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1.
Indocyanine green (ICG) is clinically approved for the determination of liver function, cardiac output and plasma volume. In this pilot study, ICG was used as photosensitizer in combination with a diode laser to treat AIDS-associated Kaposi's sarcoma (KS) in three patients. Directly and up to 50 min after intravenous administration of ICG (2-4 mg kg(-1) body weight), KS (n=57), mainly plaque-type, were irradiated using a diode laser (lambda em=805 nm, 100 J cm[-2], 0.5-5 W cm[-2]) matching the absorption maximum. Complete remission of KS (n=16) was achieved when irradiated 1-30 min after injection of the second dose of ICG (2 x 2 mg kg(-1) b.w., 30 min apart) with 3-5 W cm(-2) and 100 J cm(-2). Biopsies (n=3) revealed necrosis of the tumour 24 h and complete remission 4 weeks after therapy. In general, systemic side-effects were not observed and cosmetic results were very good. However, hyperpigmentation occurred temporarily in lesions located on the lower extremities. These findings show that AIDS-associated KS can be effectively treated after photosensitization with ICG and subsequent irradiation with an appropriate diode laser. However, additional investigations need to elucidate the exact mechanism of action of ICG-mediated phototherapy and have to show the efficacy for the treatment of other highly vascularized solid tumours.  相似文献   

2.
The deuteroporphyrin-IX derivative Dimegin [2,4-di-(alpha-methoxyethyl)-deuteroporphyrin IX] was investigated with respect to cellular uptake, intracellular localization and cell survival following photodynamic treatment in human cell lines derived from the skin (SCL1 and SCL2, squamous cell carcinoma; HaCaT keratinocytes; N1 fibroblasts). Using flow cytometry, we determined the cellular fluorescence as a marker of the uptake of Dimegin after incubation for 24 h. The intracellular localization of Dimegin was analysed using fluorescence microscopy and co-staining with fluorescent dyes specific for cell organelles. Following irradiation with an incoherent light source (580-740 nm) using a light dose of 24 J/cm2, phototoxicity was determined by means of trypan blue dye exclusion, MTT assays and growth curves. The relative Dimegin fluorescence of the different cell lines declined as follows: SCL1 > HaCaT > N1 > SCL2. Intracellular localization of Dimegin was found in the mitochondria. For all cell lines Dimegin concentrations above 15 microM yielded a significant phototoxic effect. The EC50 for SCL1 cells was 8.9 +/- 2.0 microM Dimegin. The EC50 for the cell lines increased as follows: SCL1 < HaCaT < N1 < SCL2, thus correlating with the cellular fluorescence of Dimegin. The results of the MTT assay were confirmed by trypan blue dye exclusion assay and growth curves. In conclusion, the study shows that Dimegin is an effective photosensitizer with a rapid mechanism of action in vitro, resulting in an immediate loss of plasma membrane integrity following irradiation.  相似文献   

3.
OBJECTIVE: To determine whether irradiation with a low-intensity diode laser, which produces radiation at a wavelength of 810 nm, will induce nonthermal enhancement of chondrocyte metabolism. SAMPLE POPULATION: 144 grossly normal articular cartilage explants aseptically harvested from the femoral condyles of 6 adult horses. PROCEDURE: Treated cartilage explants were irradiated with a diode laser at 1 of 7 fluence levels that ranged from 8 to 1,600 J/cm2. Explants were incubated for 24 or 72 hours, labeled for 24 hours with [35S]Na2SO4, and assayed for newly synthesized sulfated glycosaminoglycan (GAG; measured incorporation of 35SO4) and endogenous GAG, chondroitin 6-sulfate (CS), and keratan sulfate (KS) content, using a dimethylmethylene blue assay. Laser-induced temperature changes were measured during irradiation with a diode laser and a neodymium:yttrium aluminum garnet (Nd:YAG) laser, which produces radiation at a wavelength of 1,064 nm, using conditions that were reported in previous studies to increase explant metabolism. RESULTS: After incubation for 24 or 72 hours, rate of 33SO4 uptake or endogenous GAG, CS, or KS content in irradiated explants was not significantly different than in nonirradiated explants. Cartilage temperature increased < 4.75 C during diode laser application. Cartilage temperature increased 5 to 12 C during Nd:YAG laser application. CONCLUSIONS: Minimal thermal increases in cartilage explants with use of a low-intensity diode laser resulted in no change in proteoglycan metabolism of chondrocytes. An increase in tissue temperature over a narrow range with use of a Nd:YAG laser may have contributed to the metabolic alteration of chondrocytes reported in previous studies.  相似文献   

4.
BACKGROUND AND OBJECTIVE: The purpose of this study was to determine irradiation parameters of a 780 nm low power CW diode laser (6.5 mW) leading to enhanced proliferation of cultured normal human keratinocytes (NHK). The possible role of reactive oxygen species (ROS) in this response was evaluated. STUDY DESIGN/MATERIALS AND METHODS: NHK were exposed to a single dose of 0 to 3.6 J/cm2 (0-180 sec) of irradiation. Proliferation parameters studied were: incorporation of 3H-thymidine during 6-24 hr following irradiation; percentage of dividing cells and number of cells, 24 hr and 48 hr following irradiation, respectively. RESULTS: Proliferation of NHK exposed to 0.45-0.95 J/cm2 was significantly enhanced by 1.3-1.9-folds relative to sham-irradiated controls, as inferred from parameters studied. Exposure to other energy densities was considerably less effective in enhancing proliferation parameters. Added enzymatic antioxidants, superoxide dismutase or catalase, scavenging superoxide anions and H2O2, suppressed this enhanced proliferation. Added scavengers (alpha-tocopherol acetate, scavenging lipid peroxidation, or sodium azide, histidine, mannitol, scavenging singlet oxygen, superoxide anions, and hydroxyl radicals, respectively), or N-acetyl cysteine, the thiol-reducing agent, suppressed the response, but to different extents. CONCLUSIONS: The results indicate that 780 nm low power diode laser irradiation enhanced keratinocytes proliferation in vitro, with an apparent involvement of ROS in this response, and comparably, might be used to promote their proliferation in vivo to enhance wound healing.  相似文献   

5.
Apoptosis represents an active form of cell death that is involved in the control of tissue homeostasis and in the deletion of DNA-damaged cells. Because the product of the tumor suppressor gene p53 has been demonstrated to be crucial for the induction of apoptosis in certain cell types, the present study was aimed at elucidating its role in ultraviolet-induced apoptosis in HaCaT keratinocytes. After in vitro ultraviolet B irradiation, p53 protein levels were noted to increase prior to the induction of apoptosis in a time- and concentration-dependent fashion. This increase could not be inhibited by the protein synthesis inhibitor cycloheximide. Because HaCaT keratinocytes are known to bear two p53 point mutations and because it is unclear whether p53 in HaCaT cells is still functional regarding induction of apoptosis, HaCaT cells were stably transfected with wild-type p53 cDNA inserted into the expression vector pCMV-Neo-Bam in sense (pC53-SN3) and anti-sense (pC53-ASN) direction. After selection with geniticin, growing colonies were screened for the presence of the transfected cDNA constructs by polymerase chain reaction. Cell clones bearing the anti-sense product were further analyzed for p53 expression by western blotting. Clones showing reduced p53 protein levels were irradiated with ultraviolet B light, and there was a clear reduction of apoptosis in the pC53-ASN bearing cell clones compared with the parental HaCaT cells. These studies demonstrate that blocking mutated p53 can partially block apoptosis in HaCaT keratinocytes and furthermore can confirm the key role for p53 in ultraviolet-induced apoptosis in human keratinocytes. Moreover, HaCaT keratinocytes and their p53-transfectants provide a convenient model that allows for further detailed analyses of apoptosis-associated biochemical and molecular events in human keratinocytes.  相似文献   

6.
The intensity dependence of the rose bengal (RB)-photosensitized inhibition of red blood cell acetylcholinesterase has been studied experimentally and the results compared to a quantitative excitation/deactivation model of RB photochemistry. Red blood cell membrane suspensions containing 5 microM RB were irradiated with 532 nm, 8 ns laser pulses with energies between 1 and 98.5 mJ. A constant dose (7 J) was delivered to all samples by varying the total number of pulses. At incident energies greater than approximately 4.5 mJ/pulse, the efficiency for photosensitized enzyme inhibition decreased as the energy/pulse increased. The generation of RB triplet state was monitored as a function of laser energy and the triplet-triplet absorption coefficient was determined to be 1.9 x 10(4) M-1 cm-1 at 530 nm. The number of singlet oxygen molecules produced at each intensity was calculated from both the physico-mathematical model and from laser flash photolysis results. The results indicated that the photosensitized inhibition of acetylcholinesterase was exclusively mediated by singlet oxygen, even at the highest laser intensities employed.  相似文献   

7.
Signaling through the epidermal growth factor receptor (EGFR) has been primarily implicated in the growth of epithelial cells including keratinocytes. However, the mechanism by which EGFR stimulation promotes keratinocyte cell growth is poorly understood. Here we report that human keratinocytes undergo apoptosis when incubated with the blocking EGFR monoclonal antibody 225 IgG, or PD153035, a highly specific EGFR tyrosine kinase inhibitor. Endogenous mRNA and protein levels of Bcl-XL, a member the Bcl-2 family which suppresses apoptosis, were specifically inhibited by EGFR blockade. Furthermore, stimulation of EGFR signaling through two natural ligands, transforming growth factor (TGF)-alpha and epidermal growth factor (EGF), increased the expression of Bcl-XL in quiescent keratinocytes and HaCaT cells. Finally, ectopic expression of Bcl-XL in HaCaT cells increased survival after EGFR blockade when compared to untransfected cells or HaCaT keratinocytes transfected with empty vector. These results suggest that the anti-apoptotic protein Bcl-XL plays an important role in the maintenance of keratinocyte survival in response to EGFR signaling.  相似文献   

8.
BACKGROUND AND OBJECTIVE: The aim of this study was to assess CO2 laser ability to eliminate bacteria from titanium implant surfaces. The changes of the surface structure, the rise in temperature, and the damage of connective tissue cells after laser irradiation were also considered. STUDY DESIGN/MATERIALS AND METHODS: Streptococcus sanguis and Porphyromonas gingivalis on titanium discs were irradiated by an expanded beam of CO2 laser. Surface alteration was observed by a light, and a scanning electron, microscope. Temperature was measured with a thermograph. Damage of fibroblastic (L-929) and osteoblastic (MC3T3-E1) cells outside the irradiation spot and adhesion of the cells to the irradiated area were also estimated. RESULTS: All the organisms (10(8)) of S. sanguis and P. gingivalis were killed by the irradiation at 286 J/cm2 and 245 J/cm2, respectively. Furthermore, laser irradiation did not cause surface alteration, rise of temperature, serious damage of connective tissue cells located outside the irradiation spot, or inhibition of cell adhesion to the irradiated area. CONCLUSION: CO2 laser irradiation with expanded beam may be useful in removing bacterial contaminants from implant surface.  相似文献   

9.
Extensive documentation has validated the role of UV irradiation as a tumor initiator and promoter, inducing both squamous and basal cell carcinomas. Human epidermis is a tissue which undergoes active metabolism of arachidonic acid to prostaglandins which is regulated by the action of prostaglandin H synthase (also known as cyclooxygenase). One mechanism for the promotional activity of UV light may involve its ability to induce prostaglandin formation. Work in our laboratory has demonstrated that acute exposure of human keratinocytes to UVB irradiation results in increased production of prostaglandin E2 (PGE2). When cultured human keratinocytes were examined after irradiation with 30 mJ/cm2 UVB in vitro, Western blot analysis showed a 6-fold increase in COX-2 protein which was evident at 6 h and peaked 24 h after irradiation. Furthermore, when human subjects were irradiated on sun-protected skin with up to four times their minimal erythema dosage (MED) and biopsied 24 h later, upregulation of COX-2 protein expression was observed via immunofluorescence microscopy. RNAase protection assays supported this observation, showing induction of COX-2 message which peaked at approximately 12 h following irradiation in vitro. Furthermore, human squamous cell carcinoma biopsies exhibited strongly enhanced staining for COX-2 protein via immunohistochemistry and Western analysis when compared to normal non-sun-exposed control skin. Together, these data demonstrate acute upregulation of COX-2 via UVB irradiation and suggest the need for further studies of COX-2 expression as a potential pharmacological target mediating human skin tumor development.  相似文献   

10.
Cultures of human embryonic fibroblasts in vitro were subjected to helium-neon laser single and double irradiation to investigate the influence of low-energy laser irradiation on fibroblast proliferation. Mean increase in the cell number values of irradiated cells were compared with increase values of non-irradiated control samples of fibroblasts. He-Ne laser was used as a coherent source of monochromatic radiation at 632.8 nm, and Petri-dishes with cultured fibroblasts were irradiated in way to receive radiation of energy doses of 0.5; 1; 1.5 and 2J/cm2. Single He-Ne laser irradiation exhibited a significant stimulation effect on human fibroblast proliferation in cell-culture.  相似文献   

11.
BACKGROUND AND OBJECTIVES: To study tissue effects and thresholds of efficacy in producing a full-thickness scleral fistula in human eyes obtained from cadavers. The effect of laser sclerostomies created with indocyanine green (ICG) was also evaluated. MATERIALS AND METHODS: Ab externo laser sclerostomies were produced in 12 fresh human eyes obtained from cadavers using a 200-micron diameter fiber optic connected to a diode laser system. Power settings were 500, 750, 1000, 1250, 1500, and 2000 mW with a constant duration of 100 and 200 ms. The same diode laser settings were repeated in the tissues injected with ICG. RESULTS: The laser sclerostomies were associated with heat coagulation damage adjacent to the burn margins, with disruption of stromal collagen. Tissue damage was greater at higher power and longer duration. Scleral injection of ICG prior to laser sclerostomy did not enhance laser penetration. CONCLUSION: The diode laser can create a sclerostomy in human sclera with an optimum level of 1500 mW and 100 ms. ICG did not significantly enhance the ease of penetration or reduce the association thermal damage to the sclera.  相似文献   

12.
The bidirectional transepithelial fluxes of ciprofloxacin, an antibacterial fluoroquinolone, across the human intestinal epithelial Caco-2 cell-line show marked asymmetry. Basal-to-apical flux of ciprofloxacin (10 microM) exceeds apical-to-basal flux indicating net secretion. Net ciprofloxacin secretion is abolished by azide/2-deoxy-D-glucose treatment, displays saturation kinetics (Km = 0.89 +/- 0.23 mM, Vmax 44.3 +/- 4.9 nmol cm-2.h) and competition by other fluoroquinolones. A specific, active secretion in Caco-2 epithelia may explain the transintestinal elimination of ciprofloxacin observed in pharmacokinetic studies in man.  相似文献   

13.
Release of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) generated by phospholipase C (PLC) upon receptor stimulation plays an important role in the regulation of cell growth and differentiation. A second, completely different, signal transduction system involves retinoic acid (RA) and related derivatives. Binding to intracellular receptor sites can modulate keratinocyte growth and inhibits differentiation. The present study was aimed at characterizing possible interactions between the two signalling pathways in HaCaT keratinocytes. As determined by anion exchange chromatography and HPLC analysis, HaCaT keratinocytes treated with 1 microM RA for up to 72 h showed a marked decrease in Ins(1,4,5)P3 release upon stimulation with 10 microM bradykinin or 10 microM ionomycin. Thin-layer chromatography of phosphatidylinositol phosphates, the substrates of PLC, revealed no differences between RA-treated and untreated cells. Western blot analysis of the PLC isozymes present in HaCaT cells, PLC beta 3 and PLC gamma 1, showed no alterations in the expression of these proteins in RA-treated cells as compared to vehicle-treated controls. In addition, expression of the PLC-activating G protein G alpha q was not affected by RA treatment. Our results show that RA downregulates the PLC-mediated signaling system. The point of interference of this signal transduction crosstalk has yet to be elucidated. Our results suggest, furthermore, that RA-induced attenuation of keratinocyte differentiation might be mediated at least in part by the downregulation of Ins(1,4,5)P3 release.  相似文献   

14.
Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells   总被引:1,自引:0,他引:1  
There is little information concerning the intracellular function of inositol 1,3,4,5,6-pentakis- and hexakisphosphate, despite their being the most abundant inositol polyphosphates. Current opinions that they play passive roles as antioxidants (Graf, E., Mahoney, J. R., Bryant, R. G., and Eaton, J. W. (1987) J. Biol. Chem. 259, 3620-3624) or "housekeeping" molecules (Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205) arises from belief in their metabolic lethargy. However, we have discovered that cell homogenates, incubated with 5 mM fluoride and 5 mM ATP, converted both inositol hexakisphosphate (Km = 2 +/- 0.5 microM, Vmax = 9 +/- 2 pmol/mg of protein/min) and inositol 1,3,4,5,6-pentakisphosphate (Km = 13 +/- 4 microM, Vmax = 11 +/- 5 pmol/mg of protein/min) to more polar products. These reactions were also observed in intact cells treated with 0.5-20 mM fluoride, and the precursor/product relationships were confirmed by comparing the effects of fluoride on cells differentially labeled with [3H]inositol in either short-term or pulse-chase protocols. The novel products were determined to be inositol pyrophosphates because of their relatively specific hydrolysis by tobacco pyrophosphatase and alkaline phosphatase. The pyrophosphates were metabolized rapidly by cell homogenates back to their pentakisphosphate and hexakisphosphate precursors. This endogenous pyrophosphatase activity was inhibited by up to 99% by 5 mM fluoride in vitro. In intact cells incubated with 10 mM fluoride, about 20% of the inositol 1,3,4,5,6-pentakisphosphate pool, and 50% of the inositol hexakisphosphate pool were each converted to pyrophosphate derivatives within 1 h.  相似文献   

15.
A high-powered semiconductor diode laser (805 nm) has recently been developed for medical use. The laser-tissue interactions of this wavelength have been compared with Nd:YAG (1064 nm). When used in the contact mode, the extent of tissue vaporization and zones of thermal necrosis produced by these two lasers were similar. The diode laser was also an effective and haemostatic laser scalpel. This compact laser unit has potential advantages over existing Nd:YAG lasers.  相似文献   

16.
1. The modulatory effects of L-glutamate and its structural analogues, and of gamma-aminobutyric acid (GABA), on sympathetic co-transmission were studied in the rat isolated vas deferens exposed to electrical field stimulation (EFS). 2. Application of exogenous L-glutamate caused a concentration-dependent (1 microM-3 mM) inhibition of the rapid twitch component of the biphasic EFS contraction. However, L-glutamate (1 microM-3 mM) had a minimal effect on the phasic contraction induced by exogenous adenosine 5'-triphosphate (ATP, 150 microM) and noradrenaline (50 microM). Unlike L-glutamate, D-glutamate had no effect on the EFS contraction. 3. The L-glutamate-induced inhibition of the EFS contractions was significantly attenuated by the glutamate decarboxylase (GAD) inhibitor 3-mercapto-propionic acid (150 microM) and was abolished in the presence of the GABA transaminase (GABA-T) inhibitor, 2-aminoethyl hydrogen sulphate (500 microM). 4. The L-glutamate-induced inhibition of the electrically evoked contraction was not affected by the adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)(30 nM), reactive blue 2 (30 microM) or the GABAA receptor antagonist bicuculline (50 microM). However, the GABAB receptor antagonist 2-hydroxysaclofen (50 microM) significantly inhibited the L-glutamate effect. 5. Similar to L-glutamate, GABA also caused a concentration-dependent (0.1-100 microM) inhibition of the EFS contractions. This GABA-induced inhibition was not affected by either the GABAA receptor antagonist bicuculline (50 microM) or reactive blue 2 (30 microM). However, a significant attenuation of the GABA-mediated effect was recorded with the GABAB receptor antagonist 2-hydroxysaclofen (50 microM). Contractions of the vas deferens induced by exogenous ATP and noradrenaline were not affected by GABA (0.1-100 microM). 6. The L-glutamate analogues, N-methyl-D-aspartate (NMDA) (1 microM-1 mM) and quisqualate (Quis 0.1 microM-0.3 mM) had no effect, whilst kainate (Kain, 1 microM-1 mM) caused an inhibition of the EFS-induced contractions. Effects of Kain could be abolished by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dioxine (CNQX, 10 microM). NMDA, Quis and Kain had no effect on the exogenous ATP- or noradrenaline-induced contractions. 7. It is concluded that the excitatory amino acid L-glutamate modulates the electrically evoked vas deferens contraction through conversion to the inhibitory amino acid GABA by a specific GABA transaminase. The GABA formed may then act on GABAB receptors and cause inhibition of the contraction through a presynaptic mechanism.  相似文献   

17.
Apoptosis is a form of programmed cell death that is involved in cell turnover. In the present study we show that the tricyclic antidepressants (TCAS) imipramine, clomipramine and citalopram induce apoptosis in human peripheral lymphocytes. Lymphocytes were incubated with these three drugs for up to 48 h. Apoptosis was characterized by typical nucleosomal DNA fragmentation on agarose gel, as well as quantitated using 4'-6-diamidino-2-phenylindole (DAPI) staining and 3'-OH end-labeling of fragmented DNA at the single cell level. Apoptosis induced by TCAs was shown to be dose-dependent and could be detected after a 24 h incubation. The optimal concentrations of the three TCAs found to induce apoptosis were 50 microM imipramine, 20 microM clomipramine and 180 microM citalopram. Furthermore, immunofluorescence and three-color flow cytometry were used to identify the phenotype of apoptotic cells. TCA-induced apoptosis was shown to involve exclusively T-lymphocytes. Cytotoxic T-lymphocytes were more prone to undergo apoptosis than were T-helper cells. In conclusion, the present investigation clearly demonstrates that TCAs exert cell biological effects upon human T-lymphocytes. Further studies are required to determine the possible clinical relevance of these findings.  相似文献   

18.
Porphycene photosensitizers bearing two or four methoxyethyl side chains were synthesized in nine steps from commercially available starting materials. Ether cleavage led to (hydroxyethyl)- and (bromoethyl)porphycenes that were converted to vinyl and benzo derivatives. Five of the side chain-functionalized porphycenes were biologically studied in comparison with two tetra-n-propylporphycenes. Porphycenes were incorporated in small unilamellar liposomes and incubated with cultivated SSK2 murine fibrosarcoma cells. Cellular uptake and phototoxicity 24 h after 5 J/cm2 laser light treatment were determined. The porphycenes tested were between 17 and 220 times more photodynamically active than the currently clinically used sensitizer Photofrin, although extinction coefficients of the porphycenes' irradiated bands are only approximately 10-fold higher. The LD50 concentration for SSK2 cells in the incubation medium was as low as (8.5 +/- 2.8) x 10(-9) M for tetrakis(methoxyethyl)porphycene. Two methoxy or hydroxy groups enhanced cellular uptake, three or four methoxy groups both enhanced and accelerated cellular uptake of tetraalkylporphycenes. Half-life times of the uptake processes varied between (0.14 +/- 0.04) and (14 +/- 4) h and cellular saturation levels between (1.2 +/- 0.2) and (26 +/- 3) pmol/10(5) cells. When individual uptake rates were accounted for, all porphycenes had a similar "cellular" phototoxicity, pointing toward a common mechanism of action. Evidence is presented for the assumption that cell membranes are the primary targets of the tested porphycenes and that membrane solubility may play a critical role in their photodynamic efficiency. The results show that nonionic polar side chain functionalities can strongly enhance cellular uptake and antitumor activity of lipophilic porphyrinoids and thus that the known lipophilicity/activity relationship can be reversed for very hydrophobic sensitizers.  相似文献   

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