Study On The Screening Of Human Keratinocyte Clones With CCL20 Gene Modification And Their Stem Cell Transcription Factors In Vitro

Background:It is one of the key imperative issues in treatment of large-area burn to relieve allograft skin rejection and prolong the survival of burn wound grafts. Langerhans cells (LC) are the cells that trigger skin allograft rejection. CCL20, mainly produced by activated keratinocytes, is a potent chemotactic factor that induces LC precursors derived from CD34+ hematopoietic progenitors to migrate to the epidermis. Inhibition of CCL20 gene expressed by human keratinocytes may reduce migration of recipient LCs into grafts, impair indirect antigen presenting routes, thus relieving recipient rejection of grafts.The human immortal keratinocyte line (HaCaT) has a phenotype, and proliferation and differentiation properties similar with those of epidermal stem cells. Its hereditary traits are stable, and it has no tumorigenicity. Therefore, this cell line is an ideal candidate for seed cells of tissue engineered skin. A prolonged, stable modification of HaCaT CCL20 gene (knockout or interference) may modify seed cells of tissue engineered skin. Hence, in the present study, CCL20 gene knockout/replacement targeting vector was constructed, and the targeting vector DNA and human keratinocytic DNA were then subjected to homologous recombination. The recombinants were identified and transfection methods of targeting vector were investigated. The mRNA and protein expression levels of human stem cell related transcription factors were determined in HaCaT and gene knockdown cell clones, thus providing new theoretical basis for screening epidermal seed cells for CCL20 gene modified tissue engineered skin with low rejection potential.Objective:1. To construct CCL20 gene knockout/replacement targeting vector ploxP-hCCL20, and the targeting vector DNA and human keratinocyte DNA were subjected to homologous recombination. The recombinants were identified to screen CCL20 gene knockout cells. 2. Based on the results in the previous section, ploxP-hCCL20-EGFP fluorescence expression plasmid was constructed, and various transfection methods for high molecular vector were explored. Fluorescence expression in HaCaT cells was observed.3. The mRNA and protein expression levels of human stem cell related transcription factors were observed in CCL20 gene knockdown cell clones, so as to provide new theoretical basis for screening seed cells of tissue engineered skin with mild rejection.Methods:1. Primers were designed and synthesized. The CCL20 gene fragment was cloned from HaCaT cell genomic DNA by long-distance PCR. Then, using CCL20 gene as template, a homologous short arm and a homologous long arm with restriction sites were amplified, and inserted into the upstream and downstream of Neo gene in ploxP vector. The recombinant plasmid ploxP-hCCL20 replacement targeting vector for exon 2 of human CCL20 gene was constructed using T vector and gene cloning technology.2. The targeting vector was linearized by digestion with EcoR I restriction endonuclease, and transfected into HaCaT cells. The clones were screened by G418 and GANC positive and negative pressure and cultured.3. A pair of primers was designed for the upstream of the homologous short arm and the downstream of the homologous long arm of the targeting vector, acrossing Neo gene. Using cloning screened HaCaT genomic DNA as template, the clones were identified by PCR.4. A homologous probe was designed for exon 3 of human CCL20 gene. After analysis with Primer Premier software, SacI/KpnI or AccI/KpnI were selected to digest cell genes, followed by Southern blotting identification.5. The promoter CMV IE and EGFP fragments of pEGFP-N2 plasmid were cloned into between Asp718 and EcoRI restriction sites of ploxP-hCCL20 recombinant plasmid, to construct ploxP-hCCL20–EGFP fluorescence expression plasmid.6. ploxP-hCCL20–EGFP fluorescence expression plasmid was transfected into 293FT or HaCaT cells using jetPEI liposome and electroporation combined with nucleofection reagent method, and then intracellular fluorescence expression was observed and the cell transfection efficiency was calculated.7. Using flow cytometry, changes of percentage expression in human stem cell related transcription factors Oct-4, Sox-2, Nanog, FGF-4, Rex-1 and TERT were observed in HaCaT cells and 11 CCL20 gene knockdown cell clones including 1-2, 1-7, 1-4, 1-3, 1-5, 2-4, 2-6, 2-11, 2-9, 2-10 and 2-3.8. Using GAPDH as internal control of fluorescence quantitative PCR, the relative expression of Oct-4, Sox-2, and Nanog mRNA was determined in HaCaT and 11 clone cells, to observe the changes in related factors in various clones.9. Using GAPDH as internal control of fluorescence quantitative PCR, stem cell transcription factors expressed by HaCaT, human keratinocytes and amnion tissue were examined.10. Based on the preliminary screening results, the mRNA and protein levels of Oct-4, Sox-2, Nanog, FGF-4, Rex-1 and TERT were detected by fluorescence quantitative PCR and Western blotting in HaCaT, 1-4, 1-2, 2-9 and 2-10 clones, respectively.Results:1. CCL20 DNA fragment (4459bp) was cloned from genomic DNA of HaCaT cells by long-distance PCR, and the homologous short arm (1969bp) with restriction sites NotI/XhoI and homologous long arm (2356bp) with restriction sites Xbal/ClaI were obtained. The replacement targeting vector ploxP-hCCL20 (11.9 kb) was constructed for exon 2 of human CCL20 gene, which was identified to be correct by PCR and digestion with two endonucleases. The sequencing results were compared with the full-length sequence of human CCL20 gene registered with Genbank (Accession number, NT₀05403). It was suggested that there were two point mutations in the homologous short arm (115G→A, 1274A→G), and three point mutations in the homologous long arm (2228T→C, 3310T→A, 4208A→G). The mutated bases were not in the exon sequence; hence, synonymous single nucleotide polymorphism mutations were considered. The homologous short arm comprised exon 1 and some introns, and the homologous long arm comprises exons 3, 4 and some introns.2. After screening with G418 and GANC positive and negative pressure, a total of 18 clones were obtained. PCR identification indicated a single 409bp band, demonstrating the clones to be wild-type. Five Southern blotting tests were performed for 5 clones, and no 2943bp and 4684bp bands representing heterozygotes were found, suggesting no gene knockout clones. 3. Sequencing results indicated that ploxP-hCCL20–EGFP fluorescence expression plasmid (13.3 kb) was constructed successfully. The linearized plasmid was transfected into 293FT cells using jetPEI liposome, and the cells exhibited strong green fluorescence expression, with a transfection efficiency of 75.1% and a HaCaT cell transfection efficiency of 1.3%. HaCaT cells were transfected by electroporation combined with nucleofection reagent method, indicating that the transfection efficiency was 38.3% in the positive control pmaxGFP. In contrast, ploxP-hCCL20-EGFP plasmid transfection of HaCaT cells resulted in a low transfection efficiency of 0.3% and very weak fluorescence expression.4. Flow cytometric data indicated that expression percentages of Oct-4+Sox-2+ double labeled 11 clones were less than HaCaT cells, and Oct-4+Sox-2+ expression of 2-6 and 2-9 clones were lower than HaCaT (25.51±2.25% vs 94.58±1.08%, P=0.000); (22.95±3.31% vs 94.58±1.08%, P=0.002), respectively.5. Flow cytometric results of Nanog, FGF-4, Rex-1 and TERT single labeled cells indicated that:In the Nanog group, Nanog was expressed in more HaCaT (59.94±8.49%) than in 2-6 clone (8.23±4.91%, P=0.027). Nanog expression changed insignificantly in the other clones (13.51%-60.79 %, P>0.05).In the FGF-4 group, FGF-4 was more higher expressed in HaCaT (99.27±0.38%) than that of the other clone cells (76.51%-95.31%). Compared with the control group, FGF-4 expression was downregulated very markedly in 2-9 (79.81±0.41%) and 2-10 clones (92.75±0.46%)(P<0.01).In the Rex-1 group, Rex-1 expression percentage was the highest in HaCaT cells (99.32±0.32%), and Rex-1 expression was downregulated significantly in 2-9 clone (78.76±1.23%), when compared with HaCaT cells (P=0.005). The expression percentage ranged between 87.02% and 98.10% in the other clones, which differed insignificantly from that of the control group (P>0.05).In the TERT group, TERT expression percentage was the lowest in HaCaT cells (1.00±0.82%), which had very weak fluorescence. The highest expression rate (only 50.31±0.93%) was observed with 2-4 clone. The expression rate differed insignificantly between most of the other clones (P>0.05). These findings suggested that HaCaT cells and the clone cells exhibited certain properties of stem cells. 6. Fluorescence quantitative PCR screening of Oct-4, Sox-2 and Nanog in 11 clones: Oct-4 mRNA: the clones (09) included 2-6. Sox-2 mRNA: the clones (09) included 1-7, 1-3, 1-5, 2-9, 2-6, 2-10, and 2-3.7. Compared with HaCaT (RQ=1.000), the expressions of Oct-4, Sox-2, Nanog and Rex-1 in human keratinocytes and amnion tissue were very higher, varying from 24.549 to 4886.8. Fluorescence quantitative PCR of 2-9 and 2-10 clones indicated that compared with HaCaT, 2-9 clone showed a significant increase in Nanog expression (6.50±1.76, P=0.007), but no significant changes in mRNA expression of other transcription factors (Oct-4, Sox-2 and Rex-1). There were no significant differences in Oct-4, Sox-2, Nanog and Rex-1 mRNA expression between 2-10 clone and HaCaT (P>0.05).9. Western blotting results of 2-9 and 2-10 clones indicated that compared with HaCaT, Rex-1 protein expression changed insignificantly, but Oct-4, Sox-2, Nanog and TERT protein expression increased significantly in these two clones (P<0.05).10. Fluorescence quantitative PCR of 1-4 and 1-2 clones indicated that compared with HaCaT, 1-4 clone showed a significant decrease in Sox-2 and Rex-1 mRNA expression (P=0.017; P=0.043), but no significant changes in Oct-4 and Nanog mRNA expression. In contrast, compared with HaCaT, 1-2 clone showed a significant increase in Oct-4 and Nanog mRNA expression (P=0.047; P=0.029), but no significant changes in Sox-2 and Rex-1 mRNA expression.11. Western blotting results of 1-4 and 1-2 clones indicated that Oct-4, Sox-2 and TERT protein expression was extremely low in HaCaT, 1-4 and 1-2 clones, and there were no significant differences between different clones (P>0.05). Compared with HaCaT, Nanog and Rex-1 protein expression decreased significantly in 1-4 and 1-2 clones (P<0.05). Conclusions:1. The human replacement targeting vector ploxP-hCCL20 for exon 2 of CCL20 gene is constructed successfully, and no heterozygote knockout clone is identified by PCR and Southern blotting after transfection of HaCaT cells with the targeting vector.2. ploxP-hCCL20–EGFP fluorescence expression plasmid is constructed successfully, but the transfected cells do not express high levels of green fluorescence, possibly due to high molecular weight of the vector or low transfection efficiency of HaCaT cells. Modification of the transfection method might lead to obtaining gene knockout clones.3. There are different changes in the expression percentage and mRNA level of related stem cell transcription factors of 11 clones with CCL20 gene knockdown, suggesting gene modification interferes the expression of Oct-4, Sox-2, Nanog, FGF-4, Rex-1 and TERT at the level of mRNA and protein.4. The expression of stem cell transcription factors including Oct-4, Sox-2, Nanog and Rex-1 in HaCaT is obviously lower than that in adult human keratinocytes and amnion tissue.5. Compared with HaCaT, 2-9 and 2-10 clones show significant increase in the protein expression of Oct-4, Sox-2, Nanog and TERT, suggesting that stem cell transcription factors expressions of these CCL20 knockdown clones have changed. The mechanism involved needs further investigation.6. Compared with HaCaT, 1-2 and 1-4 clones show no significant change in the expression percentage of Oct-4+Sox-2+, Nanog, Rex-1 and TERT, and also low or extremely low expression levels of corresponding proteins. Hence, these two clones can be further investigated as candidate seed cells for tissue-engineered skin.