CN101824397A

CN101824397A - Hepatoma cell line EHBC-512 and application thereof

Info

Publication number: CN101824397A
Application number: CN200910047025A
Authority: CN
Inventors: 邹奇飞; 沈锋; 侯瑛; 葛瑞良; 刘健
Original assignee: Oriental Hepatobiliary Surgery Hospital Second Military Medical University Of Chinese Pla
Current assignee: Oriental Hepatobiliary Surgery Hospital Second Military Medical University Of Chinese Pla; Second Military Medical University SMMU
Priority date: 2009-03-04
Filing date: 2009-03-04
Publication date: 2010-09-08
Anticipated expiration: 2029-03-04
Also published as: CN101824397B

Abstract

The invention relates to a hepatoma cell line EHBC-512, which is preserved in the China Center for Type Culture Collection and has a preservation number of CCTCC No: C200911. The invention also provides application of the hepatoma cell line EHBC-512. The hepatoma cell line EHBC-512 has the advantages that the hepatoma cell line EHBC-512 established by the invention is cultured in vitro for a long time and can grow stably for transfer of culture. The cellular morphology is polygonal, and the size atypia is obvious. Cells lose contact inhibitions, can grow in an overlapping mode after overgrowing, and can form a clone easily. Under an electron microscope, cell nucleuses are big and obvious, the karyoplasmic ratio is inverted, and a large quantity of plastosomes shows the vigorous growing power and the metabolic capability of the cells. After a long time of in vitro culture, the expression of AFP can also be detected in an anchorage-dependent cell supernatant, and the expression level is high, which shows that the EHBC-512 reserves characteristics of hepatocellular carcinoma and is an ideal new experimental material for liver cancer foundation and clinical researches.

Description

A kind of hepatoma cell line EHBC-512 and application thereof

[technical field]

The present invention relates to microorganism zooblast field, be specifically related to a kind of hepatoma cell line EHBC-512 and application thereof.

[background technology]

Primary hepatocarcinoma is one of modal malignant tumour of China, often merges in liver cirrhosis and the liver to shift the prognosis extreme difference.Operation remains the most effective methods of treatment of present liver cancer, but because most of patient has belonged to middle and advanced stage when going to a doctor, has taken place that liver is inside and outside to be shifted, so the excision rate is low, the postoperative recurrence rate is higher.Therefore, press for the pathogenesis of further investigation liver cancer, seek effective liver cancer treatment means.Hepatoma cell line is current to have crucial value in liver cancer theory and applied research.But since the heterogeneity and the complicacy of tumour, not agnate tumour cell of originating with the region, and its some characteristic is not quite similar.

The experimental study of liver cancer can not directly carry out at human body, and therefore, need to set up and be convenient to the interior liver cancer animal model of studying of body and the hepatoma cell line of in vitro study, be the indispensable means of laboratory study liver cancer and set up external human hepatoma cell system.Chen Ruiming equals to set up the first strain hepatoma cell line in the world in 1962, also sets up HepG2, Hep3B, PLC/PRF/5 abroad in succession.In addition, the researchist has also set up some animal liver cancerous cell lines, as bringing out the RLC-801 that the Wistar rat is set up with the chemical carcinogens butter yellow, and the ascitic type liver cancer MH of the mouse of bringing out with tetracol phenixin ₁₃₄But along with deepening continuously of liver cancer research, requirement heterogeneous to tumour and the complicacy aspect is more and more higher, the while hepatoma cell line is subculture in vitro separately constantly, some features of its liver cancer fade away, therefore, need constantly set up new hepatoma cell line and embody its complicacy, enrich our research means.

CN1338512A discloses and has utilized people's liver cancer in-situ inoculating to the nude mice liver, draws materials from the lung metastasis of nude mice and has set up the Bel7402, and mechanism and the transfer of control lung shifted for the liver cancer lung provide suitable experiment material.CN1232874A discloses and utilized the high nude mice model transplanted tumor that shifts of people's liver cancer is the knurl source as building, and adopts vitro culture to hocket in conjunction with the mode that inoculates growth in the animal body, builds up the clone MHCC97 with high metastatic potential.And do not appear in the newspapers in the hepatoma cell line aspect that derives from people's liver cancer.

[summary of the invention]

The objective of the invention is provides a kind of hepatoma cell line EHBC-512 at deficiency of the prior art.

One purpose more of the present invention is that a kind of purposes of hepatoma cell line EHBC-512 is provided.

For achieving the above object, the technical scheme that the present invention takes is: a kind of hepatoma cell line EHBC-512, and wherein, described hepatoma cell line EHBC-512 is deposited in Chinese typical culture collection center, and deposit number is: CCTCC NO:C200911

For realizing above-mentioned second purpose, the technical scheme that the present invention takes is: a kind of hepatoma cell line EHBC-512 is as the application of experiment material in liver cancer basis and clinical study.

The hepatoma cell line EHBC-512 preservation that the present invention sets up.Preservation date: on February 25th, 2009, depositary institution: Chinese typical culture collection center.Depositary institution address: Wuhan University.Deposit number: CCTCC-C200911.

The invention has the advantages that: the hepatoma cell line EHBC-512 that the present invention sets up, external long-term cultivation can go down to posterity by stable growth.Cellular form is a Polygons, and the big or small opposite sex is obvious.Cell loses the contact inhibition phenomenon, can overlapping growth, easily formation clone after covering with.Nucleus is big and obvious under the Electronic Speculum, the karyoplasmic ratio inversion, and a large amount of plastosomes shows growth and the metabolic capacity that it is vigorous.Chromosome karyotype analysis shows that it derives from the people, has tangible chromosome structure and quantity unusual, and mode is about 120.Analyze that it is tissue-derived, immune fluorescence grouping shows that liver cell source mark CK18 strong positive is expressed, and biliary tract mark CK19 does not see Table and reaches, simultaneously, most of cell all has the strong positive of AFP to express, and shows that EHBC-512 is the hepatoma cell line in strain liver cell source.Become in the nude mouse in the knurl experiment, get the tumor tissue pathology detection of subcutaneous growth, and find relatively that with primary tumo(u)r both have identical cellular form and histological structure, further specify, this cell is a hepatoma cell line.Through the vitro culture of long period, in the attached cell supernatant, still can detect the expression of AFP, and expression amount is higher, illustrate that EHBC-512 is keeping the characteristic of hepatocellular carcinoma, be the new experiment material of an ideal liver cancer basis and clinical study.

[description of drawings]

Accompanying drawing 1A is the EHBC512 normal morphology, (viable cell).

Accompanying drawing 1B is the EHBC512 normal morphology, HE * 10 (HE dyeing, 10 times of amplifications).

Accompanying drawing 1C is the EHBC512 normal morphology, HE * 40 (HE dyeing, 40 times of amplifications).

Accompanying drawing 2A is the EHBC512 transmission electron microscope photo, (nucleus, arrow) (* 3000).

Accompanying drawing 2B is the EHBC512 transmission electron microscope photo, (smooth endoplasmic reticulum, arrow) (* 5000).

Accompanying drawing 2C is the EHBC512 transmission electron microscope photo, (plastosome, arrow) (* 8000).

Accompanying drawing 3 is growth curves.

Accompanying drawing 4 is adherent rate curves.

Accompanying drawing 5 is cell cycles.

Accompanying drawing 6 is chromosome karyotype analysis.

Accompanying drawing 7 is that the EHBC-512 plate clone forms.

Accompanying drawing 8 is that the tumour index detects.

Accompanying drawing 9A is the nude mice subcutaneous transplantation, (subcutaneous transplantation).

Accompanying drawing 9B is the nude mice subcutaneous transplantation, (xenotransplantation) (HE * 40).

Accompanying drawing 9C is the nude mice subcutaneous transplantation, (patient's primary tumo(u)r) (HE * 40).

[embodiment]

Below in conjunction with accompanying drawing the specific embodiment of the present invention is elaborated.

Embodiment

1, materials and methods

1.1 experiment reagent

High sugared DMEM (Gibco), foetal calf serum, IV Collagen Type VI enzyme (Sigma), the anti-people AFP of rabbit antibody, mouse-anti people CEA antibody, mouse-anti people CK18 antibody, mouse-anti people CK19 antibody (Shanghai biological products company)

1.2 it is tissue-derived

Liver cancer tissue is taken from the east 2008-05-12 of hospital of liver and gall surgical department excision sample, and the male sex 31 years old, finds to be admitted to hospital liver 2 weeks of occupy-place CT prompting right lobe of liver primary hepatocarcinoma, Zhi Shuansai behind the portal vein, AFP＞1750ug/l, the HBsAg positive.Pathological diagnoses is three grades of hepatocellular carcinomas, thick beam type, brief summary nodal pattern liver cirrhosis, no coating, TTPV.

1.3 primary cell culture

Cut the operation of liver cancer sample under the aseptic condition, clean for several times with adding two anti-training liquid, aseptic operation cuts into 1mm ³The size tissue block, 37 ℃ of digestion of IV Collagen Type VI 1 hour, sterile gauze filters.Erythrocyte cracked liquid is removed red corpuscle, and DMEM adds 37 ℃ of 10% foetal calf serums, 5%CO ₂Vitro culture was changed fresh training liquid in every 2-3 days, and adherent method is removed inoblast repeatedly.Occur several clones after January, select bigger clone, go down to posterity after the trysinization.Carry out index of correlation when reaching for the 10th generation and detect, and called after EHBC-512.

1.4 cellular form is observed

Cultured cell in vitro is observed with inverted microscope; The cell 5 * 10 in vegetative period of taking the logarithm ⁵, PBS washes for several times, and centrifuging and taking precipitation glutaraldehyde is fixed, and the Electronic Speculum making method is carried out embedding, ultrathin section(ing) routinely, dyeing and transmission electron microscope observing.

1.5 growth curve

Adjust cell concn, get 1 * 10 ⁵Cell inoculation is in six orifice plates, and each sample repeats 3 holes.Counted continuous counter 5 days in per 24 hours.Getting incubation time is X-coordinate, and cell count is an ordinate zou, draws growth curve.

1.6 adherent rate

Get 1 * 10 ⁵The cell kind is cultivated under the standard conditions in six orifice plates, and every group is repeated 3 holes.Get one group in per 2 hours and digest attached cell counting, continuous counter 24 hours.Getting incubation time is X-coordinate, and attached cell number and inoculating cell number are drawn the adherent rate curve than being ordinate zou.

1.7 the cell cycle is detected

The cell in vegetative period of taking the logarithm, trysinization, centrifugal, precooling PBS washes for several times, gets precipitation after centrifugal, 75% alcohol that adds 4 ℃ of precoolings, ice bath 30 minutes, centrifugal, PBS is resuspended, add Hoechst33258,30 minutes on ice, centrifugal, carry out the fluidic cell cycle detection after PBS is resuspended.

1.8 nucleus type analysis

In being logarithmic growth the 10th generation cell, add colchicine, add toughener after 30 minutes, cross digestion in 30 minutes, centrifugal, remove supernatant, add the KCL of 0.075mmol/l, 37 ℃ 30 minutes, add the 1ml stationary liquid, piping and druming evenly, the centrifugal 10min of 2000r/min, centrifugal, remove supernatant, add stationary liquid 6-8ml, room temperature was put 30 minutes, fixed twice, and collecting cell carries out chromosome karyotype analysis.

1.9 plate clone forms experiment

The every hole of six orifice plates adds 1 * 10 ³Cell is done 3 multiple holes for every group.Cultivate 2-3 week, occur stopping to cultivate after as seen naked eyes observe the clone, clone counting after fixing, dyeing.Cloning efficiency=clone's number/inoculating cell number * 100%

1.10 the tumour index detects

Get the 30th generation the cell kind in the circle slide on, 4% Paraformaldehyde 96 is fixed 20 minutes, 1%triton rupture of membranes room temperature 5 minutes, 10% lowlenthal serum sealing 30 minutes, one anti-ly is respectively AFP, CEA, CK18, CK19, and 4 ℃ are spent the night, it is anti-that PBS washes back adding fluorescence two, room temperature 1 hour, Hoechst redyes, and microscopy is taken the photograph sheet after the water-based mountant mounting.After cell attachment covers with the culture dish bottom, collect the supernatant liquor chemoluminescence method and detect the AFP secretion.

1.11 become knurl in the nude mouse

4-5 week is the BALB/c-nu/nu nude mice greatly, and the SPF environment is raised down.Got for the 30th generation 5 * 10 ⁶/ 200ul injection cell is subcutaneous in the nude mice back, the back observation of 4 weeks.After generating, tumour cuts that the part tumor tissue is done HE dyeing and former patient's tumor tissue compares.

2, the result

2.1 morphological observation

Inverted microscope is observed, and cell is polygon, and is not of uniform size, and iuntercellular is arranged closely, visible polykaryocyte, and be the lumps growth, the confluent culture ware has overlapping growth (Figure 1A) behind the bottom.Cell HE dyeing showed cell nuclear increases, and kernel is (Figure 1B, Fig. 1 C) obviously.Electron microscopic observation, nucleus is big, and heteromorphism is obvious, and kernel gathers in nuclear membrane, and endochylema is less, and more plastosome and lysosome are arranged, visible smooth endoplasmic reticulum and rrna (Fig. 2 A, Fig. 2 B, Fig. 2 C).

2.2 growth curve as shown in Figure 3.

2.3 the adherent rate curve as shown in Figure 4.

2.4 the cell cycle.

The DNA cycle analysis: the G1 phase is 39.25%, and the G2 phase 24.87%, the S phase is 15.09% (Fig. 5).

2.5 chromosome analysis

Choose metacinesis phase cell and observe, karyological character is a pentaploid, mode 110-120.Karyomit(e) R shows band and analyzes 5p+, 9p+, and 17p+, yq+, other has 3 Marker karyomit(e)s (Fig. 6).

2.6 the cloning efficiency cloning efficiency is 20%-30% (Fig. 7).

2.7 the tumour index detects

Immune fluorescence grouping shows that cell AFP expresses stronger, and CEA does not then see obvious expression, most of cell expressing CK18, and do not express CK19 (Fig. 8).In addition, the detection of AFP is pointed out greater than 1210ug/L in the cell conditioned medium liquid, and EHBC512 is a strain hepatocellular carcinoma cells system.

2.8 become knurl in the nude mouse

Become knurl, 5 * 10 in the nude mouse ⁶Cell inoculation is subcutaneous in the nude mice back, and 1/5 nude mice has tumour to generate (Fig. 9 A) in two months.Tumor tissue staining pathologic section and former patient compare demonstration, both cellular fories and organizational structure similar (Fig. 9 B, Fig. 9 C).

The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; can also make some improvement and replenish, these improvement and replenish and also should be considered as protection scope of the present invention.

Claims

1. a hepatoma cell line EHBC-512 is characterized in that, described hepatoma cell line EHBC-512 is deposited in Chinese typical culture collection center, and deposit number is: CCTCC NO:C200911.

2. hepatoma cell line EHBC-512 according to claim 1 is as the application of experiment material in liver cancer basis and clinical study.