Application of chick embryo allantoic membrane model in lung adenocarcinoma drug resistance mechanism research

Abstract

The invention belongs to the technical field of biological medicine, and discloses application of a chick embryo allantoic membrane model in lung adenocarcinoma drug resistance mechanism research. The chick embryo allantoic membrane model is obtained by hatching fertilized white-shell eggs in a bird incubator at 37.5 DEG C and 65% humidity. After cancer cells are transplanted to Y-shaped blood vessels of the chorioallantoic membrane, H2228 cells continuously expressing Venus fluorescent protein are transplanted to the chorioallantoic membrane so as to confirm the implantation condition. Before and after medicine injection, all organs of the cut tumor and embryo are shot under fluorescence, and the fluorescence intensity of all the organs is quantitatively analyzed through a fluorescein filter. According to the invention, a chick embryo allantoic membrane model is used for verifying the cancer inhibition effect of Chb-M'in ALK mutant lung adenocarcinoma cells for the first time, the experiment cost is relatively low, and the experiment period is relatively short.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to application of a chicken embryo chorioallantoic membrane model in research on drug resistance mechanisms of lung adenocarcinoma. Background Art

[0002] Previous in vivo studies investigating drug resistance mechanisms in lung adenocarcinoma have primarily used nude mice, which is costly and time-consuming. The chick chorioallantoic membrane assay (CAM), a classic developmental model, has been widely used in developmental studies, virology, parasitology, and in vivo studies of various tumor and non-neoplastic diseases. During development, the chick embryo forms abundant chorioallantoic membrane vessels, providing excellent conditions for studying vascular development and angiogenesis. Therefore, the CAM is widely used to screen and evaluate drugs for their effects on angiogenesis. The CAM assay has applications in in vivo studies of cardiovascular disease drugs, anti-angiogenic tumor drugs, and the vascular irritation of test substances in the conjunctiva, genitals, lips, nasal cavity, anus, and skin. In recent years, with the deepening of research on gene function, the CAM assay, combined with gene delivery, has also been widely applied to in vivo studies of gene function. Summary of the Invention

[0003] To overcome the technical problems of using nude mice as experimental animals, the present invention provides an application of the chicken embryo chorioallantoic membrane model in the study of drug resistance mechanisms of lung adenocarcinoma. For the first time, the chicken embryo chorioallantoic membrane model is used to verify the tumor suppressor effect of Chb-M' in the ALK mutant lung adenocarcinoma cell line H2228.

[0004] The present invention discloses application of a chicken embryo chorioallantoic membrane model in research on drug resistance mechanism of lung adenocarcinoma.

[0005] Preferably, the chick chorioallantoic membrane model is derived from: fertilized white-shell chicken eggs incubated in a bird incubator at 37.5° C. and 65% humidity.

[0006] Preferably, after the cancer cells are transplanted into the Y-shaped vessels of the chorioallantoic membrane, H2228 cells that continuously express Venus fluorescent protein are transplanted into the chorioallantoic membrane to confirm the implantation.

[0007] Preferably, before and after drug injection, the excised tumor and various organs of the embryo are photographed under fluorescence, and the fluorescence intensity of each organ is quantitatively analyzed using a fluorescein filter.

[0008] Compared with the prior art, the present invention has the following beneficial effects: The present invention is the first to use the chicken embryo chorioallantoic membrane model to verify the tumor suppressor effect of Chb-M' in the ALK mutant lung adenocarcinoma cell line H2228, with low experimental cost and short experimental cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Schematic diagram of the molecular structure of Chb-S, FITC-Chb-S, Chb-M', and FITC-Chb-M'; Figure 2 are the dose-response curves of Chb, Chb-S, and Chb-M' in PC9 and H2228 cells; Figure 3-Figure 5 is a schematic diagram of the nuclear localization experiment of Chb-M'; Figures 6-10 This is a schematic diagram of the effective uptake experiment of FITC-Chb-M' in H2228 cancer tissue; Figures 11-16 Schematic diagram of the experiment showing preferential accumulation of FITC-Chb-M' in CAM cancers. DETAILED DESCRIPTION

[0010] The following describes the embodiments of the present invention in detail. Unless otherwise specified, the raw materials and equipment used are commercially available or commonly used in the art. The methods in the embodiments, unless otherwise specified, are conventional methods in the art. The embodiments are illustrative only and are not to be construed as limiting the present invention.

[0011] Drug molecular structure The molecular structures of Chb-S, FITC-Chb-S, Chb-M', and FITC-Chb-M' are as follows Figure 1 shown.

[0012] Chb-M' is a DNA methylation drug targeting the consensus sequence (5'-TGTGGT-3') in RUNX, while Chb-S is a mismatch drug that lacks a binding motif within the consensus sequence in the RUN chick embryo chorioallantoic membrane model for studying drug resistance mechanisms in lung adenocarcinoma. FITC-Chb-S and FITC-Chb-M' are fluorescently labeled with FITC at the C-termini of Chb-S and Chb-M', respectively. N-methylpyrrole and N-methylimidazole are indicated in blue and red, respectively. Figure 1 ).

[0013] The main contents of this study are as follows: First, in vitro cell experiments showed that ( Figure 2 ), Chb-M' showed good tumor suppressor effect in ALK mutant lung adenocarcinoma cell lines PC9 and H2228, which was further verified by in vivo experiments. The chicken embryo chorioallantoic membrane model was selected for this in vivo verification.

[0014] In vitro assay: Cells were treated with the indicated concentrations of Chb, Chb-S, and Chb-M'. Cell counts were determined by absorbance after 72 hours of treatment (n = 3).

[0015] Nuclear localization of FITC-Chb-M' See Figure 3 , Nuclear localization of Chb-M'. PC9 (left) and H2228 (right) cells were treated with 1 μM FITC-Chb-M', FITC-Chb-S, or FITC, respectively. DMSO served as a negative control. Two hours after treatment, cell nuclei were stained with DAPI (red). Fluorescence images (green) were captured under a confocal laser microscope (magnification: 400x). Merged images are shown in yellow. Scale bar represents 50 μm. Histograms show mean FITC fluorescence intensity. The bottom panel shows images at higher magnification. Only one cell is shown. Scale bar represents 5 μm.

[0016] See Figure 4 Quantification of signal intensity. PC9 and H2228 cells were treated as above. Two hours after treatment, whole-cell lysates were prepared, and fluorescence intensity was measured using a fluorescence plate reader (n = 3). Data are mean ± standard error. The signal intensities generated by FITC-Chb-S and FITC-Chb-M' in PC9 (left) and H2228 (right) cells were tested for significant differences. *p < 0.05, **p < 0.01 (two-sided Student's t-test).

[0017] See Figure 5 PC9 and H2228 cells were treated as above. Two hours after treatment, nuclear lysates were prepared and fluorescence intensity was measured using a fluorescence plate reader (n = 3). Data are mean ± standard error. Each value was normalized to the DMSO control. *p < 0.05, **p < 0.01 (two-sided Student's t-test).

[0018] CAM construction method: Fertilized white-shelled eggs were incubated in an avian incubator at 37.5°C and 65% humidity and turned every hour.

[0019] After ten days of incubation, the indicated cancer cells were transplanted onto the eggs: First, a deep incision is made at the blunt end of the eggshell (above the air cell) using a diamond cutter. Next, a window is created in the eggshell without damaging the shell membrane. The shell membrane is removed, and the chorioallantoic membrane is retrieved. To transplant cancer cells, a sterile ring is placed over the Y-shaped vessels of the chorioallantoic membrane. Cancer cells (2 × 10^6 cells / 20 μl of culture medium / egg) are added to the ring, and the window is covered with film. After returning the egg to the incubator, the egg flipping function is turned off.

[0020] The rings were removed from the chorioallantoic membrane on day 12. To confirm engraftment, H2228 cells transduced with a lentiviral vector (CS-RfA-ETV) that constitutively expressed Venus fluorescent protein were transplanted onto the chorioallantoic membrane.

[0021] On day 13, a small window was opened in the egg without damaging the eggshell membrane. FITC-Chb-M' and FITC-Chb-S were administered at 32 μg / egg and 32 μg / egg, respectively, followed by an equal volume of dimethyl sulfoxide (DMSO) intravenous injection.

[0022] On days 14 and 15 (before injection and 3, 6, 12, and 24 hours after injection), excised tumors and various embryonic organs (heart, liver, spleen, lungs, kidneys, brain, intestines, and stomach) were photographed under fluorescence microscope. Fluorescence intensity of each organ was quantified using a fluorescein filter. The excitation and emission wavelengths of fluorescein are 460-490 nm and 518-546 nm, respectively. Furthermore, representative cancers formed on the CAM were fixed in 4% paraformaldehyde at 4°C overnight, treated with 99.8% methanol at -80°C for 30 minutes, incubated in 20% sucrose solution at 4°C overnight, and embedded in OCT compound at -80°C.

[0023] CAM model verifies the effectiveness of Chb-M' in suppressing tumors 1. FITC-Chb-M' is effectively taken up in H2228 cancer tissues. The uptake of FITC-Chb-M' in cancer tissues of fertilized chicken embryos transplanted with H2228 cancer cells demonstrated that Chb-M' gradually accumulated in the tumor after injection.

[0024] See Figure 6 , graphical representation of the chick embryo cancer model. To examine the cancer cell aggregation of Chb-M' and Chb-S, FITC-labeled Chb-M' or FITC-labeled Chb-S were intravenously injected into day-14 chick embryos. The chick embryo window, excised cancer tissue, and various organs were observed using a stereofluorescence microscope before injection and 3, 6, 12, and 24 hours after injection.

[0025] See Figure 7 , Fluorescence images (taken using a GFP filter) of Venus-expressing H2228 cells transplanted onto the CAM (day 13).

[0026] See Figure 8 Representative brightfield and FITC fluorescence images (taken using a GFP filter) from the chick embryo window from the indicated control and experimental groups, taken before and 3, 6, 12, and 24 hours after injection. The bar graph on the right shows the temporal changes in FITC fluorescence intensity of cancerous tissue on the CAM in each experimental group (n = 5). Data are expressed as mean ± standard error. The signal intensity generated by FITC-Chb-S and FITC-Chb-M' was tested for significant differences (*p < 0.05, NS). See Figure 9Representative bright field and FITC fluorescence images (taken using a GFP filter) of resected tumors from the control and experimental groups, 24 hours after injection. The values ​​below indicate FITC fluorescence intensity.

[0027] See Figure 10 , Cryosections of H2228 tumors resected from the CAM were stained with DAPI and imaged with FITC fluorescence. The lower panel shows the merged image (yellow). The scale bar indicates 50 μm.

[0028] 2. The preferential accumulation of FITC-Chb-M' in CAM cancers demonstrates that Chb-M' has a stronger anti-tumor effect than the control groups DMSO and Chb-S. Figures 11 to 16 .

[0029] Targeting FITC-Chb-M' ( Figure 11 ) and FITC-Chb-S ( Figure 12 ) reaction, and the FITC fluorescence intensity from various organs and CAM cancers was quantitatively analyzed (n=11).

[0030] Data are presented as mean ± standard error. P values ​​were obtained by two-tailed t-test, with *p < 0.05, **p < 0.01, and ***p < 0.001.

[0031] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to the above embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. Application of the chick embryo chorioallantoic membrane model in the study of drug resistance mechanisms in lung adenocarcinoma.

2. The application of the chicken embryo chorioallantoic membrane model in the study of drug resistance mechanism of lung adenocarcinoma according to claim 1, characterized in that: The chick chorioallantoic membrane model was derived from fertilized white-shell chicken eggs incubated in an avian incubator at 37.5°C and 65% humidity.

3. The use of the chicken embryo chorioallantoic membrane model in the study of drug resistance mechanism of lung adenocarcinoma according to claim 1, characterized in that: After cancer cells were transplanted into the Y-shaped vessels of the chorioallantoic membrane, H2228 cells that continuously expressed Venus fluorescent protein were transplanted into the chorioallantoic membrane to confirm the engraftment.

4. The use of the chicken embryo chorioallantoic membrane model in the study of drug resistance mechanism of lung adenocarcinoma according to claim 3, characterized in that: Before and after drug injection, the excised tumor and embryonic organs were photographed under fluorescence, and the fluorescence intensity of each organ was quantitatively analyzed using a fluorescein filter.

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