Study On The Screening Of Human Keratinocyte Clones With CCL20 Gene Knockdown And Their Long-term Effects In Vitro

Background: Extensive burns and other skin lesions of patients due to limited sources of autologous skin will require a lot of skin allograft or xenograft for wound coverage to prevent the microbial contamination and reduce the body fluid loss. Although the allogeneic tissue-engineered skin products have been used in patients with skin ulcer for more than a decade, the immune rejection of the body against them is still the most critical bottleneck that restricts their expended applications in severely burned patients. This type of skin immune rejection is initiated only by the indirect antigen presentation of recipients'Langerhans cells because there are no donors'Langerhans cells in these products. CCL20 is produced by epidermal cells and attracts the recipients'Langerhans cells into allogeneic tissue engineeringed skin. Therefore, CCL20 plays a key role in the rejection of allogeneic tissue engineeringed skin and can be served as the target gene for genetic manipulation in epidermal seed cells, i.e. the keratinocytes. Human immortal keratinocyte line (HaCaT) has genetically stable characteristics and high percentage of epidermal stem cell phenotypes without tumorigenicity. HaCaT with manipulated CCL20 gene might be used as seed cells for novel tissue-engineered skin with lower rejection.Objective:1. The study will firstly explore the gene transfer efficiencies of various vectors into human keratinocyes by different approaches, and provide helpful experimental evidences for the effective gene transfer of the tough epidermal cells.2. Based on the results of gene transfer approaches, the previously constructed lentiviral vectors with CCL20 gene specific and mismatched shRNA in our lab will be applied to infect HaCaT. The corresponding genetically modified HaCaT clones will be screened out under the pressure of G418. Their interference effects on the CCL20 mRNA expression will be primarily assessed by quantitative RT-PCR. 3. After these clones are subculured for more than thirty five passages, their long-term interferencing effects on the mRNA and protein levels of CCL20 gene will be determined by by Real-time RT-PCR and ELISA, respectively.4. The protocol of inducing Langerhans cells from CD34+ cells will optimized from various combinations of cytokines. The CD34+ cells separated from human umbilical cord blood will be induced into Langerhans cells. Their long-term chemotactic effects of these CCL20 genetically manipulated HaCaT clones will be further evaluated in the chemotaxis model of Langerhans cells in vitro.Methods1. HaCaT,human Cervical cancer cell line (HeLa) and human embryo kidney cell line (Hek 293FT) were transfected with four different molecular weight plasmids by liposome and cation polymerizer. The expressions of GFP or EGFP reporter gene in tranfected cells were observed by fluorescence microscope.2. HaCaT was transfected with two bigger plasmids by electroporation combined with cell nuclear transfer reagents Cell Line Nucleofector Kit V Solution. The GFP or EGFP expressions of tranfected cells were observed by fluorescence microscope.3. The three previously constructed pHSER-CCL20-shRNA-GFP vectors were transfected into the virus packaging cell line 293FT by using CaCl2 methods to produce lectiviral particles. After the viral titers of these three harvested cell supernatants were determined by flow cytometry, HaCaT cells were transfected by them.4. HaCaT was cultivated with G418 medium of different concentrations, to get the minimum cytotoxic concentration of G418. The transfected HaCaT was added in to 96-well plates by limited dilution method, and screened under the pressure of G418 for 5-8weeks.5. In order to assess the CCL20 gene knockdown effects on the CCL20 expressions of mRNA and protein, these clones were cultured with or without inflammatory cytokines, i.e. TNF-αand IL-1βin 6-well plates. After 24 hours, the total RNA from these clones was extracted with Trizol reagent for CCL20 mRNA analysis. After 48 hours, the culture supernatant was also collected and stored at -20°C for CCL20 assay.6. Quantitative PCR(qPCR): After the RNA samples were transcripted into cDNA by reverse transcriptase, the CCL20 mRNA expressions of HaCaT clones were detected by Real-time RT-PCR. GAPDH gene was used as an internal control. Compared to the non-infected HaCaT, the CCL20 gene knockdown effects on the relative CCL20 mRNA expressions of G418 selected positive HaCaT clones were observed.7. ELISA: The OD450 values of standard preparations and supernatant samples were recorded by microplate reader. Based on the r2 values of all the equations, the best fitting standard curve and equation were chosen. The CCL20 protein contents in the supernatants can be calculated according their OD450 values and the standard curve equation.8. The mononuclear cells(MNCs) were separated from human umbilical cord blood by using human lymphocyte separation medium. The CD34+ cells were then further purified from MNCs by direct CD34 immunomagnetic beads and MACS.9. The CD34+ cells were cultured with various combinations of five types of cytokines in vitro for 6 days and 12 day, and then induced into Langerhans cells. Langerhans cells with CD1a phenotype were identified by flow cytometry analysis, and the protocol with high yield of CD1a + Langerhans cells was optimized. The other cell phenotypes, such as HLA-DR, CD40 and CD80, were also measured.10. These Langerhans cells can be served as effect cells in the chemotaxis model of CCL20 detection. The chemotaxis effect of CCL20 standard preparation on Langerhans cells was measured to verify the stability of this model. The culture supernatants from CCL20 genetically manipulated HaCaT clones were further evaluated for their long-term chemotactic effects in this newly established model.Results:1. Minimum cytotoxic concentration of G418 for nonmodified HaCaT was 400μg/ml. The initial and later concentrations of G418 for the selection of CCL20 gene modified HaCaT clones were based on this minimum cytotoxic concentration.2. The transfection efficiencies of HaCaT transfected by three approaches (liposome, cation polymerizer and electroporation) with four different molecular weight plasmids (pEGFP-N2,pSUPER-EGFP,pHSER-GFP and ploxP-EGFP) were 1.0~3.3%, 1.0~3.7% and 3.3%～22.3%, respectively. The transfection efficiencies of HeLa transfected by liposome and cation polymerizer with these four plasmids were 30.3%～35.7% and 33.7%～36.7%, respectively. The 293FT has much higher transfection efficiencies than HeLa, i.e. 80.0%～84.7% and 81.3%～86.7%. The transfection efficiencies of HaCaT transfected with pHSER-GFP by lentivirus was 97.0%. 3. HaCaT was transfected by these lentiviruses and the positive clones were selected under the pressure of G418. Thirty eight HaCaT clones (28 clones from CCL20 gene specific shRNA lentivector, 10 clones from CCL20 gene mismacthed shRNA lentivector,) were obtained after G418 screening for 5-8 weeks. The relative quantity of CCL20 mRNA expressions in 28 clones was measured by real-time PCR. Compared to nonmodified HaCaT, the inhibition rates of CCL20 mRNA expressions in 9 out of 28 clones were 70%～100% ,those 4 out of 28 clones were 50%～70% when they were cultured with inflammatory cytokines.4. The HaCaT clones with inhibitory rate of CCL20 mRNA expression over 50% were further evaluated for their long-term CCL20 gene knockdown effects at the mRNA and protein levels. There were 4 clones of which mRNA expression and protein level of CCL20 gene were down regulated significantly even after 35～45 passages.5. After 6 days cultured with five protocols of various combinations of cytokines, CD1a+ cells induced from CD34+ cells by protocols(I, II, III, IV and V) were 19.47%, 17.20%, 14.03%, 34.39% and 38.90%, respectively. After 12 days, the CD1a+ cells by protocols(I, II, III, IV and V) were about 31.81%, 24.87%, 26.24%, 51.61% and 52.94%, respectively. The protocols of IV and V induced more CD1a+ cells than the other three protocols.The expressions of CD80 and CD40 on CD34 + cells after culture with protocol V for 6 days were more than that of protocol IV. The protocol IV increased the expression of HLA-DR on CD34 + cells after 12 days'culture when compared to protocol V.6. The CD1a+ cells derived from protocol IV were used in the chemotaxis model of Langerhans cells. The CCL20 gene knockdowned HaCaT clones showed significant suppression of chemotaxis for Langerhans cells after 35～45 passages.Conclusions:1. It's difficult to introduce exogenous gene into human keratinocytes by routine chemical methods. Lentiviral transfection has a highest transfection efficiency among these four methods. Different size of plasmids can also greatly affect the transfection efficiency of gene transfer.2. There are 38 positive HaCaT clones with recombinant lentiviral vectors after G418 selection. There are 11 clones with a CCL20 mRNA inhibition rates over 70% from the 28 clones of 38 clones after the primary qPCR screening. 3. Four HaCaT clones have been indentified from the 11 clones with long-term CCL20 gene knockdown effects at the mRNA and protein levels.4. Four cytokines'combination in protocol IV can induce more Langerhans cells(CD1a+ cells) and HLA-DR+ cells from CD34 + cells after 12 days'culture in vitro.5. These CD1a+ cells show normal chemotactic behaviors to CCL20, as well as deeply inhibited chemotaxtis to the HaCaT clones with a stable CCL20 knockdown effect.