Use of GTTN-containing reagent in preparation of kit for diagnosing gastrointestinal cancer

Abstract

A use of a GTTN-containing reagent in preparation of a kit for diagnosing gastrointestinal cancer, wherein the GTTN-containing reagent can be used for specifically diagnosing gastrointestinal cancer, especially colon cancer. The GTTN-containing reagent exhibits good imaging performance at colon cancer sites, provides accurate detection results, and has good clinical application prospects in the diagnosis of colon cancer.

Description

Application of GTTN-containing reagents in the preparation of diagnostic kits for gastrointestinal cancer Technical Field

[0001] This invention belongs to the field of diagnostic reagents for gastrointestinal cancer, and particularly relates to the application of a GTTN-containing reagent in the preparation of a diagnostic reagent kit for gastrointestinal cancer. Background Technology

[0002] Gastrointestinal malignancies are the most common digestive system tumors, and their high incidence and mortality rates seriously threaten public health. Colorectal cancer, as a malignant tumor of the digestive tract, ranks third in incidence worldwide. Once colorectal cancer cells metastasize or spread, the five-year survival rate is extremely low. Currently, radical surgery remains the primary treatment for colorectal cancer; however, due to the poor efficacy of chemotherapy after metastasis, recurrence still occurs in some patients. Therefore, colorectal cancer screening and diagnosis are crucial for its prevention and treatment, and the development of effective reagents and kits for monitoring colorectal cancer recurrence and metastasis is an urgent and critical issue that needs to be addressed.

[0003] If colon cancer originates in the gastrointestinal tract, its insidious location can make early diagnosis difficult, and it can even be easily missed during endoscopic examinations. Fluorescence imaging technology has been used for a long time, with indocyanine green (ICG) being a typical example, widely applied in breast cancer, hepatobiliary, and gastrointestinal tumor surgeries. Depending on the hospital's resources and the intended use, it can be injected preoperatively endoscopically, laparoscopically into the subserosal region, or intravenously to locate the tumor, visualize lymph nodes, aid in assessing anastomotic blood supply, and confirm liver metastases. If a tumor is found intraoperatively, ICG can be injected directly into the subserosal region to aid in assessing lymph node distribution; however, this may result in poor imaging. In such cases, a second injection may lead to excessive diffusion, making it difficult to effectively distinguish tumors from normal tissue under fluorescence imaging. Furthermore, the diagnosis of colon cancer within or outside the gastrointestinal tract remains a major challenge for clinicians, with almost no reliable imaging diagnostic methods available. Therefore, there is an urgent need to find a reagent for diagnosing colon cancer, especially colon cancer within the gastrointestinal tract. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide an application of GTTN-containing reagents in the preparation of diagnostic kits for gastrointestinal cancer.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides the application of a graphene-based tumor cell nucleus-targeting fluorescent nanoprobe reagent in the preparation of a diagnostic kit for gastrointestinal cancer.

[0007] Preferably, the preparation method of the graphene-based tumor cell nuclear targeting fluorescent nanoprobe reagent includes adding 2.5 mL GTTN, 0.024 g potassium dihydrogen phosphate, 0.0144 g disodium hydrogen phosphate, 0.08 g sodium chloride, and 0.002 g potassium chloride to deionized water to 10 mL and mixing them evenly.

[0008] Preferably, the pH of the graphene-based tumor cell nuclear targeting fluorescent nanoprobe reagent is adjusted to 7.0 with dilute hydrochloric acid.

[0009] Preferably, the gastrointestinal cancer is colon cancer.

[0010] Preferably, the colon cancer is a colon cancer model constructed from the VX2 tumor cell line.

[0011] This invention provides the application of a graphene-based tumor cell nuclear-targeting fluorescent nanoprobe reagent in the preparation of a kit for screening animal models of gastrointestinal cancer or in the preparation of animal models of gastrointestinal cancer.

[0012] In the above application of preparing a kit for screening animal models of colon cancer, the gastrointestinal cancer is colon cancer.

[0013] The present invention also provides a diagnostic kit for gastrointestinal cancer, the kit comprising a graphene-based tumor cell nuclear-targeting fluorescent nanoprobe reagent, wherein the graphene-based tumor cell nuclear-targeting fluorescent nanoprobe reagent comprises 2.5 mL GTTN, 0.024 g potassium dihydrogen phosphate, 0.0144 g disodium hydrogen phosphate, 0.08 g sodium chloride, 0.002 g potassium chloride and 10 mL deionized water.

[0014] Preferably, the pH of the graphene-based tumor cell nuclear-targeting fluorescent nanoprobe reagent is 7.0.

[0015] Preferably, the kit further includes a negative control reagent, which is PBS.

[0016] The present invention also provides a method for screening animal models of gastrointestinal cancer, including the step of detecting fluorescence signals in recipient animals using the above-described kit.

[0017] Preferably, the method includes the step of treating the recipient animal with the negative control reagent described above.

[0018] Preferably, if the fluorescence signal of the recipient animal is significantly increased compared with the fluorescence signal of the negative control reagent, it indicates that the gastrointestinal cancer animal model has been successfully established.

[0019] Preferably, the animal includes a rabbit.

[0020] The present invention also provides a method for diagnosing gastrointestinal cancer, comprising the step of detecting fluorescent signals in intestinal tissue using the above-described kit.

[0021] Preferably, the gastrointestinal cancer is colon cancer.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention provides an application of GTTN-containing reagents in the preparation of diagnostic kits for gastrointestinal cancer. Studies have found that GTTN-containing reagents can specifically diagnose gastrointestinal cancers, especially colon cancer, and that GTTN-containing reagents provide good imaging results and accurate detection at colon cancer sites, showing promising clinical application prospects in the preparation of diagnostic kits for colon cancer. Furthermore, using GTTN-containing reagents to detect intestinal tissues in recipient animals can also specifically screen for gastrointestinal cancer animal models. Attached Figure Description

[0024] Figure 1 shows the results of detecting colon cancer in the gastrointestinal tract using GTTN reagent. a) is the fluorescence imaging result of colon cancer in the gastrointestinal tract 4 hours after injection of GTTN reagent; b) is the comparison of fluorescence signals in normal tissue and colon cancer in the gastrointestinal tract; c) is the fluorescence imaging result of colon cancer in the gastrointestinal tract after injection of GTTN reagent at different times. Detailed Implementation

[0025] This invention provides the application of a graphene-based tumor cell nucleus-targeting fluorescent nanoprobe reagent in the preparation of a diagnostic kit for gastrointestinal cancer.

[0026] In this invention, the preparation method of the graphene-based tumor cell nuclear-targeting fluorescent nanoprobe reagent includes adding 2.5 mL of GTTN, 0.024 g of potassium dihydrogen phosphate, 0.0144 g of disodium hydrogen phosphate, 0.08 g of sodium chloride, and 0.002 g of potassium chloride to deionized water to a final volume of 10 mL, and mixing thoroughly to obtain a GTTN-containing reagent. The pH of the graphene-based tumor cell nuclear-targeting fluorescent nanoprobe reagent is 7.0, and the pH of the GTTN-containing reagent is adjusted using 3M dilute hydrochloric acid. The preparation method of the GTTN-containing reagent of this invention is simple to operate, low in cost, and easily scalable for industrial production.

[0027] The GTTN-containing reagent of this invention can image gastrointestinal cancer with good imaging results, thereby effectively detecting gastrointestinal cancer. As a preferred embodiment, the gastrointestinal cancer is colon cancer, and more specifically, the colon cancer is a colon cancer model constructed from VX2 cells. Furthermore, the GTTN-containing reagent of this invention can directly penetrate the cell membrane of gastrointestinal cancer cells and specifically stain the tumor cell nuclei without staining normal tissue cells, thereby achieving the purpose of specifically detecting gastrointestinal cancer.

[0028] This invention provides the application of a graphene-based tumor cell nuclear-targeting fluorescent nanoprobe reagent in the preparation of a kit for screening animal models of gastrointestinal cancer or in the preparation of animal models of gastrointestinal cancer.

[0029] In the above application of the kit for preparing and screening animal models of colon cancer, the gastrointestinal cancer is colon cancer, and further, the colon cancer is a colon cancer model constructed from the VX2 tumor line, and the model animal is a rabbit.

[0030] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] The preparation method of the graphene-based tumor cell nuclear-targeting fluorescent nanoprobe (GTTN) is described in the authorized patents published by CN 111467510 A and application number 202010278513.4. Specifically, the preparation method of GTTN includes the following steps: 0.5 g of pyrene powder is added to 25 mL of nitric acid (concentration 65-68 wt%) at 80 °C and reacted for 24 h; after the reaction is completed, the mixture is cooled, washed with 150 mL of deionized water, and filtered through a 0.22 μm filter membrane; the filtrate is added to 50 mL of Na2SO3 aqueous solution (concentration 0.5 mol / L) and stirred for 0.5 h, then transferred to a 150 mL ceramic autoclave and heated at 130 °C for 12 h; after cooling to room temperature, the resulting material is transferred to an autoclave lined with polytetrafluoroethylene and placed in a vacuum drying chamber at 200 °C for 12 h; after the reaction is completed, the mixture is cooled, and the resulting system is filtered. The filtrate contains GTTN, and this filtrate is referred to as GTTN stock solution.

[0033] Example 1

[0034] A method for preparing a GTTN-containing reagent: 2.5 mL of GTTN, 0.024 g of potassium dihydrogen phosphate, 0.0144 g of disodium hydrogen phosphate, 0.08 g of sodium chloride, and 0.002 g of potassium chloride are added to deionized water to a final volume of 10 mL. The mixture is stirred until homogeneous, and the pH is adjusted to 7.0 with 3 M dilute hydrochloric acid to obtain the GTTN-containing reagent.

[0035] Application of GTTN-containing reagents in the detection of colon cancer in the gastrointestinal tract:

[0036] (1) Construction of a colon cancer model:

[0037] VX2 tumor strains were obtained by taking fish-flesh-like tumor tissue with vigorous growth at the tumor margin, cutting it into a homogenate with ophthalmic scissors, adding an appropriate amount of physiological saline to make a suspension, and using a 5mL syringe needle (to prevent tissue from clogging the needle) and a 1mL syringe to extract the tumor tissue suspension for later use.

[0038] (2) 3% sodium pentobarbital was injected into the marginal ear vein of rabbits at a dose of 30 mg / kg, with an injection volume of 1 mL / kg. The rabbits were placed supine on the operating table, and the abdomen was prepared and routinely disinfected. The colon was dissected from the abdominal cavity, and a 1.0 mL syringe was inserted obliquely into the submucosal space of the intestine to inject 0.1 mL of rabbit-derived VX2 tumor tissue suspension to construct a colon cancer model. The colon cancer model rabbit was successfully established two weeks later.

[0039] (2) The colon cancer model rabbits were fasted for 24 hours in advance to cleanse their intestines;

[0040] (3) The colon cancer model rabbits were divided into two groups: a blank control group (Control) and a GTTN-containing reagent treatment group. The GTTN-containing reagent treatment group was treated by administering 35.9 mg / kg of GTTN-containing reagent via a single ear vein, while the blank control group was treated by administering an equal volume of PBS via a single ear vein. Fluorescence signals in the intestines were observed using fluorescence endoscopy at 0.5 h, 1 h, 4 h, 5 h, 6 h, 8 h, and 10 h. The fluorescence signals of tumor sites and normal tissues in the colon cancer model rabbits were also statistically analyzed. Tissues labeled with GTTN fluorescence (green light) were defined as tumor tissues.

[0041] Figure 1 shows that, compared with the blank control group, the GTTN-containing reagent administered via the marginal ear vein to a colon cancer model rabbit enabled efficient imaging of colon cancer within the colon. Furthermore, the fluorescence signal of colon cancer within the colon was significantly higher than that of normal tissue, indicating that the GTTN-containing reagent has good specificity and high diagnostic accuracy for colon cancer detection. The fluorescence signal intensity of colon cancer within the colon at different time points shows that the imaging effect is best within 0.5 h to 4 h after injection, while the fluorescence signal intensity is weak from 5 h to 10 h, and the cancer is basically metabolized out of the intestine after 10 h.

[0042] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of a graphene-based tumor cell nucleus-targeting fluorescent nanoprobe reagent in the preparation of a diagnostic kit for gastrointestinal cancer.

2. The application according to claim 1, characterized in that, The preparation method of the graphene-based tumor cell nuclear targeting fluorescent nanoprobe reagent includes 2.5 mL GTTN, 0.024 g potassium dihydrogen phosphate, 0.0144 g disodium hydrogen phosphate, 0.08 g sodium chloride, 0.002 g potassium chloride, and deionized water to 10 mL, and then mixing them evenly.

3. The application according to claim 2, characterized in that, The pH of the reagent containing the graphene-based tumor cell nucleus-targeting fluorescent nanoprobe is 7.

0.

4. The application according to claim 1, characterized in that, The gastrointestinal cancer mentioned is colon cancer.

5. The application according to claim 4, characterized in that, The colon cancer described is a colon cancer model constructed from the VX2 tumor cell line.

6. The application of a graphene-based tumor cell nuclear-targeting fluorescent nanoprobe reagent in the preparation of a kit for screening animal models of gastrointestinal cancer or in the screening of animal models of gastrointestinal cancer.

7. The application according to claim 6, characterized in that, The gastrointestinal cancer mentioned is colon cancer.

8. A diagnostic kit for gastrointestinal cancer, characterized in that, The kit includes a graphene-based tumor cell nuclear-targeting fluorescent nanoprobe reagent, which comprises 2.5 mL GTTN, 0.024 g potassium dihydrogen phosphate, 0.0144 g disodium hydrogen phosphate, 0.08 g sodium chloride, 0.002 g potassium chloride and 10 mL deionized water.

9. The reagent kit according to claim 8, characterized in that, The pH of the reagent containing the graphene-based tumor cell nucleus-targeting fluorescent nanoprobe is 7.

0.

10. The reagent kit according to claim 8, characterized in that, The kit also includes a negative control reagent, which is PBS.

11. A method for screening animal models of gastrointestinal cancer, characterized in that, The method includes the step of detecting fluorescence signals in recipient animals using the kit described in any one of claims 8 to 10.

12. The method according to claim 11, characterized in that, The method includes the step of treating the recipient animal with the negative control reagent as described in claim 10.

13. The method according to claim 12, characterized in that, If the fluorescence signal of the recipient animal is significantly increased compared with that of the negative control reagent, it indicates that the gastrointestinal cancer animal model has been successfully established.

14. The method according to any one of claims 11 to 13, characterized in that, The animals mentioned include rabbits.

15. A method for diagnosing gastrointestinal cancer, characterized in that, The method includes the step of detecting fluorescent signals in intestinal tissue using the kit described in any one of claims 8 to 10.

16. The method according to claim 15, characterized in that, The gastrointestinal cancer mentioned is colon cancer.

17. The method according to claim 16, characterized in that, The colon cancer described is a colon cancer model constructed from the VX2 tumor cell line.

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