CN106947739A

CN106947739A - The method that pancreatic cancer cell is cultivated under anaerobic environment

Info

Publication number: CN106947739A
Application number: CN201710291218.0A
Authority: CN
Inventors: 孙诚谊; 张宏; 潘耀振; 喻超; 田舍; 何志伟; 陈玲
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-04-28
Filing date: 2017-04-28
Publication date: 2017-07-14
Also published as: WO2018196344A1

Abstract

The invention discloses a kind of method that pancreatic cancer cell is cultivated under anaerobic environment.The present invention is using the true environment for simulating pancreatic tumor growth in body, three kinds of conventional pancreatic tumor cells are cultivated under anaerobic environment, the cancer cell that obtains is cultivated closer to the cancer cell grown under full-scale condition, the accuracy for research experiment provides cancer cell material that good basic material makes acquisition in follow-up scientific experiment and has more real convincingness.

Description

The method that pancreatic cancer cell is cultivated under anaerobic environment

Technical field

The present invention relates to medicine technology field, especially a kind of method that pancreatic cancer cell is cultivated under anaerobic environment.

Background technology

Cancer of pancreas grade malignancy height, 5 years survival rates have the title of king of cancer less than 5%.Most of entity tumors are all Tumour cell is by constantly breeding formation, and often blood supply is enriched very much for tumour and its surrounding tissue.Tumour is imbued with the life of blood supply Thing characteristic is also the main cause of its fast-growth, while also providing thinking to antitumor for us.Doctor by medicine, The various means such as operation are blocked after the blood supply of tumor tissues, often can " hungry to death " tumour obtain good therapeutic effect.But this A series of remedy measures seem that the effect in face of cancer of pancreas is poor, fail to the life span of significantly extension Pancreas cancer patients.

Why cancer of pancreas meeting " armsproof "Research finds that in the tumor tissues of cancer of pancreas, tumour cell is accounted for not Foot 30%, is more inflammation interstitial cell and fibr tissue, and this phenomenon causes the spy inside cancer of pancreas with the weary oxygen of weary blood Point.In this unique tumor microenvironment, not only antineoplastic is difficult to reach tumor by local, and limitation by blood vessel Identification and removing of the normal immunocyte for tumour cell, conventional " hunger cure " also can be overshadowed in addition, because Cancer of pancreas is not just afraid of " starvation " originally.

There is a class hypoxia inducible factor in human body, they are by continuous degraded under normal circumstances and maintain for a long time low Level, but research finds that micro-environmental hypoxia occurs in many tumor tissues, still, at present for pancreatic cancer cell culture according to Be so using conventional training method, the cancer cell obtained in such formula mode be likely to under true micro-environmental hypoxia The cancer cell of production has differences.

The content of the invention

The purpose of the present invention is：There is provided a kind of method that pancreatic cancer cell is cultivated under anaerobic environment, which culture The cancer cell of acquisition is closer to the cancer cell grown under full-scale condition, and the accuracy for research experiment provides good basic material Material.

What the present invention was realized in：The method that pancreatic cancer cell is cultivated under anaerobic environment, makes AsP C-1 or BxPC-3 Cell growth is aided with 37 DEG C of wet air 10% hyclone (V in rpmi -1640 culture mediums_Hyclone： V_{DMEM culture mediums}=1：10, level of endotoxin≤10EU/ml. hemoglobin levels≤30mg/dl), 100U/ml benzyl penicillins and BxPC-3 And 100 μ g/ml streptomysins；By PANC-1 cells in DMEM medium cultures；Three of the above pancreatic cancer cell is in hypoxic cell Cultivated in incubator, wherein O₂、CO₂And N₂Volume ratio be 1.5:5:93.5.

Compared with prior art, the present invention is using the true environment for simulating pancreatic tumor growth in body, in anoxic Three kinds of conventional pancreatic tumor cells are cultivated under environment, the cancer cell for cultivating acquisition is thin closer to the cancer grown under full-scale condition Born of the same parents, the accuracy for research experiment provides cancer cell material that good basic material makes acquisition in follow-up scientific experiment more With real convincingness.

Brief description of the drawings

Fig. 1 is mRNA relative expression levels figure respectively after normal oxygen and anoxic culture 16h；By extracting pancreatic cancer cell RNA, stem-cell marker after stem cell markers, (BMI-1/OCT4A/Nanog/SOX2) mRNA expression, anoxic is detected by PCR Thing mRNA expressions are raised, and difference has statistical significance (P<0.05)；

Fig. 2 is the thin fluidic cell figure after normal oxygen and anoxic culture 16h of cancer of pancreas；Pass through Flow cytometry CD133 surface markers positive rates, cancer of pancreas surface markers CD133 positive rates are significantly raised under anoxic culture, and difference has statistics Learn meaning (P<0.05)；

Fig. 3 is that, respectively in normal oxygen and anoxic low suspension culture Pancreatic Cancer Pancreatic carcinoma cell, cell gradually forms suspension ball life It is long, represent the feature of tumour cell " dryness ".Cultivate after one week, compared with normal oxygen, the tumour cell cultivated under anaerobic condition hangs Ball float is more normal, and oxygen group is bigger, and the more (P of balling-up<0.05)；

Fig. 4 is laser confocal microscope testing result；Observed and exported after sample, image procossing using 40 times of oil mirrors； CD133 (red) is located in cell membrane surface and cytoplasm, blue indicator cells core；Compared with normal oxygen group, anoxic culture 16h is thin Red light intensity in born of the same parents substantially increases；Show through statistical analysis, two groups of differences have statistical significance (P<0.05)；

After Fig. 5 is transfection si-HIF-1 α and NC siRNA respectively, 16h, rapid extraction cell egg are cultivated under anoxic In vain, detect that cancer of pancreas stem cell markers albumen changes by western blot, under anoxic conditions, lower after HIF-1 α, BMI-1, OCT4A, Nanog, SOX2 expression are notable to lower, and shows that micro-environmental hypoxia may be by HIF-1 α to regulate and control pancreas The dryness biological behaviour of cancer cell.

Embodiment

Embodiments of the invention：The method that pancreatic cancer cell is cultivated under anaerobic environment

1.1:Material source:Human pancreatic cancer cell PANC-1, AsPC is the Qin of Tongji Medical College, Huazhong Science and Technology Univ. kindheartedness and righteousness Professor give.DMEM in high glucose nutrient solution, RPMI-1640 nutrient solutions, superfine hyclone, dual anti-solution and OPTI-MEM are purchased from U.S. Gibco companies of state.Si-RNA-HIF-1a, nonspecific NC-RNA are designed and synthesized by Guangzhou Rui Bo biotech firms.HIF- 1a, CD133, BMI-1, OCT4A, Nanog, sox2 are purchased from abcam companies.GAPDH primary antibodies and goat-anti rabbit and sheep anti mouse secondary antibody, it is glimmering Light secondary antibody is purchased from Wuhan Boster Biological Technology Co., Ltd..Lip03000 reagents are purchased from Invitrogen companies of the U.S.. CD133-PE streaming antibody is purchased from German You Ningwei companies.Primer is designed and synthesized by Shanghai life work.

1.2 cell culture：PANC1 and AsPC cells are used respectively contains 10% hyclone, 100U/ml penicillin, strepto- 37 DEG C, 5%CO2 karyon humidified incubator culture are placed under the DMEM in high glucose and RPMI-1640 of element, normal oxygen.It is placed under hypoxemia 37 DEG C, 5%CO2,1%O2 karyon humidified incubator culture.0.25% trypsin solution is nitrified, passed on, given birth to from logarithm Long-term cell is tested.

1.3PCR detections mRNA expression extracts RNA in various types of cells according to Trizol specifications, is tried further according to reverse transcription Agent box specification, respectively reverse transcription be cDNA using GAPDH as internal reference, fluorescence real-time quantitative PCR, using relative quantification method (relative quantitative, RQ) represents mRNA expression quantity.

1.4Western Blotting detect the expression of albumen：PANC-1 the and AsPC cells in logarithmic growth are taken respectively Albumen is extracted, 30ug total protein of cell row polyacrylamide gel electrophoresises is taken, goes on polyvinylene double oxide (PVDF) film, Vibration closing 1h, adds the secondary antibody (1: 5 000) of primary antibody (1: 1000), 4 DEG C of overnight incubations, plus horseradish peroxidase-labeled, 37 DEG C of incubation 1h, wash and luminous developer solution are added after film, and electrochemical luminescence kit light post-exposure, the control by internal reference of GAPDH.

1.5 Flow cytometry cell surface marker positive rates：Collect different disposal group cell 1X10⁶In centrifuge tube, 4 DEG C of addition precooling PBS cell 3 times after centrifugation, 800r/min centrifugation 5min, abandons supernatant.With 200 μ l PBS suspension cells. Often pipe sequentially adds 10ulCD133-PE streaming antibody, mixes；Room temperature lucifuge reacts 30min；Add the resuspended cleaning cells 3 of PBS It is secondary, abandon supernatant.It is resuspended with 200ulPBS again.Machine testing on flow cytometer, records result.

The observation CD133 expression of 1.6 immunofluorescences and positioning take the pancreatic cancer cell 2X10 in logarithmic growth⁵It is individual, it is incubated at Copolymerization Jiao's ware, adherent rear many base formaldehyde, which are fixed, adds CD133 primary antibodies (1 after 30min, triton ruptures of membranes：200), 4 DEG C were incubated At night, fluorescence secondary antibody is added, PBS 3 times, DAPI dye core 5min are cleaned 3 times, seen after mounting under Laser Scanning Confocal Microscope again Examine and take pictures.

1.7 statistical procedures：Data analysis is carried out using the statistics softwares of SPSS 22.0.Measurement data is examined using t, Enumeration data uses x²Examine, P ＜ 0.05 are that difference is statistically significant.

As a result

HIF1 α and CD133 expression under 2.1 cancers of pancreas difference anoxic different time：By to pancreatic cancer cell PANC-1, AsPC cells HIF1- α and CD133 after normal oxygen and anoxic 4h, 8h, 16h, 24,30h expression.With the extension of hypoxic exposure, HIF-1a, CD133 expression are gradually increasing, and in 16h, HIF-1a expression is in peak.After hypoxic exposure is more than 16h, with After HIF-1a expression reduction, CD133 expression is reduced simultaneously；

2.2 respectively after normal oxygen and anoxic culture 16h, extracts pancreatic cancer cell RNA, stem-cell marker is detected by PCR Thing (BMI-1/OCT4A/Nanog/SOX2) mRNA is expressed, and stem cell markers mRNA expressions are raised after anoxic, difference tool Statistically significant (P<0.05 Fig. 1)

2.3 pancreatic cancer cells pass through Flow cytometry CD133 surface markers sun after normal oxygen and anoxic culture 16h Property rate, cancer of pancreas surface markers CD133 positive rates are significantly raised under anoxic culture, and difference has statistical significance (P<0.05, figure 2)。

2.4 respectively in normal oxygen and anoxic low suspension culture Pancreatic Cancer Pancreatic carcinoma cell, and cell gradually forms suspension ball growth, Represent the feature of tumour cell " dryness ".Cultivate after one week, compared with normal oxygen, the tumour cell suspension ball cultivated under anaerobic condition More normal oxygen group is bigger, and the more (P of balling-up<0.05, Fig. 3).

2.5 laser confocal microscopes are detected:Observed and exported after sample, image procossing using 40 times of oil mirrors.As a result as schemed. CD133 (red) is located in cell membrane surface and cytoplasm, blue indicator cells core.As a result as schemed, compared with normal oxygen group, anoxic Red light intensity in culture 16h cells substantially increases.Show through statistical analysis, two groups of differences have statistical significance (P< 0.05, Fig. 4).

2.6 respectively after transfection si-HIF-1 α and NC siRNAs, cultivate 16h under anoxic, rapid extraction cell protein, Detect that cancer of pancreas stem cell markers albumen changes by western blot, under anoxic conditions, lower after HIF-1 α, BMI- 1st, OCT4A, Nanog, SOX2 expression are notable lowers, and shows that micro-environmental hypoxia may be by HIF-1a thin so as to regulate and control cancer of pancreas The dryness biological behaviour of born of the same parents.(Fig. 5)

Conclusion

Micro-environmental hypoxia is generally existing in the generation evolution of human malignancies.Recent studies have shown that lacking There are complicated regulation and control to the oncobiology such as the metabolism of tumour, propagation, invasion and attack phenomenon in oxygen microenvironment.Micro-environmental hypoxia is to tumour Propagation and the biology malignant activity such as invasion and attack play an important role.The micro-environmental hypoxia of inside tumor is conducive to tumor stem cell The maintenance of special phenotype, that is, contribute to the malignant progression of tumour.And tumor stem cell is likely to be the high invasion of cancer of pancreas, Gao Fu One of hair rate, major reason of high mortality.Current tumor stem cell theory is considered as that tumour cell effectively carries out self more The most key subgroup of new and various biological behaviours.

The result of the present embodiment confirms that the processing of anoxic can be ground with the maintenance and enhancing of inducing pancreatic cancer cancer cell " dryness " Study carefully CD133 under confirmation anoxia condition and express consistent with HIF-1 α expression, HIF-1 alpha expressions level reaches peak after anoxic 16h Value, CD133 also reaches peak value simultaneously.The pancreatic cancer cell cultivated under anoxic conditions, CD133⁺The positive tumour of surface markers Cell positive rate increase, while tumor stem cell associated transcription factor protein B MI-1, OCT4A, Nanog, SOX2 is being transcribed and turned over Level is translated to increase.In summary, anoxic can induce the maintenance and enhancing of pancreatic tumour " dryness ".We study further It was found that, under anoxic conditions, after the expression for lowering HIF-1 α, this change of hypoxia inducible is suppressed, dryness associated retroviral because Sublist is up to lowering, and it is most important that this shows that HIF-1 α are served in the regulation and control of hypoxia inducible human pancreatic carcinoma cell " dryness " Effect.But the further related mechanism and signal path being directed to, which need further research, to be confirmed.Grind at present Study carefully and show, during anoxic culture, the propagation and invasive ability of pancreatic cancer cell are raised, and this data proves anoxic simultaneously to be promoted Enter the expression rise of pancreatic cancer cell dryness related biological phenomenon and marker protein.

Claims

1. a kind of method that pancreatic cancer cell is cultivated under anaerobic environment, it is characterised in that：Give birth to AsP C-1 or BxPC-3 cell It is long in rpmi -1640 culture mediums, and be aided with 37 DEG C of wet air 10% hyclone, V_Hyclone：V_{DMEM culture mediums}=1： 10, level of endotoxin≤10EU/ml. hemoglobin levels≤30mg/dl, 100U/ml benzyl penicillins and BxPC-3 and 100 μ g/ml chains Mycin；By PANC-1 cells in DMEM medium cultures；Three of the above pancreatic cancer cell is cultivated in hypoxic cell incubator, Wherein O₂、CO₂And N₂Volume ratio be 1.5:5:93.5.