| Background and purposeHemophagocytic syndrome(HPS),also known as hemophagocytic lymphohistiocytosis(HLH),is a complex and rare hyperinflammatory response syndrome.The onset of HLH is rapid,the condition is critical,the prognosis is poor,the number of natural killer(NK)cells is often significantly reduced,and the activity is reduced during acute attacks.Its pathogenesis is often associated with NK cells and cytotoxic T lymphocytes(CTL)mediated perforin-dependent defects in cytotoxicity,resulting in the excessive proliferation and activation of T lymphocytes and macrophages,and the large release of cytokines(commonly known as cytokine storm).Therefore,NK cells,as an important mechanism cell in the occurrence and development of HLH disease,have already become an important carrier cell for studying the relevant mechanism of the disease.At the same time,NK cells are a group of cells characterized by CD3~-CD56~+CD16~+.They are innate immune cells that can identify and kill abnormal cells that are not restricted or unsensitized by major histocompatibility complex(MHC).It is considered to be the most effective immune cell subgroup to monitor and clear diseased cells in vivo.Therefore,as the key cells of immunotherapy,NK cells have become a research focus and attracted much attention.However,NK cells only account for5%-10%of normal human peripheral blood mononuclear cells(PBMCs),and it is difficult for them to be expanded and cultured in vitro.Current research shows that cytokines such as interleukin(IL)-2,IL-4,IL-9,IL-15 and IL-21 play a role in expanding NK cells in vitro.IL-21 belongs to the IL-2 family and is a common gamma chain cytokine.It is expressed by activated CD4~+T cells and plays an important role in the activation,maturation and proliferation of NK cells.In humans,IL-21 has a proliferation effect on NK cells,and stimulates the cytotoxic activity of NK cells by increasing the expression of perforin and granzyme B.Studies have shown that IL-21combined withIL-2 and IL-15 can have a positive effect on NK cell proliferation,cytotoxicity and IFN-γproduction.In view of the fact that NK cells have great application potential in tumor immunity and play an important role in the pathogenesis of HLH,the current research focus is to amplify NK cells in vitro,enhance their anti-tumor activity and further study the pathogenesis of HLH.A variety of culture systems have been used for in vitro amplification,most of which use intermittently added NK production factors such as IL-2,IL-15,IL-21 and some target antigens such as co-incubation with K562 cells,but there still exist some problems.Some studies have suggested that NK cells can expand,but their killing activity is reduced.It may maintain or increase the killing activity,but the amplification efficiency is reduced.The reason may be due to the imperfect culture system and the intermittent addition of NK growth factors to provide pulsed growth stimulation of NK cells,which is related to the obvious difference in physiological state.We hypothesized that the use of continuously secreting il-21 cell lines as feeder cells for the proliferation of NK cells in vitro might address their proliferation efficiency in vitro and maintain their killing activity.This study intends to construct a CHO-hIL21 cell line and explore the effects of its continuously secreted IL-21 based culture system on the in vitro expansion and killing function of NK cells.This study is divided into the following two parts:(1)establishment and expression of CHO-hIL21 cell line;(2)Effect of CHO-hIL21 on natural killer(NK)cells.Methods1. Establishment and expression of CHO-hIL21 cell line1.1 Construction of pLenti-IL-21 lentivirus overexpression vector.Look up the data,retrieve the Pubmed Genebank,find the IL-21 gene sequence,the total length of the reading frame is 489 bps,according to the requirements of plasmid construction,design restriction sites Bam H I at both ends of the reading frame,together with the restriction site and reading frame sequence,the total length is 501 bps,Sent to the company for full gene synthesis,and synthesized with pLenti plasmid vector.The returned plasmid was verified for gene sequence by coating,picking,shaking,and sequencing.1.2 Transfect CHO to determine the expression of IL-21.Extract pLenti-IL-21transfection grade plasmid,transfect CHO cells with liposome method(Roche reagent),add Blasticidin for screening and maintain the optimal concentration after 24h,collect the culture supernatant after 48h,the expression of IL-21 was detected by Cytometric Bead Array(CBA)technology.1.3 Lentiviral packaging.A large number of shuttle plasmids pLenti,pLenti-IL-21 and packaging plasmids PSPAX2 and Pmd2g were extracted and transfected into 293T cells by liposome method(Lipofectamine 3000 reagent),Centrifugal filtration,concentration,limiting dilution method to measure virus titer.1.4 Lentivirus infects CHO cells.The above packaged complete lentivirus infected CHO cells,while adding Polybrene(final concentration:5μg/ml)to enhance the infection efficiency,and 72 hours after infection,Blasticidin was added for screening.CHO-hIL21 cell line was selected by multiple cloning to observe the fluorescence intensity and CBA technology to detect IL-21 concentration.1.5 IL-21 gene expression and protein expression identification.RNA was extracted from CHO-hIL21 cells and reverse-transcribed into c DNA.RT-PCR was used to detect the expression of IL-21 gene in CHO-hIL21 cells.The primary antibody was rabbit anti-IL-21 antibody,and the secondary antibody was goat anti-rabbit FITC.The immunofluorescence method was used to detect the expression of IL-21 protein in CHO-hIL21 cells;the flow cytometry method was used to quantitatively detect the expression of IL-21 in the culture supernatant of CHO-hIL21 cells.2.Effect of CHO-hIL21 on NK cellsFlow cytometry was used to detect the targeted killing ability of normal NK cells to K562 cells.CCK-8 test to detect the optimal concentration of Mitomycin C to inhibit the proliferation of CHO-hIL21 cells.Mitomycin C treated CHO-hIL21 cells to inhibit cell proliferation,co-culture with NK cells,counted NK cell proliferation ability with cell counter,flow cytometry detection Effect of IL-21 on the killing function of NK cells.Flow cytometric CBA technology was used to quantitatively detect the changes of Th1/Th2 cytokine levels in NK cells mediated by IL-21.Results1.Establishment and expression of CHO-hIL21 cell line1.1 Construction of pLenti-IL-21 lentivirus overexpression vector.Determine the IL-21 gene sequence by consulting relevant materials and literature,and entrust the company to synthesize the gene.The sequencing results were compared with the designed sequence,and it was completely consistent with the designed sequence,indicating that the pLenti-IL-21 lentivirus overexpression vector was successfully constructed.1.2 Transfect CHO to determine the expression of IL-21.Transient transfection of CHO cells and quantitative detection of IL-21 concentration showed that the secretion of IL-21 could reach more than 800 pg/ml,suggesting that the plasmid was constructed correctly.1.3 Lentiviral packaging.The lentivirus overexpression vector containing the target gene of IL-21 was successfully packaged,the supernatant of the virus was collected and concentrated by 50 times.The titer measurement results showed that the titer of pLenti-IL-21 was 4×10~8/ml,and the titer of pLenti was 4.5×10~8/ml.1.4 Lentivirus infects CHO cells.Lentivirus successfully infected CHO cells.The results of multiple cloning screening and quantitative detection by CBA method showed that after each cloning,the concentration of IL-21 was increased by multiple,up to3721.13pg/ml,so as to determine the CHO-hIL21 cell line with stable expression of IL-21.1.5 IL-21 gene expression and protein expression identification.The results of RT-PCR showed that the band size of IL-21(approximately 300 bps)was consistent with the expected result(298 bps),suggesting that the successfully selected CHO-hIL21cells were transferred into pLenti-IL-21 plasmid;the results of immunofluorescence showed that the green fluorescent protein was expressed in the GREEN fluorescence field under the excitation of 488nm excitation light,but not in the no-load group,suggesting that the IL-21 protein was successfully expressed in the CHO-hIL21 cells after screening;Western-blot results showed that the IL-21 protein band was between15-20 KDa,which was consistent with the molecular weight of IL-21 at 18 KDa,which also indicated that the IL-21 protein was successfully expressed in the CHO-hIL21 cells after screening.2. Effect of CHO-hIL21 on NK cellsThe results of CCK-8 experiments showed that the optimal inhibitory concentration of Mitomycin C on CHO cells was 10μg/ml.After CHO-hIL21 was inhibited by Mitomycin C,the secretion of cytokine IL-21 was continuously increased,suggesting that after Mitomycin C inhibiting the amplification of CHO-hIL21,the secretion of IL-21 gradually increased in the short term,reaching 4842±444.3 pg/ml.The co-culture results of CHO-hIL21 and NK cells showed that there was no expansion of NK cells in PBMC,and the proliferation efficiency of purified NK cells was lower than that of recombinant human IL-21+IL-2 group(P<0.01).Compared with the IL-2culture alone,the survival rate of NK cells was higher until the 9th day(P<0.05).The results of cytokine levels showed that the level of IL-6 in PBMC was significantly increased(P<0.001),and the level of IFN-γin the IL-21+IL-2 group was slightly increased in purified NK cells(P<0.05).However,there was no change before and after co-culture in terms of killing activity.Conclusions1.Successfully constructed the lentiviral overexpression vector of human IL-21;2. Successfully constructed and screened CHO-hIL21 cell lines with high expression of IL-21;3. The continuous secretion of CHO-hIL21 resulted in a significant increase of IL-6levels during the culture process of NK cells in PBMC;4. The continuous secretion of CHO-hIL21 has a weak ability to amplify NK cells,but it can improve the survival rate of NK cells. |
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