| Objective:To investigate whether the extraction of the infective larvae of Ascaris lumbricoides could induce apoptosis of human alveolar epithelial cells and how the extraction concentration and inducing time could be related to the apoptosis.Methods:1. Ascaris lumbricoides adult worms were collected from children at the primary school of Changyi Township, Xingjian County, Jiangxi Province, China, after an oral administration of pyrantel pamoate at one dosage of 30 mg/kg of body weight. 2. Eggs were collected from the uteri of female Ascaris lumbricoides. Eggs were then decoated in 0.5% sodium hypochlorite, washed and cultured until the infective stage for 2-3 weeks in 0.1N sulfuric acid at 30±1℃.3. The infective-stage eggs were transferred into an Erlenmeyer flask containing glass beads (diameter 2 mm) withsmooth surface and the egg suspension was stirred at 60 rotations/min for 2-3 min to remove shells. Larvae suspension were transferred onto the surface of Ficoll-Hypaque in a tube, and centrifuged at 1000rpm for 10 min. The larvae were harvested from the sediment and washed with normal saline and homogenized in a tissue grinder in icebath, followed by three times of repeated freezing and thaw, the supernatants after centrifuged at 1000rpm for 10 min were extraction and stored at -30℃ for later use.4. According to the result of cytotoxicity experiment (MTT), five different concentrations of the extraction of larvae (i.e. 10(g/ml, 30(g/ml, 100(g/ml, 300(g/ml and 600(g/ml) were chosen to induce A549 cells. Meanwhile, control groups without the inducement were set up. For each group, observations were made at five time points after the inducement, to assess the existence of apoptosis and percentages of cells showing characteristics of apoptosis.5. HE stain and diphenylamine reaction methods were mainly used to assess apoptosis of A549 cells. Furthermore, for some group, transmissional electron mocroscope, agarose gel electrophoresis of DNA and Flow cytometry were also employed to confirm apoptosis.Results:1. With HE stain method, characteristic morphological changes related to apoptosis were observed in a large part of A549 cells induced by the extraction of infective larvae at different concentrations, such as intensive condensation of chromatin into dense masses, dramatic cell shrinkage, concentration of cytoplasm and formation of apoptotic bodies. But these were not seen in the most of cells in control groups. Observations under transmissional electron microscope also showed these similar characteristic changes. The agarose gel electrophoresis demonstrated a characteristic ladder-like DNA degradation pattern from induced A549 cells but not in control groups. Flow cytometric examination revealed a typical diploid DNA peak in control A549 cells, but the extraction induced cells showed a hypodiploid DNA peak and an impressive reduction in diploid DNA peak. 2. The observation from HE stain as well as diphenylamine reaction methods indicated that the higher percentages of apoptotic cells were related to the inducement with higher concentration of extraction in all time points. For all the five groups with different concentrations, the ratios of apoptosis were increasing as the inducing time prolonged up to 5 hours sincethe induce started, but decreasing after that time. Conclusions:1. The extraction of infective larvae of Ascaris lumbricoides can induce apoptosis of human alveolar epithelia in vitro.2. Apoptosis of A549 cells induced by Ascaris larvae extraction implies a concentration-dependent pattern, namely, higher extraction concentration always induced higher apoptosis ratio. With regard to the relationship between the intensity of apoptosis and time of inducement, two kinds of trend revealed. That is, with the inducing time increased up to 5 hours of post-inducement, the ratio of apoptosis increased also, but after that time point, the ratios decreased gradually. This kind of relationship was also found to be influenced by the concentra... |
PDF Full Text Request