| Objective:To explore the dose conditions and time conditions for the steady amplification of oncolytic vaccinia virus S6,so as to realize the efficient production and stable titration of oncolytic vaccinia virus S6,to meet the requirements of subsequent experiments.Method:1)First of all,20μl,40μl,60μl,80μl,100μl of oncolytic vaccinia virus S6 was inoculated into He La cells,by comparing the average fluorescence intensity and infection rate of the different dose groups above to select the appropriate inoculation dose;2)Then select the best harvest time by comparing average fluorescence intensity and infection rate of 12h,24h,36h,48h,60h in He La cells of the same density with the same dose of oncolytic vaccinia virus S6,The optimized virus amplification conditions were further verified by comparing the virus titration at different harvest times with the Plaque experiment.Results:The initial inoculation dose of 20μl in the 36h and the differences in average fluorescence intensity and infection rate in the 40μl,60μl,80μl,100μl group were not statistically significant(P>0.05),so the initial viral dose of 20ul was selected,and the difference in average fluorescence intensity and infection rate at 20μl in time was statistically significant(P<0.05),48h to the highest,preliminary speculation 48h this point in time is suitable for harvesting virus point in time,the 60h of the detection results of the virus titration in the 10~5 PFU range,the 48h harvest of the virus titration in the range of 10~6 PFU,two time points virus titration is statistically significant(P<0.05).Conclusion::After dose optimization and time optimization,we can concluded that the highest virus titration was harvested in the 48h and 20ul,the titration can improved five to ten fold,eventually,the titration can reach about 10~6PFU。Objective: To Explore oncolytic vaccinia virus S6 can be used to detect bladder cancer cell in a large number of white blood cell populations,and whether circulating tumor cells can be detected from bladder cancer patients,in order to providing an auxiliary means for clinical diagnosis and treatment of bladder cancer patients.Method: Cell meter evaluates the difference in the size of bladder cancer cells and white blood cells,by using fluorescent microscope to evaluate the infection rate and average fluorescence intensity of oncolytic vaccinia virus S6 at all points in time of T24 cells,flow cytometer was used to detect infection of bladder cancer cells and white blood cells,and high content imaging system evaluates oncolytic vaccinia virus S6 infection of bladder cancer cells and white blood cells,fluorescent microscope qualitative evaluation of the identification of T24 bladder cancer cells in a large number of white blood cell populations,using the remaining 3 types of bladder cancer cells RT4,UMUC3,SW780 verified evaluation results,and the high-content cell imaging system identifies circulating tumor cells in blood samples of bladder cancer patients,thus inferring the feasibility of using oncolytic vaccinia virus S6 to detect circulating tumor cells in patients with bladder cancer.Cellomics TM can detected three CTCs,one of which was mesenchymal,and the other two were epithelial.Results: The difference between bladder cancer cells and white blood cell size was statistically significant(P<0.05),and the infection rate and average fluorescence intensity(P>0.05)of T24 cells were assessed under a fluorescent microscope,but the average infection rate and average fluorescence intensity were slightly higher than 12 h in 16 h,The infection rate of bladder cancer cells was detected by flow cytometers is about 70%,and there is strong PE signal interference in the white blood cell population The oncolytic vaccinia virus S6 is almost non-infected to white blood cells under the high content imaging system,the infection rate and infection spectrum of bladder cancer cells are evaluated under high content imaging system,and the infection rate of T24 cells can reach more than 80% at MOI 1,and the infection rate of T24 cell is up to 90% at MOI 2 The above infection rate,2 groups of infection rate difference is statistically significant(P<0.05),at MOI 1,by comparing infection rate of virus S6 to bladder cancer cells of T24,UMUC3,SW780,RT4,The difference is statistically significant,under the fluorescent microscope can identify bladder cancer cells with a diameter greater than the white blood cell population and express PE,in the high-content instrument using virus S6 in 2 bladder cancer patients 4 ml blood samples can detect PE-positive cells,initially thought PE-positive cells may be CTC.Cellomics TM can detected three CTCs,one of which was mesenchymal,and the other two were epithelialConclusion: The detection limition of oncolytic vaccinia virus S6 on bladder cancer cells is high,white blood cells almost no infection,increase the dose of the virus can increase the infection rate of bladder cancer cells,the initial judgment of the oncolytic vaccinia virus S6 maybe detecte circulating tumor cells from the blood samples of bladder cancer patients,taken as a whole,the use of oncolytic vaccinia virus S6 detection of bladder cancer cells is promising. |
