| Phosphorus is an indispensable element for the metabolism and life of organisms. There is no deficiency of phosphorus in human and animal because phosphorus widely distributed in nature. However, high phosphorus content is harmful to the health of human. The hyperphosphate dietary can cause imbalance of calcium and phosphorus, leading to osteoporosis and hyperphosphatemia. It can also increase the risk of cardiovascular disease in normal person, especially in patients with chronic kidney disease. Hyperphosphatemia is the leading cause of cardiovascular calcification in CKD patients. For person with renal insufficiency, reduction of phosphorus intake is not only reducing kidney failure but also effectively prevention from kidney disease. Besides, high phosphorus can also set off water pollution and result in serious environmental and health problems. In this thesis, the biological effects of high concentration of phosphorus on the cardialmyocytes from neonatal rat were investigated, including the calcification, apoptosis of cardialmyocytes. In addition, a polyphosphate accumulating bacterium was isolated from environment sample and its ability to removal phosphorus was tested. The main results are as follows:1. The enzymatic digestion and explant culture were applied to separate the primary culture of cardiomyocytes from neonatal rat. The two methods are compared based on the viability and purity of cell separated. The results showed that the myocardial cells isolated by two methods have rhythmic beat, and the cytoplasm was stained brown with blue nuclei by immunohistochemistry method, thus the cells isolated were confirmed to cardiomyocytes. Enzyme digestion method is superior to explant culture. The viability of cells obtained with two methods was above 95% (P>0.05). The purity of cell from enzymatic digestion reached to 97.638%, higher than 82.295% from explant culture (P<0.05). According to the effect of separation conditions on the activity of cardialmyocytes, the enzymatic digestion conditions were optimized as follow:trypsin concentration,0.125%; pH 6.8-7.0; digestion time, 40 min. The purity of the cardialmyocytes reached 95% above after adherent culture for 120 min. 2. Effect of three high phosphorus concentrations (3,6, and 9 mmol/L) on the cardialmyocytes was compared according to the calcium deposition, OPN content and expression, and apoptosis of cell. The results showed that in comparison with the control, the deposit of calcium in three high phosphorus groups were significantly higher (P<0.05), and the content of calcium deposition depended to the culture time and phosphorus concentration; the expression of OPN in three high phosphorus groups was significantly increased with the increase of culture time and phosphorus concentration(P<0.05).3. High phosphate led to the pro-apoptotic of cardialmyocytes. Caspase3 of the cardialmyocytes in high phosphorus groups increased after 2 d. Caspase3 content in the control group was 0.034 pmol/L while 0.344 pmol/L in the 9 mmol/L phosphorus group. After the cells cultured for 6 d, the apoptosis fragments of cardialmyocytes appeared in the groups with 6 mmol/L and 9 mmol/L of phosphate.4. A polyphosphate accumulating bacterium was isolated from wastewater and sludge samples. On the basis of physiological and biochemical poperties and 16S rDNA analysis, the isolate was identified to Arthrobacter sp. The phosphorus-removing conditions were tested in synthetic wastewater with initial phosphorus concentration of 20 mg/L. The results showed that when the temperature was 32℃, pH 7, inoculum size 10%, seed age 16 h, and a certain trace elements, total phosphorus in synthetic wastewater from 20 mg/L decreased to 0.1 mg/L after 10 h, phosphorus removing rate rechead to 99.5%. |
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