CN113151358B - Construction method and application of ZNF382 stably transfected diffuse large B cell lymphoma cell strain - Google Patents
Construction method and application of ZNF382 stably transfected diffuse large B cell lymphoma cell strain Download PDFInfo
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Abstract
The invention relates to the biomedical field, and discloses a construction method of a diffuse large B cell lymphoma cell strain stably transfected by ZNF382, which comprises slow virus packaging, virus concentration and slow virus infection of DLBCL cells; also disclosed are uses of the constructed cell lines. The cell strain constructed by the invention is helpful for widening the understanding of the pathogenesis of DLBCL, and provides a more stable and reliable experimental material for clinical application of demethylated drugs to DLBCL and clinical treatment of DLBCL.
Description
Technical Field
The invention relates to the biomedical field, in particular to a construction method and application of a diffuse large B cell lymphoma cell strain stably transfected by ZNF382.
Background
Diffuse large B-cell lymphoma (DLBCL) is a group of highly heterogeneous and invasive malignant tumors, the most common type of lymphoma. With the application of anti-CD 20 monoclonal antibody and the development of hematopoietic stem cell transplantation and CAR-T, the survival outcome of DLBCL patients is improved remarkably, but the treatment of the DLBCL patients still faces a great challenge due to the heterogeneity of the DLBCL and the problems of drug resistance, relapse and the like of the patients. Therefore, the intensive study of the pathogenesis of DLBCL and the search of new targets with therapeutic potential have important clinical practical significance. In recent years, a plurality of researches indicate that gene abnormality and biological change are important initial factors of DLBCL occurrence and evolution, and the identification of new abnormal methylation genes possibly has better understanding on the occurrence of DLBCL, thereby being beneficial to providing new theoretical basis and treatment targets for the treatment of DLBCL. The KRAB zinc finger proteins (KRAB-ZFPs) are the most common transcription factors in the body, which are highly conserved during evolution. Different KRAB-ZFP play different biological roles in tumors, and more researches indicate that various KRAB-ZFP have abnormal methylation phenomena in malignant tumors and participate in the occurrence and development processes of the malignant tumors. Therefore, the DNA methylation regulation and the transcriptional regulation molecular mechanism of the KRAB-ZFP gene are revealed to have important significance for early detection of canceration, judgment of clinical prognosis and the like. ZNF382 is one of members of the KRAB-ZFP protein family, located at 19p13.12, and the literature reports that there is abnormal methylation or down-regulation of expression of a promoter region in both leukemia and gastric cancer, which may be a potential cancer suppressor gene, but the specific mechanism has not yet been elucidated. At present, there are few reports on ZNF382 on other hematological malignant tumors, and the biological functions and molecular action mechanisms of the ZNF382 remain to be elucidated.
In order to discuss apparent regulation and biological functions of ZNF382 in DLBCL, it is necessary to detect expression of ZNF382 in DLBCL bone marrow tissue and cell lines thereof, and detect methylation status of ZNF382 in DLBCL bone marrow tissue and cell lines thereof by MSP, demethylating drug treatment and the like; the diffuse large B cell lymphoma cell strain stably transfected by ZNF382 needs to be constructed, and the influence of ZNF382 on the biological functions of DLBCL cells is discussed through a CCK-8 cell proliferation experiment, a Transwell migration experiment and the like so as to promote the clinical treatment research of DLBCL.
Disclosure of Invention
Based on the problems, the invention provides a construction method and application of a ZNF382 stably transfected diffuse large B cell lymphoma cell strain, and the constructed cell strain is helpful for widening the understanding of DLBCL pathogenesis, and provides a more stable and reliable experimental material for clinical application of demethylated drugs in DLBCL and clinical treatment of DLBCL.
In order to solve the technical problems, the invention provides a construction method of a diffuse large B cell lymphoma cell strain stably transfected by ZNF382, which comprises the following steps:
s1: lentivirus packaging and virus concentration
Culturing to obtain 25ml of 293T cells to be transfected, mixing ZNF382 slow virus over-expression plasmid containing ZNF382 gene sequence and auxiliary plasmid, mixing with 150 mu L of transfection reagent, standing at room temperature for 12min, co-transfecting 293T cells, transferring liquid after transfection for 6-8h, collecting culture medium supernatants containing viruses respectively at 24h, 48h and 72h, mixing the culture medium supernatants containing viruses collected for 3 times, centrifuging at 4 ℃ for 10min at 2000r/rnin to obtain supernatant, and precipitating viruses by a polyethylene glycol concentration method;
s2: lentivirus infection of DLBCL cells
Detecting the lentiviral titer of the ZNF382 overexpression group in the step S1, wherein the viral titer of the ZNF382 overexpression group is 5 multiplied by 10 8 TU/ml; when DLBCL cell strain is in logarithmic growth phase, adding concentrated virus solution with MOI value of 100, and making the final concentration of ploybrene be 8 mug/ml, after 12h of infection, changing the culture medium into fresh culture medium, after 48h of infection, changing the culture medium into culture medium containing 1 mug/ml puromycin condition to make screening so as to obtain positive infected cell, and finally obtaining ZNF382 stably transfected diffuse large B cell lymphoma cell strain.
Furthermore, the ZNF382 lentivirus over-expression plasmid containing the ZNF382 gene sequence is pCDH-CMV-ZNF382-GFP, and the addition amount is 21 mug; helper plasmids included 17.5. Mu.g pSPAX2 and 7. Mu.g pMD2.G.
Further, the specific method for precipitating viruses by polyethylene glycol concentration in the step S1 is as follows:
A. filtering the supernatant by using a filter membrane made of PES material with the thickness of 0.45 μm, and preparing a 5X PEG8000+NaCl mother liquor;
B. adding 5 XPEG-8000+NaCl mother liquor 7.5ml into every 30ml filtered virus initial liquor, mixing on ice for 5h, mixing once every 30min, centrifuging at 4deg.C for 2h at 4000g, absorbing supernatant, centrifuging at 4deg.C for 5min at 4000g, absorbing residual liquid, adding appropriate amount of lentivirus dissolving solution to dissolve lentivirus precipitate, packaging concentrated virus suspension, quick freezing, and storing at-80deg.C.
Further, the preparation method of the 5X PEG8000+NaCl mother liquor is as follows: weighing 8.766g of NaCl and 800050g of PEG, dissolving in 200ml of Milli-Q pure water, sterilizing at 121deg.C for 30min under moist heat, and preserving at 4deg.C.
Further, DLBCL cells in step S2 include OCI-LY10 cells and U2932 cells.
In order to solve the technical problems, the invention also provides application of the cell strain constructed by the construction method in preparation of products for researching or treating DLBCL.
Compared with the prior art, the invention has the beneficial effects that: the cell strain constructed by the invention is helpful for widening the understanding of the pathogenesis of DLBCL, and provides a more stable and reliable experimental material for clinical application of demethylated drugs to DLBCL and clinical treatment of DLBCL.
Drawings
FIG. 1 is a graph showing the correlation between the low expression of ZNF382 in a DLBCL bone marrow tissue sample and its cell lines and the abnormal hypermethylation of its promoter region;
FIG. 2 is a graph of the results of DLBCL cells stably overexpressing ZNF382 in accordance with an embodiment of the present invention;
FIG. 3 is a graph showing the effect of ZNF382 on the biological function of DLBCL cells according to the example of the present invention.
Detailed Description
For the purpose of making apparent the objects, technical solutions and advantages of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, wherein the exemplary embodiments of the present invention and the descriptions thereof are for illustrating the present invention only and are not to be construed as limiting the present invention.
Examples:
this example collected 30 cases of DLBCL bone marrow tissue specimens and 10 cases of healthy volunteer specimens, and mononuclear cells were isolated using lymphocyte separation liquid Ficoll (Invitrogen, usa). The DLBCL cell lines OCI-LY10, U2932 were selected for subsequent experiments.
The experimental materials used were as follows: total RNA was extracted using Trizol extraction kit (Japanese Takara Co., ltd.), and reverse transcription was performed using M-MLV reverse transcriptase kit (Japanese Takara Co., ltd.) to obtain cDNA; detecting mRNA expression of ZNF382 in DLBCL bone marrow tissue specimens, DLBCL cell lines (OCI-LY 10, U2932) and normal bone marrow cells by using Real-time PCR and RT-PCR experiments; DNA of DLBCL bone marrow tissue specimens, DLBCL cell lines (OCI-LY 10, U2932) and normal bone marrow cells were extracted using a DNA extraction kit (Biyun of China), bisulphite modified using EZ DNAmethylation-gold kit (ZYMO research Co., U.S.A.), ZNF382 MSP primers were designed, and the promoter methylation level of ZNF382 in DLBCL cells was detected using MSP.
This example first examined the effect of the demethylated drug DAC on ZNF382 mRNA expression levels: DLBCL cells (OCI-LY 10, U2932) were adjusted to 5X 10 5 The cells were plated in 6-well plates at a final concentration of 5. Mu. Mol/LDAC and 10. Mu. Mol/LDAC, and the drug concentration was maintained by daily pipetting. After 72h, cells were collected, RNA was extracted, and the expression level of ZNF382 mRNA was detected by RT-PCR.
The lentiviral packaging plasmid systems (pCDH-CMV-GFP, pSPAX2, pMD2. G) used in this example were purchased from Aidekang organisms and ZNF382 overexpressing lentiviral vectors (pCDH-CMV-ZNF 382-GFP) were constructed by Shanghai Biotechnology Co., ltd. According to the ZNF382CDS sequence (GCTAGCATGTCTCAGGGATCAGTGTCATTCAAGGATGTGACTGTGGACTTCACCCAGGAGGAGTGGCAGCAACTAGACCCTGCTCAGAAGGCGCTTTACAGGGATGTGATGTTGGAAAACTATTGCCACTTCGTATCTGTGGGGTTTCACATGGCTAAGCCTGATATGATCCGCAAGTTGGAACAAGGAGAAGAGCTATGGACACAGAGAATTTTTCCAAGTTACAGCTACCTAGAAGAAGATGGGAAAACTGAAGATGTCTTAGTGAAGTTCAAAGAATACCAAGACAGGCATTCTAGACCCCTCATATTCATCAACCACAAAAAACTAATTAAGGAGAGAAGTAATATTTATGGTAAAACATTTACTCTAGGCAAGAACCGTATTTCAAAAACAATACTATGTGAATATAAACCTGATGGAAAAGTTTTGAAAAATATTTCAGAACTAGTCATTAGAAATATAAGCCCCATAAAAGAGAAGTTTGGTGACAGTACTGGATGGGAGAAATCACTCCTCAATACCAAGCATGAGAAAATTCATCCTGCAGTGAATCTCCATAAACAAACAGAAAGAGTTCTCAGTGGTAAACAGGAGCTTATTCAGCATCAGAAGGTTCAAGCTCCAGAGCAACCATTTGACCATAATGAATGTGAAAAATCCTTCCTGATGAAAGGAATGCTATTTACACATACTAGAGCTCACAGAGGAGAAAGAACCTTTGAATACAATAAAGATGGAATTGCCTTCATAGAAAAGTCAAGCCTCAGTGTCCATCCAAGTAATCTTATGGAAAAGAAGCCCTCTGCCTACAACAAATATGGGAAATTCCTCTGCAGAAAGCCTGTTTTTATTATGCCTCAGAGACCTCAAACAGAAGAGAAACCCTTTCACTGTCCTTACTGTGGGAATAACTTTAGAAGGAAGTCATACCTCATTGAACATCAGCGAATTCACACAGGTGAAAAACCTTATGTTTGCAATCAATGTGGAAAGGCCTTCCGTCAGAAGACAGCCCTCACCCTTCATGAGAAAACACATATAGAGGGGAAACCCTTTATTTGTATCGATTGTGGGAAGTCCTTCCGCCAGAAGGCCACCCTCACTAGACATCACAAAACACATACGGGGGAGAAAGCCTATGAATGTCCTCAGTGTGGAAGTGCCTTTAGGAAGAAGTCATACCTCATTGATCACCAGAGAACTCACACAGGAGAGAAACCGTATCAGTGTAATGAGTGTGGGAAGGCATTTATCCAGAAGACAACCCTCACTGTTCATCAGAGAACTCACACAGGAGAGAAACCCTATATTTGCAATGAATGTGGGAAGTCCTTCTGCCAAAAGACAACCCTCACTCTCCACCAGAGAATTCACACGGGGGAAAAACCCTATATTTGTAATGAATGTGGGAAGTCCTTCCGCCAGAAGGCAATCCTCACTGTTCATCACAGAATACATACAGGAGAAAAATCCAATGGGTGTCCTCAGTGTGGGAAAGCCTTCAGTAGGAAATCAAACCTCATTCGCCATCAGAAAACTCACACAGGCGAGAAACCATATGAATGTAAACAGTGTGGGAAGTTCTTCAGTTGTAAGTCAAACCTCATTGTCCATCAGAAAACTCACAAGGTAGAAACCACGGGAATTCAGTAAGGATCC) supplied by us.
The construction conditions of diffuse large B-cell lymphoma cell lines stably transfected with ZNF382 were studied as follows:
s1: lentivirus packaging and virus concentration
293T cells with good growth state are inoculated into a cell culture dish with the diameter of 15cm, the final volume of the cell culture dish is 25mL, and the cell culture dish is placed at 37 ℃ and contains 5% CO by volume 2 Culturing in an incubator, and transfecting when the cells grow to about 70% of fusion degree; the 293T cells are randomly divided into a blank control group, an empty plasmid transfection group and a ZNF382 transfection group; the blank control cells were treated with only liquid change, empty plasmid transfected cells were added with empty lentiviral expression plasmid (21. Mu.g pCDH-CMV-GFP) and helper plasmid (17.5. Mu.g pSPAX2, 7.0. Mu.g pMD2. G), ZNF382 transfected cells were added with ZNF382 lentiviral over-expression plasmid (21. Mu.g pCDH-CMV-ZNF 382-GFP) and helper plasmid (17.5. Mu.g pSPAX2, 7.0. Mu.g pMD2. G) containing the gene sequence of ZNF382,mixing the plasmid mixture with 150 μl LVTransm transfection reagent (Aidekang organism in China) respectively, standing at room temperature for 12min, and co-transfecting 293T cells; transferring the liquid after transfection for 6-8 hours, collecting culture medium supernatants containing viruses respectively at 24 hours, 48 hours and 72 hours, mixing the culture medium supernatants containing viruses for 3 times, centrifuging at 4 ℃ for 10 minutes at 2000r/rnin, precipitating part of cell fragments, obtaining the supernatant, and precipitating the viruses by a polyethylene glycol (PEG) concentration method, wherein the specific method is as follows, (1) filtering the supernatant by a filter membrane made of a PES material of 0.45 μm; (2) 5X PEG8000+NaCl, weighing 8.766g NaCl and 800050g PEG, dissolving in 200ml Milli-Q pure water, sterilizing at 121deg.C for 30min under moist heat, and storing at 4deg.C for use; (3) Every 30ml of filtered virus initial liquid is added with 7.5ml of 5 XPEG-8000+NaCl mother liquid; (4) uniformly mixing on ice for 5 hours, and mixing every 30 minutes; (5) 4 ℃,4000g, centrifuging for 2h; (6) The supernatant was removed, centrifuged at 4℃and 4000g for 5min to remove residual liquid; (7) Adding proper amount of slow virus dissolving solution to dissolve slow virus precipitate; (8) Split charging the concentrated virus suspension into 50 μl each, storing in a finished tube, quick-freezing with crushed dry ice, and storing at-80deg.C;
s2: lentivirus titer detection in empty-load group and ZNF382 over-expression group
(1) 293T cells with good growth state were plated in 96-well plates at a concentration of 5X 10 3 Volume 100 μl per well; placed at 37 ℃ and 5% CO 2 Incubator, overnight for standby; (2) Preparing 8 clean EP tubes for each of a gradient dilution virus, an empty load group and a ZNF382 over-expression group, adding 90 mu l of serum-free culture medium, uniformly mixing 10 mu l of virus concentrate with the culture medium of 1 tube, sucking 10 mu l of the tube, adding another tube, blowing and uniformly mixing, and continuing the same operation until the final tube is 1; (3) discarding the volume of 90. Mu.l, adding 90. Mu.l of the virus solution; at 37 ℃,5% CO 2 Incubator overnight; (4) After 24h, 100 μl of complete medium was added and the procedure was gentle; after 48h, change Kong Zhongxin fresh 150 μl complete medium; after 96h, observation is performed under a fluorescence inverted microscope; (5) determination of viral titer; the number of cells with GFP positive was counted divided by the fold dilution. The viral titers of both the empty and ZNF382 overexpressing groups we obtained were 5×10 8 TU/ml；
S3: pre-experiment for lentivirus infection
(1) OCI-LY10 and U2932 cells were well grown, cultured in 96-well plates, and adjusted to a concentration of 5X 10 in a volume of 90. Mu.l 3 A/hole; (2) Preparing virus solutions with complex infection values (multiplicity of infection, MOI) of 1, 10, 50 and 100 respectively, and adding 10 μl of each group of viruses into the wells; (3) Adding 10 mu l polybrene diluent into a hole which is set to be added with polybrene, wherein the final concentration is 8 mu g/ml; (4) Gently shaking to mix the viruses thoroughly, and placing at 37deg.C with 5% CO 2 An incubator; (5) after 12h, discarding the supernatant; replacement with fresh medium; (6) After 3-4d, cells were observed under a fluorescent inverted microscope: (7) And judging the infection condition and parameters according to the cell transfection effect.
Experimental results show that OCI-LY10 cells and U2932 cells are not easy to infect, and only part of cells can be infected when the MOI value is 100, and in order to improve the infection efficiency, polybrene with the concentration of 8 mug/mL needs to be added in subsequent experiments to increase the infection effect. Lentiviruses of the empty and ZNF382 overexpressing groups described above infect DLBCL cells (OCI-LY 10, U2932): according to the result of the pre-infection test, when DLBCL cell lines (OCI-LY 10 and U2932) are in the logarithmic phase, adding corresponding concentrated virus liquid according to MOI value, enabling the final concentration of ploybrene to be 8 mug/ml, changing the culture medium to a fresh culture medium after 12 hours of infection, changing to a conditioned medium containing 1 mug/ml puromycin (U.S. Selleck Chemicals company) after 48 hours of infection, and finally obtaining positive cells.
The present example also performed the following in vitro experiments: (1) proliferation assay: taking DLBCL cells (OCI-LY 10, U2932) stably over-expressing ZNF382 and DLBCL cells (OCI-LY 10, U2932) of control group at 3×10 per well 4 Inoculating to 96-well plate with final volume of 100 μl, arranging 3 compound wells in each group, culturing for 48 hr, adding 10 μl of CCK-8 reagent (Biyundian Co., china) in dark place in each well, incubating for 2-4 hr, measuring absorbance value of each well with enzyme-labeled instrument under the condition of wavelength of 450nm, and calculating cell proliferation inhibition rate; (2) Transwell assay to detect cell migration ability: cells of OCI-LY10 and U2932 stably overexpressing ZNF382 in the logarithmic growth phase were selected, centrifuged at 900rpm, resuspended in serum-free 1640 medium, counted and cell concentration adjusted to 2.5×10 5 Individual/ml; 600ul of 1640 medium containing 15% fetal bovine serum was added to the bottom of the 24-well plate, i.e., the lower chamber; 200ul (5X 10) of cell suspension was added to the Transwell chamber 4 Individual cells); placing in a carbon dioxide incubator at 37 ℃ for culturing for 96 hours; taking out the cell, directly observing the cells passing through the lower cell under a microscope, randomly selecting a plurality of visual fields, photographing, counting and analyzing the result.
FIG. 2 is a graph showing the results of DLBCL cells stably overexpressing ZNF382, wherein (A) the fluorescence of lentiviral infected OCI-LY10 and U2932 cells is shown (200X); (B) The purity of OCI-LY10 and U2932 stable transformants was examined by flow cytometry. The result shows that OCI-LY10 cells and U2932 cells which stably and over express ZNF382 are obtained; FIG. 3 is a graph showing the effect of ZNF382 on the biological function of DLBCL cells, wherein (A) CCK-8 detects the effect of over-expressed ZNF382 on proliferation of OCI-LY10 and U2932 cells; (B) Transwell experiments detect the effect of over-expression of ZNF382 on the ability of OCI-LY10 and U2932 cells to migrate; experimental results show that the over-expression of ZNF382 can inhibit the proliferation and migration of U266 cells, and also show that the cell strain constructed in the embodiment is constructed successfully.
See FIG. 1, wherein (A) RT-PCR detects the expression level of ZNF382 in DLBCL; (B) MSP detects the methylation status of ZNF382 in DLBCL; (C) RT-PCR (reverse transcription-polymerase chain reaction) detection of influence of a demethylated drug DAC on expression level of ZNF382 mRNA in DLBCL cell strain OCI-LY 10; (D) The effect of the demethylated drug DAC on the expression level of ZNF382 mRNA in DLBCL cell line U2932 was examined by RT-PCR. Compared with a normal bone marrow tissue sample, the experimental result shows that the mRNA expression level of ZNF382 in the DLBCL bone marrow tissue sample and cell lines OCI-LY10 and U2932 thereof is obviously reduced as shown in the attached figure 1A. As shown in FIG. 1B, the methylation specific polymerase chain reaction (Methylation specific PCR, MSP) was used to detect that the DLBCL cell lines OCI-LY10 and U2932 have high methylation in the ZNF382 promoter region. Referring to fig. 1C and 1D, ZNF382 was partially restored using the demethylated drug Decitabine (DAC). Thus, the abnormal hypermethylation of the ZNF382 gene promoter region influences the expression of the ZNF382 gene in DLBCL cells, but the influence of the ZNF382 on the biological functions of the DLBCL cells is needed to be further studied. The above results also show that the ZNF382 constructed in the example is successfully constructed by stably transfected diffuse large B cell lymphoma cell lines and has value as a research material.
The above is an embodiment of the present invention. The foregoing embodiments and the specific parameters of the embodiments are only for clarity of description of the invention and are not intended to limit the scope of the invention, which is defined by the appended claims, and all equivalent structural changes made in the description and drawings of the invention are intended to be included in the scope of the invention.
SEQUENCE LISTING
<110> Luoyang City center Hospital (Zhengzhou university affiliated Luoyang center Hospital)
<120> construction method and application of diffuse large B cell lymphoma cell strain stably transfected by ZNF382
<130> 202011635086.7
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1662
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
gctagcatgt ctcagggatc agtgtcattc aaggatgtga ctgtggactt cacccaggag 60
gagtggcagc aactagaccc tgctcagaag gcgctttaca gggatgtgat gttggaaaac 120
tattgccact tcgtatctgt ggggtttcac atggctaagc ctgatatgat ccgcaagttg 180
gaacaaggag aagagctatg gacacagaga atttttccaa gttacagcta cctagaagaa 240
gatgggaaaa ctgaagatgt cttagtgaag ttcaaagaat accaagacag gcattctaga 300
cccctcatat tcatcaacca caaaaaacta attaaggaga gaagtaatat ttatggtaaa 360
acatttactc taggcaagaa ccgtatttca aaaacaatac tatgtgaata taaacctgat 420
ggaaaagttt tgaaaaatat ttcagaacta gtcattagaa atataagccc cataaaagag 480
aagtttggtg acagtactgg atgggagaaa tcactcctca ataccaagca tgagaaaatt 540
catcctgcag tgaatctcca taaacaaaca gaaagagttc tcagtggtaa acaggagctt 600
attcagcatc agaaggttca agctccagag caaccatttg accataatga atgtgaaaaa 660
tccttcctga tgaaaggaat gctatttaca catactagag ctcacagagg agaaagaacc 720
tttgaataca ataaagatgg aattgccttc atagaaaagt caagcctcag tgtccatcca 780
agtaatctta tggaaaagaa gccctctgcc tacaacaaat atgggaaatt cctctgcaga 840
aagcctgttt ttattatgcc tcagagacct caaacagaag agaaaccctt tcactgtcct 900
tactgtggga ataactttag aaggaagtca tacctcattg aacatcagcg aattcacaca 960
ggtgaaaaac cttatgtttg caatcaatgt ggaaaggcct tccgtcagaa gacagccctc 1020
acccttcatg agaaaacaca tatagagggg aaacccttta tttgtatcga ttgtgggaag 1080
tccttccgcc agaaggccac cctcactaga catcacaaaa cacatacggg ggagaaagcc 1140
tatgaatgtc ctcagtgtgg aagtgccttt aggaagaagt catacctcat tgatcaccag 1200
agaactcaca caggagagaa accgtatcag tgtaatgagt gtgggaaggc atttatccag 1260
aagacaaccc tcactgttca tcagagaact cacacaggag agaaacccta tatttgcaat 1320
gaatgtggga agtccttctg ccaaaagaca accctcactc tccaccagag aattcacacg 1380
ggggaaaaac cctatatttg taatgaatgt gggaagtcct tccgccagaa ggcaatcctc 1440
actgttcatc acagaataca tacaggagaa aaatccaatg ggtgtcctca gtgtgggaaa 1500
gccttcagta ggaaatcaaa cctcattcgc catcagaaaa ctcacacagg cgagaaacca 1560
tatgaatgta aacagtgtgg gaagttcttc agttgtaagt caaacctcat tgtccatcag 1620
aaaactcaca aggtagaaac cacgggaatt cagtaaggat cc 1662
Claims (5)
1. The construction method of the diffuse large B cell lymphoma cell strain stably transfected with ZNF382 is characterized by comprising the following steps:
s1: lentivirus packaging and virus concentration
Culturing to obtain 25ml of 293T cells to be transfected for later use;
mixing ZNF382 lentivirus over-expression plasmid containing ZNF382 gene sequence with auxiliary plasmid, mixing with 150 mu L transfection reagent, standing at room temperature for 12min, and co-transfecting 293T cells;
transferring the liquid after transfection for 6-8h, collecting culture medium supernatants containing virus at 24h, 48h and 72h respectively, mixing the culture medium supernatants containing virus collected for 3 times, centrifuging at 4deg.C for 10min at 2000r/rnin to obtain supernatant, and precipitating virus by polyethylene glycol concentration method;
the ZNF382 gene sequence refers to the CDS sequence of ZNF382, and the sequence is as follows:
GCTAGCATGTCTCAGGGATCAGTGTCATTCAAGGATGTGACTGTGGACTTCACCCAGGAGGAGTGGCAGCAACTAGACCCTGCTCAGAAGGCGCTTTACAGGGATGTGATGTTGGAAAACTATTGCCACTTCGTATCTGTGGGGTTTCACATGGCTAAGCCTGATATGATCCGCAAGTTGGAACAAGGAGAAGAGCTATGGACACAGAGAATTTTTCCAAGTTACAGCTACCTAGAAGAAGATGGGAAAACTGAAGATGTCTTAGTGAAGTTCAAAGAATACCAAGACAGGCATTCTAGACCCCTCATATTCATCAACCACAAAAAACTAATTAAGGAGAGAAGTAATATTTATGGTAAAACATTTACTCTAGGCAAGAACCGTATTTCAAAAACAATACTATGTGAATATAAACCTGATGGAAAAGTTTTGAAAAATATTTCAGAACTAGTCATTAGAAATATAAGCCCCATAAAAGAGAAGTTTGGTGACAGTACTGGATGGGAGAAATCACTCCTCAATACCAAGCATGAGAAAATTCATCCTGCAGTGAATCTCCATAAACAAACAGAAAGAGTTCTCAGTGGTAAACAGGAGCTTATTCAGCATCAGAAGGTTCAAGCTCCAGAGCAACCATTTGACCATAATGAATGTGAAAAATCCTTCCTGATGAAAGGAATGCTATTTACACATACTAGAGCTCACAGAGGAGAAAGAACCTTTGAATACAATAAAGATGGAATTGCCTTCATAGAAAAGTCAAGCCTCAGTGTCCATCCAAGTAATCTTATGGAAAAGAAGCCCTCTGCCTACAACAAATATGGGAAATTCCTCTGCAGAAAGCCTGTTTTTATTATGCCTCAGAGACCTCAAACAGAAGAGAAACCCTTTCACTGTCCTTACTGTGGGAATAACTTTAGAAGGAAGTCATACCTCATTGAACATCAGCGAATTCACACAGGTGAAAAACCTTATGTTTGCAATCAATGTGGAAAGGCCTTCCGTCAGAAGACAGCCCTCACCCTTCATGAGAAAACACATATAGAGGGGAAACCCTTTATTTGTATCGATTGTGGGAAGTCCTTCCGCCAGAAGGCCACCCTCACTAGACATCACAAAACACATACGGGGGAGAAAGCCTATGAATGTCCTCAGTGTGGAAGTGCCTTTAGGAAGAAGTCATACCTCATTGATCACCAGAGAACTCACACAGGAGAGAAACCGTATCAGTGTAATGAGTGTGGGAAGGCATTTATCCAGAAGACAACCCTCACTGTTCATCAGAGAACTCACACAGGAGAGAAACCCTATATTTGCAATGAATGTGGGAAGTCCTTCTGCCAAAAGACAACCCTCACTCTCCACCAGAGAATTCACACGGGGGAAAAACCCTATATTTGTAATGAATGTGGGAAGTCCTTCCGCCAGAAGGCAATCCTCACTGTTCATCACAGA
ATACATACAGGAGAAAAATCCAATGGGTGTCCTCAGTGTGGGAAAGCCTTCAGTAGGAAATCAAACCTCATTCGCCATCAGAAAACTCACACAGGCGAGAAACCATATGAATGTAAACAGTGTGGGAAGTTCTTCAGTTGTAAGTCAAACCTCATTGTCCATCAGAAAACTCACAAGGTAGAAACCACGGGAATTCAGTAAGGATCC；
s2: lentivirus infection of DLBCL cells
Detecting the slow virus titer of the ZNF382 over-expression group in the step S1, and adjusting the virus titer of the ZNF382 over-expression group to be 5 multiplied by 10 8 TU/ml；
When DLBCL cell strain is in logarithmic growth phase, adding concentrated virus solution with MOI value of 100, and making the final concentration of ploybrene be 8 μg/ml;
after 12h of infection, the medium was replaced with fresh medium;
after 48h of infection, changing the culture medium into a culture medium containing 1 mug/ml puromycin condition for screening, and finally obtaining positive infected cells, namely obtaining diffuse large B cell lymphoma cell lines stably transfected by ZNF 382;
the DLBCL cell line is OCI-LY10 or U2932 cell line cell.
2. The construction method according to claim 1, wherein the ZNF382 lentivirus overexpressing plasmid containing the ZNF382 gene sequence is pCDH-CMV-ZNF382-GFP, added in an amount of 21 μg; helper plasmids included 17.5. Mu.g pSPAX2 and 7. Mu.g pMD2.G.
3. The construction method according to claim 1, wherein the specific method for precipitating viruses by polyethylene glycol concentration in step S1 is as follows:
A. filtering the supernatant by using a filter membrane made of PES material with the thickness of 0.45 μm, and preparing a 5X PEG8000+NaCl mother solution;
B. adding 7.5ml of 5 XPEG-8000+NaCl mother liquor into every 30ml of filtered virus initial liquor, uniformly mixing for 5h on ice, mixing every 30min, centrifuging at 4 ℃ for 2h at 4000g, absorbing supernatant, centrifuging at 4 ℃ for 5min at 4000g, absorbing residual liquid, adding a proper amount of lentivirus dissolving liquor to dissolve lentivirus precipitate, packaging concentrated virus suspension, quick freezing and storing at-80 ℃.
4. The construction method according to claim 3, wherein the preparation method of the 5x peg8000+nacl mother liquor is as follows: 8.766g of NaCl and 800050g of PEG are weighed and dissolved in 200ml of Milli-Q pure water, sterilized at 121 ℃ for 30min and stored at 4 ℃.
5. The use of a cell line constructed by the construction method of any one of claims 1 to 4 in the preparation of a medicament for studying or treating DLBCL.
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| WO2015157291A1 (en) * | 2014-04-07 | 2015-10-15 | The Brigham And Women's Hospital, Inc. | Classification of myc-driven b-cell lymphomas |
| CN107043786A (en) * | 2017-05-11 | 2017-08-15 | 合肥知恩生物技术有限公司 | A kind of stable cell line construction method of secreting, expressing albumen |
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