| BackgroundLeukemia is a type malignant diseases originating in hematopoietic stem cells. In cancer's mortalities, leukemia ranks sixth (male) and eighth (female), and in children and adults under the age of 35, it ranked first. So it is a very serious threat to people's lives and health. At present, the leukemia treatment methods, including radiotherapy and chemotherapy, biological therapy, hematopoietic stem cell transplantation, and other modern treatment methods, have made great progress. But there are still many phenomenas, including drug resistance, relapse, and other phenomenas, which affect the patient's long-term survival rate. So we need explore the new treatment methods further.In the last 30 years, photodynamic therapy (PDT) has developed new tumor treatment methods. Photosensitizes, selectively combining with the malignant cells and tissues, in the irradiation of particular wavelength and particular power of the exposure light source, have a cytotoxic effect, thus play the role of killing malignant cells and tissues. The three elements of PDT are light, oxygen and photosensitizer. In recent years, PDT in the treatment of variouss diseases, particularly in solid tumors, has scored remarkable achievements But the domestic reports of PDT for the treatment of leukemia are rare. It is still unclear that "5 - aminolevulinic acid Hexyl -photodynamic therapy (He-ALA-PDT)" can selectively kill leukemia cells and reverse their resistance.ObjectiveThis paper was designed to study the selective killing effects of He-ALA-PDT on the multi-drug resistance human erythroleukemia cell line K562/ADM and its Parental cell line K562 and human normal cord blood mononuclear cell (CBMC). To discuss if He-ALA-PDT was an effective way to diminish the anti-multi-drug resistant leukemia cells without affecting the normal functions of hematopoietic stem cell; and to find the best photodynamic role conditions of He-ALA-PDT on leukemia cells, and to further explore the models of leukemia cell death, to explore the new ways for removing multi-drug resistant leukemia cells and reducing the disease's recurrence rate.MethodsChapterâ… The research of the best photodynamic conditions of laser exciting 5 - amino acid Hexyl selectively killing human multi-drug resistant leukemia cell line K562/ADM and parental cell line K562Comparison the similarities and differences of the killing effects of different concentrations of mitoxantrone and daunorubicin on K562, K562/ADM cell lines first. Then cells were divided into four groups: PDT group(united photosensitizer with light irradiation), laser group(only light irradiation), dark cytotoxic group(only photosensitizer), normal control group(neither photosensitizer nor light irradiation). K562 and K562/ADM cell lines were incubated with various concentrations of He-ALA(0.025,0.1,0.4,1.6mmol/L) for 4 hours, then illuminated with different light doses(4.5,9,18,36 J/cm2) using a laser at 630nm. After 12 hours' incubation, the survival rates of cells were analyzed. Comparison the similarities and differences of a single factor (laser or photosensitizer) and the Joint factors (laser + photosensitizer) on the effects of anti-leukemia cells. The cell's morphological changes were observed; the best photodynamic conditions were selected. Under the selective best conditions, the effect of PDT on CBMC was studied.Chapterâ…¡The research of the killing effect of laser exciting 5 - amino acid Hexyl on human erythroleukemia cell line K562 and drug-resistance cell line K562/ADM irradiated in vitroCells were divided into PDT group and normal control group, treated by the conditions selected in chapterâ… . Wright's staining method was used to observe the leukemia cell's morphological changes after PDT, the fluorescence intensities of photosensitizer in K562 and K562/ADM cell lines were assayed by FCM, and the changes of colony-forming abilities of leukemia cells were analyzed by methylcellulose colony-forming assay, and the changes of colony-forming abilities into BFU-E,CFU-GM,CFU-MIX of CBMC were analyzed by Cord Blood colony composite medium(MethoCultTM GF H4434) colony-forming assay. Using Annexin V-FITC/PI double - staining method for detecting of the death models of K562 and K562/ADM cell lines after PDT 2h,4h,8h.Statistical analysisThe calculation was performed using SPSS 11.5 software package. The data represented as the mean±standard deviation. Comparison in experiments was performed using Factorial Analysis, One-Way Analysis of Variance(ANOVA), Paired-Sample t-test to assess the statistical significance of differences between groups. Differences with P<0.05 were considered to be significant. ResultsChapterâ… The research of the best photodynamic conditions of laser exciting 5 - amino acid Hexyl selectively killing human multi-drug resistant leukemia cell line K562/ADM and parental cell line K5621. Comparison the similarities and differences of the killing effects of different concentrations of mitoxantrone and daunorubicin on K562, K562/ADM cell lines: The killing efficiencies of mitoxantrone and daunorubicin of K562 were significantly higher than that of K562/ADM(P<0.001)2 . The average survival rates of dark cytotoxic group and laser group: The average survival rate of dark cytotoxic group was all more than 96 percent; the one of laser group was all more than 86 percent.3 . The killing effects of various concentrations of He-ALA and different light doses on K562 and K562/ADM cell lines:3.1 The influence of various concentrations of He-ALA on the viability of K562 and K562/ADM cell lines: Under the same light dose condition, as the photosensitizer concentrations gradually increased from 0.025 mmol/L to 0.4 mmol/L, the viabilities of K562, K562/ADM cell lines decreased. Two of each group compared with significant differences(P<0.001). Under the various light doses conditions, when the photosensitizer concentrations were 0.4mmol/L and 1.6mmol/L, there was no significant difference between the survival rates K562/ADM cell line(P=0.158). while the survival rates of K562 cell line keep on decreaseing(P=0.001).3.2 The influence of different light doses on the viability of K562 and K562/ADM cell lines: Under the same photosensitizer concentration condition, as the light doses gradually increased from 4.5J/cm2 to 18J/cm2, the viabilities of K562, K562/ADM cell lines decreased. Two of each group compared with significant differences(P<0.001). Under the various photosensitizer concentrations conditions, when the light doses were 18J/cm2 and 32J/cm2, there was significant difference between the survival rate of K562 cell line(P<0.001), while there was no significant difference between the survival rate of K562/ADM cell line(P=0.077).3.3 The contrast of the viability of K562 and K562/ADM cell lines: When the photosensitizer concentration was 0.4 mmol/L, as the light doses gradually increased from 9.0J/cm2 to 36J/cm2, the viability of K562/ADM cell line was significantly higher that of K562(P<0.001); When the laser dose was 18J/cm2, as the photosensitizer concentration increased gradually from 0.1 J/cm2 to 1.6 J/cm2, the viability of K562/ADM cell line was also significantly higher that of K562(P<0.001)4 . The influence of PDT on the viability of CBMC: The survival rates of normal control group, 0.4mmol/L dark cytotoxic group, 1.6mmol/L dark cytotoxic group, 18J/cm2 laser group, 36J/cm2 laser group, 0.4mmol/L+18J/cm2 PDT group, 0.4mmol/L +36J/cm2 PDT group, 1.6mmol/L+18J/cm2 PDT group, 1.6mmol/L +36J/cm2 PDT group respectively were as follows: 100.00±2.12%,99.00±2.16%,96.12±3.56%,94.17±2.25%,91.13±1.18%,92.17±2.14%,83.53±2.64%,90.57±4.61%,81.76±3.78%Chapterâ…¡The research of the killing effect of laser exciting 5 - amino acid Hexyl on human erythroleukemia cell line K562 and drug-resistance cell line K562/ADM irradiated in vitro1. The fluorescence intensities of photosensitizer in K562 and K562/ADM cell lines assayed by FCM: The fluorescence intensities of photosensitizer in K562 and K562/ADM cell lines were 86.7% and 59.8%.2 . The colony forming rates of K562 and K562/ADM cell line changed after PDT: The colony-forming rates of K562 normal control group and PDT group respectively were 34.53±0.81% and 1.03±0.06%; The colony-forming rates of K562/ADM normal control group and PDT group respectively were 53.00±0.66% and 12.20±0.95%. PDT could destruct both leukemia cell lines significantly(PK562 <0.001, PK562/ADM), while there were more significantly destructions on K562 than K562/ADM cell lines(P=0.003).3. The colony forming rates of CBMC: The colony forming rates into BFU-E,CFU-GM and CFU-MIX of normal control group and PDT group respectively were 35.33±2.08% and 34.67±1.15%,29.00±4.36%,27.33±2.31%,47.00±2.00% and 44.00±3.00%. Contrast with control group, there were no significant differences.4. Analyzing of the death model of K562 and K562/ADM cell lines after PDT: Contrast total viabilities of both, the mortality of K562 is significantly higher than that of K562/ADM. For K562 cell line, the part of apoptosis was major, while the part of necrosis was minor; For K562/ADM. the ratio of apoptosis was very close to that of necrosis. As the time of cultivation extended after PDT, the viabilities of both were further reduced, and the proportion of apoptotic cells reduced, while the percentage of necrotic extension increased.SummaryHe-ALA-PDT can clearly kill human leukemia cell line K562 and multi-drug resistance cell line K562/ADM, lead to leukemia cells' apoptosis and necrosis, reduce leukemia cells' colony forming rates, and has little effect to the CBMC normal activity and self-replication and multi-functional differentiation. So it won't affect the hematopoietic reconstructing function of normal stem cell. There are different degrees of sensitivity to the PDT between two kinds of cells, and K562 cell line is more sensitive than K562/ADM. Photosensitizer concentration of 0.4 mmol/L and light dose of 18J/cm2 are the best conditions of PDT for K562 and K562/ADM cell lines. Our experiments prove that He-ALA-PDT is a selective and efficient anti-leukemia cells method. It provides an experimental basis for the applying PDT to the field of graft purification of autologous hematopoietic stem cell transplantation, and to lowing the relapse rate of the desease.
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