Biological Effects Of ALA-PDT On PBMCs From Healthy And Psoriatic Patients

In the present study, we obsearved the effects of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) on the peripheral blood mononuclear cells (PBMCs) from healthy and psoriatic patients, and explored the immune mechanism, feasibility and suitable treatment parameter of ALA-PDT on psoriasis, which might provide new information for the treatment of psoriasis.1. Peripheral blood mononuclear cells were isolated from healthy people and psoriatic patients, respectively.2. MTT were used to detect the survival of PBMCs from healthy after ALA-PDT at indicated concentration of ALA (0.1 mmol/L,0.6 mmol/L,1.2 mmol/L,1.8 mmol/L,3.6mmol/L) and incubation time (0.5, 1h,3h,5h), than the suitable concentration and incubation time were selected.3. Annexin V/PI double-staining combined with flow cytometry(FCM) were used to detect the apoptosis of PBMCs from healthy after ALA-PDT at indicated concentration of ALA (0.1 mmol/L,0.6 mmol/L,1.2 mmol/L,1.8 mmol/L,3.6mmol/L) and incubation time (0.5, 1h,3h, 5h), to furdher verify the selected concentration and incubation time.4. Transmission electron microscope (TEM) were used to observe the ultra-structure changes of PBMCs from healthy after ALA-PDT (0.6 mmol/L ALA, incubated 1h).5. RT-PCR was used to detect the mRNA expression of T helper 1/T helper2 (Th1/Th2)-type cytokines of PBMCs from healthy and psoriatic patients.1. PBMCs from healthy were incubated with ALA at indicated concentration (0.1,0.6,1.2,1.8, 3.6mmol/L) for 0.5h, 1h,3h or 5h separately at 37℃in the darkness before treatment with 635nm laser. MTT results showed that the survival rate was lowest in ALA-PDT group incubated with 0.6 mmol/L ALA for 1 hour, when compared with control group.2. The ALA-PDT group incubated with 0.6 mmol/L ALA for 1 hour showed the highest PBMCs apoptosis rate as 8.5±0.5%(P<0.05), compared with control group (4.3±0.1%). 3. Apoptotic PBMCs were observed by TEM after treatment with ALA-PDT, but none in control group.4. RT-PCR showed that mRNA expression of IFN-γand TNF-αin psoriatic PBMCs was significantly higher than that in healthy control. Treatment with ALA-PDT clearly decerased mRNA expression of IFN-γand TNF-α, moreover mRNA expression of IFN-γreached to the basic level. In addition, IL-4 mRNA in psoriatic PBMCs was lower than that in healthy control, ALA-PDT markedly increased mRNA expression of IL-4, no reaching to control level. IL-10 mRNA in psoriatic PBMCs was a little bit lower than that in healthy control, and raised some after ALA-PDT.1. The suitable concentration of ALA was 0.6 mmol/L and incubation time was 1 hour for ALA-PDT on human PBMCs.2. ALA-PDT could markedly promote cell apoptosis of PBMCs from healthy at 0.6mmol/L concentration and 1 hour incubation time.3. Psoriatic PBMCs showed abnormal Th1 immune responses was downregulated after ALA-PDT, meanwhile Th2 immune responses was upregulated.