1.1 Bacterial Strain Salmonella typhimurium Standard strain CMCC 50115 was purchased from the National Institute for the Control of Pharmaceutical and Biological Products. Using the flat paper method, piperacillin drug paper was used to induce Salmonella typhimurium into L forms [2]. Subculture to obtain fresh culture of Salmonella typhimurium L forms, and use sterile 3% NaCl hypertonic saline to make 108 CFU/ml bacterial solution. Counting adopts McDonald's turbidimetric method. Because bacterial L forms have no cell wall, they can pass through the sterile filter. While the bacterial prototype has a cell wall, they cannot pass through the sterile filter. The bacterial liquid passed through the sterilizing filter to ensure that the bacterial L form liquid was obtained for subsequent experiments.
1.2 Cell line According to the reference methods [3,4], Human umbilical vein endothelial cells were used as vascular endothelial cells in the experiment.
1.3 Main reagents Annexin V-FITC/PI double staining cell apoptosis detection kit was purchased from Nanjing KaiGi Bioengineering Co., Ltd.; Caspase-9 activity detection kit was purchased from Shanghai Beibo Biological Company.
1.4 Main instruments Fluorescence inverted microscope, Nikon, Japan; flow cytometer, BD company; microplate reader, Bio-Rad Model 550.
1.5 Cell culture Place the vascular endothelial cells in fresh RPMI-1640+10% FBS culture medium, and place them in a 37℃, 5% CO2 incubator, and harvest the cells at a concentration of 105/ml. Inoculate a 6-well cell culture plate with 2 ml cell suspension per well. After 12 hours of regular culture, discard the old culture medium and replace it with fresh RPMI-1640 culture medium without calf serum for testing.
1.6 Experimental grouping and induction
The experimental groups were as follows: group A: L-form induction group, 100μl of Salmonella typhimurium L forms bacterial solution made by adding sterile hypertonic saline to each hole of the cell culture plate; group B: 3% NaCl hypertonic saline control group, adding 100μl of sterile 3% NaCl hypertonic saline to the control well; group C : Normal saline control group, adding 100μl of sterile normal saline to the control well. The cells of each group were collected 8 hours after the inducing factors were added, and the Annexin V-FITC/PI double staining flow cytometry was performed to detect the apoptosis of vascular endothelial cells induced by Salmonella typhimurium L-form, and the level of Caspase-9 activation was detected by spectrophotometry. Make another cell slide, pre-place clean sterile small slides in each well of the cell culture plate, and then perform the experiment. After 8 hours of induction, the cell slides were taken out for Annexin V-FITC/PI double staining fluorescence microscopy.
1.7 Apoptosis detection
1.7.1 Annexin V staining flow cytometry AnnexinV-FITC/PI double staining flow cytometry was used to detect apoptotic cells. Collect the cell suspension and operate according to the instructions. After the cells are double-stained with AnnexinV-FITC/PI, they are washed twice with PBS, and tested and analyzed on the machine.
1.7.2 Direct fluorescence method Use AnnexinV-FITC/PI double stain fluorescence method to detect apoptotic cells. Take cell slides, double stain with AnnexinV-FITC/PI, wash twice with PBS, observe and take pictures with inverted fluorescence microscope.
1.7.3 Spectrophotometry Caspase-9 activity detection kit is based on Caspase-9 which can catalyze the substrate Ac-LEHD-pNA to produce yellow pNA. The activity of Caspase-9 can be detected by measuring the absorbance of the reaction solution. Operate according to the kit instructions, collect the cell suspension, lyse the cells to collect the protein, add detection reagents, measure the absorbance value of A405 with a microplate reader, and subtract the absorbance value of A405 of the blank control without pNA to calculate the actual absorbance produced by pNA, Then to calculate the ratio of the experimental group and the control group to determine the activity of Caspase-9.
1.8 Statistical methods to use t test and SPSS10.0 statistical analysis software for data processing.