CN113355278A - Culture method and application of spleen reticular fibroblast immortalized cell line of I-type interferon receptor gene knockout mouse - Google Patents
Culture method and application of spleen reticular fibroblast immortalized cell line of I-type interferon receptor gene knockout mouse Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine, in particular to a culture method and application of a mouse spleen reticular fibroblast immortalized cell line knocked out by type I interferon receptor genes, which solves the problems that although the virus infection rate of a mouse FRCs primary immortalized cell line established in the prior art is high, the virus content of a supernatant after virus infection is very low, the expression of key cytokines is not high and the like; a step-by-step sorting method of magnetic bead sorting enrichment and flow cytometry accurate sorting is applied; and finally infecting FRCs (FRCs) by using SV40 large T antigen, screening, cloning and culturing for more than 50 generations to obtain a stable cell line. The cell line of the invention has short separation and culture period, low cost and no magnetic bead enrichment process.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a culture method and application of a spleen reticular fibroblast immortalized cell line of a type I interferon receptor gene knockout mouse.
Background
Fever with thrombocytopenia syndrome (SFTS) is an important tick-borne disease found by scientists in China, and the pathogen is fever with thrombocytopenia syndrome bunyavirus (SFTSV), which is called new bunyavirus for short. The disease is widely distributed, the average fatality rate is 10% -15% in hilly areas of more than 20 provinces, and the partial areas can reach 30%, thus seriously threatening the health of human beings. The infectious disease is an acute onset disease, the main clinical manifestations are fever, the body temperature is more than 38 ℃, serious patients have persistent high fever, the temperature can reach more than 40 ℃, the heat course of some cases can reach more than 10 days, and the patients are accompanied by hypodynamia, obvious anorexia, nausea, vomiting and the like; some cases have symptoms such as headache, muscle ache, diarrhea, etc.; the body is often marked by swelling of superficial lymph nodes such as neck and groin with tenderness, tenderness in the upper abdomen and relatively slow vessels; in a few cases, the condition is critical, and consciousness disturbance, skin ecchymosis, gingival bleeding, gastrointestinal bleeding, pulmonary bleeding, multiple organ bleeding and the like appear; patients may die due to shock, respiratory failure, Disseminated Intravascular Coagulation (DIC) and other organ failure.
There is no report on the lethal mechanism of the virus, and animal model studies thereof confirm that the main target cell of the SFTSV causing lethal infection is spleen reticulocyte (FRCs); this finding was confirmed by the national NIH virus laboratory master Hideki Ebihara and japanese scientists in animal models and human autopsy studies, respectively. This important finding indicates that SFTSV infection of FRCs is closely related to death, but the specific mechanism is not reported. Therefore, the interaction relationship between the SFTSV and the FRCs of the target cells is an urgent problem to be solved in the research of the virus lethal infection.
Mouse primary FRCs cells have very low spleen content, no more than three thousandths. In the prior art, the separation cost of FRCs cells in mouse spleen is very high, the separation technology is very complex, the culture period is long, and the stability is poor; in addition, although the mouse FRCs primary immortalized cell line established in the prior art has high virus infection rate, the virus content of the supernatant after virus infection is very low, and the expression of key cell factors is not high. Based on the statement, the invention provides a culture method and application of a spleen reticular fibroblast immortalized cell line of a type I interferon receptor gene knockout mouse.
Disclosure of Invention
The invention aims to solve the problems that although the virus infection rate of a mouse FRCs primary immortalized cell line established in the prior art is high, the virus content of a supernatant after virus infection is very low, the expression of key cytokines is not high and the like, and provides a culture method and application of a mouse spleen reticular fibroblast immortalized cell line knocked out by type I interferon receptor genes.
The culture method of the spleen reticular fibroblast immortalized cell line of the I-type interferon receptor gene knockout mouse comprises the following steps:
s1, knocking out a mouse spleen by using an I-type interferon receptor, adding digestive juice to digest fragments of the mouse spleen, and releasing reticulocytes of the mouse spleen from a tightly combined matrix to obtain a digestion product I;
s2, blowing and beating the digestion product I by using a 5ml suction head for 1 minute, standing for 1 minute, sucking the supernatant of the digestion product I, adding the supernatant into 5ml of nutrient solution precooled at 4 ℃, adding 5ml of digestion solution into the precipitate of the residual digestion product I, digesting for 15 minutes at 37 ℃, obtaining a digestion product II after digestion, blowing and beating the digestion product II by using a 5ml suction head for 1 minute, standing for 1 minute, sucking the supernatant of the digestion product II, and adding the supernatant into 5ml of nutrient solution precooled at 4 ℃;
s3, centrifuging the supernatant liquid added with the nutrient solution twice, setting the centrifugation temperature at 4 ℃ and the centrifugation speed at 300g, centrifuging for 3 minutes, discarding the supernatant liquid to obtain cell precipitates, merging and re-suspending the cell precipitates with the nutrient solution, filtering, dyeing and counting the placenta blue, and then inoculating the cell precipitates to 25cm2In flask I, 5% CO at 37 ℃2After 12 hours of adherence, the culture solution is discarded, after the adherence cells are washed twice by DMEM, 6ml of nutrient solution is added at 37 ℃ and 5% CO2Culturing, wherein the nutrient solution is changed every 3-7 days in the culture process, and adherent cells are washed twice by DMEM before changing the nutrient solution every time;
s4, after the flask I was filled with the cells (after culturing for 2-3 weeks) in step S3, DMEM was used to wash the cells twice, and then the mixture was added to the flask I containing 5X 106Maintenance solution I2ml of live SV40 virus (carrying large T antigen and puromycin resistance), 37 ℃, 5% CO2Infecting for 4 hours, removing supernatant, adding maintenance fluid I6ml, and continuing culturing for 72 hours;
s5, discarding the culture medium, replacing with maintenance liquid II6ml, 37 deg.C, 5% CO2Screening infected cells, and changing the maintenance solution II once in the fourth day during the screening period;
s6, screening for 7 days, changing into nutrient solution for continuous culture, carrying out passage for three times after the cell state is stable, carrying out culture on the fourth generation of cells in a 96-well plate by using a MOFLO XDP sorting flow cytometer and CYCLONE sorting CD45-CD31-podoplanin +, wherein 100 mu l of nutrient solution is pre-added into each well, and only one cell is received;
s7, culturing for 3-6 weeks while marking the hole with single clone, changing culture medium once per week, 200 μ l nutrient solution each time, transferring the hole full of cells to one hole of 6-hole culture plate, and transferring to 25cm after the 6-hole plate is full2And (5) culturing in a culture bottle, and after seed preservation, carrying out passage for more than 50 times to obtain the stable immortalized cell line.
Preferably, the specific operation of step S1 is as follows: (1) selecting a 4-week-old I-type interferon knockout mouse, dislocating and killing cervical vertebrae, disinfecting and peeling, and taking a spleen; (2) after two washes with sterile PBS at 4 deg.CCutting spleen to 1mm in sterile penicillin bottle using sterile ophthalmic scissors3The small pieces were washed twice with 4 ℃ sterile PBS again, 5ml of the digestion solution was added, and the digestion was carried out at 37 ℃ for 15 minutes, thereby obtaining a digested product I.
Preferably, the digestion solution in the culture method is DMEM containing 0.8mg/ml metalloenzyme, 0.2mg/ml collagenase P and 0.1mg/ml DNase I.
Preferably, the nutrient solution in the culture method is DMEM containing 10% FBS and 1% double antibody.
Preferably, in the culture method, the maintenance solution I is DMEM containing 2% FBS and 1% double antibody, and the maintenance solution II is DMEM containing 2 mug/ml puromycin, 2% FBS and 1% double antibody.
Preferably, in the step S6, the passage time is once every three days, the passage mode is one-to-two or one-to-one, the specific passage operation is to digest with 0.05% trypsin for 30-120 seconds, and the original bottle is discarded.
The invention also provides application of the mouse spleen reticular fibroblast immortalized cell line knocked out by the I-type interferon receptor gene, and the immortalized cell line is applied to the research of cell identification, seed preservation and hemorrhagic fever virus infection.
Preferably, the hemorrhagic fever virus infection specifically refers to the cell line infected by using a new bunyas standard virus strain given by the hot house of the national virus institute, and the virus infection rate, the virus content in culture supernatant and the expression of a key cytokine IL-6 in infected cell species are detected.
The culture method of the I-type interferon receptor gene knockout mouse spleen reticular fibroblast immortalized cell line provided by the invention has the following beneficial effects:
1. the invention separately cultures the FRCs immortalized cell line of the I-type interferon knockout mouse on the basis of repeated optimization for many years, the virus content of the obtained cell line supernatant is increased by nearly 100 times, the expression of various inflammation related cell factors is obviously increased, the invention is more suitable for the culture and research of the virus, the separation culture period is short, the cost is low, and the invention only uses a flow cytometer to sort to a 96-pore plate without the enrichment process of magnetic beads.
2. The invention provides a research of an I-type interferon receptor gene knockout mouse spleen FRCs immortalized cell line applied to an infection mechanism and a disease treatment mechanism of a blood heat virus, and virus infection experiments show that compared with the FRCs immortalized cell line without gene knockout, the virus content of infected supernatant is improved by about 100 times and is closer to that of a normally used vero cell, the output of a key cell factor IL-6 is improved by more than 10 times, and the cell line provided by the invention provides a new platform for in vitro culture of the blood heat virus and mechanism research of patient infection.
Drawings
FIG. 1 is a flow identification chart of FRCs immortalized cell line in the culture method and application of I-type interferon receptor gene knockout mouse spleen reticulocyte immortalized cell line;
in the figure: type I interferon receptor negative, purity greater than 99%, upper left: CD45-Percp-Cy5.5 and CD31-BB515 isotropic controls; upper right: analyzing that the cells after culture and purification do not express CD45 and CD 31; the following left: normal FRCs cells express type I interferon receptor IFNAR1, FRCs after the system is established for the time do not express IFNAR 1; the following steps: the positive rate of the specific antibody podoplanin-PE antibody isotype control and the expression map is 99.9%.
FIG. 2 is a laser confocal identification chart of an FRCs immortalized cell line in the culture method and application of a mouse spleen reticular fibroblast immortalized cell line with type I interferon receptor gene knockout provided by the invention;
in the figure: from left to right: dapi (blue), anti-fibroplast (green), podoplanin (red) and Sanzhong colors are combined, and completely accord with FRCs phenotypes.
FIG. 3 is a phase contrast microscopic morphological diagram of an immortalized cell line of FRCs in the culture method and application of the spleen reticulocyte immortalized cell line of the type I interferon receptor gene knockout mouse provided by the invention.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
Example one
The invention provides a culture method of a spleen reticular fibroblast immortalized cell line of a type I interferon receptor gene knockout mouse, which comprises the following steps:
s1, selecting a 4-week-old I-type interferon knockout mouse, dislocating and killing cervical vertebrae, disinfecting and peeling, and taking a spleen; after two washes with sterile PBS at 4 deg.C, spleens were minced to 1mm in sterile penicillin bottles using sterile ophthalmic scissors3Washing the small blocks twice with 4 ℃ sterile PBS again, adding 5ml of digestive juice (DMEM containing 0.8mg/ml metalloenzyme, 0.2mg/ml collagenase P and 0.1mg/ml DNase I, the same below), digesting for 15 minutes at 37 ℃, and obtaining a digested product I after digestion;
s2, blowing and beating the digestion product I by using a 5ml suction head for 1 minute, standing for 1 minute, sucking the supernatant of the digestion product I, adding the supernatant into 5ml of nutrient solution precooled at 4 ℃, adding 5ml of digestion solution into the precipitate of the residual digestion product I, digesting for 15 minutes at 37 ℃, obtaining a digestion product II after digestion, blowing and beating the digestion product II by using a 5ml suction head for 1 minute, standing for 1 minute, sucking the supernatant of the digestion product II, and adding the supernatant into 5ml of nutrient solution precooled at 4 ℃;
s3, centrifuging the supernatant liquid added with the nutrient solution twice, setting the centrifugation temperature at 4 ℃ and the centrifugation speed at 300g, centrifuging for 3 minutes, discarding the supernatant liquid to obtain cell precipitates, merging and re-suspending the cell precipitates with the nutrient solution, filtering, dyeing and counting the placenta blue, and then inoculating the cell precipitates to 25cm2In flask I, 5% CO at 37 ℃2After 12 hours of adherence, the culture solution is discarded, after the adherence cells are washed twice by DMEM, 6ml of nutrient solution is added at 37 ℃ and 5% CO2Culturing, wherein the nutrient solution is changed every 3-7 days in the culture process, and adherent cells are washed twice by DMEM before changing the nutrient solution every time;
s4, after the flask I was filled with the cells (after culturing for 2-3 weeks) in step S3, DMEM was used to wash the cells twice, and then the mixture was added to the flask I containing 5X 106Maintenance solution I (DMEM containing 2% FBS and 1% double antibody, the same applies hereinafter) of individual SV40 virus (carrying large T antigen and puromycin resistance) 2ml, 37 ℃, 5% CO2Infecting for 4 hours, removing supernatant, adding maintenance fluid I6ml, and continuing culturing for 72 hours;
s5, discarding the medium, and changing to maintenance solution II (DMEM containing 2. mu.g/ml puromycin, 2% FBS and 1% diabody) 6ml, 37 ℃, 5% CO2For after infectionThe cells are screened, and the maintenance solution II is changed once in the fourth day of the screening period;
s6, screening for 7 days, changing into nutrient solution for continuous culture, carrying out passage three times after the cell state is stable (passage once every three days, one passage two or one passage one, digesting for 30-120 seconds by 0.05% trypsin, abandoning the original bottle), using a MOFLO XDP sorting flow cytometer for the fourth generation cell, sorting CD45-CD31-podoplanin + cells by CYCLONE into a 96-well plate for culture, adding 100 mu l of nutrient solution into each well in advance, and receiving only one cell;
s7, culturing for 3-6 weeks while marking the hole with single clone, changing culture medium once per week, 200 μ l nutrient solution each time, transferring the hole full of cells to one hole of 6-hole culture plate, and transferring to 25cm after the 6-hole plate is full2And (5) culturing in a culture bottle, and after seed preservation, carrying out passage for more than 50 times to obtain the stable immortalized cell line.
The invention also provides application of the mouse spleen reticular fibroblast immortalized cell line knocked out by the I-type interferon receptor gene, and the immortalized cell line is applied to the research of cell identification, seed preservation and hemorrhagic fever virus infection.
The hemorrhagic fever virus infection provided by the invention specifically refers to the infection of the cell line by using a new bunyas standard virus strain given by the hot house of the national virus institute, and the detection of the virus infection rate, the virus content in the culture supernatant and the expression of a key cytokine IL-6 in the infected cell species.
The virus content in the supernatant and IFNAR-FRCs in the fourth day (peak value) after infection are detected by fluorescent quantitative PCR, which are nearly hundred times higher than the virus in the supernatant of FRCs and close to VERO cells, and the specific results are shown in Table 1:
after infection for 3 days, the expression of mRNA of key cytokines such as IFNAR-FRCs expressing IL-6 is obviously increased, and the specific result is shown in a table 2:
the above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (8)
1. The culture method of the spleen reticular fibroblast immortalized cell line of the I-type interferon receptor gene knockout mouse is characterized by comprising the following steps:
s1, knocking out a mouse spleen by using an I-type interferon receptor, adding digestive juice to digest fragments of the mouse spleen, and releasing reticulocytes of the mouse spleen from a tightly combined matrix to obtain a digestion product I;
s2, blowing and beating the digestion product I by using a 5ml suction head for 1 minute, standing for 1 minute, sucking the supernatant of the digestion product I, adding the supernatant into 5ml of nutrient solution precooled at 4 ℃, adding 5ml of digestion solution into the precipitate of the residual digestion product I, digesting for 15 minutes at 37 ℃, obtaining a digestion product II after digestion, blowing and beating the digestion product II by using a 5ml suction head for 1 minute, standing for 1 minute, sucking the supernatant of the digestion product II, and adding the supernatant into 5ml of nutrient solution precooled at 4 ℃;
s3, centrifuging the supernatant liquid added with the nutrient solution twice, setting the centrifugation temperature at 4 ℃ and the centrifugation speed at 300g, centrifuging for 3 minutes, discarding the supernatant liquid to obtain cell precipitates, merging and re-suspending the cell precipitates with the nutrient solution, filtering, dyeing and counting the placenta blue, and then inoculating the cell precipitates to 25cm2In flask I, 5% CO at 37 ℃2After 12 hours of adherence, the culture solution is discarded, after the adherence cells are washed twice by DMEM, 6ml of nutrient solution is added at 37 ℃ and 5% CO2Culturing, wherein the nutrient solution is changed every 3-7 days in the culture process, and adherent cells are washed twice by DMEM before changing the nutrient solution every time;
s4, after the culture flask I was filled with the cells in the step S3, the cells were washed twice with DMEM, and then the solution containing 5X 10 cells was added6Maintenance liquid I2ml, 37 ℃ and 5% CO of live SV40 virus2Infecting for 4 hours, discarding the supernatant, adding maintenance solution I6ml, and culturing for 72 hours;
s5, discarding the culture medium, and changing to maintenance solution II6ml, 37 deg.C, 5% CO2Screening infected cells, and changing the maintenance solution II once in the fourth day during the screening period;
s6, screening for 7 days, changing into nutrient solution for continuous culture, carrying out passage for three times after the cell state is stable, using a MOFLOXDP sorting flow cytometer for the fourth generation of cells, sorting CD45-CD31-podoplanin + cells by CYCLONE into a 96-well plate for culture, adding 100 mu l of nutrient solution into each well in advance, and receiving only one cell;
s7, culturing for 3-6 weeks while marking the hole with single clone, changing culture medium once per week, 200 μ l nutrient solution each time, transferring the hole full of cells to one hole of 6-hole culture plate, and transferring to 25cm after the 6-hole plate is full2And (5) culturing in a culture bottle, and after seed preservation, carrying out passage for more than 50 times to obtain the stable immortalized cell line.
2. The method for culturing spleen reticulocyte immortalized cell line of type i interferon receptor knock-out mouse according to claim 1, wherein the step S1 is performed as follows: (1) selecting a 4-week-old I-type interferon knockout mouse, dislocating and killing cervical vertebrae, disinfecting and peeling, and taking a spleen; (2) after two washes with sterile PBS at 4 deg.C, spleens were minced to 1mm in sterile penicillin bottles using sterile ophthalmic scissors3The small pieces were washed twice with 4 ℃ sterile PBS again, 5ml of the digestion solution was added, and the digestion was carried out at 37 ℃ for 15 minutes, thereby obtaining a digested product I.
3. The method for culturing the spleen reticulocyte immortalized cell line of type I interferon receptor gene knockout mouse according to claim 1, wherein the digestion solution is DMEM containing 0.8mg/ml metalloenzyme, 0.2mg/ml collagenase P and 0.1mg/ml DNase I.
4. The method for culturing the spleen reticulocyte immortalized cell line of type I interferon receptor gene knockout mouse according to claim 1, wherein the nutrient solution in the method is DMEM containing 10% FBS and 1% double antibody.
5. The method for culturing the spleen reticulocyte immortalized cell line of type I interferon receptor gene knockout mouse according to claim 1, wherein the maintenance fluid I is DMEM containing 2% FBS and 1% double antibody, and the maintenance fluid II is DMEM containing 2 μ g/ml puromycin, 2% FBS and 1% double antibody.
6. The method for culturing the spleen reticulocyte immortalized cell line of type I interferon receptor knock-out mouse according to claim 1, wherein the passage time in step S6 is one passage every three days, the passage mode is one-to-two or one-to-one, the passage operation is digestion with 0.05% trypsin for 30-120 seconds, and the original bottle is discarded.
7. The use of the spleen reticulocyte fibroblast immortalized cell line of type i interferon receptor knock-out mouse according to any one of claims 1 to 6, wherein said immortalized cell line is used in the study of cell identification, conservation and infection with hemorrhagic fever virus.
8. The use of the spleen reticulocyte immortalized cell line of type I interferon receptor knock-out mouse according to claim 7, wherein the hemorrhagic fever virus infection is specifically characterized in that the cell line is infected by using a new bunyas standard virus strain given by the hot house of the national virus, and the virus infection rate, the virus content in the culture supernatant and the expression of the key cytokine IL-6 in the infected cell species are detected.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160250208A1 (en) * | 2013-10-11 | 2016-09-01 | University Of Massachusetts | Treating organ-specific t cell mediated autoimmune diseases |
| CN111164223A (en) * | 2017-09-28 | 2020-05-15 | 欧莱雅 | Molecular signatures of three sub-populations of dermal fibroblasts and dermal equivalents comprising one of these sub-populations |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160250208A1 (en) * | 2013-10-11 | 2016-09-01 | University Of Massachusetts | Treating organ-specific t cell mediated autoimmune diseases |
| CN111164223A (en) * | 2017-09-28 | 2020-05-15 | 欧莱雅 | Molecular signatures of three sub-populations of dermal fibroblasts and dermal equivalents comprising one of these sub-populations |
Non-Patent Citations (3)
| Title |
|---|
| CHERIE T. NG ET AL.: "Immortalized clones of fibroblastic reticular cells activate vrus-specific T cells during virus infection", 《PNAS》 * |
| CHRISTIAN PEREZ-SHIBAYAMAET AL.: "Type I interferon signaling in fibroblastic reticular cells prevents exhaustive activation of antiviral CD8+ T cells", 《SCIENCE IMMUNOLOGY》 * |
| 李嘉嘉: "SFTSV感染靶细胞系的建立及转录组测序分析", 《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》 * |
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