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

Abstract

The invention provides application of a GTTN-containing reagent in preparation of a gastrointestinal cancer diagnosis kit, and belongs to the technical field of gastrointestinal cancer diagnosis reagents. The research finds that the GTTN-containing reagent can specifically diagnose gastrointestinal cancer, especially colon cancer, and the GTTN-containing reagent has a good imaging effect and an accurate detection result at the colon cancer part, and has a good clinical application prospect in the colon cancer diagnosis reagent.

Description

Technical Field

[0001] The invention belongs to the technical field of gastrointestinal cancer diagnostic reagents, and in particular relates to an application of a GTTN reagent in preparing a gastrointestinal cancer diagnostic kit. Background Art

[0002] Gastrointestinal malignancies are the most common digestive system tumors, and their high incidence and mortality rates seriously threaten people's health. As a malignant tumor of the digestive tract, colon cancer is the third most common malignant tumor in the world. When colon cancer cells metastasize or spread, the five-year survival rate of patients is extremely low. At present, the treatment of colon cancer is still mainly radical surgery, but due to the poor chemotherapy effect after colon cancer metastasis, some patients still experience recurrence after surgery. Therefore, the screening and diagnosis of colon cancer is very important for the prevention and treatment of colon cancer, and the development of effective reagents and kits for monitoring the recurrence and metastasis of colon cancer is an important issue that needs to be solved urgently.

[0003] If colon cancer originates in the gastrointestinal tract, it will be difficult to diagnose early due to the hidden site of onset, and it may even be easily missed during endoscopic examination. Fluorescence imaging technology has been used for a long time, and indocyanine green (ICG) is a typical representative of it. It has been widely used in breast cancer, hepatobiliary and gastrointestinal tumor surgery. Depending on the conditions of each hospital and the purpose of use, it can be injected under endoscopy before surgery, injected into the subserosa through laparoscopy or injected intravenously to locate the tumor, display lymph nodes, assist in judging the blood supply of the anastomosis, confirm liver metastasis, etc. If a tumor is found during surgery, ICG can be directly injected into the subserosa during surgery to assist in judging the distribution of lymph nodes, but the imaging effect may be poor. At this time, if the injection is supplemented again, the diffusion area may be too large, and the tumor cannot be effectively distinguished from normal tissue under the fluorescence imaging mode. In addition, the diagnosis of colon cancer in the gastrointestinal tract or colon cancer outside the gastrointestinal tract has been a major problem that has troubled clinicians so far, and there are almost no reliable imaging diagnostic methods. Therefore, it is urgent to find a reagent for diagnosing colon cancer, especially colon cancer in the gastrointestinal tract. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a use of a GTTN reagent in the preparation of a kit for diagnosing gastrointestinal cancer.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an application of a tumor cell nucleus-targeted fluorescent nanoprobe reagent based on graphene in the preparation of a kit for diagnosing gastrointestinal cancer.

[0007] Preferably, the preparation method of the reagent containing the graphene-based tumor cell nucleus-targeted fluorescent nanoprobe comprises: 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, 0.002 g of potassium chloride, and adding deionized water to 10 mL, and mixing evenly.

[0008] Preferably, the pH of the reagent containing the graphene-based tumor cell nucleus-targeted fluorescent nanoprobe 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 strain.

[0011] The present invention provides an application of a reagent containing a graphene-based tumor cell nucleus-targeted fluorescent nanoprobe in the preparation of a kit for screening a gastrointestinal cancer animal model or in the preparation of a gastrointestinal cancer animal model.

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

[0013] The present invention also provides a method for screening a gastrointestinal cancer animal model, comprising the step of detecting the fluorescence signal in a recipient animal using the above-mentioned reagent containing a graphene-based tumor cell nucleus-targeted fluorescent nanoprobe.

[0014] Preferably, the method comprises the step of treating the recipient animal with a negative control reagent.

[0015] Preferably, the negative control reagent is PBS; compared with the fluorescence signal of the negative control reagent, if the fluorescence signal of the recipient animal is significantly increased, it indicates that the construction of the gastrointestinal cancer animal model is successful.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides an application of a reagent containing GTTN in the preparation of a kit for diagnosing gastrointestinal cancer. The present invention has found that the reagent containing GTTN can specifically diagnose gastrointestinal cancer, especially colon cancer, and has good imaging effect and accurate detection results at the colon cancer site, and has good clinical application prospects in the preparation of reagents for diagnosing colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1Results of detecting colon cancer in the gastrointestinal tract using the GTTN reagent. A shows the fluorescence imaging results of colon cancer in the gastrointestinal tract 4 hours after injecting the GTTN reagent. B shows the comparison results of fluorescence signals between normal tissues and colon cancer in the gastrointestinal tract. C shows the fluorescence imaging results of colon cancer in the gastrointestinal tract at different times after injecting the GTTN reagent. Detailed implementation mode

[0019] The present invention provides an application of a graphene-based tumor cell nucleus-targeted fluorescent nanoprobe reagent in the preparation of a kit for diagnosing gastrointestinal cancer.

[0020] In the present invention, the preparation method of the graphene-based tumor cell nucleus-targeted 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, and adding deionized water to 10 mL, and mixing evenly to obtain the GTTN reagent. The pH of the graphene-based tumor cell nucleus-targeted fluorescent nanoprobe reagent is 7.0, and the pH of the GTTN reagent is adjusted with dilute hydrochloric acid with a concentration of 3 M. The preparation method of the GTTN reagent of the present invention has simple operation, low cost, and is easy to realize industrial production.

[0021] The GTTN reagent of the present invention can be used for imaging gastrointestinal cancer, and has good imaging effect, so as to effectively detect gastrointestinal cancer. As a preferred mode, the gastrointestinal cancer is colon cancer. Further, the colon cancer is a colon cancer model constructed from VX2 cells. In addition, the GTTN reagent of the present invention can directly penetrate the cell membrane of gastrointestinal cancer and specifically stain the tumor cell nucleus, without staining normal tissue cells, so as to achieve the purpose of specifically detecting gastrointestinal cancer.

[0022] The present invention provides an application of a graphene-based tumor cell nucleus-targeted 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.

[0023] In the above application of preparing a kit for screening a colon cancer animal model, the gastrointestinal cancer is colon cancer. Further, the colon cancer is a colon cancer model constructed from VX2 tumor cells, and the model animal is a rabbit.

[0024] The present invention also provides a method for screening an animal model of gastrointestinal cancer, including the step of detecting the fluorescence signal in a receptor animal using the above-mentioned graphene-based tumor cell nucleus-targeted fluorescent nanoprobe reagent.

[0025] In the present invention, the method includes the step of treating recipient animals with a negative control reagent. The recipient animals include rabbits. The negative control reagent is PBS; compared with the fluorescence signal of the negative control reagent, if the fluorescence signal of the recipient animal is significantly increased, it indicates that the construction of the gastrointestinal cancer animal model is successful. In the present invention, GTTN or the negative control reagent is administered to the recipient animal by a one-time ear marginal vein injection. The imaging effect of the injection of the GTTN reagent within 0.5 h to 4 h is good, which indicates that the present invention can quickly screen the gastrointestinal cancer animal model.

[0026] In the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art.

[0027] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] The preparation method of the graphene-based tumor cell nucleus-targeted fluorescent nanoprobe (GTTN) can be referred to the authorized patent with the publication number CN111467510A and the application number 202010278513.4. Specifically, the preparation method of the GTTN includes the following steps: adding pyrene powder (0.5 g) to nitric acid (25 mL, with a concentration of 65-68 wt%) at 80 °C and reacting for 24 h; after the reaction is completed, cooling, washing the obtained system with 150 mL of deionized water, and filtering with a 0.22 μm filter membrane; adding the obtained filtrate to an aqueous solution of Na2SO3 (50 mL, with a concentration of 0.5 mol / L), stirring for 0.5 h, then transferring it to a 150 mL ceramic autoclave, and heating at 130 °C for 12 h; then cooling to room temperature, transferring the obtained material to an autoclave with a polytetrafluoroethylene lining, and reacting in a 200 °C vacuum drying chamber for 12 h; after the reaction is completed, cooling, filtering the obtained system, and the obtained filtrate contains GTTN, and this filtrate is denoted as the GTTN stock solution.

[0029] Example 1

[0030] A preparation method of a reagent containing GTTN: Mix 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, add deionized water to 10 mL, mix evenly, and adjust the pH to 7.0 with 3M dilute hydrochloric acid to obtain the reagent containing GTTN.

[0031] Application of the reagent containing GTTN in detecting colon cancer in the gastrointestinal tract:

[0032] (1) Construction of the colon cancer model:

[0033] The VX2 tumor strain was used to obtain fish-like tumor tissues with vigorous growth at the edge of the tumor mass. The tissues were cut into a homogenate with ophthalmic scissors and an appropriate amount of normal saline was added to make a suspension. A 5 mL syringe needle (to prevent tissue blockage of the needle) and a 1 mL syringe were used to draw the tumor tissue suspension for standby.

[0034] (2) Rabbits were intravenously injected with 3% sodium pentobarbital at the marginal ear vein, and the anesthetic dose was 30 mg / kg, with an injection volume of 1 mL / kg. The rabbits were placed supine and fixed on the operating table, and the abdominal skin was prepared and routinely surgically disinfected. The colon part was isolated in the abdominal cavity of the rabbit. A 1.0 mL syringe was obliquely inserted into the submucosa of the intestine, and 0.1 mL of the rabbit-derived VX2 tumor tissue suspension was injected to construct a colon cancer model. After two weeks, the colon cancer model rabbits were successfully modeled, and colon cancer model rabbits were obtained.

[0035] (2) The colon cancer model rabbits were fasted 24 h in advance to clean the intestine;

[0036] (3) The colon cancer model rabbits were divided into two groups, namely the blank control group (Control) and the group treated with the GTTN reagent. The treatment method for the group treated with the GTTN reagent was to intravenously administer 35.9 mg / kg of the GTTN reagent to the colon cancer model rabbits at the marginal ear vein once. The treatment method for the blank control group was to intravenously administer an equal amount of PBS to the colon cancer model rabbits at the marginal ear vein once. The fluorescence signals in the intestine were observed at 0.5 h, 1 h, 4 h, 5 h, 6 h, 8 h, and 10 h using the fluorescence endoscope mode. At the same time, the fluorescence signals of the tumor sites and normal tissues of the colon cancer model rabbits were counted. Among them, the tissues labeled with GTTN fluorescence (green light) were tumor tissues.

[0037] Figure 1 The results showed that compared with the blank control group, after the colon cancer model rabbits were intravenously administered with the GTTN reagent at the marginal ear vein, high-efficiency imaging of colon cancer in the colon could be achieved. Moreover, compared with normal tissues, the fluorescence signals of colon cancer in the colon were significantly higher than those of normal tissues, indicating that the GTTN reagent had good specificity and high diagnostic accuracy for the detection of colon cancer in the colon. From the fluorescence signal intensities of colon cancer in the colon at different times of the GTTN reagent, the imaging effect was good within 0.5 h to 4 h after the injection of the GTTN reagent, while the fluorescence signal intensity was weak at 5 - 10 h, and it was basically metabolized out of the intestine at 10 h.

[0038] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

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

2. The use according to claim 1, characterized in that: The preparation method of the graphene-based tumor cell nucleus targeting fluorescent nanoprobe reagent comprises 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, 0.002 g of potassium chloride, adding deionized water to 10 mL, and mixing evenly.

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

0.

4. The use according to claim 1, characterized in that: The gastrointestinal cancer is colon cancer.

5. The use according to claim 4, characterized in that: The colon cancer model is constructed from the VX2 tumor strain.

6. Use of a graphene-based tumor cell nucleus-targeted fluorescent nanoprobe reagent in the preparation of a kit for screening gastrointestinal cancer animal models or in screening gastrointestinal cancer animal models.

7. The use according to claim 6, characterized in that: The gastrointestinal cancer is colon cancer.

8. A method for screening gastrointestinal cancer animal models, characterized in that: The method comprises the step of using the graphene-based tumor cell nucleus-targeted fluorescent nanoprobe reagent as described in any one of claims 1 to 3 to detect fluorescent signals in a recipient animal.

9. The method according to claim 8, characterized in that The method includes the step of treating a recipient animal with a negative control agent.

10. The method according to claim 9, characterized in that The negative control reagent is PBS; 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 is successfully constructed.

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