CN104962548A - Construction method of lung cancer radiation resistance cell strain - Google Patents
Construction method of lung cancer radiation resistance cell strain Download PDFInfo
- Publication number
- CN104962548A CN104962548A CN201510367859.0A CN201510367859A CN104962548A CN 104962548 A CN104962548 A CN 104962548A CN 201510367859 A CN201510367859 A CN 201510367859A CN 104962548 A CN104962548 A CN 104962548A
- Authority
- CN
- China
- Prior art keywords
- lung cancer
- radiation resistance
- cell
- dose
- cell strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention discloses a construction method of a lung cancer radiation resistance cell strain. The method includes the following steps of inoculating lung cancer cells in a six-hole plate when the lung cancer cells are conventionally bred to the logarithmic phase, placing the six-hole plate at the center of an irradiation field, covering the bottom face of the six-hole plate with tissue equivalent filler 1.5 cm thick, perpendicularly transmitting 6MV-X rays according to the dose rate of 300 cGy/min and the source skin distance of 100 cm, sequentially conducting irradiation according to the dose of 2 Gy, 4 Gy, 6 Gy, 8 Gy and 10 Gy each for two times till the total dose reaches 60 Gy, continuing to conduct breeding and passage to five or more generations till the cellular morphology and multiplication are stable, and conducting cryopreservation for standby application. The construction method is small in irradiation time number, short in construction time, small in cell contamination probability and superior to a small-dose equal-segmentation irradiation method; the colony formation experimental result shows that the radiation resistance of the lung cancer radiation resistance cell strain constructed according to the method is higher than that of lung cancer radiation resistance cell strains constructed according to the sublethal dose irradiation method.
Description
Technical field
The invention belongs to cell biology, specifically relate to a kind of construction process with the lung cancer radiation resistance cell strain of stable radiation resistance.
Background technology
Lung cancer is one of modal malignant tumour of China, and within 2014, National Cancer Center issues " Chinese tumour registration annual report " report, and lung cancer sickness rate in China's malignant tumour occupies first, annual new cases about 600,000, death about 490,000.Radiotherapy is one of important means of current lung cancer multidisciplinary synthesis treatment, and the patient of statistics display more than 70% will accept radiotherapy in diagnosis and treatment process.But though through various means complex therapy, lung cancer curative effect is totally barely satisfactory, within 5 years, survival rate is hovered for a long time in about 15%, urgently improves.Tumor recurrence and transfer are the major causes of radiotherapy of lung cancer failure, and existing research is thought, residual cell after radiotherapy of lung cancer, and namely lung cancer radiation resistance cell invasion power and transfer activity strengthen, and are the roots of tumor recurrence and transfer.Therefore, build lung cancer radiation resistance In vitro cell model, significant to raising lung cancer therapy curative effect.
Mainly contain low dose of grade for the method building lung cancer radiation resistance cell at present and split irradiation and Sublethal Doses method, its small decile cuts irradiation, and because irradiating, number of times is more, construction schedule needs more than 1 year, cell contamination probability is large, and stable going down to posterity into is more difficult.Sublethal Doses method adopts sublethal dose 1 property making the splitting of chain of lung carcinoma cell DNA single to irradiate, and 1 property such as giving lung cancer A549 and H1299 cell sublethal dose 6.5Gy and 5.5Gy is respectively irradiated.But current routine clinical dose fractionation radiotherapy total dose should be not less than 60Gy, Sublethal Doses method differs comparatively large with existing clinical radiation therapy pattern, the lung cancer radiation resistance cell therefore adopting this method to build cannot simulate clinical recurrence cancer radiobiology characteristic to greatest extent.
Summary of the invention
The object of this invention is to provide a kind of construction process with the lung cancer radiation resistance cell strain of stable radiation resistance, irradiate number of times few, construction schedule is short, and cell contamination probability is little, and the radiation resistance cell strain of structure can simulate clinical recurrence cancer radiobiology characteristic.
To achieve these goals, the present invention adopts following technical scheme: a kind of construction process of lung cancer radiation resistance cell strain, comprises the following steps:
1. lung carcinoma cell is placed in 37 DEG C, 5%CO
2cellar culture in incubator, cover culturing bottle 60 ~ 70% when it and when being in logarithmic phase, being inoculated in six orifice plates, six orifice plates are placed in irradiation field center, six orifice plate bottom surfaces cover the thick tissue equivalent's weighting material of 1.5cm, irradiating source is clinac, 6MV-X line, and frame rotates 180 °, ray vertical incidence from below at ambient temperature, dose rate 300cGy/min, ource-skin Distance SSD=100cm, give 2Gy for the 1st time and irradiate;
2. change fresh culture after irradiating, when cell covers culturing bottle more than 80%, trysinization, goes down to posterity more than twice;
3. go down to posterity and be placed on 37 DEG C, 5%CO
2cultivate 2 ~ 3 days in incubator, again cover after culturing bottle 60 ~ 70% until cell, again give 2Gy and irradiate;
4. 2. postradiation cell all goes down to posterity with step method 3. by above-mentioned steps and irradiates after being cultured to steady state again each time, increase irradiation dose successively and reach 4Gy, 6Gy, 8Gy and 10Gy, every dosage all irradiates 2 times, total dose reaches 60Gy, continue cultivation and 5 generations of going down to posterity more than to stablize to cellular form, propagation, frozen for subsequent use.
Further, in described six orifice plates, every porocyte inoculum size is 5 × 10
5individual.
Further, described clinac is Varian 23-EX clinac.
Further, the irradiation field size of described x-ray is 30cm × 30cm.
Technical scheme of the present invention has following beneficial effect:
1, utilize the present invention to build lung cancer radiation resistance cell strain, altogether carry out radiation exposure 10 times, irradiate number of times few; Construction schedule about 6 months, the time is short; And the number of times being placed on radiotherapy room from cell Sterile culture room because of cell is less, cell contamination probability is little, is better than low dose ofly waiting segmentation irradiation;
2, Colony forming experiment is the gold standard of current internationally recognized detection Cell irradiation resistance.The present invention adopts Colony forming to test the radiation resistance detecting cell, and result shows the average lethal dose D of the lung cancer radiation resistance cell strain adopting the present invention to build
0, Dq Dq and 2Gy survive mark SF
2all higher than Sublethal Doses method, the present invention is namely adopted to build the radiation resistance of lung cancer radiation resistance cell strain higher than Sublethal Doses method;
3, total dose is irradiated in the present invention is 60Gy, close with existing clinical radiation therapy pattern, and the radiation resistance cell strain of structure can simulate clinical recurrence cancer radiobiology characteristic to greatest extent.
Accompanying drawing explanation
Fig. 1 is the schema of construction process of the present invention;
Fig. 2 is A549 cell and the aspect graph of radiation resistance A549R cell under inverted light phase microscope (100 times are amplified), and wherein, A is A549 cellular form figure, B is A549R cellular form figure;
Fig. 3 is the A549 of Colony forming experiment drafting and the cells survival graphic representation of radiation resistance cell, and wherein A is the multi-hit model of single target, and B is quadratic linear model;
Fig. 4 is H1299 cell and the aspect graph of radiation resistance H1299R cell under inverted light phase microscope (100 times are amplified), and wherein, C is H1299 cellular form figure, D is H1299R cellular form figure;
Fig. 5 is the H1299 of Colony forming experiment drafting and the cells survival graphic representation of radiation resistance cell, and wherein C is the multi-hit model of single target, and D is quadratic linear model.
Embodiment
Human A459 lung cancer cell line and H1299, be purchased from the biological company limited of the triumphant base in Nanjing, substratum is 1640 substratum (flying generation that biochemistry corporation,Ltd. purchased from Sai Mo) containing 10% foetal calf serum (being purchased from folium ilicis chinensis company), is placed in 37 DEG C, 5%CO respectively
2cultivate in incubator.
Embodiment 1
As shown in Figure 1, a kind of construction process of lung cancer radiation resistance cell strain, comprises the following steps:
1. human lung carcinoma cell line A549 is placed in 37 DEG C, 5%CO
2cellar culture in incubator, when it covers culturing bottle 60 ~ 70% when being in logarithmic phase, with every hole 5 × 10
5individually be inoculated in six orifice plates, six orifice plates are placed in irradiation field center, six orifice plate bottom surfaces cover the thick tissue equivalent's weighting material of 1.5cm, and irradiating source is Varian23-EX clinac, 6MV-X line, frame rotates 180 °, ray vertical incidence from below at ambient temperature, dose rate 300cGy/min, ource-skin Distance SSD=100cm, irradiation field size is 30 × 30cm, gives 2Gy for the 1st time and irradiates;
2. change fresh culture after irradiating, when cell covers culturing bottle more than 80%, trysinization, goes down to posterity more than twice;
3. go down to posterity and be placed on 37 DEG C, 5%CO
2cultivate 2 ~ 3 days in incubator, again cover after culturing bottle 60 ~ 70% until cell, again give 2Gy and irradiate;
4. 2. postradiation cell all goes down to posterity with step method 3. by above-mentioned steps and irradiates after being cultured to steady state again each time, increase irradiation dose successively and reach 4Gy, 6Gy, 8Gy and 10Gy, every dosage all irradiates 2 times, total dose reaches 60Gy, continue cultivation and 5 generations of going down to posterity more than to stablize to cellular form, propagation, namely A549 lung cancer radiation resistance cell is obtained, called after A549R (A549-radioresistance) cell, frozen for subsequent use.
Observation of cell form under inverted light phase microscope (906 Nikon Ti inverted fluorescence microscope).As shown in Figure 2, A is A549 cellular form, and B is the radiation resistance A549R cellular form that the present invention obtains.It can thus be appreciated that A549R cell volume becomes large, iuntercellular is apart from increasing, and cell polarity disappears, and to spindle sample mesenchymal cell morphologic change, and has many and long pseudopodium to be formed.
The display of Colony forming experimental result is along with the increase of dosage, A549 respectively organizes cell colony rate of formation and reduces gradually, with illuminating method gained of the same race under dose radiation resistance group cell comparatively control group growth all very fast, cell number is more, colony forming efficiency is large, existence mark high (see table 1).Show that the sublethal dose of A549 is 6Gy through screening.Fig. 3 is the cells survival graphic representation according to one-hit multitarget and quadratic linear model-fitting, and wherein A is the multi-hit model of single target, and B is quadratic linear model, and draws each biological parameter as shown in table 2, it can thus be appreciated that A549R group is A549 group and sublethal dose group D comparatively
0, Dq, SF
2all increase, α, α/β all reduce, and can show that the radiation resistance of the lung carcinoma cell resistance A549R cell built by the present invention is stronger.
Table 1 irradiates the existence mark of rear A549 and radiation resistance cell
Note: ▲ P<0.05 A549R VS A549
Table 2 A549 and radiation resistance cells survival curve biological parameter
Embodiment 2
A construction process for lung cancer radiation resistance cell strain, comprises the following steps:
1. human lung carcinoma cell line H1299 is placed in 37 DEG C, 5%CO
2cellar culture in incubator, when it covers culturing bottle 60 ~ 70% when being in logarithmic phase, with every hole 5 × 10
5individually be inoculated in six orifice plates, six orifice plates are placed in irradiation field center, six orifice plate bottom surfaces cover the thick tissue equivalent's weighting material of 1.5cm, and irradiating source is Varian23-EX clinac, 6MV-X line, frame rotates 180 °, ray vertical incidence from below at ambient temperature, dose rate 300cGy/min, ource-skin Distance SSD=100cm, irradiation field size is 30 × 30cm, gives 2Gy for the 1st time and irradiates;
2. change fresh culture after irradiating, when cell covers culturing bottle more than 80%, trysinization, goes down to posterity more than twice;
3. go down to posterity and be placed on 37 DEG C, 5%CO
2cultivate 2 ~ 3 days in incubator, again cover after culturing bottle 60 ~ 70% until cell, again give 2Gy and irradiate;
4. 2. postradiation cell all goes down to posterity with step method 3. by above-mentioned steps and irradiates after being cultured to steady state again each time, increase irradiation dose successively and reach 4Gy, 6Gy, 8Gy and 10Gy, every dosage all irradiates 2 times, total dose reaches 60Gy, continue cultivation and 5 generations of going down to posterity more than to stablize to cellular form, propagation, namely H1299 lung cancer radiation resistance cell is obtained, called after H1299R (H1299-radioresistance) cell, frozen for subsequent use.
Observation of cell form under inverted light phase microscope (906 Nikon Ti inverted fluorescence microscope).As shown in Figure 4, C is H1299 cellular form, and D is the radiation resistance H1299R cellular form that the present invention obtains.It can thus be appreciated that H1299R cell volume becomes large, iuntercellular is apart from increasing, and cell polarity disappears, and to spindle sample mesenchymal cell morphologic change, and has many and long pseudopodium to be formed.
The display of Colony forming experimental result is along with the increase of dosage, H1299 respectively organizes cell colony rate of formation and reduces gradually, with illuminating method gained of the same race under dose radiation resistance group cell comparatively control group growth all very fast, cell number is more, colony forming efficiency is large, existence mark high (see table 3).Through our experiment screening, show that the sublethal dose of A549 is 6Gy.Fig. 5 is the cells survival graphic representation according to one-hit multitarget and quadratic linear model-fitting, and wherein C is the multi-hit model of single target, and D is quadratic linear model, and draws each biological parameter as shown in table 4, it can thus be appreciated that H1299R is H1299 group and sublethal dose group D comparatively
0, Dq, SF
2all increase, α, α/β all reduce, and can show that the radiation resistance of the lung carcinoma cell resistance H1299R cell built by the present invention is stronger.
Table 3 irradiates the existence mark of rear H1299 and radiation resistance cell
Note: ▲ P<0.05 H1299R VS H1299
Table 4 H1299 and radiation resistance cells survival curve biological parameter
Claims (5)
1. a construction process for lung cancer radiation resistance cell strain, is characterized in that, comprises the following steps:
1. lung carcinoma cell is placed in 37 DEG C, 5%CO
2cellar culture in incubator, cover culturing bottle 60 ~ 70% when it and when being in logarithmic phase, being inoculated in six orifice plates, six orifice plates are placed in irradiation field center, six orifice plate bottom surfaces cover the thick tissue equivalent's weighting material of 1.5cm, irradiating source is clinac, 6MV-X line, and frame rotates 180 °, ray vertical incidence from below at ambient temperature, dose rate 300cGy/min, ource-skin Distance SSD=100cm, give 2Gy for the 1st time and irradiate;
2. change fresh culture after irradiating, when cell covers culturing bottle more than 80%, trysinization, goes down to posterity more than twice;
3. go down to posterity and be placed on 37 DEG C, 5%CO
2cultivate 2 ~ 3 days in incubator, again cover after culturing bottle 60 ~ 70% until cell, again give 2Gy and irradiate;
4. 2. postradiation cell all goes down to posterity with step method 3. by above-mentioned steps and irradiates after being cultured to steady state again each time, increase irradiation dose successively and reach 4Gy, 6Gy, 8Gy and 10Gy, every dosage all irradiates 2 times, total dose reaches 60Gy, continue cultivation and 5 generations of going down to posterity more than to stablize to cellular form, propagation, frozen for subsequent use.
2. the construction process of lung cancer radiation resistance cell strain as claimed in claim 1, it is characterized in that, in described six orifice plates, every porocyte inoculum size is 5 × 10
5individual.
3. the construction process of lung cancer radiation resistance cell strain as claimed in claim 1 or 2, it is characterized in that, described clinac is Varian 23-EX clinac.
4. the construction process of lung cancer radiation resistance cell strain as claimed in claim 1 or 2, it is characterized in that, the irradiation field size of described x-ray is 30cm × 30cm.
5. the construction process of lung cancer radiation resistance cell strain as claimed in claim 3, it is characterized in that, the irradiation field size of described x-ray is 30cm × 30cm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510367859.0A CN104962548A (en) | 2015-06-29 | 2015-06-29 | Construction method of lung cancer radiation resistance cell strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510367859.0A CN104962548A (en) | 2015-06-29 | 2015-06-29 | Construction method of lung cancer radiation resistance cell strain |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104962548A true CN104962548A (en) | 2015-10-07 |
Family
ID=54216639
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510367859.0A Pending CN104962548A (en) | 2015-06-29 | 2015-06-29 | Construction method of lung cancer radiation resistance cell strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104962548A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107779438A (en) * | 2017-09-25 | 2018-03-09 | 杭州市第人民医院 | A kind of radiotherapy tolerance lung cancer cell line and its construction method and application |
| CN109735495A (en) * | 2018-12-26 | 2019-05-10 | 台州市中心医院 | A kind of construction method of the clinically relevant Radioresistance cell strain of nasopharyngeal carcinoma |
| CN110878284A (en) * | 2019-10-12 | 2020-03-13 | 南方医科大学南方医院 | Model of lung cancer cell surviving or resisting by equal difference dose gradient radiation and construction and application |
| CN112662629A (en) * | 2021-01-26 | 2021-04-16 | 兰州大学 | Construction method of prostate cancer radiation-resistant cell strain |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1386848A (en) * | 2001-05-23 | 2002-12-25 | 中国人民解放军军事医学科学院放射医学研究所 | Human cerebral transfer cell line of lung cancer |
| CN104017799A (en) * | 2013-08-08 | 2014-09-03 | 中南大学湘雅医院 | Method for screening nasopharyngeal carcinoma radiotherapy resistant cells by using radioactive irradiation mode |
-
2015
- 2015-06-29 CN CN201510367859.0A patent/CN104962548A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1386848A (en) * | 2001-05-23 | 2002-12-25 | 中国人民解放军军事医学科学院放射医学研究所 | Human cerebral transfer cell line of lung cancer |
| CN104017799A (en) * | 2013-08-08 | 2014-09-03 | 中南大学湘雅医院 | Method for screening nasopharyngeal carcinoma radiotherapy resistant cells by using radioactive irradiation mode |
Non-Patent Citations (2)
| Title |
|---|
| 刘静等: "放射耐受性人小细胞肺癌亚株的建立", 《中国比较医学杂志》 * |
| 李果: "鼻咽癌放疗抵抗相关microRNA差异表达谱的建立", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107779438A (en) * | 2017-09-25 | 2018-03-09 | 杭州市第人民医院 | A kind of radiotherapy tolerance lung cancer cell line and its construction method and application |
| CN109735495A (en) * | 2018-12-26 | 2019-05-10 | 台州市中心医院 | A kind of construction method of the clinically relevant Radioresistance cell strain of nasopharyngeal carcinoma |
| CN110878284A (en) * | 2019-10-12 | 2020-03-13 | 南方医科大学南方医院 | Model of lung cancer cell surviving or resisting by equal difference dose gradient radiation and construction and application |
| CN112662629A (en) * | 2021-01-26 | 2021-04-16 | 兰州大学 | Construction method of prostate cancer radiation-resistant cell strain |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fernandez-Palomo et al. | Bystander effects in tumor-free and tumor-bearing rat brains following irradiation by synchrotron X-rays | |
| CN102174469B (en) | Method for effectively culturing tumor infiltrating lymphocytes (TILs) | |
| CN104962548A (en) | Construction method of lung cancer radiation resistance cell strain | |
| Bettega, P. Calzolari, P. Chauvel, A. Courdi, J. Herault, N. Iborra, R. Marchesini, P. Massariello, GL Poli, L. Tallone | Radiobiological studies on the 65MeV therapeutic proton beam at Nice using human tumour cells | |
| Weichselbaum et al. | The differential response of human tumours to fractionated radiation may be due to a post-irradiation repair process | |
| CN105233292A (en) | Application of ORY-1001 to treatment of triple negative breast cancer through combined radiotherapy and chemotherapy | |
| Fujisawa et al. | Comparison of human chordoma cell-kill for 290 MeV/n carbon ions versus 70 MeV protons in vitro | |
| Wang et al. | The direct biologic effects of radioactive 125I seeds on pancreatic cancer cells PANC-1, at continuous low-dose rates | |
| CN107779438A (en) | A kind of radiotherapy tolerance lung cancer cell line and its construction method and application | |
| Gragg et al. | The Response of Chinese Hamster Ovary Cells to Fast-Neutron Radiotherapy Beams: II. Sublethal and Potentially Lethal Damage Recovery Capabilities | |
| CN110878284A (en) | Model of lung cancer cell surviving or resisting by equal difference dose gradient radiation and construction and application | |
| CN104017799A (en) | Method for screening nasopharyngeal carcinoma radiotherapy resistant cells by using radioactive irradiation mode | |
| CN106367391A (en) | Rectal cancer radiotherapy resistance cell model and construction method therefor | |
| Yang et al. | Mutation effects of C2+ ion irradiation on the greasy Nitzschia sp. | |
| CN102363044B (en) | Application of three anthraquinone substances of targeted mitochondria as nasopharyngeal darcinoma radiosensitizers | |
| CN105087489A (en) | DC cell culture reagent and culture method thereof | |
| Qin et al. | Biological effects of protons targeted to different ranges in Arabidopsis seeds | |
| CN106047701B (en) | A kind of short distance Induced by Radioactive Ray cancer cell-apoptosis model equipment | |
| CN103898043B (en) | Cell feeder layer and the application in cultivation people primary tumor cell thereof | |
| Pakniyat et al. | Enhanced response of radioresistant carcinoma cell line to heterogeneous dose distribution of grid; the role of high-dose bystander effect | |
| Pinho et al. | Assessing patient characteristics and radiation-induced non-targeted effects in vivo for high dose-rate (HDR) brachytherapy | |
| CN102908356A (en) | Application of geniposide | |
| CN112662629A (en) | Construction method of prostate cancer radiation-resistant cell strain | |
| CN107217013A (en) | A kind of strange coccus T1R5 and its application in anti-radiation protection | |
| Bakeine et al. | Feasibility study on the utilization of boron neutron capture therapy (BNCT) in a rat model of diffuse lung metastases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151007 |
|
| RJ01 | Rejection of invention patent application after publication |