism of gastric cancer；Establish gastric cancer hair
Raw, development or transfer cell model；Establish animal modal of gastric carcinoma；Research gastric cancer differentiation mechanism, cellular morphology and dysfunction,
Tumor invasion and metastasis mechanism and the cell model for instructing Comprehensive Clinical diagnosis and treatment.The method for building up and concrete application of model are all abilities
Known to domain, it can be designed and be used according to specific situation.
Beneficial effects of the present invention are as follows: by providing a kind of signet ring cell gastric carcinoma cell line gc- of novel stabilising passage
006-03, to study the generation of stomach signet ring cell cancer from now on, development is shifted, and then the pathogenesis for disclosing stomach signet ring cell cancer mentions
For certain research clue and experimental model.Meanwhile it establishing gc-006-03 cell strain and not being only gastric cancer molecular biology, be immunized
It learns and the researchs such as Comprehensive Clinical diagnosis and treatment provides advantage, and the platform provided convenience for screening prevention and treatment gastric cancer medicament.
In addition, the method high survival rate of separation adherent growth cell strain of the present invention, clones weaker adherent growth to being formed
Cell separation has obvious help.
Detailed description of the invention
For above and other purpose, feature, advantage and examples of implementation of the invention can be clearer and more comprehensible, to institute's attached drawing
It is described as follows:
Fig. 1 is morphological observation figure (× 40) of the gc-006-03 growth conditions under phase contrast microscope.
The HE that Fig. 2 is gc-006-03 dyes aspect graph (X100).
Fig. 3 is gc-006-03 transmission electron microscope aspect graph (X10000).
Fig. 4 is that gc-006-03 keratin immunohistochemistry is positive (X20).
Fig. 5 is that gc-006-03 vimentin immunohistochemistry is negative (X20).
The ck7 immunohistochemistry that Fig. 6 is gc-006-03 is positive (X20).
The ck20 immunohistochemistry that Fig. 7 is gc-006-03 is negative (X20).
The ttf1 immunohistochemistry that Fig. 8 is gc-006-03 is negative (X20).
The Li-cad immunohistochemistry that Fig. 9 is gc-006-03 is positive (X20).
The cdx2 immunohistochemistry that Figure 10 is gc-006-03 is positive (X20).
The CEA immunohistochemistry that Figure 11 is gc-006-03 is in strong positive (X20).
Figure 12 is the ki-67 immunohistochemistry positive rate about 45% (X20) of gc-006-03.
Figure 13 is the cell growth curve of gc-006-03: abscissa is time (hour), and ordinate is degrees of fusion (%).
Figure 14 is the kind plate efficiency violet staining figure of gc-06-03.
Figure 15 a and 15b are gc-006-03 chromosome karyotype analysis figure.
Figure 16 is Tumor formation experimental result in the immunodeficient mouse body of gc-006-03.
Figure 17 is that tumor formation sample is sliced HE colored graph (X100) in the nude mouse of gc-006-03.
Figure 18 is that this patient stomach neoplasm tissue biopsy HE dyes picture (X40).
Specific embodiment
In order to keep the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with the drawings and the specific embodiments,
The present invention will be described in further detail.It should be appreciated that the specific embodiments described herein are only used to explain this hair
It is bright, it is not intended to limit the present invention.Experimental method described in following embodiments is unless otherwise specified conventional method.
The gc-006-03 of 1 signet ring cell gastric carcinoma cell line of embodiment is established
Stomach signet ring cell cancer patient's ascites is extracted, cell is separated using the percoll solution of 1.077g density.Through 1640 trainings
It supports base and (contains 10% fetal calf serum, 10mM HEPES, 100U/ml benzyl penicillin, 100 μ g/ml streptomycin sulphates and 0.25 μ g/ml two
Property mycin B), 37 DEG C are set, 5%CO2Cell in vitro secondary culture is carried out in incubator, establishes stomach signet ring cell cancerous cell line
gc-006。
Establish cell strain: we, which take, is first serially diluted the cell of gc-006 cell line, with 100-2000/ware
It is seeded in 5 10cm culture dishes respectively, with 1640 culture mediums (ibid), sets 37 DEG C, 5%CO2It is cultivated 7-10 days in incubator.
Cell is in disperse and dynamic monoclonal shape at this time.Then monoclonal cell is precisely selected, chooses clone without adding pancreatin, directly
It connects and picks out monoclonal cell with pipette tips.It is first cultivated in coated 24 orifice plate of poly-L-sine, makes cell is easily adherent to survive.
Then changed according to culture solution pH, half amount was carried out every 2-3 days and changes liquid.Cell covers with the 80-90% of bottom of bottle, passes in time, passes
For ratio 1:4~1:2.It is digested 5 minutes when passage cell with 0.05% pancreatin, cell rounding pats culture bottle 2 times, makes cell
De- wall is rapidly added 10-20 times of HBSS liquid (Hank ' the s liquid of calcium-magnesium-containing) and terminates digestion, is centrifuged 1000g, 3min, reject supernatant
Liquid is resuspended cell, is transferred to new culture medium.Gradually amplification cultivation is to 108-109Cell concentration, using 10%DMSO cryoprotector, liquid
Nitrogen saves.Above method has been successfully set up one plant of signet ring cell gastric carcinoma cell line for capableing of the high-purity of continuous passage, name
For gc-006-03.It is verified by experiments, gc-006-03 cell strain, which can pass on, reached for > 100 generations, and cell quality still keeps stable.
It is realApply the biological characteristics detection of 2 signet ring cell gastric carcinoma cell line gc-006-03 of example
1, cytomorphology:
(1) living cells is observed: after cell passage growth is stablized, being observed under phase contrast microscope.Cell is given birth in adherent overlapping
It is long, contactless suppression (Fig. 1).
(2) HE is dyed: cell reached for 31 generations, and cell number is up to 106-107When, specimens paraffin embedding slices are carried out, are seen under HE dyeing mirror
It examines.Cell is seen in pernicious class Epithelial form, and nuclear proportion is big, and kernel is multiple, and cell has the core to differ in size, is clear to nuclear fission
Phase, still visible a certain number of signet ring cells (Fig. 2).
(3) transmission electron microscope observing: intracellular ribosomes is few, and rough surfaced endoplasmic reticulum (RER) is abundant, Golgi complex saccular dilatation, shape
State is changeable, and feature outstanding is relatively abundant for mucus particles in endochylema；Euchromatin is abundant, and kernel is obvious.There is microvillus on surface, row
Arrange it is in disorder, it is (Fig. 3) different in size.
2 ﹑ cellular immunity groups:
The Abnormal Cytokeratin Expression in Hepatocytes positive (Fig. 4), Cytokeratin dye sepia, after birth karyon not brown；Waveform
Protein expression is negative, and endochylema dyes (Fig. 5) without sepia, meets epithelial origin characteristic.
Due to gc-006-03 have drawn from the stomach signet ring cell cancer patient for having lung and lymphatic metastasis ascites in, ck7,
Ck20 and ttf1 joint-detection is difference one of lung source or the important method of stomach source cell, and ck7 (2+) (figure is presented in this cell
6), ck20 (-) (Fig. 7) and ttf1 (-) (Fig. 8), as a result meets the tumour cell of stomach source property.In addition it also observes Li-cad (+)
(Fig. 9), cdx2 (2+) (Figure 10), CEA (3+) (Figure 11), Ki-67 positive rate about 45% (Figure 12), further show the cell
Related neoplasms biological nature.
3, cell growth curve measures: the 7th generation cell in logarithmic growth phase being taken to be made 1 × 103/ ml's is unicellular outstanding
Then liquid is inoculated in 24 orifice plates according to the hole 1000ul/, done 5 multiple holes.It is enterprising to be placed in JuLi Stage real-time cell calculating instrument
Row cell count, record is primary per hour, records 8 days altogether.Growth curve (Figure 13) is drawn with cell fusion degree and time.By public affairs
Formula (PDT)=(t-t0)〔lg2/(lgNt-lgN0)) doubling time is calculated, gc-006-03 cell mean doubling time is about 36h.
4, soft-fractrue rock mass is tested: with the covering 6cm culture dish preparation of -1640 culture medium of 0.6% low melting-point agarose
Bottom-layer agar, it is culture medium top-layer agar that 0.3% low melting point agar -1640 of the 0.5ml containing 1000 cells, which is added,.Set 37
DEG C, 5%CO2It is cultivated 14 days in incubator, counts the colony for containing 50 or more cells, calculate cell colony formation rate.Colony shape
At rate=clone's number/inoculating cell number × 100%.It is as the result is shown 70%.
5, plant plate efficiency: the finely dispersed 4th generation cell of inoculation, in every 100 cells/6cm plate, be arranged 3 it is parallel
Control is cultivated 9 days.It is first fixed with methanol after culture, then plus violet staining, counting contain gram of 50 or more cells in right amount
Grand number.Kind plate efficiency=formation colony number/inoculating cell number × 100%.As the result is shown: kind plate efficiency is approximately equal to 88% (figure
14)。
6, chromosome analysis: the 5th, 10,25 generation cells of logarithmic growth phase make karyotyping respectively.Cell is through colchicum
Element effect 2h, methanol-glacial acetic acid fixing process hypotonic through 0.075mol/L KCl, ice humidity strip drip piece, use pancreas after aged at room temperature
Enzymatic treatment, the aobvious band analysis of Giemsa dyeing.As the result is shown: this cell line modal number is between 38~57.Diploid
And hypodiploid cells (23 < chromosome number≤46) account for 17%, triploid and hypo-triploid cell (46 < chromosome number≤69) account for
86%, for stable hypo-triploid cell line, mostly 50~53 chromosomes (accounting for 58.5%).54 chromosome karyotype analysis:
Xxx ,+6 ,+7+7 ,+11 ,+18 ,+20, it is seen that cell chromosome dystopy, derivative chromosome, dicentromere and unknown sources dyeing
Phenomena such as body (Figure 15 a and 15b).There are also+8 ,+9 ,+9 ,+10 for other karyotypings.It can be seen that this cell strain has malignant tumour thin
The feature of born of the same parents.
7 ﹑ short tandem repeat (short tandem repeat, STR) are also known as microsatellite DNA, refer to chromosome
On, by several base-pairs as core unit (2-6 base-pair), (number of repetition is for a kind of DNA sequence dna that tandem sequence repeats are formed
More than 10~60 times, genetic fragment is below 400 base-pairs)；Each duplicate number of core unit will appear individual difference, thus
Form the different allele of fragment length.Therefore, the number of repetition of one group of STR sequence is almost unique in Different Individual
, it is the gene identities feature of individual and the main method that cell biology identifies cell identity and source.This is thin
The STR cellular identification of born of the same parents' strain is to carry out Capillary Electrophoresis using ABI3730xd instrument using Goldeneye20A kit,
GeneMapper4.0 software is analyzed and is obtained.
8, tumor formation is tested in immunodeficient mouse body: being taken 4 week old male BALB/C mices 5, is infused respectively under mouse butt
Penetrate side shoulder and opposite side buttocks, each site 5 × 107A/ml cell infusion 100ul, 1 week after nude mice by subcutaneous inoculating cell,
I.e. visible transplantable tumor is grown.5 nude mice whole tumor formations, transplantable tumor are in nodositas, have adhesion (Figure 16) with skin.Cervical dislocation
Nude mice is put to death, transplantable tumor is taken out, formalin is fixed, specimens paraffin embedding slices, HE dyeing.Tumor formation sample microscopic observation in nude mouse
Cell growth is vigorous, and nucleus increases, deep to contaminate, and atypia is obvious, it is seen that a small amount of signet ring shape cell (Figure 17).
9, the pathological section of tumour: patient's stomach neoplasm tissue is taken to carry out biopsy, after the fixation of formalin routine, paraffin embedding
Slice and HE dyeing.As the result is shown: without apparent cancer nests, in diffusing infiltrative growth.It cancer cells secrete mucus but does not arrange mostly
It arrives out extracellularly, since mucus increases in endochylema, nucleus is extruded to a side periphery of cell more, makes cancer cell in signet ring
Shape.This signet ring cell > 50% meets the stomach signet ring cell cancer diagnostic criteria (Figure 18) of WHO.
The foregoing is merely the preferred embodiment of the present invention, are not intended to limit the invention, and the scope of the present invention is by weighing
Sharp claim and its equivalent replacement mode are limited, it is done within the spirit and principles of the present invention it is any modification, etc.
With replacement and improvement etc., should all be included in the protection scope of the present invention.
Claims (6)
1. it is a kind of derived from advanced gastric signet ring cell cancer can continuous passage cell strain gc-006-03, passage > 100 can be stablized
Generation, the cell strain are deposited in China typical culture collection center, and deposit number is CCTCC NO:C2016152.
2. application of the cell strain gc-006-03 as described in claim 1 in screening prevention and treatment gastric cancer medicament.
3. application of the cell strain gc-006-03 as described in claim 1 in the genesis mechanism of research gastric cancer.
4. cell strain gc-006-03 as described in claim 1 is in establishing the cell model that gastric cancer occurs, develops or shifts
Using.
5. cell strain gc-006-03 as described in claim 1 is in the application established in animal modal of gastric carcinoma.
6. cell strain gc-006-03 as described in claim 1 is different in foundation research gastric cancer differentiation mechanism, cellular morphology and function
Often, tumor invasion and metastasis mechanism and the application in the cell models of Comprehensive Clinical diagnosis and treatment is instructed.
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Non-Patent Citations (3)
| Title |
|---|
| Establishment and characterization of human signet ring cell gastric carcinoma cell lines with amplification of the c-myc oncogene;Yanaqihara K.,et al;《Cancer Res.》;19910101;第51卷(第1期);381-386 * |
| 分化及未分化胃癌细胞株诱导癌相关成纤维细胞能力的比较;李云成;《中国优秀硕士学位论文全文数据库》;20140515(第2014/05期);E072-242 * |
| 新的胃癌细胞系的建立和胃癌干细胞异质性研究;徐馨;《中国博士学位论文全文数据库》;20160315(第2016/03期);第E072-84页 * |
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