The Effects Of Keratinocyte Growth Factor And It's Receptor's Antisense Oligonucleotides On Proliferation Of HaCaT Cells

Objective 1. To investigate the effect of keratinocyte growth factor(KGF) on HaCaT cells proliferation, cell cycle, apoptosis, expression of proliferating cell nuclear antigen(PCNA), and to explore the correlation of KGF with psoriatic epidermal proliferation and apoptosis. 2. To study the suppressing effect of antisense oligonucleotides (ASODN) targeting to KGFR mRNA on HaCaT cells proliferation induced by KGF, and to explore the possibility of KGFR ASODN being used for psoriasis treatment.Methods 1. HaCaT cells were cultured in vitro. 2. Two sequences of antisense oligonucleotides targeting to KGFR mRNA (ASODN1, ASODN2) and one sequence of sense oligonucleotides (SODN) as control were designed and synthesized, cationic liposomes agent was used to conduct these oligonucleotides transfecting HaCaT cells, and laser confocal microscopy was employed to measure the transfecting efficiency. 3. MTT assay, colone forming experiment as well as cell growth curve test were used to determine the proliferating effect of KGF on HaCaT cells and the inhibiting effect of KGFR ASODN on the proliferation induced by KGF. 4. Flow cytometer (FC) was used to investigate cell cycle, apoptosis, expression levels of KGFR and PCNA after the treatment with KGF and KGFR ASODN.Results 1. Fluorescence granules were found in the plasma of positive cells after transfected with ASODN1, ASODN2 and SODN, the transfection rate was increased in a dose depended manner (EC₅₀=2.7μg/ml, 2.8μg/ml and 2.8μg/ml, respectively); 2. MTT assay showed that after the treatment with KGF for 24h, HaCaT cell proliferation was increased significantly in a dose dependent manner (EC₅₀=5.6ng/ml), the proliferation induced by KGF was inhibited significantly by ASODN1 as well as ASODN2 at more than 0.25μg/ml concentration (P<0.05), and the inhibiting effect was showed in a dose dependent manner (IC₅₀=3.2μg/ml and 3.55μg/ml respectively). SODN had no inhibiting effect on HaCaT cells proliferation (P>0.05); 3. Cell growth curve showed that HaCaT cells growed more rapidly after the treatment with 10ng/ml KGF compared with control group(P<0.05), the up-regulated growth rate induced by KGF has been suppressed by ASODN1 and ASODN2 since the second day(P<0.05); 4. Clone forming experiment showed that HaCaT cell clone forming rate was up-regulated after the treatment with 10ng/ml KGF significantly (P<0.05), the increased clone...