Preliminary Study On MACS Of Human KSC And The Effect Of CCL20-siRNA

Objectives:1. Keratinocytes were isolated from human foreskins and cultured in serum free medium in vitro to investigate its growth feature. Separating keratinocyte stem cells (KSC) by magnetic activated cell sorting(MACS)combined human placenta collagen typeⅣadhesion, the cells were cultured in serum free medium to found an ideal method of KSC'sorting and culture.2. Our group had succeeded in finishing the construction of two small interference RNA (siRNA) lentiviral vectors specific to human CCL20 gene. At this basis, we will confirm by DNA sequencing another siRNA lentiviral vector for negative control. After then, the packaging cells 293FT will be transduced by these recombinant lentiviral vectors. At last, the human keratinocyte cell line HaCaT cells and KSC will been infected by lentivral particles to detect the effect of siRNA to human CCL20 gene.Method:1. Keratinocytes were isolated from human foreskins by dispase-trypsin two step combined dissociation. We pre-coated the dishes with human placenta collagen typeⅣaccording to the density of 5μg/cm2 to facilitate keratinocytes to adhere, then the keratinocytes were cultured in Defined Keratinocyte Serum Free Medium(DK-SFM). After that, we observed the morphological change, colony-forming and expression of surface CD71, CD49f(integrinα6) and CD29 (integrinβ1) of KSC.2. We separated KSC by the MACS according expression of surface CD49f(integrinα6) and CD71(CD49fbriCD71dim )and analyzed the purity and ratio of KSC. Then we also had these KSC cultured in serum free medium by human placenta collagen typeⅣadhesion to observe these morphological change, colony-forming.3. The competent E. coli. DH5αcells bacterium liquid that the recombinant plasmid had been transformed successfully into were seeded on LB culture plate with Amp (60μg/ml). The positive recombinant colony was selected by ampicillin medium agar and identified by DNA sequencing.4. The packaging cells 293FT were cotransduced by jetPEI and the mixture of the recombinant lentiviral expression vector pHSER-CCL20-siRNA-SIN , the packaging plasmid Lentipack and the envelop plasmid Lentienv in a certain proportion., after 48h, 72h, and 96 h, the lentiviral particles in the 293FT media supernatant were collected and frozen under -80℃.5. HaCaT cells and KSC were infected by lentiviral particles and set up blank group for control. After 24 hours,we added the mixture of stimulating factor TNF-α, IL-1βand DK-SFM in all cell groups, in whicn the final concentration of TNF-αand IL-1βboth were 100ng/ml. 48 hours later, the cell media supernatant were collected and detected the CCL20 protein content by human CCL20 linked-immuno-sorbent assay (ELISA) kit: firstly, the OD450 of both standard preparation and sample were reported by microplate reader. And then,the fitting curve and equation were got according the OD450 of standard preparation. Lastly,the CCL20 protein content can be calculated according the OD450 of sample and the curve equation. Comparing the CCL20 protein content of the lentiviral particles groups and control group, we observed and judged whether the effect of siRNA to human keratinocyte CCL20 gene was down regulation.Result:1.Keratinocytes could be cultured in serum-free medium for 35.13±7.59 days in vitro and passaged to the 3.87±0.83 th generation, and the primary cells'colony-forming could be observed after 5.75±0.90 days. There was no difference on colony forming time and passage time among the primary and passage cultured cells (p>0.05). The number of cells had not risen after they were passaged.2. In primary cultured keratinocytes, about 46.6±2.1%cells were CD49fbriCD71dim positive and 65.8±2.3% cells were CD29 positive.3. After MACS, about (12.2±7.1)% cells were CD49fbriCD71dim positive. We could observe that the cells were typical epithelium cells, just like pebbles. These cells were close-up and had high karyoplasmic ratio and clear circumscription. And then their holoclone could be observed after 5-7 days. All these are characteristic of KSC.4 The recombinant siRNA lentiviral negative control vector was confirmed by DNA sequencing.5. The lentiviral particles in the 293FT media supernatant were collected successfully. After the HaCaT cells and KSC were infected by the lentiviral particles, we observed that the effect of siRNA to human CCL20 gene of keratinocytes was down regulation by human CCL20 ELISA.Conclusion:1. Keratinocytes can be cultured successfully in serum free medium in vitro.2. By combining MACS and human placenta collagen typeⅣadhesion, we can harvest more KSC and it was an effective method for KSC'sorting.3. Lentiviral vector-based siRNA negative control plasmids against human CCL20 gene have been successfully constructed and identified by DNA sequencing.4. HaCaT cells and KSC were infected successfully by using CCL20-siRNA lentiviral particles specific human CCL20 gene and the effect of siRNA to human keratinocyte CCL20 gene was down regulation.