CN102676456A - Method for separating lung cancer cell from hydrothorax - Google Patents
Method for separating lung cancer cell from hydrothorax Download PDFInfo
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- CN102676456A CN102676456A CN2012101437721A CN201210143772A CN102676456A CN 102676456 A CN102676456 A CN 102676456A CN 2012101437721 A CN2012101437721 A CN 2012101437721A CN 201210143772 A CN201210143772 A CN 201210143772A CN 102676456 A CN102676456 A CN 102676456A
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Abstract
The invention discloses a method for separating lung cancer cell from hydrothorax. The method comprises the following steps of: (1) preparing Percoll solutions with different concentrations; (2) preparing hydrothorax suspension; (3) preparing Percoll layers with discontinuous density gradients; (4) centrifuging; (5) sampling; and (6) performing cell culture. The method disclosed by the invention is simple and stable and has the advantages of low cost and good effect.
Description
Technical field
The present invention relates to the isolating method of a kind of tumour cell, be specifically related to the method for separation and purification lung carcinoma cell in a kind of hydrothorax, belong to the cellular biology of tumor technical field.
Background technology
The closure lacuna that the thoracic cavity is made up of partial pleura and visceral pleura; It in it negative pressure; The liquid institute lubricate that has very small amount (about 1 ~ 30 milliliter) under the normal circumstances between the two-layer pleura; Minimizing is the friction between the two-layer pleura in the respiratory activity process, is beneficial to lung easypro contracting in the thoracic cavity.This liquid produces from partial pleura, is absorbed by visceral pleura, and the part that constantly circulates is in running balance, and it is constant that amount of liquid keeps.No matter have influence on pleura when certain situation takes place, be that partial pleura produces the speed that hydrothorax or visceral pleura absorb hydrothorax and changes, and can make all that liquid increases in the thoracic cavity, just so-called hydrothorax (hydrops).
Cells in pleural fluids from lung cancer cases be the medium and advanced lung cancer patient than common complication, at first maybe be also little, but, can cause symptoms such as expiratory dyspnea, cough, pectoralgia along with course of disease progress to quality of life influence, to patient's harm even surpassed lung cancer itself.Cancerous hydrothorax is courageous and upright often, except that the tumour cell that comes off, includes a large amount of red white corpuscles and cell debris.
Tumor research needs lots of clinical tumor tissues and cell; Present most of investigator mainly selects clinical tumor patient's specimens from pri for use, though it is more to originate, specimen amount is big; But, be difficult for obtaining corresponding specimens from pri because the treatment of advanced lung cancer is main with expectant treatment often.
The advanced lung cancer patient is more complication such as hydrothorax to occur, and extracting hydrothorax is clinical conventional treatments, and separating tumor cell has easy, economic characteristics in hydrothorax, can obtain the lung carcinoma cell of a large amount of high late cases of grade of malignancy simultaneously again.
Though adopted direct centrifugation method simple, other cell and fragment of tissue are more in the past, the purity of tumour cell is lower, and practical application has little significance.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of hydrothorax to separate the method for lung carcinoma cell, to solve the many weak points of existing in prior technology.
Principle of the present invention:
Use the method for gradient centrifugation can the cell of different sizes, proportion be carried out layering and separate; We are under the guiding of this method; Through exploring repeatedly and testing; Utilize big, the heavy characteristics of other normal cell volume of tumor cell ratio, set up in the hydrothorax and separated the method for lung carcinoma cell, and be used for the foundation of collection, cultivation and the clone of lung carcinoma cell.
Percoll is a kind of silica gel particle that is surrounded by V-Pyrol RC.Osmotic pressure very low (<20mosm/>kg H
2O), viscosity is also very little, can form the density up to 1.3g/ml, can (200～1000g) reach satisfied cellular segregation result in several branches in to tens of minutes at low cf-when adopting preformed density gradient.Because the Percoll diffusion constant is low, formed gradient is very stable.In addition, Percoll does not penetrate microbial film, and therefore the pair cell toxicological harmless is used for isolated cell, bacterium and virus.But do not see that the centrifugal method that utilizes the corresponding density gradient separates lung carcinoma cell in hydrothorax, be correlated with simultaneously former be commissioned to train to support and build be.
The technical problem that will solve required for the present invention, can realize through following technical scheme:
A kind of hydrothorax is separated the method for lung carcinoma cell, it is characterized in that, may further comprise the steps:
(1) preparation of different concns Percoll solution: the first mixing with 1 part of 8.5% NaCl or 1.5M PBS with 9 parts of Percoll reaches physiological osmotic pressure, is diluted to 60 ~ 70%wt, 40 ~ 50%wt with physiological solution then;
(2) preparation of hydrothorax suspension: centrifugal 5 minutes of hydrothorax sample 1500rpm abandons supernatant, and 1% PBS cleans one time, and centrifugal 5 minutes of 1500rpm abandons supernatant, and 1%PBS is resuspended; Contain various kinds of cell composition and cell tissue fragment in the hydrothorax suspension this moment;
(3) preparation of discontinuous density gradient Percoll layer:
60 ~ 70%wt, 40 ~ 50%wt, hydrothorax suspension are successively disposed to low density by high-density according to the ratio of 1:1:1, form 3 density gradients;
(4) centrifugal: the centrifugal 25-40min of 2800-3200rpm;
(5) sampling: the isolated cells of is positioned at two bed interfaces, draws the intermediate cell layer carefully, and 1% PBS cleans 1 time, and the centrifugal 5min of 1500rpm abandons supernatant, harvested cell; And
(6) cell cultures: the cell of acquisition is cultivated with the DMEM of 10%FBS is conventional.
Wherein, the physiological solution described in the step (1) is 0.85%WT NaCl or 0.15M PBS.
The concentration of said Percoll solution is 60%wt, 40%wt.
Wherein, each gradient 3ml in the step (3), 9ml adds the 10ml centrifuge tube from the bottom to top altogether, according to how many definite how many centrifuge tubes of hydrothorax suspension.
Wherein, centrifugal described in the step (4) is the centrifugal 30min of 3000rpm.
Beneficial effect of the present invention:
Method of the present invention has simply, stablizes, low, the effective advantage of cost.
Description of drawings
Further specify the present invention below in conjunction with accompanying drawing and embodiment.
Fig. 1 is the aspect graph after the cellular segregation.
Fig. 2 is the aspect graph after the cellular segregation.
Fig. 3 is the aspect graph after the cellular segregation.
Fig. 4 is the aspect graph after the cellular segregation.
Embodiment
In order to make technique means of the present invention, creation characteristic, to reach purpose and effect and be easy to understand and understand,, further set forth the present invention below in conjunction with concrete diagram.
Embodiment
Experimental procedure:
1. the preparation of different concns (density) Percoll solution:
The first mixing with 1 part of 8.5% NaCl or 1.5M PBS with 9 parts of Percoll reaches physiological osmotic pressure, uses physiological solution (0.85% NaCl or 0.15M PBS) to be diluted to desired concn then.What we adopted is 60% and 40% concentration.This concentration gradient is simple and effective through relatively finding, separating effect is better.
2. the preparation of hydrothorax suspension:
Centrifugal 5 minutes of hydrothorax sample 1500rpm abandons supernatant, and 1% PBS cleans one time, and centrifugal 5 minutes of 1500rpm abandons supernatant, and 1%PBS is resuspended; Contain various kinds of cell composition and cell tissue fragment in the hydrothorax suspension this moment.
3. the preparation of discontinuous density gradient Percoll layer:
60%, 40%, the hydrothorax suspension successively disposes to low density by high-density according to the ratio of 1:1:1, forms 3 density gradients.Slowly soft as far as possible when specific requirement is liquid feeding, in order to avoid influence original density gradient.General each gradient 3ml, 9ml adds the 10ml centrifuge tube from the bottom to top altogether, according to how many definite how many centrifuge tubes of hydrothorax suspension.
4. centrifugal:
The centrifugal 25-40min of 2800-3200rpm, tumour cell are very fast and the time is longer than the die centrifugal speed, and we select the centrifugal 30min of 3000rpm for use usually, because the density difference is little between 3 layers, so whizzer quickens, want slow during reduction of speed, steadily.That the experiment table that whizzer is placed is wanted is firm, preferably import of whizzer.
5. sampling:
The isolating lung carcinoma cell of wanting because the bigger proportion of volume slightly heavily is positioned at the nearly intermediate interface of centrifuge tube, form the pearl cellular layer; Other cell and fragment are less to sink to the pipe end, if the red cell mass at the visible pipe of the bloody pleural fluid end.The isolated cells of wanting is positioned at two bed interfaces, draws the intermediate cell layer carefully, and 1% PBS cleans 1 time, and the centrifugal 5min of 1500rpm abandons supernatant, the pearl lung carcinoma cell at the results pipe end, microscopically observation of cell form.
6. cell cultures:
Since in the hydrothorax through the lung carcinoma cell grade of malignancy (late period just occurring hydrothorax) of separation and purification after the density gradient centrifugation and purity (little other cell) all than specimens from pri (general early stage consideration operative treatment; In the sample except that tumour cell; Also have more normal cell, non-viable non-apoptotic cell and fragment of tissue) to get well, be the good material of cell cultures.Isolating primary cell can carry out routine with the DMEM of 10%FBS and cultivate.Perhaps cultured continuously goes down to posterity and sets up corresponding lung cancer cell line as required.
The cell that obtains is cultivated with the DMEM of 10%FBS is conventional.
Hydrothorax lung carcinoma cell separation case, detailed results is seen table 1.
Table 1 is a hydrothorax lung carcinoma cell separation case
| Numbering | Sex | Age | The acquisition time | Remarks |
| 1 | The man | 48 | 2010.10.29 | It is few that hydrothorax is separated the acquisition cell concentration |
| 2 | The man | 56 | 2010.11.11 | Cellular segregation is cultivated unsuccessful |
| 3 | The man | 44 | 2010.12.3 | Find cancer cells in the hydrothorax |
| 4 | The man | 56 | 2010.12.9 | Find adenocarcinoma cell in the hydrothorax |
| 5 | The man | 38 | 2010.12.15 | Find adenocarcinoma cell in the hydrothorax |
| 6 | The man | 30 | 2010.12.21 | Find adenocarcinoma cell in the hydrothorax |
| 7 | The woman | 44 | 2010.12.27 | Find adenocarcinoma cell in the hydrothorax |
| 8 | The woman | 49 | 2011.1.5 | Separation and Culture is unsuccessful |
| 9 | The woman | 67 | 2011.1.6 | Find adenocarcinoma cell in the hydrothorax |
| 10 | The man | 67 | 2011.1.11 | Cellular segregation is cultivated unsuccessful |
| 11 | The man | 60 | 2011.1.14 | Find adenocarcinoma cell in the hydrothorax |
| 12 | The man | 72 | 2011.1.17 | Find adenocarcinoma cell in the hydrothorax |
| 13 | The woman | 73 | 2011.1.17 | Find gland cancer in the hydrothorax |
| 14 | The man | 78 | 2011.3.24 | Separation and Culture is unsuccessful |
| 15 | The woman | 66 | 2011.3.31 | Find gland cancer in the hydrothorax |
| 16 | The man | 65 | 2011.4.7 | Find adenocarcinoma cell in the hydrothorax |
| 17 | The man | 51 | 2011.4.19 | Bloody pleural fluid, separation and Culture is unsuccessful |
| 18 | The woman | 39 | 2011.4.27 | Faint yellow hydrothorax, separation and Culture is unsuccessful |
| 19 | The man | 54 | 2011.5.3 | Find cancer cells in the hydrothorax |
| 20 | The man | 53 | 2011.5.3 | Find cancer cells in the hydrothorax |
| 21 | The man | 49 | 2011.5.17 | Bloody pleural fluid finds adenocarcinoma cell in the hydrothorax, subcutaneous one-tenth knurl, and tumor tissue is successfully built and is |
| 22 | The man | 49 | 2011.5.26 | Find gland cancer in the hydrothorax |
| 23 | The woman | 38 | 2011.6.21 | Separation and Culture is unsuccessful |
| 24 | The woman | 46 | 2011.8.24 | Separation and Culture is unsuccessful |
16 examples successfully are separated to lung carcinoma cell in the 24 routine hydrothorax, and big multipotency is cultivated and gone down to posterity 5-10 generation, and wherein 1 example is built and is.8 routine separation and Culture are unsuccessful mainly to be not have cancer cells or cell concentration few in the hydrothorax.
Part hydrothorax extracting cell is unsuccessful, and the part cell reaches natural apoptosis about 6～10 generations, all can't become knurl after most of cell subcutaneous vaccination.
Fig. 1 ~ 4 are the aspect graph after the cellular segregation.
As shown in Figure 1, the aspect graph after No. 11 the cellular segregation.
As shown in Figure 2, the aspect graph after No. 12 cellular segregation.
As shown in Figure 3, the aspect graph after No. 9 cellular segregation.
As shown in Figure 4, No. 21 are directly built with hydrothorax extracting cell is unsuccessful, but becomes knurl after collecting all cells subcutaneous vaccination, successfully builds with tumor tissue to be.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; The present invention is not restricted to the described embodiments; That describes in the foregoing description and the specification sheets just explains principle of the present invention; The present invention also has various changes and modifications under the prerequisite that does not break away from spirit and scope of the invention, and these variations and improvement all fall in the scope of the invention that requires protection.The present invention requires protection domain to be defined by appending claims and equivalent thereof.
Claims (5)
1. the method that hydrothorax is separated lung carcinoma cell is characterized in that, may further comprise the steps:
(1) preparation of different concns Percoll solution: the first mixing with 1 part of 8.5% NaCl or 1.5M PBS with 9 parts of Percoll reaches physiological osmotic pressure, is diluted to 60 ~ 70%wt, 40 ~ 50%wt with physiological solution then;
(2) preparation of hydrothorax suspension: centrifugal 5 minutes of hydrothorax sample 1500rpm abandons supernatant, and 1% PBS cleans one time, and centrifugal 5 minutes of 1500rpm abandons supernatant, and 1%PBS is resuspended; Contain various kinds of cell composition and cell tissue fragment in the hydrothorax suspension this moment;
(3) preparation of discontinuous density gradient Percoll layer:
60 ~ 70%wt, 40 ~ 50%wt, hydrothorax suspension are successively disposed to low density by high-density according to the ratio of 1:1:1, form 3 density gradients;
(4) centrifugal: the centrifugal 25-40min of 2800-3200rpm;
(5) sampling: the isolated cells of is positioned at two bed interfaces, draws the intermediate cell layer carefully, and 1% PBS cleans 1 time, and the centrifugal 5min of 1500rpm abandons supernatant, harvested cell; And
(6) cell cultures: the cell of acquisition is cultivated with the DMEM of 10%FBS is conventional.
2. method according to claim 1 is characterized in that: the physiological solution described in the step (1) is 0.85%WT NaCl or 0.15M PBS.
3. method according to claim 1 is characterized in that: the concentration of said Percoll solution is 60%wt, 40%wt.
4. method according to claim 1 is characterized in that: each gradient 3ml in the step (3), 9ml adds the 10ml centrifuge tube from the bottom to top altogether, according to how many definite how many centrifuge tubes of hydrothorax suspension.
5. method according to claim 1 is characterized in that: centrifugal for the centrifugal 30min of 3000rpm described in the step (4).
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107254446A (en) * | 2017-08-04 | 2017-10-17 | 北京世纪劲得生物技术有限公司 | A kind of method for separating and preparing of people's primary tumor cell |
| CN107988159A (en) * | 2017-12-14 | 2018-05-04 | 武汉大学深圳研究院 | A kind of method that primary tumor cell is separately cultured using malignant pleural effusion |
| CN112899228A (en) * | 2021-02-04 | 2021-06-04 | 广州医科大学附属第一医院 | Method for separating and extracting cells from bronchoalveolar lavage fluid |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1232181A (en) * | 1999-03-19 | 1999-10-20 | 孙小蓉 | Smear preparing method for selective increase of diagnosis cell quantity |
| CN101050452A (en) * | 2007-03-27 | 2007-10-10 | 江苏省人民医院 | Method for separating stem cell of breast cancer |
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2012
- 2012-05-10 CN CN2012101437721A patent/CN102676456A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1232181A (en) * | 1999-03-19 | 1999-10-20 | 孙小蓉 | Smear preparing method for selective increase of diagnosis cell quantity |
| CN101050452A (en) * | 2007-03-27 | 2007-10-10 | 江苏省人民医院 | Method for separating stem cell of breast cancer |
Non-Patent Citations (1)
| Title |
|---|
| 马吉勇: "胸腔积液端粒酶活性和催化亚单位检测的诊断价值研究", 《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107254446A (en) * | 2017-08-04 | 2017-10-17 | 北京世纪劲得生物技术有限公司 | A kind of method for separating and preparing of people's primary tumor cell |
| CN107988159A (en) * | 2017-12-14 | 2018-05-04 | 武汉大学深圳研究院 | A kind of method that primary tumor cell is separately cultured using malignant pleural effusion |
| CN112899228A (en) * | 2021-02-04 | 2021-06-04 | 广州医科大学附属第一医院 | Method for separating and extracting cells from bronchoalveolar lavage fluid |
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Application publication date: 20120919 |