| Melatonin is an indole compound Synthetic secretionby human and mammalian pineal gland,Its chemical name is N-acetyl-5-methoxytryptamine.In addition to pineal gland,MLT synthesis was found in several other organs,including retina,gastrointestinal tract,skin,bone marrow and lymphocyte.MLT was found to be associated with circadian rhythms of organisms,and later found to have significant antineoplastic effects.However,it is unknown whether MLT can affect TAMs cells in TME to relieve immune escape from tumors.Method1.High performance liquid chromatography-mass spectrometry(LC/MS/MS) method for analyzing the concentration of melatonin(MLT)in biological sample was established.2.Pathological changes of tumor tissues in tumor-bearing mice were detected by HE.3.The purpose of this study was to detect the fluctuation of melatonin in different organs of the Gastric Tumor-Bearing Mice within one day by LC/MS method. Including serum,pineal gland,thymus and spleen,in order to clarify the changes of endogenous MLT after tumorigenesis.4.The purpose of this study was to detect the fluctuation of Exogenous melatonin different organs of the gastric Tumor-Bearing Mice within one day by LC/MS method.Including serum,pineal gland,thymus and spleen,in order to clarify the changes of endogenous MLT after tumorigenesis.5.The concentrations of melatonin in serum and organs,including pineal gland, thymus,spleen,gastrointestinal tract,liver and tumor body,were measured by LC/MS/MS at different time after injection of melatonin in gastric cancer mice in order to determine the retention of exogenous melatonin in different organs after tumor formation.6.From the perspective of TEM,flow cytometry was used to analyze the proportion of m1-type and m2-type macrophages in tumor tissues of tumor-bearing mice to determine the changes of m1-type and m2-type macrophages in tumor tissues after melatonin intervention.Results1.The LC/MS/MS method for the detection of biological samples of melatonin has been preliminarily established,which is accurate,reliable and stable,and suitable for the concentration detection of biological samples.The standard melatonin had good linearity in the range of 4-1000 pg/ml Under the determined chromatographic conditions,the melatonin in the biological samples of mice had a good separation effect with no impurity interference,and the retention time was moderate(t _R=3.0 min).The separation degree was greater than 1.5,and the minimum quantitative concentration is 4 pg/ml.2.He staining results showed that there were a large number of hyperchromatic megakaryocytes in the transplanted tumor.The tumor cells were diffusely distributed,large in size,irregular in shape,distinct in heteromorphosis,rich in blood vessels and distinct in necrotic areas.3.The results of LC/MS/MS showed that 615 mice could synthesize and secrete MLT and had circadian rhythm.The concentration of MLT in pineal gland was relatively low and did not fluctuate much during the day(6:00-18:00),the peak was reached at 22:00,and the lowest concentration appeared at 10:00;the concentration of MLT in gastroduodenum was relatively low at night(22:00-6:00),the peak was reached at 10:00 and 18:00 in the day(14:00);the concentration of MLT in spleen was relatively low at 22:00-6:00,and the peak was reached at 10:00 and 18:00 in the day,The lowest MLT concentration appeared at 2:00;the MLT concentration in thymus was relatively low at night(18:00-2:00),peaked at 10:00 in daytime,and the lowest MLT concentration appeared at 14:00;the MLT concentration in liver was relatively high at night (18:00-2:00),peaked at 10:00 in daytime and 2:00 in nighttime,and the lowest MLT concentration appeared at 6:00;the MLT concentration in serum peaked at18:00,and the lowest MLT concentration appeared at 14:00 In the rest time,MLT concentration fluctuated smoothly.4.The results of LC/MS/MS showed that MLT could be synthesized and secreted by gastric cancer mice with circadian rhythm.MLT concentration in gastroduodenum was relatively low at night(22:00-2:00),and reached the peak at day time(10:00),with the lowest concentration at 2:00;MLT concentration in spleen was relatively low at 14:00-6:00,and reached the peak at day time(10:00),with the lowest concentration at 2:00;MLT concentration in thoracic gland was relatively low at night(22:00-2:00),and reached the peak at day time(14:00),with the lowest concentration at night(22:00-2:00)The concentration of MLT in the pineal gland was relatively low and fluctuated little in the daytime(6:00-14:00),peaked at night(22:00),and the lowest concentration appeared at14:00;the concentration of MLT in the liver was relatively low at night(22:00-2:00),peaked at day(6:00),the second peaked at 14:00,and the lowest concentration appeared at 2:00;the concentration of MLT in the serum peaked at18:00,The lowest concentration appeared at 14:00,and the concentration of MLT fluctuated greatly in the rest time.5.The results of LC/MS/MS showed that the concentration of MLT in thymus of normal mice was relatively high.Except for 14:00 and 18:00,the concentration of MLT in thymus of mice with gastric cancer was significantly higher than that of other organs.The concentration of MLT in thymus of mice with gastric cancer was higher than that of other organs at any time.The concentration of MLT in spleen and gastroduodenum of normal mice and mice with gastric cancer was relatively high The MLT concentration in spleen and gastroduodenum of normal mice was higher than that in stomach and duodenum except 2:00 and 6:00.The MLT concentration in spleen of gastric cancer bearing mice was similar to that in thymus at 10:00.Except 14:00 and 18:00,the MLT concentration in spleen of normal mice was higher than that in stomach and duodenum.The MLT concentration in liver of normal mice was the same as that of gastric cancer bearing mice The concentration was at the lowest level at any time.6.The results of LC/MS/MS showed that the MLT concentration in serum,pineal gland,thymus,spleen,gastroduodenum and liver of mice with gastric cancer was higher than that of normal mice.The MLT concentration in pineal gland was higher than that in normal mice at 6:00,10:00,18:00,22:00 and 2:00;the MLT concentration in serum was higher than that in normal mice at 6:00,10:00,18:00,22:00 and 2:00;the MLT concentration in thymus was higher than that in normal mice at 6:00,14:00,18:00 and 2:00;the MLT concentration in spleen was higher than that in normal mice at 6:00 and 10:00;the MLT concentration in liver was higher than that in normal mice at 6:00,14:00,18:00 and 2:00 The concentration of MLT in gastroduodenum was higher at 6:00,14:00 and 2:00 than that in normal mice.7.The results of LC/MS/MS showed that the MLT concentration in the organs of mice with gastric cancer was in the order of tumor tissue,spleen,liver,gastroduodenum and thymus from high to low 15 min after MLT injection;the MLT concentration in the organs of mice with gastric cancer was in the order of gastroduodenum,thymus,tumor tissue,liver and spleen from high to low 1 h after MLT injection;4 h The concentrations of MLT in each organ were thymus,tumor tissue,spleen,gastroduodenal liver from high to low after H.8.The results of flow cytometry showed that:after one week of MLT intervention, compared with the control group,M1 macrophages had a significant decrease trend,and the proportion of M2 macrophages had no significant change.Conclusion1.The LC/MS/MS detection method of MLT in different biological samples was established.The method is accurate,reliable,stable,highly sensitive and reproducible.It is suitable for MLT concentration detection in biological samples.2.615 mice can synthesize and secrete MLT,which presents its own rhythmic fluctuation in pineal gland,thymus,spleen,gastroduodenum,liver and blood.3.the MLT concentration in different organs of 615 mice was the highest in thymus,the second in spleen,stomach and duodenum,and the lowest in liver.4.The circadian rhythms of MLT in the pineal gland,spleen and blood did not change significantly after gastric cancer transplantation.The highest and lowest MLT in thymus moved,and the circadian rhythms of MLT in gastroduodenum and liver changed significantly.5.The MLT concentration in the pineal gland,thymus,spleen,gastroduodenum,liver and blood increased after gastric cancer transplantation.6.The MLT concentration in tumor tissue was the highest 15 minutes after exogenous melatonin was applied to mice with gastric cancer,the MLT concentration in gastroduodenum was the highest 1 hour later,and the MLT concentration in tumor tissue was the highest 4 hours later.7.MLT significantly decreased M1 macrophages in tumor tissues of mice with gastric cancer,but had no effect on M2 macrophages. |
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