Experimental Study Correlation Between DWI?IVIM And Ki-67 In Mice With Rhabdomyosarcoma

Objective To investigate the ADC value of DWI in mouse rhabdomyosarcoma,the correlation between the D value,D* value and f value of IVIM and the expression level of immunohistochemical index Ki-67.Materials and Methods 1.The experimental materials 1.1 Experimental animals and cells 27 female Balb/c nu/nu nude mice,embryonic rhabdomyosarcoma cells RD.1.2experimental reagents DMEM medium,fetal bovine serum,Ki-67 antibody.1.3 Experimental equipment 1.3.1 Cell processing equipment: ultra-clean workbench,high-speed centrifuge,cell culture incubator.1.3.2.MRI equipment: US GE Discovery 750 W 3.0T MRI scanner,four-channel wrist phased array RF coil.2.the experimental method 2.1 Establishing a mouse rhabdomyosarcoma model 2.1.1 Rhabdomyosarcoma cell culture The cells were cultured in a sterile medium containing 10% fetal bovine serum DMEM in a sterile incubator at 37 ° C,5% CO 2 and humidity.2.1.2 Nude mouse vaccination Each nude mouse was injected with 1*107 cells/ml of cell suspension 0.15 ml on the right back.2.1.3 Nude mouse breeding Nude mice are housed in the IVC system of the University Model Animal Research Institute of Dalian Medical University.2.1.4 Observation of tumor formation and growth in nude mice Tumor growth and mental status of nude mice were observed every 2-4 days after inoculation,and the tumor volume of each nude mouse was calculated(V=0.5 *ab2).2.1.5.The tumor formation standard The inoculated site of the mouse can be touched and toughened,and its volume is more than 1000mm3.The subcutaneous soft tissue mass can be seen on MRI T1 WI and T2 WI.The pathological microscopic observation is consistent with the diagnostic criteria of rhabdomyosarcoma.2.2 MRI examination technique,MRI image processing and observation method of rhabdomyosarcoma tumor-bearing mice 2.2.1 MRI examination technique of rhabdomyosarcoma tumor-bearing mice FSE-T1 WI,FSE-T2 WI,EPI-DWI,and EPI-IVIM images of rhabdomyosarcoma tumor-bearing mice were collected,and all images were uploaded to the ADW4.7 workstation after the scan was completed.2.2.2 MRI image processing and observation method of rhabdomyosarcoma tumorbearing mice On the DWI and IVIM maps,the highest signal is selected as the ROI,and each ADC pseudo-color map,D value map,D* value map and f-value map are repeatedly measured and averaged three times.2.3 MRI-pathological comparison method for mouse rhabdomyosarcoma The experimental study of the MRI-pathological precision control method of soft tissue mimics in the early stage of our group was used.The main steps are as follows.2.3.1 Preparation for rhabdomyosarcoma tumor-bearing mice before scanning 10% chloral hydrate was anesthetized by intraperitoneal injection at 0.05/10 g,followed by labeling of the surface of the tumor-bearing mouse.2.3.2 MRI examination of rhabdomyosarcoma tumor-bearing mice The experimental part was mainly to collect T1 WI,T2WI,DWI and IVIM images of rhabdomyosarcoma tumor-bearing mice.2.3.3 MRI level of rhabdomyosarcoma tumor-bearing mice-pathological section control method 2.3.3.1 Cut the skin of the tumor surface along the long axis of the mouse 2.3.3.2 After the tumor is exposed during surgery,it overlaps with the A line,and a surgical marker line B is placed in the center of the exposed surface of the tumor.2.3.3.3 overlapping with the A line,a surgical marking line on the left side of the exposed surface of the tumor,which is C;2.3.3.4 overlapping with the A line,a surgical marking line on the right side of the exposed surface of the tumor,which is D;2.3.3.5 On the edge of the exposed surface of the tumor directly above the head line of the B line,a surgical marker line E Use the pathological material to cut along the positions of the B,C,and D lines to obtain the material.2.3.4 Image surface ROI and pathological material comparison method The image surface is printed according to the actual size of the tumor 1:1,and the region of interest(ROI)is completely cut to form a translucent area,and the printing paper containing the translucent area is matched with the pathological material B,and the blank area is the pathological observation area.2.4 HE slice preparation and immunohistochemical sectioning technology 2.4.1 HE section preparation: The tissue block was subjected to gradient alcohol dehydration and immersed in paraffin for 3 hours.The tissue blocks after waxing were embedded in paraffin and 4 mm sections,and baked for use.The slice after baking was subjected to hematoxylin and eosin staining in turn,and finally the neutral gum was sealed.2.4.2 KI67 immunohistochemical section preparation: the steps before baking were made with HE slices,and the slices after baking were placed in xylene and gradient alcohol for dewaxing and rehydration.After completion,the antigen was repaired at a high temperature of 100 degrees.Finally,the antigen-antibody reaction and neutral gum seal were performed.2.5 HE section and immunohistochemical observation method The immunohistochemical expression of HE sections and KI67 was observed independently by two pathologists under the microscope.3.the observation indicators 3.1Tumor formation and tumor growth of rhabdomyosarcoma tumor-bearing mice;3.2Routine MRI findings of mouse rhabdomyosarcoma: tumor location,morphology,size,border,signal,presence or absence of capsule,presence or absence of lobulation,intratumoral separation;3.3The indexes obtained by DWI and IVIM double-index model: apparent diffusion coefficient(ADC)value,simple diffusion coefficient(D)value,pseudo-diffusion coefficient(D*)value,perfusion score(f)value;3.4 Histopathological features: tissue structure,cell morphology,cell atypia,necrosis,etc.;3.5 Ki-67 expression: 1000 tumor cells were counted,and the percentage of positively stained tumor cells was counted.4.statistical methods Statistical analysis was performed using SPSS 24.0 statistical software(version 24.0,SPSS Inc,Chicago)for apparent diffusion coefficient(ADC)values,simple diffusion coefficient(D)values,pseudo diffusion coefficients(D*)values,perfusion scores.The(f)value is subject to a person-related test with a Ki-67 percentage;if one is not obeyed,a spearman correlation test is performed.result 1.the establishment of mouse rhabdomyosarcoma model After each inoculation of the nude mice,the formation of the ridges can be reached and the texture is soft.The time to tumor formation in 27 cases was 7.9 ± 3.9 days.2.MRI findings of rhabdomyosarcoma-bearing mice 2.1 T1 WI,T2WI performance: 27 tumors were located subcutaneously in the right side of the mouse.Compared with the muscle signal,T1 WI showed that the tumor was a uniform signal or a slightly higher signal,and T2 WI showed that the tumor was a high signal.2.2 DWI,IVIM performance: 27 cases of tumor DWI showed uneven high signal,IVIM showed high signal.3.rhabdomyosarcoma tumor-bearing mice ADC,D,D* and f values MRI images of 27 cases of rhabdomyosarcoma tumor-bearing mice were postprocessed,and ADC,D,D* and f values were measured at their ROI.The average ADC value was 1.0470±0.2036×10-3mm2/s;the average D value was 0.7237± 0.0971×10-3 mm 2 /s;average D* value is 4.8497±1.6293×10-3 mm 2 /s,and f value is 0.1020-0.2290.4.Routine pathological findings of rhabdomyosarcoma-bearing mice In 27 cases,the rhabdomyosarcoma cells were small in shape,the nucleus was deeply stained,the cytoplasm was not obvious,and mitosis was common.5.Expression of Ki-67 in rhabdomyosarcoma tumor-bearing mice The scores of the Ki-67 sections of 27 rhabdomyosarcoma-bearing mice were observed,and the average Ki-67 expression was 84.6±4.8%.6.Correlation between DWI,IVIM and Ki-67 in mouse rhabdomyosarcoma Pearson correlation test was performed on 27 cases of mouse rhabdomyosarcoma with DWI and IVIM parameters and Ki-67.The results showed that the ADC values of 27 cases of mouse rhabdomyosarcoma were moderately negatively correlated with Ki-67,and the difference was significant(P<0.05);D value was negatively correlated with Ki-67,the difference was significant(P<0.05);f value was weakly negatively correlated with Ki-67,the difference was not significant(P>0.05);D* There is no significant correlation with Ki-67.conclusion Through this study we can draw the following conclusions: 1.the embryonic rhabdomyosarcoma cell line was implanted subcutaneously in the back of nude mice.The established mouse rhabdomyosarcoma model has a high tumor formation rate and can be repeated and can be used for the experimental study of soft tissue tumor imaging-pathology.2.using the three-step control method of the body sign-image surface-taking surface method on the MRI image surface of the rhabdomyosarcoma of mice,the pathological material surface,the region of interest-pathological material control point,the MR function image DWI ADC value of mouse rhabdomyosarcoma The IVIM D value was correlated with the pathological immunohistochemical index Ki-67 expression,and it was negatively correlated.That is,the higher the ADC value and the D value,the lower the expression of Ki-67,and vice versa.The mouse rhabdomyosarcoma There was no correlation between D* and f values of MR functional image IVIM and Ki-67 expression.