CN111733136B - Method for improving separation efficiency of CD90posi cells - Google Patents
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Abstract
The invention belongs to the technical field of cell engineering, and particularly relates to a method for improving separation efficiency of CD90posi cells. The method directly extracts the CD90posi cells from the liver cancer tissues, and during extraction, the cell promoter is added, so that the aggregation of the CD90posi cells is effectively promoted, the cell separation efficiency is improved, and the subsequent research is facilitated.
Description
Technical Field
The invention belongs to the technical field of cell engineering, and particularly relates to a method for improving separation efficiency of CD90posi cells.
Background
The primary liver cancer is one of the most common malignant tumors in China, has high morbidity and high mortality, is the second place of domestic tumors in the disease mortality, and seriously threatens the life health of people. The liver cancer is hidden, the clinical manifestations lack specificity, the liver cancer is usually in the middle and late stage of the liver cancer when hospitalized, patients in the early stage of the liver cancer are difficult to find, and the treatment effect of the liver cancer is seriously influenced.
The existing literature indicates that the clinical signs of primary liver cancer are extremely atypical, and the symptoms are usually much less obvious, especially in the early stage of the disease process. Usually, about 70% of small liver cancers below 5cm are asymptomatic, and about 70% of asymptomatic subclinical liver cancers are small liver cancers. Once symptoms appear, indicating that the tumor is already large, the progression of the disease is generally much more rapid, usually exhibiting cachexia within weeks, often failing to die within months to 1 year. The clinical picture is mainly the pathological changes of two aspects: the manifestations of cirrhosis, such as ascites, collateral circulation, hematemesis and edema of limbs; ② the symptoms caused by the tumor itself, such as weight loss, weakness of the whole body, pain of the liver area, enlargement of the liver, and the like.
Therefore, how to deeply research the liver cancer-related cells to obtain the best treatment means for liver cancer and avoid the harm caused by delayed treatment is a problem which is most concerned by all people at present.
In the research process, the CD90posi cells are found to be obviously related to the occurrence of liver cancer and can be used for detecting the liver cancer, but how to better and more obtain the CD90posi cells for subsequent research is a difficult point.
The existing cell separation methods mainly comprise a tissue block method, an enzymolysis tissue method, an enzyme digestion method and the like, although the cells can be separated by the methods, the separated cells are more doped with other histiocytes, so that the cell purity is low, the separation efficiency is low, and a large amount of cell waste is caused.
Disclosure of Invention
Aiming at the defects generally existing in the prior art, the invention provides a method for improving the separation efficiency of CD90posi cells. The separation method provided by the invention can directly separate the CD90posi cells from the liver cancer tissues, has high separation efficiency and simple preparation process, and is beneficial to the subsequent research on the CD90posi cells.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method of increasing the efficiency of CD90posi cell isolation comprising the steps of:
s1, taking the sterilized dishes, and adding a PBS solution containing 1.2-1.5% of double-antibody for later use;
s2, rinsing the obtained small tissue blocks with PBS, removing blood stain on the surface of the tissue, cutting off other attached tissues such as fat and redundant fibers, cutting into small blocks, placing the small blocks in the prepared dish of the step S1, and cutting into pieces of 1-2 mm by using a surgical knife3Obtaining processed crushed tissue blocks;
s3, adding a cell promoter into the processed minced tissue block prepared in the step S2, and uniformly mixing to obtain a minced tissue block containing the cell promoter and a PBS solution;
s4, placing the crushed tissue blocks containing the cell promoter and the PBS solution containing 1.2-1.5% of double antibody obtained in the step S3 into a tissue grinding homogenizer, and slowly grinding for 6-8 times by hands to prepare homogenate dispersed in cells;
and S5, screening the homogenate dispersed in the cells prepared in the step S4 by a 60-80-mesh sieve, standing the cell homogenate suspension for 5-10 min, removing the precipitate, taking the upper cell suspension, washing the upper cell suspension for 3-5 times by using a PBS (phosphate buffer solution) solution, adding the PBS solution containing 0.6-0.8% of double antibody, centrifuging, and collecting the cells.
Preferably, the dish used in the step S1 is a circular dish with a diameter of 3-6 cm.
Preferably, the PBS solution prepared in steps S1, S3, S4 and S5 is prepared from 1.9734g of disodium hydrogen phosphate and 0.2245g of monopotassium phosphate by adding water to dissolve the disodium hydrogen phosphate and the monopotassium hydrogen phosphate into 1000ml and adjusting the pH value to 7.3.
Preferably, the diabesin of steps S1, S4, S5 consists of penicillin and chloramphenicol in a mass ratio of 1: 1.
Preferably, the tissue is cut into 6-8 mm in step S23Small pieces of (a).
Preferably, the addition amount of the cell promoter in the step S3 is 0.3-0.5 g, and the weight ratio of gamma-globulin to vitamin B2 is 9-11: 2 to 5.
Preferably, the amount of the cell promoter added in step S3 is 0.4g, and the ratio of gamma-globulin to vitamin B2 is 10: 3, and (3).
Preferably, the centrifugation condition in step S5 is stirring at 2000-3000 rpm for 10-20 min.
In the research of hepatocellular carcinoma microenvironment cell groups, a team screens out CD90posi tumor cells, and finds that the CD90posi cells promote the proliferation and migration of liver cancer cells. Meanwhile, the CD90posi cell shows strong proliferation potential and cell dryness and is a key factor for proliferation and growth of hepatocellular carcinoma.
In order to further prove the function of the CD90posi cell, the invention provides a method for directly separating the CD90posi cell from the liver cancer tissue, the method takes PBS buffer solution as a solvent for separation, and in the actual separation process, the inventor also unexpectedly discovers that the cell promoter which is formed by mixing gamma-globulin and vitamin B2 according to a certain mass ratio is added into the cell promoter, the CD90posi cell aggregation can be promoted, the separation efficiency is improved, and in the process of adding the double antibody, the invention finally screens out the adding amount of the double antibody with a certain content, and the separation efficiency of the CD90posi cell is further improved.
Meanwhile, after the cell homogenate is finally prepared, the cell homogenate is kept stand for 5-10 min, so that the separation of CD90posi cells from other substances can be promoted, the improvement of the cell purity is facilitated, and the separation method provided by the invention is simple in extraction process, low in production cost and convenient to apply.
In summary, compared with the prior art, the method for improving the separation efficiency of the CD90posi cells provided by the invention has the following advantages:
(1) according to the method for improving the separation efficiency of the CD90posi cells, the cell promoter and a proper amount of double antibodies are added, so that the separation efficiency of the CD90posi cells is effectively improved;
(2) according to the method for improving the separation efficiency of the CD90posi cells, the cell homogenate is kept still for 5-10 min, so that the separation of the cells from other components is facilitated, and the purity of the cells is improved;
(3) the method for improving the separation efficiency of the CD90posi cells, provided by the invention, has the advantages that the operation process is extremely simple, too many expensive components are not required to be added, the production cost is reduced, and the subsequent research on the CD90posi cells is facilitated.
Detailed Description
The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The gamma-globulin can be purchased from shanghai shifeng biotechnology limited; the vitamin B2 can be purchased from Shanghai leaf Biotech, Inc.; the disodium hydrogen phosphate and the potassium dihydrogen phosphate can be purchased from Hunan Polystoichiometries, Inc.
Example 1A method for increasing the efficiency of CD90posi cell separation
The method for improving the separation efficiency of the CD90posi cells comprises the following steps:
s1, taking a sterilized round dish with the diameter of 3cm, and adding a PBS solution containing 1.2% of double antibody (consisting of penicillin and chloramphenicol in a mass ratio of 1: 1) for later use; the PBS solution is prepared by adding water into 1.9734g of disodium hydrogen phosphate and 0.2245g of monopotassium phosphate to dissolve the disodium hydrogen phosphate and the monopotassium hydrogen phosphate into 1000mL of solution, and adjusting the pH value to 7.3;
s2, rinsing the small tissue with PBS solution, removing blood stain on the tissue surface, cutting off other tissues such as fat and redundant fiber, and cutting into 6mm pieces3The small blocks are put into the prepared dish in the step S1 and cut into 1mm by a scalpel3Obtaining processed crushed tissue blocks;
s3, adding 0.3g of cell promoter into the processed minced tissue block prepared in the step S2, and uniformly mixing to obtain a minced tissue block containing the cell promoter and a PBS solution; the cell promoter is prepared from gamma-globulin and vitamin B2 according to the mass ratio of 9: 2, preparing a composition;
s4, placing the crushed tissue blocks containing the cell promoter and the PBS solution containing 1.2 percent of double antibody (consisting of penicillin and chloramphenicol in a mass ratio of 1: 1) obtained in the step S3 into a tissue grinding homogenizer, and slowly grinding the crushed tissue blocks for 6 times by hands to prepare homogenate dispersed in cells;
s5, screening the homogenate dispersed in the cells prepared in the step S4 by a 60-mesh sieve, standing the cell homogenate suspension for 5min, removing precipitates, taking the upper cell suspension, washing the upper cell suspension for 3 times by using a PBS solution, adding the PBS solution containing 0.6% of double antibody (consisting of penicillin and chloramphenicol in a mass ratio of 1: 1), centrifuging the mixture at a rotating speed of 2000rpm for 10min, and collecting the cells to obtain the cell suspension.
Example 2A method for increasing the efficiency of CD90posi cell separation
The method for improving the separation efficiency of the CD90posi cells comprises the following steps:
s1, taking a sterilized circular dish with the diameter of 6cm, and adding a PBS solution containing 1.5% of double antibody (consisting of penicillin and chloramphenicol in a mass ratio of 1: 1) for later use; the preparation process of the PBS solution is the same as the example;
s2, rinsing the small tissue with PBS solution, removing blood stain on the tissue surface, cutting off other tissues such as fat and redundant fiber, and cutting into pieces of 8mm3The small blocks are put into the prepared dish in the step S1 and cut into 2mm by a scalpel3Obtaining processed crushed tissue blocks;
s3, adding 0.5g of cell promoter into the processed minced tissue block prepared in the step S2, and uniformly mixing to obtain a minced tissue block containing the cell promoter and a PBS solution; the cell promoter is prepared from gamma-globulin and vitamin B2 in a mass ratio of 11: 5, preparing a composition;
s4, placing the crushed tissue blocks containing the cell promoter and the PBS solution containing 1.5 percent of double antibody (consisting of penicillin and chloramphenicol in a mass ratio of 1: 1) obtained in the step S3 into a tissue grinding homogenizer, and slowly grinding the crushed tissue blocks by hands for 8 times to prepare homogenate dispersed in cells;
s5, screening the homogenate of the cells scattered in the step S4 by a 80-mesh sieve, standing the cell homogenate suspension for 10min, discarding the precipitate, taking the upper cell suspension, washing the upper cell suspension for 5 times by using a PBS solution, adding the PBS solution containing 0.8% of double antibody (consisting of penicillin and chloramphenicol in a mass ratio of 1: 1), centrifuging the mixture at the rotating speed of 3000rpm for 20min, and collecting the cells to obtain the cell suspension.
Example 3A method for increasing the efficiency of CD90posi cell separation
The method for improving the separation efficiency of the CD90posi cells comprises the following steps:
s1, taking a sterilized circular dish with the diameter of 5cm, and adding a PBS solution containing 1.3% of double antibody (consisting of penicillin and chloramphenicol in a mass ratio of 1: 1) for later use; the PBS solution was prepared in the same manner as in example 1;
s2, rinsing the small tissue with PBS, removing blood stain on the surface of the tissue, cutting off other tissues such as fat and redundant fiber, and cutting into 7mm pieces3The small blocks are put into the prepared dish in the step S1 and cut into 1-2 mm by a scalpel3Obtaining processed crushed tissue blocks;
s3, adding 0.4g of cell promoter into the processed crushed tissue block prepared in the step S2, and uniformly mixing to obtain a crushed tissue block containing the cell promoter and a PBS solution; the cell promoter is prepared from gamma-globulin and vitamin B2 in a mass ratio of 10: 3, preparing a composition;
s4, placing the crushed tissue blocks containing the cell promoter and the PBS solution containing 1.3 percent of double antibody (consisting of penicillin and chloramphenicol in a mass ratio of 1: 1) obtained in the step S3 into a tissue grinding homogenizer, and slowly grinding the crushed tissue blocks for 7 times by hands to prepare homogenate dispersed in cells;
s5, screening the homogenate dispersed in the cells prepared in the step S4 by a 70-mesh sieve, standing the cell homogenate suspension for 8min, discarding the precipitate, taking the upper cell suspension, washing the upper cell suspension for 4 times by using a PBS solution, adding the PBS solution containing 0.7% of double antibody (consisting of penicillin and chloramphenicol in a mass ratio of 1: 1), centrifuging the mixture for 15min at the rotating speed of 2500rpm, and collecting the cells to obtain the cell suspension.
Comparative example 1 separation method of CD90posi cell
The CD90posi cells were isolated in a similar manner to example 3;
the difference from example 3 is that the cell promoter in comparative example 1 is prepared from gamma-globulin and vitamin B2 in a mass ratio of 1: 1.
Comparative example 2 separation method of CD90posi cell
The CD90posi cells were isolated in a similar manner to example 3;
the difference from example 3 is that comparative example 2 does not contain a cell promoter.
Comparative example 3 separation method of CD90posi cell
The CD90posi cells were isolated in a similar manner to example 3;
the difference from example 3 is that the double antibody content in comparative example 3 is 1%.
Comparative example 4 separation method of CD90posi cell
The CD90posi cells were isolated in a similar manner to example 3;
the difference from example 3 is that comparative example 3 removed the process of leaving the cell homogenate to stand for 8min in step S5.
Comparative example 5 separation method of CD90posi cell
The CD90posi cells were isolated in a similar manner to example 3;
the difference from example 3 is that comparative example 4 was left standing for 1min on the cell homogenate in step S5.
Test example 1 measurement of cell separation efficiency
1. Test samples: the upper cell suspensions obtained in step S5 of examples 1 to 3 and comparative examples 1 to 5;
2. the test method comprises the following steps: adding goat anti-rabbit IgG antibody labeled by horseradish peroxidase as fluorescent antibody into the test sample for specific screening of CD90posi cell, placing at 37 deg.C, saturation humidity, and 5% CO2And (3) incubating in an incubator for 30min, discarding the supernatant, washing for 2 times by using PBS solution, adding a small amount of PBS buffer solution, shaking up, flaking, blowing dry, observing cell morphology under a fluorescence microscope, and counting the number of cells.
Cell separation efficiency (number of fluorescent cells/total number of cells) × 100%
3. And (3) test results: the specific test results are shown in Table 2.
TABLE 2 comparison of cell separation efficiency of different test samples
| Test sample | Total number of cells/cell | Number of fluorescent cells/ | Cell separation efficiency/%) |
| Example 1 | 1.6×105 | 1.48×105 | 92.5％ |
| Example 2 | 1.52×105 | 1.42×105 | 93.4％ |
| Example 3 | 1.63×105 | 1.55×105 | 95.1％ |
| Comparative example 1 | 1.55×105 | 1.11×105 | 71.6％ |
| Comparative example 2 | 1.65×105 | 1.04×105 | 63.1％ |
| Comparative example 3 | 1.6×105 | 1.23×105 | 75.6％ |
| Comparative example 4 | 1.68×105 | 1.11×105 | 65.7％ |
| Comparative example 5 | 1.63×105 | 1.15×105 | 70.5％ |
As shown in Table 2, the cell separation efficiency of the cells obtained by the methods described in examples 1-3 of the present invention is as high as 90% or more, especially, the cell separation efficiency of example 3 group is 95.1%, so example 3 is the best example of the present invention; correspondingly, the groups of comparative examples 1 to 3 have significantly reduced cell separation efficiency due to the change in the content of the cell-promoting agent or the removal of the cell-promoting agent, and the change in the content of the diabase, which laterally confirms the synergistic interaction between the components of the present application; in contrast, the groups of comparative examples 4 to 5 changed the standing time of the cell homogenate, and also significantly reduced the cell separation efficiency, and reduced the purity of the CD90posi cells, which reflected the importance of the selection of the standing time of the present invention.
Finally, it should be noted that the above-mentioned embodiments are merely illustrative of the principles of the present invention and its efficacy, and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
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