Method of improving efficiency of low temperature plasma to kill cancer cells through targeted protective autophagy
A low-temperature plasma and cell technology, applied in the field of biomedicine, can solve problems such as large side effects, unsatisfactory prognosis, and easy drug resistance, and achieve the effect of improving killing efficiency and cancer killing efficiency
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Embodiment 1
[0036] Example 1 CCK-8 detects the killing effect of low temperature plasma on cancer cells
[0037] 1. Experimental device:
[0038] A dielectric barrier discharge low temperature plasma device was used (J.Ma,H.Zhang,C.Cheng,J.Shen,L.Bao,W.Han.Contribution of hydrogen peroxide to non-thermal atmospheric pressure plasma induced A549 lung cancer cell damage. Plasma Process Polym. DOI: 10.1002 / ppap.201600162; J.Hou # , J.Ma #, K.N.Yu, W.Li, C.Cheng, L.Bao, W.Han.Non-thermal plasmatreatment altered gene expression profiling in non-small-cell lung cancerA549 cells.BMC Genomics 16(2015)435.), the device The structure diagram of the main components is as follows figure 1 As shown, the dielectric barrier discharge low-temperature plasma device is mainly composed of 3 parts, a high-voltage power supply, a plasma reaction chamber, and a working gas; wherein, the plasma reaction chamber is composed of 4 pairs of electrodes, and all embodiments of the application select helium as wor...
Embodiment 2
[0049] Example 2 Annexin-V / PI double staining method to detect apoptosis induced by low temperature plasma
[0050] The experimental device, experimental materials and low-temperature plasma treatment method are the same as in Example 1. After the low-temperature plasma treatment, the cells were further cultured for 24 hours. After the cells were digested with EDTA-free trypsin, the cells were collected by centrifugation at 1000 rpm for 5 minutes. Wash the cells twice with pre-cooled PBS to pellet the cells. Add 500 μL of Binding Buffer (1×) to resuspend the cells. Add 5 μL Annexin V-FITC and 5 μL Propidium Iodide respectively, mix well; stain at room temperature for 15 minutes in the dark. Place on ice and perform flow cytometry within 1 hour.
[0051] The Annexin-V / PI double staining method was used to detect the occurrence of low-temperature plasma-induced apoptosis by flow cytometry, and the results were as follows: image 3 As shown in a, the results show that after l...
Embodiment 3
[0053] Example 3 Detection of Low-temperature Plasma-Induced Autophagosome Formation by Immunofluorescence
[0054] This embodiment adopts The autophagy detection kit detects autophagic vesicles and autophagic flow in live cells. The kit can selectively label the autophagic vesicles accumulated in cells and not accumulate in lysosomes, specifically indicating the occurrence of autophagy . Experimental device and experimental material are with embodiment 1, and concrete detection method is as follows:
[0055] Place a special cell slide with a diameter of 16 mm in a cell culture dish (35 mm). After enzymatic hydrolysis and counting of non-small cell lung cancer cells A549 and breast cancer cells MDA-MB-231, they are inoculated in a culture dish and placed in an incubator (37 mm). °C, 5% CO 2 ) for 24 hours. After low temperature plasma treatment for 0, 30, 60, 90, 120s, 37°C, 5% CO 2 Conditions continued for 24 hours. Prepare microscope double-staining dyes: Hoechst 3334...
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