Rat Liver Targeting Of Microwave Assisted Mediated Gene Transfection Experiment Research

ObjectTo determine the tissue demage and morphological change of rat liver after micowave ablation, transfect liver tissue with plasmid following micowave ablation, analyze the effect of microwave ablation on gene transfection.MethodsIn this study, microwave ablation was applied to rat liver after laparotomy.The ablation area can be divided into needle tract carbonization zone, coagulation necrosis zone, inflammatory reaction zone and normal liver tissue zone. In order to prove that a selectively permeable membrane change of the inflammatory reaction zone (target area) caused by thermal damage is conducive to gene transfection, different power cell ablation with its physiological function and morphology ablation zone size can be selected. Microwave ablation under specific power make liver ablation model established. According to the literature, experimental ablation power was20W,25W and30W, ablation time is set to60S. The abdomen was closed after the end of ablation, respectively, in the first three, five, and seven postoperative days, rats were sacrificed by cutting femoral artery. Hepatic ablation specimens were taken. Along ablation needle tract and vertical directions, specified diameter of2mm liver specimens were taken for HE staining to determine the targeted range of areas both directions.Next, along the ablation needle tract and longitudinal direction, transfection was accomplished by local and intravenous injection to liver target area with green fluorescent protein (GFP) positive plasmid and Renilla luciferase positive plasmid. Using frozen section technique, with the inverted fluorescence microscope to observe green fluorescent protein expression. The degree of expression plasmid was determined by versatile full wavelength microplate reader; plasmids expression levels were also measured by full wavelength microplate reader versatility combined Renilla luciferase plasmid kit to assess the situation and the transfection efficiency of expression of target genes. Results1. Rat liver ablation is accomplished by three different microwave power, liver ablation samples were taken to HE staining to determine the range of the inflammatory reaction zone (target area of each region) On first day and the third day. Range in the diameter of the first day of the final direction of30W,25W,20W power after the formation of the different target areas identified for ablation (0.892±0.097,1.183±0.113) cm,(0.583±0.052,0.783±0.068) cm,(0.567±0.082,0.775±0.076) cm; diameter in the vertical direction (0.750±0.055,1.017±0.075) cm,(0.583±0.098,0.792±0.092) cm,(0.550±0.055,0.758±0.058) cm; In the diameter direction of the third range30W,25W,20W power are formed of different power target area is (0.833±0.137,1.175±0.154) cm,(0.667±0.103,0.908±0.086) cm,(0.567±0.052,0.767±0.041) cm; diameter in the vertical direction (0.800±0.110,1.067±0.121) cm,(0.700±0.089,0.933±0.075) cm,(0.550±0.084,0.775±0.082) cm.2. Frozen sections of MWA plus empty plasmid group and GFP expression plasmid injection group showed no fluorescence expression on the first, thrid, fifth, seventh day. Positive control group injected with portal hypertension showed obvious fluorescence expression, the fluorescence intensity was slightly lower than the experimental group.3. Compared with the other groups, frozen section of MWA plus GFP plasmid injection group showed visible green fluorescence in targeted area on the first day, the third day, fifth day, the seventh day under inverted fluorescence microscope. Fluorescence degree of the third day was stronger than that of the first, fifth and seventh day, from the fifth day fluorescence started to fade, on the seventh day, fluorescence showed obvious weakness.4. Full wavelength multifunctional microplate luminescence test value of green fluorescent protein:The results showed that, on the first day and on the third day, the fluorescence intensity (the first day of:360.7±78.31,6422.7±1666.37day:323.7±37.00,8837.2±3268.07)of transfection control group (MWA+empty plasmid group, GFP expression plasmid injection alone group) was significantly lower than the experimental group (the first day:10754.2±1242.91day:24163.3± 1522.90)(P<0.05) and a positive control group portal hypertension injection group (the first day:10428.0±1025.09, the third day:22172.83±2122.00)(P <0.05), no significant difference between the experimental group and positive control group plasmid (first day:P=0.631third days:P=0.282)(P>0.05).5.(1)Renilla luciferase assay showed that on the first postoperative day (POD1) after Renilla luciferase plasmid injection, luminescence value of the experimental group (36.2±1.33)RLU has statistically significant differences(P<0.05) with the control group, Renilla luciferase plasmid injection group (23.0±0.63)RLU, microwave ablation+luminescence value empty plasmid group (20.2±2.23) RLU, higher than the control group. It also has significant differences with the first day of the positive control group (portal hypertension injection Renilla luciferase plasmid vector group) luminescence values (46.7±2.25) RLU. The experimental value is lower than the positive control group.(2) On the third day after transfection (POD3), Renilla luciferase plasmid luminous values of the experimental group (46.7±2.25) RLU were statistically significant differences (P<0.05) from the third control group(32.0±2.60) RLU, microwave ablation plus empty plasmid group (23.2±1.94) RLU, a value higher than the control group. It also showed significantly difference (P<0.05) from the third positive control group injected with portal hypertension group luminous values (92.7±4.27) RLU. The experimental value is lower than the positive control group.(3) The Renilla luciferase luminescence value from the first day after transfection (POD1) of experimental group (36.2±1.33) RLU and portal hypertension injection group (46.7±2.25) RLU showed statistically significant differences (P<0.05) with it from the third day (POD3) of the experimental group (46.7±2.25)RLU and injection luminescence value of portal hypertension group (92.7±4.27)RLU, POD3emitting luciferase plasmid was significantly stronger than POD1.(4) the Renilla luciferase luminescence value from the fifth day after transfection (POD5) of experimental group and portal hypertension injection group showed statistically significant differences (P<0.05) from it from the seventh day(POD7) of the experimental group and injection luminescence value of portal hypertension group. At the same time, value of the experimental group is lower than the portal hypertension injection group.ConclusionDirect injection of plasmid vector to inflammatory reaction zone after microwave ablation (target area) can accomplish effective gene transfection, but the transfection efficiency is lower than portal hypertension injection. Which indicated that certain degree of thermal damage is benefit to promote gene transfection of the liver targeting area. This experiment aims to provides some experimental basis for exploring the mechanism of how thermal damage promote liver cells gene transfection.