Effects of Negative Pressure on Keratinocyte Migration in Hyperglycemic Environment

The limb amputation occurs 10 to 30 times more often in diabetic patients than in general population. The 5-year mortality rate is 16-80% in the leg-amputated diabetic patients. Many reports showed encouraging results that the negative-pressure wound therapy (NPWT) could promote wound healing. However, detailed cellular mechanisms are not fully clarified. Electric cell-substrate impedance sensing (ECIS) obtains cell migration information through monitoring the electrode impedance changes. The purpose of the study attempts to evaluate negative pressure effects on wound-healing process of injured keratinocytes at high glucose environment with the ECIS technology. First year: In the negative pressure incubator (NPI), O2 and water will be removed from the incubator. In addition to CO2 gas cylinder, an oxygen bottle is indicated to allow O2 tension is equal to 159 mmHg in the airtight chamber at ambient pressure (AP), -75 mmHg (NP75), -125 mmHg (NP125) and -175 mmHg (NP175). Human keratinocytes (HaCaT) will be inoculated on the ECIS electrodes for quantifying migration speed at the above conditions and chambered coverglasses for creating traditional wound-healing assays (TWHA) incubated at the above conditions for 6 hours. Thereafter, Flow cytometer examination (FCE) for cell vitality and immunofluorescent microscopic examination (IFM) for actins, E-cadherins and TRPC1 will also be evaluated. Second year: The culture medium glucose concentration is 5 mM (≈ 90 mg/dL) in the control group and 25 mM (≈ 450 mg/dL) in the high glucose group by respectively adding adequate amount of 45% glucose in the medium. The HaCaT cells will be cultured in normal and high glucose medium for 7 days. The cells will be used for ECIS and TWHA analyses as the experiment in the first year. FCE for cell vitality and mitochondria activities, and enzyme-linked immunosorbent assay for expression of advanced glycation end product amounts will be evaluated. IFM for actin and E-cadherin will also be performed. A better understanding for effects of high glucose on wound-healing process can then be made. Third year: The wounded normal and glycosylated HaCaT cells will be respectively harvested at AP, NP75, NP125 and NP175. ECIS and TWHA analyses will be done as the experiment in the first year. IFM for actin and E-cadherin is going to do following procedures developed in the first year. Cell vitality, mitochondria and AGE studies will be performed as steps in the second year. Western blot for Cdc42, N-WASp, Arp 2, E-cadherin and actin protein amounts in the two groups of cells at different conditions may help understanding impacts of high glucose and negative pressures on the actin polymerization.

Project ID:PC10008-0742
External Project ID:NSC100-2314-B182-053-MY3