CN114468997A - Multi-mode detection method for in-situ transplantation lung cancer model - Google Patents
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Abstract
The invention discloses a multi-modal detection method of an in-situ transplanted lung cancer model, which comprises the following steps: 1. taking the orthotopic transplantation lung cancer model, carrying out fluorescence imaging on the orthotopic transplantation lung cancer model, and determining whether the tumor of the orthotopic transplantation lung cancer model is successfully orthotopic transplantation or not according to the fluorescence imaging result; 2. determining the approximate position of the tumor according to the fluorescence imaging result of the orthotopic transplantation lung cancer model; 3. continuously carrying out micro-CT imaging on the in-situ transplanted lung cancer model, and determining the specific position, size and depth of the tumor according to the micro-CT imaging; 4. the in-situ implantation of the lung cancer model is continued129Xe MRI imaging, based on129And obtaining the ventilation condition of the lung, the lung gas-blood exchange function parameter and the microstructure parameter of the lung of the in-situ transplantation lung cancer model through the Xe MRI imaging result. The method uses multi-modal imaging means to treat tumorsThe growth condition and the lung function of the lung cancer are detected, thereby providing a research basis for the development of an early diagnosis technology of the lung cancer, the screening of therapeutic drugs and the evaluation of curative effect.
Description
Technical Field
The invention belongs to the technical field of lung cancer model detection, and particularly relates to a multi-modal detection method for an in-situ transplanted lung cancer model.
Background
Globally, lung cancer is the most serious malignant tumor with the highest mortality and the second highest morbidity, and greatly threatens the life health of human beings.
The current animal models of lung cancer are mainly divided into three types: induced lung cancer models, transgenic lung cancer models, and transplant lung cancer models. Although the induced lung cancer model is close to the actual evolution law and mechanism of human lung cancer, the induction time is long and inconsistent, the tumor formation rate is low, and the induced lung cancer model is difficult to be applied to large-scale research. The transgenic lung cancer model is similar to the human lung cancer subtype with corresponding gene mutation to a certain extent in mechanism and morphology, but the cost is high, the time consumption is long, and the application of the method is limited. In the transplanted lung cancer model, the subcutaneous transplanted tumor model has the most extensive application, simple operation, high tumor formation rate and can dynamically monitor the tumor volume, however, the microenvironment difference between the subcutaneous part and the lung is huge, so that the difference between the result obtained when the model is applied to the drug efficacy evaluation and the actual clinical situation is larger. The orthotopic transplantation of the lung cancer model refers to a method for inoculating tumor cells in the respiratory system of an animal and then forming tumors in situ, the method is closer to the generation process of clinical lung cancer, can simulate the microenvironment of the tumors, and has important significance and position in the in vivo research of the lung cancer.
In the process of constructing the lung cancer model, the growth condition of the tumor needs to be regularly monitored to determine the size and the position of the tumor, so as to determine the time required by the model construction. The most common detection method at present is living body fluorescence imaging, the method can detect the bioluminescence of cells marked by Luciferase (Luciferase) genes in animal bodies, and has the advantages of simple operation, intuitive result and high sensitivity, so the method is widely applied to the identification of lung cancer models. However, the method has the inherent disadvantages of fluorescence imaging, namely low spatial resolution and shallow penetration depth, so that the method can only detect the approximate position of the tumor, estimate the relative size of the tumor according to the fluorescence intensity, cannot determine the specific size, depth and position of the lung tumor, and cannot monitor the ventilation and qi-blood exchange function change of the lung.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a multi-modal detection method for an in-situ transplanted lung cancer model, which uses a multi-modal imaging means to detect the growth condition and the lung function of a tumor, thereby providing a research basis for the development of an early diagnosis technology of lung cancer, the research of an evolution molecular mechanism, the screening of therapeutic drugs and the evaluation of curative effect.
The technical scheme adopted for realizing the above purpose of the invention is as follows:
a multi-modal detection method of an orthotopic transplantation lung cancer model comprises the following steps:
1. taking the orthotopic transplantation lung cancer model, carrying out fluorescence imaging on the orthotopic transplantation lung cancer model, and determining whether the tumor of the orthotopic transplantation lung cancer model is successfully orthotopic transplantation or not according to the fluorescence imaging result; if the transplantation is successful, carrying out subsequent detection, and if the transplantation is unsuccessful, replacing the orthotopic transplantation lung cancer model and continuing carrying out fluorescence imaging detection until the orthotopic transplantation lung cancer model with successful tumor transplantation is detected;
2. determining the approximate position of the tumor in the lung part of the orthotopic transplantation lung cancer model according to the fluorescence imaging result of the orthotopic transplantation lung cancer model with successful tumor transplantation;
3. continuously carrying out micro-CT imaging on the orthotopic transplantation lung cancer model, and determining the specific position, size and depth of the tumor in the orthotopic transplantation lung cancer model lung according to the micro-CT imaging;
4. the in-situ implantation of the lung cancer model is continued129Xe MRI, based on129Obtaining ventilation condition, lung gas-blood exchange function parameters and microstructure parameters of the lung of the in-situ transplantation lung cancer model according to the Xe MRI result;
the pulmonary air-blood exchange function parameters comprise pulmonary parenchyma/alveolar volume ratio VS/VA, blood residence time in capillary Tx and lung interval thickness d, and the microstructure parameters comprise alveolar duct inner diameter R, alveolar duct outer diameter R, alveolar depth h, alveolar mean linear intercept Lm, alveolar surface volume ratio SVR and apparent diffusion coefficient ADC.
Further, the animal selected by the orthotopic transplantation lung cancer model construction is a mouse.
Compared with the prior art, the invention has the advantages and beneficial effects that:
1. the method comprises the steps of firstly detecting an orthotopic transplantation lung cancer model by using a fluorescence imaging method, qualitatively judging whether a tumor is successfully transplanted or not by detecting fluorescence of a lung, determining whether the tumor is transplanted to a required position or not according to the fluorescence position, then detecting the orthotopic transplantation lung cancer model by using a micro-CT imaging method, determining the specific size, position and depth of the lung tumor according to the micro-CT imaging result, and finally using129The Xe MRI detects the in-situ transplantation lung cancer model, determines the ventilation condition of the lung and acquires the lung microstructure parameters and the gas-blood exchange function parameters. Compared with the fluorescence imaging method and the micro-CT imaging method which are most commonly used before,129xe MRI can provide functional information of the lungs, which is not available with the first two methods.
2. The method comprises the following steps129The Xe MRI is combined with two traditional lung cancer monitoring means of living body fluorescence and CT, can more comprehensively and multi-dimensionally evaluate the tumor growth condition and the lung function of a lung cancer model, and has important significance in the application of the lung cancer model to the development of new drugs, the evaluation of curative effect, the molecular mechanism and other related researches. In addition, in the early stage of lung cancer, the lung function is damaged earlier than the substantive lesion, so that the multi-modal detection method is beneficial to the research related to the early stage lung cancer.
Drawings
FIG. 1 is a live fluorescence image of the lung of a nude mouse successfully modeled by the primary site-specific lung cancer model in example 1.
FIG. 2 is a micro-CT image of the lung of a nude mouse successfully modeled by the primary site-shifted lung cancer model in example 1.
FIG. 3 is a graph of lung of nude mice successfully modeled by the primary site-shifted lung cancer model in example 1129Xe aeration image
FIG. 4 is a microstructure parameter distribution diagram of the lung of a nude mouse successfully modeled by the primary transplantation lung cancer model in example 1.
Detailed Description
The present invention will be described in detail with reference to specific examples.
Example 1
Taking luciferase-labeled A549 cells (A549-Luc) out of a liquid nitrogen tank, recovering and culturing the A549-Luc cells by using a screening culture medium, digesting the A549-Luc cells by using pancreatin when the A549-Luc cells are cultured to a logarithmic growth phase to obtain a cell suspension, mixing the cell suspension with an equal volume of matrigel to obtain a cell concentration of 2 x 107The seed/ml LA549-Luc cell inoculation liquid.
Weighing 10 BALB/C nude mice of 6 weeks old, injecting a tribromoethanol solution (400 mg/kg) into the abdominal cavity of the nude mice after weighing, anesthetizing the mice, fixing in a prone position, cutting the skin of the left lung, injecting 40 mu LA549-Luc cell inoculum into the left lung intercostals of the mice, slowly pulling out the needle after stopping the needle for about 3 seconds, suturing, and inoculating for 3 weeks to obtain 10 nude mice of the in-situ transplantation lung cancer model, wherein the nude mice are respectively numbered as 7.7-1, 7.7-2, 7.7-3, 7.7-4, 7.7-5, 7.7-6, 7.7-7, 7.7-8, 7.7-9 and 7.7-10.
Test I, test for detecting nude mice in situ-planted lung cancer model
1. Living body fluorescence imaging test of nude mouse in-situ implanted lung cancer model
1.1, test method:
preparing D-luciferin working solution (15mg/mL) by PBS (phosphate buffer solution), taking 10 nude mice transplanted with a lung cancer model in situ, injecting the D-luciferin working solution into the abdominal cavity of each nude mouse according to the weight of 10 mu L/g, after injecting for 10-15 minutes, anesthetizing each nude mouse by isoflurane gas, after anesthetizing, detecting the bioluminescence condition of the lung of each nude mouse by a PerkinElmer mouse living body fluorescence imaging system, and imaging the prone position and the right lateral decubitus position of each nude mouse;
1.2, test results:
of 10 nude mice, 2 nude mice died within 3 weeks, 1 nude mouse did not detect fluorescence, 7 nude mice numbered 7.7-1, 7.7-2, 7.7-3, 7.7-5, 7.7-7, 7.7-9, and 7.7-10 all fluoresced, and the success rate of orthotopic transplantation of the lung cancer model was 70%;
living body fluorescence imaging picture of 7 nude mice successfully modeledAs shown in FIG. 1, it can be seen from FIG. 1 that the fluorescence of the nude mice except the nude mice numbered 7.7-1 is concentrated in the left lung, and the fluorescence intensity ranges from 1 to 4X 105p/sec/cm2The reason why the fluorescence was analyzed was that the lung was transferred in the nude mice with 7.7-1/sr, both right and left lungs were fluorescent.
2. micro-CT imaging test of nude mouse in-situ implanted lung cancer model
2.1, test method:
3 nude mice are selected from 7 nude mice successfully molded and detected in the test 1, the numbers are respectively 7.7-3, 7.7-9 and 7.7-10, the three nude mice are anesthetized by isoflurane gas, are fixed on an animal bed in a supine position, and after each nude mouse breathes slowly and stably, the lung of each nude mouse is scanned by using micro-CT (Bruker SkyScan 1176; Germany), and the parameters of the micro-CT are as follows: the filter is Al-0.5mm and the X-ray voltage is 50 kV; the image resolution was 35 μm and coronal and transversal reconstructions were performed using nreco software, selecting the layer of the largest section of the tumor for each surface to be displayed.
2.2, test results:
the micro-CT images of 3 nude mice are shown in FIG. 2, the position pointed by the yellow arrow in the micro-CT image of each nude mouse is the tumor position, as shown in FIG. 2, the tumors of the nude mice numbered 7.7-3 and 7.7-10 are located in the inferior lobe of the left lung and close to the left chest wall, while the tumors of the nude mice numbered 7.7-9 are located in the tip of the lung and close to the dorsal chest wall, and the difference of the tumor positions of each nude mouse may be caused by slight difference of the needle insertion position and depth. Therefore, the position information of the tumor cannot be acquired in the fluorescence imaging in the experiment 1, and the specific position of the tumor can be acquired by the CT imaging, which shows that the CT imaging plays a good supplementary role.
3. In-situ implantation of lung cancer model in nude mice129Xe MRI test
3.1, test method:
anaesthetizing 3 nude mice selected in experiment 2 with isoflurane gas, inserting trachea of the 3 nude mice to control their breathing, fixing the nude mice on the animal bed in a supine manner, and scanning with 7T animal MRI after each nude mouse breathes slowly and stablyThe apparatus (Bruker Biospec 70/20 USR; Germany)129Xe MRI experiments using 31mm ID double tuning (1H/129Xe) birdcage coil, hyperpolarized sequentially for each nude mouse129Acquiring Xe ventilation images, CSSR and DWI pulse sequences;
hyperpolarisation of129In the Xe ventilation image acquisition test, each mouse performs one xenon pre-respiration, and then samples are taken during the second hyperpolarized xenon breath-hold, the ventilation image employs a FLASH sequence, and the acquisition parameters are as follows: single slice coronal imaging; the number of layers is 10, and the thickness of the layers is 1.5 cm; FOV 30X 30mm2(ii) a FA-20 °; the imaging matrix is 64 × 64; BW is 50 kHz; TE/TR is 3.522ms/88.97ms, Matlab is used for arranging the original data to k space through center coding, and then two-dimensional Fourier transform is carried out to obtain a ventilation image;
in CSSR pulse sequence acquisition test, for dissolving state129The two Gaussian pulse lengths of Xe signal saturation and excitation are 0.5ms and 0.3ms respectively. 25 exchange time points are set between 1.4 ms and 200ms for acquiring lung dynamic magnetic resonance spectrum (1.4, 3, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 and 200ms), and each nude mouse is subjected to xenon pre-respiration twice to improve the dissolution state before formal breath holding sampling129Xe signal, CSSR acquisition experiment of each mouse was repeated 5 times, and the original FID data was one-dimensional Fourier transformed using Matlab to obtain hyperpolarization at 25 different crossover times129A Xe magnetic resonance spectrum, further performing fitting and normalization of Xe dissolved state and gaseous state signals, and finally obtaining gas-blood exchange functional parameters [ maiH, Kimura A, Iguchi S, Hori Y, Masuda S, Fujiwara H. Noninivastic detection of pure tissue determination in a mouse model of analysis used hyper polarized 129Xe under sponge formation, Magn resonance Med. 2010; 64(4) 929-938.doi 10.1002/mrm.22437]；
The parameters of the DWI pulse sequence acquisition experiment are as follows: the imaging matrix is 64 × 64; FOV 50X 50mm2(ii) a FA-10 °; BW is 50 kHz; TE/TR 4.31ms/23.401 ms; 10 b values (4, 8, 1)2. 16, 20, 24, 28, 32, 36 and 40s/cm2) Data fitting for DWI models, where each b-value image was acquired in a separate breath hold, with and without diffusion gradients applied (i.e., b 0, x, 0 s/cm) in one breath hold, respectively2) The method comprises the steps of acquiring image data of 3 images, adopting an interleaved sampling strategy so as to minimize signal fluctuation influence caused by T1 relaxation, performing two-dimensional Fourier transform on original k-space data by using Matlab to obtain images, and acquiring three images in each breath, wherein b is x s/cm2Is 0s/cm with b2Dividing the mean values of the two images, then segmenting the images, separating lung regions from background noise, eliminating pixel points with SNR less than 3 and pixel points of a main trachea, taking the final segmentation mask as the intersection of the results of the three steps, applying the intersection to Sb/S0 images in each breath, and finally performing point-to-point fitting on the images [ Sukstanski AL, Yablonskiy DA. Lung morphology with hyperpolarized 129Xe: the organic background. 67(3) 856-866 doi 10.1002/mrm 23056]And finally obtaining a distribution graph of the lung microstructure parameters, and obtaining an integral mean value of the lung microstructure parameters by counting pixel points in the distribution graph.
3.2, test results:
lungs of 3 nude mice129Xe ventilation pattern As shown in FIG. 3, lungs of each nude mouse129The area pointed by yellow arrows in the Xe ventilation image is an obvious ventilation defect area, the ventilation defect position is highly consistent with the tumor position shown by CT in figure 2, and moreover, the ventilation conditions of 3 mice are all relatively uneven, and the ventilation defect caused by local lung cancer can cause physiological compensation of other parts of the lung;
the pulmonary microstructure parameter distribution map of 3 nude mice is shown in fig. 4, and the pulmonary gas-blood exchange function parameters and microstructure parameters of 3 nude mice are shown in the following table 1:
TABLE 1
In Table 1, VS/VA is the pulmonary parenchyma/alveolar volume ratio, Tx is the blood residence time in the capillaries, d is the lung space thickness, R is the alveolar pipe inner diameter, R is the alveolar pipe outer diameter, h is the alveolar depth, Lm is the alveolar mean linear intercept, SVR is the alveolar surface volume ratio, and ADC is the apparent diffusion coefficient. As can be seen from Table 1, the VS/VA, Tx and d values of the nude mice numbered 7.7-3 are all significantly higher than those of the other two nude mice, thereby indicating that the lungs of the nude mice numbered 7.7-3 may have the conditions of interstitial thickening, inflammatory cell infiltration and capillary vessel blockage, and the function of qi-blood exchange is seriously impaired. From the live fluorescence imaging and micro-CT imaging, it can also be seen that the lung cancer volume of nude mice numbered 7.7-3 is the largest of the 3 nude mice, and the results of the above lung function parameters are also confirmed.
Claims (2)
1. A multi-modal detection method of an orthotopic transplantation lung cancer model is characterized by comprising the following steps:
1.1, taking the orthotopic transplantation lung cancer model, carrying out fluorescence imaging on the orthotopic transplantation lung cancer model, and determining whether the tumor of the orthotopic transplantation lung cancer model is successfully orthotopic transplantation or not according to the fluorescence imaging result; if the transplantation is successful, carrying out subsequent detection, and if the transplantation is unsuccessful, replacing the orthotopic transplantation lung cancer model and continuing carrying out fluorescence imaging detection until the orthotopic transplantation lung cancer model with successful tumor transplantation is detected;
1.2, determining the approximate position of the tumor in the lung part of the orthotopic transplantation lung cancer model according to the fluorescence imaging result of the orthotopic transplantation lung cancer model with successful tumor transplantation;
1.3, continuously carrying out micro-CT imaging on the orthotopic transplantation lung cancer model, and determining the specific position, size and depth of the tumor in the orthotopic transplantation lung cancer model lung according to the micro-CT imaging;
1.4, continuing to plant the lung cancer model in situ129Xe MRI imaging, based on129Obtaining ventilation condition, lung gas-blood exchange function parameters and microstructure parameters of the lung of the in-situ transplantation lung cancer model according to the Xe MRI imaging result;
the lung air-blood exchange function parameters comprise lung parenchyma/alveolar volume ratio VS/VA, blood residence time in capillary Tx and lung interval thickness d, and the microstructure parameters comprise alveolar pipe inner diameter R, alveolar pipe outer diameter R, alveolar depth h, alveolar average linear intercept Lm, alveolar surface volume ratio SVR and apparent diffusion coefficient ADC.
2. The multi-modal detection method of orthotopic transplantation lung cancer model according to claim 1, characterized in that: the selected animal constructed by the orthotopic transplantation lung cancer model is a mouse.
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