| Objectives: To establish the method of Rhodamine 123 (Rhl23)-mediatedphotodynamic therapy, and observe the effects of photodynamic therapy on proliferation and activation function of mice and human lymphocytes, on the Colony Forming Unit-Granulocyte Macrophage colony formation of mice bone marrow(BM), to evaluate the property of photodynamic therapy on selective purging host-reactive mice and human T lymphocytes and the property of preserving response of resting T lymphocytes to third-party antigen, to observe the prevention of acute graft versus host disease (aGVHD) in mice allogeneic bone marrow transplantation(BMT) model, and then to evaluate the efficacy and safety of photodynamic therapy for the prevention of aGVHD.Methods:1 Establishment of the experimental condition of photodynamic therapy: Argon laser device (wavelength 514 nm) was used for light source. BALB/c mice spleen mononuclear cells (MNCs) were used as stimulator cells , inactivated by mitomycin C, and C57BL/6 mice spleen MNCs were used as as responder cells. Mixed lymphocyte culture (MLC) was performed at 37℃ in a fully humidified 5 %CO2 incubator for 72 h, then (1) RM23 (50 nmol/L) was added , cocultured for 30 min, 40 min, 50 min7and 60 min respectively , uptake rates of Rhl23 by MLC-MNCs and C57BL/6 spleen cells at different time points were tested by flow cytometry. (2)) Mixed lymphocytes were plated in 96-well round-bottom plate, cocultured with Rhl23( with final concentrations 10 nmol/L -60 nmol/L respectively); (3) Mixed lymphocytes received laser irradiation(power intensitiy 10 mW/cm2-60 mW/cm2, irradiation time 3 min or 4 min respectively); (4) Mixed lymphocytes cocultured with Rhl23 (50 nmol/L ) for 40 min, then received laser irradiation (power intensitiy 10 mW/cm2-60 mW/cm2 , 3 min or 4 min respectively) . Above three groups continued to coculture for 24 h, As7o were tested by MTT assay.2 Impact of photodynamic therapy on colony formation of mice bone marrow (BM) progenitor cells: C57BL/6 BM MNCs were cocultured with Rhl23 (concentrations 30 nmol/L-60 nmol/L respectively)for 40 min, then received laser irradiation for 3 min (power intensitiy 10 mW/cm2-50 mW/cm2 respectively) , cultured at 37 in a fully humidified 5%CO2 incubator for 14 days. Colony Forming Unit of Granulocyte Macrophage ( CFU-GM) were observed and counted under a multifunction inverted microscope.3 Effect of photodynamic therapy on CD69 expression on early activated mice T lymphocytes: C57BL/6 mice and BALB/c mice mixed lymphocytes cocultured for 24 ru then treated with photodynamic therapy : coincubated with Rhl23 (50 nmol/L), then received laser irradiation for 3 min (30 mW/cm2) , CD69 expression of T lymphocytes was tested by flow cytometry.4 Influence of photodynamic therapy on proliferation of human lymphocytes: Three samples of peripheral blood MNC were obtained from three healthy donors A,B and C. MNC from individual A and MNC from individual C were inactivated by mitomycin C and used as stimulator cells , MNC from individual B was used as responder cells. Mixed lymphocytes culture was conducted ( S/R 1:1 ratio) in 96-well round-bottom plate at 37, in a fully humidified 5%CC<2 incubator for 72 h, then MNCs were resuspended with Rhl23 (50 nmol/L) for 40 min, received laser irradiation (power intensitiy 10 mW/cm2, 30 mW/cm2, 50 mW/cm2 for 3 min respectively) . TheseMNCs treated with photodynamic therapy were divided into two groups: (1) presented with the same stimulator cells from individual A for the second time for more 24 h coculture; (2) presented with the third- party stimulator cells from individual C for more 24 h coculture . ASVQ were tested by MTT assay.5 Establishment of aGVHD mice model of allogeneic BMT: C57B/6 H'2b donor mice BM-MNCs (4X 106) together with spleen-MNCs( 4X 106) were injected intravenously via the tail vein into BALB/c H~2d recipient mice conditioned with total body irradiation (TBI, 60Co, 8.0 Gy, dose rate 0.7513 cGy/min). Normal saline group, syngeneic BM group... |
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