اسم المؤلف:
الهام سعيد عبد الكريم بنو
اسم المشرف:
رشيد محجوب مصلح
ناهي يوسف ياسين
المستخلص:
his study aimed investigating the cytotoxic effect of crude enterotoxins on normal and cancer cells both invitro and invivo. the present study included isolation and identification of Pathogenic Escherichia coli, From Children (under 3 - years for both sexes) infected with severe diarrhea, from March to June 2005, and as follows : *Sixtysix Islates (60%) were obtained from 110 samples. These Isolates were Identified according to morphological and biochemical tests, and for confirmation by Api - 20E System. *Serological identification for these Isolates( 66 isoleates )showed that only 12 (18%) isolates were belonged to Enteropathogenic group of E. coli.(EPEC).* The results of using Suckling Mouse Assay(SMA) showed that only 13 (19.6%) out of 66 Isolates were capable of producing heat - stable (ST) enterotoxin, therefore these isolates belonged to Enerotoxigenic group of E.coli ( ETEC).Whereas the EPEC isolates were all negative for this test. According to the toxin activily that was evaluated by (SMA) the Isolate No. 99 determined as the most efficient Isolate in producing the (ST). The same Isolate (99) of E. coli showed its ability to produce heat Labile enterotoxin (LT), by using Rabbit lileal Loops.The Isolate (99) revealed for sensitivity to Ampicillin, Gentamicin, Nalidixic acid and Nitrofurantoin antibiotics, but resistance to Amoxicillin , Cefixime, Cephallothin, Ciprofloxacin and trimethoprim.This Isolate also showed its ability to adhere by using the special media that contain the congo red stain, and by using hemagglutination test because it posseses the colonization factors antigen (CFA/1) and CFA /III However it failed in producing hemolysin enzyme that hemolyze red blood cells The toxin reserved Its activity at temperatures (20,40,60 and 80)c0 , but loss some of its effect at 100 c0 and kept its activity at 4 c0 for (24 - 48) hrs, It was found that the highly toxic activity reduced in the PH 5,9.5. The time ofmice response to enterotoxin was determined. At 90 minutes, as the maximum of response and 180 minutes as the optimum time of response. Enterotoxin was purified partially by using sepharose CL - 6B, and the molecular weight for (ST) was 17378 dalton. LD50 of both bacterial Suspension and crude enterotoxin in mice was 2.13 × 108 cell/ mice, and 48.75 mg/ mice respectively. The crude enterotoxin showed more severe effect than the bacterial suspension in spleen, Intestine, and Lung, while the effect of bacteria suspension was more severe in Liver.The therapeutic dose of crude enterotoxin was determined according to LD50 in mice it was revealed that the concentration of 390 mg/kg have the activity in reducing the tumor volume when injected directly in tumor, with an inhibition ratio between(83 - 89)% beginning at 8th day of the 25th injecting days. While when the toxin was injected intraperitonealy, the inhibition ratio of tumor was Less than the injection in tumor it self. The dose 97.5 mg/kg that given daily for 25 days showed more efficiectly in reducing the tumor in percent of 73.3. The Comparitive study between the relative volume of tumor in treated group and the relative volume of tumor in control group revealed that there was significant difference statistically important all over the treatment time.. The necrosis and fibrosis were the most important characteristics in treated groups after histopathological examination which appearantly with the progress of treatment associated with volume decrease of tumor, so it was found that the last stage of treatment showed the cancer cells presented like small Land Surrounded by dense fibrous tissue. The treatment by using all toxin concentration in both ways of injections on murine bone marrow cells, showed significant increase in blast Index (BI) and mitotic Index (MI) when compared with control.The toxic effect of crude extraction was studied in tumor cell lines (in vitro), Hep - 2 and AMN - 3 and the normal cell line REF, the study showed that the toxic effect depend on the type of cells, the dose and the time of exposure. This study revealed that the AMN - 3 Cells were more Susceptible from that of Hep - 2 Cells and the high concentration caused inhibition to the growth of the tumor cells, Specially the concentration of 60000 Mg/ml, Also growth and multiplication of REF cells. Whereas the concentrations of 1875and 3750 Mg/ml were found as an inhibitor to REF Cells. Partially purified enterotoxin (ST) showed that it's effect was found to in hibit Hep - 2, AMN - 3 and REF cells at 72 hrs of exposure and has an inducer effect to growth of cells at 24 hrs of exposure in all concentrations,but the effect was differ in the time of exposure at 48hrs,that the three concentrations (1.986,3.965and 7.95) Mg/ml showed inducing effect ,while the three hight concentrations(15.86,158.6 and1586) Mg/ml showed inhibition effect to AMN - 3 cells.Also the hight concentrations(30000 and 60000) Mg/ml werefound as inhibitor to REF cells at( 48,72)hrs,but they were inducer of growth at 24hrs of exposure. The toxicity effects of crude enterotoxin were studied in human Lymphocyte multiplication (in vitro) , and the higher concencentration showed decline in Mitotic index (MI), but It was induced cells to transform in present of mitogen, so there was inverse in blast index (BI) when compare with the control.
