A method for constructing a nasopharyngeal carcinoma tumor-bearing rat model with increased nasal cavity secretion and uses thereof

Abstract

The application provides a method for constructing a nasopharyngeal carcinoma tumor-bearing rat model with increased nasal cavity secretion, and the rat model is constructed by subcutaneous injection of murine nasopharyngeal carcinoma cells (FAT) and local irradiation by X-ray irradiation. The rat model constructed by the method has both the tumor-bearing state of nasopharyngeal carcinoma and the side effect state of increased nasal cavity secretion after radiotherapy, and the immune system still exists, and is suitable for the research on the effect and mechanism of drugs, especially traditional Chinese medicines, for exerting a therapeutic effect by immunity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal model construction, and particularly relates to a construction method and application of a nasopharyngeal carcinoma tumor-bearing rat model with increased nasal cavity secretion. BACKGROUND

[0002] Nasopharyngeal carcinoma is one of the common head and neck malignancies, and its incidence is closely related to genetic, EB virus and environmental factors. The environmental factors are mainly divided into three categories: biological (mainly virus), physical (mainly radiation) and chemical substances. Due to the close relationship between the incidence of nasopharyngeal carcinoma and environmental factors and genetic factors, at present, the construction of nasopharyngeal carcinoma animal models mostly uses the method of long-term stimulation of chemical substances and inoculation of tumor cells in immunodeficient nude mice.

[0003] In 1973, the tumor research room of Hunan Medical College used diethyl nitrosamine, dimethyl benzanthracene or a combination of the two to induce nasopharyngeal carcinoma in rats. In 1981, they used the method of subcutaneous injection of 0.5% di-nitrosopiperazine aqueous solution to induce nasopharyngeal carcinoma in rats. The earliest time of nasopharyngeal carcinoma was found in the 4 groups of animals, which were 217, 192, 206 and 245 days, respectively (Yao Kaitai, Pan Shikai, Huang Jiliang, et al. Further study on di-nitrosopiperazine-induced nasopharyngeal carcinoma in rats [J]. Journal of Hunan Medical College, 1981(01): 1-6+97.). Since then, the method of di-nitrosopiperazine-induced construction of rat nasopharyngeal carcinoma model has been widely used, and after improvement, the modeling time is shortened to 90 days (Yang Jing, Xu Mingjun, Wang Yuli, et al. Preventive and therapeutic effects of stephania root alkaloids on nasopharyngeal carcinoma in rats via the MAPK / ERK signaling pathway and their effects on the expression of Survivin, XIAP and Bcl-2 [J]. Journal of Xi'an Jiaotong University (Medical Science), 2021, 42(04): 633-638). However, although the modeling time is greatly shortened, the time cost of 90 days of modeling cycle is still very high, which limits the use of this method.

[0004] In 1995, the Guangxi People's Hospital and the Chinese Academy of Preventive Medicine transplanted human nasopharyngeal carcinoma specimens into T lymphocyte immunodeficient nude mice subcutaneously, and nasopharyngeal carcinoma tumors could be formed in as fast as one month (Jiao Wei, Zhou Weiya, Zhang Xing, et al. Establishment and characteristics of a nude mouse model of human nasopharyngeal carcinoma liver metastasis (CNT-1) [J]. Guangxi Medicine, 1995, 17(01): 10-12.). The method of transplanting nasopharyngeal carcinoma tumor cells into the subcutaneous tissue of nude mice or other immunodeficient mice is simple to operate and fast to form a model, so it has also been widely used and continuously optimized. It has been reported that 9 days after the nasopharyngeal carcinoma tumor cells were transplanted and inoculated, soft nodules could be seen with the naked eye at the inoculation site, indicating that the model was successfully constructed (Chen Wubing, Wang Yilong, Ying Yongjie, Tao Baohong, Cai Zhiyi. Inhibition of nasopharyngeal carcinoma 5-8F cell growth in nude mice by overexpression of cell mitosis checkpoint and its mechanism [J]. Chinese Journal of Otorhinolaryngology Head and Neck Surgery, 2023, 04: 241-245+265). However, although the nude mouse tumor model is convenient to operate and fast to form a tumor, it has many limitations in actual application.

[0005] The immune system of the body has the function of immune surveillance. When malignant cells appear in the body, the immune system can recognize and specifically eliminate these "non-self" cells through immune mechanisms, resisting the occurrence and development of tumors. However, malignant cells can escape the body's immune surveillance in some cases through various mechanisms, rapidly proliferate in the body, and form tumors. Whether a tumor occurs and how it turns out depends on the overall effect of the two. For this reason, the study of the influence of tumor cells on the immune system is one of the focuses of tumor research, and stimulating and enhancing the body's immune system to kill tumor cells is one of the main ways of anti-tumor treatment. Because immunodeficient nude mice lack thymuses and cannot form T cells, they cannot perform normal immune functions, so they cannot be used for research on the intrinsic relationship between tumor cells and the immune system and drugs that exert anti-tumor effects through the immune system.

[0006] Currently, the treatment of nasopharyngeal carcinoma is mainly radiotherapy. Radiotherapy can control and kill tumor cells by damaging the genomes of tumor cells and interfering with the division and proliferation of tumor cells. However, radiotherapy can also cause damage to normal tissues and organs, leading to a number of side effects, including damage to the normal tissues of the nasal mucosa, increased inflammatory secretions, skin reactions (redness, scaling, itching), changes in taste, throat, smell, and hearing, dry mouth and difficulty swallowing, fatigue, and others.

[0007] Traditional Chinese medicine has the characteristics of multi-component and multi-target, and can play a comprehensive role in the treatment of nasopharyngeal carcinoma and the reduction of radiotherapy damage. Since traditional Chinese medicine can inhibit tumors and improve the quality of life by improving the function of the immune system, and the function of traditional Chinese medicine in reducing the side effects of nasopharyngeal carcinoma radiotherapy is closely related to its anti-tumor effect, therefore, when studying traditional Chinese medicine in the treatment of nasopharyngeal carcinoma, the function effects and mechanisms of the two aspects need to be studied simultaneously. Therefore, an animal model that can retain the immune system is needed for related research. The rat model is one of the most commonly used animal models in drug research, and the existing rat model modeling method cannot meet the needs of traditional Chinese medicine anti-nasopharyngeal carcinoma research due to the long modeling time or the lack of immune function of the obtained animal model. SUMMARY

[0008] The purpose of the present application is to overcome the deficiencies in the prior art, and to provide a construction method and use of a nasopharyngeal carcinoma tumor-bearing rat model with convenient preparation and short modeling time. The rat model prepared by the method can retain the immune system and can be used to study the treatment effect of nasopharyngeal carcinoma and the improvement effect of radiotherapy damage at the same time.

[0009] The purpose of the present application is achieved by the following technical solutions:

[0010] On the one hand, the present application provides a construction method of a nasopharyngeal carcinoma tumor-bearing rat model with increased nasal secretions, comprising the following steps:

[0011] (1) preparing a FAT cell suspension after culturing murine nasopharyngeal carcinoma cells;

[0012] (2) using the FAT cell suspension prepared in step (1) to subcutaneously inject the rat 2-4 times;

[0013] (3) using X-rays to locally irradiate the nasopharynx of the rat after subcutaneous injection in step (2).

[0014] Preferably, the cell suspension in step (1) has a density of 4×10 7 cells / mL.

[0015] Preferably, the single injection dose of the subcutaneous injection in step (2) is 2×10 7 -4×10 7 cells per rat.

[0016] Further, the single injection dose of the subcutaneous injection in step (2) is 4×10 7 cells per rat.

[0017] Preferably, the subcutaneous injection in step (2) is injected once every 5 days at the same location, for a total of 3 injections.

[0018] Preferably, the X-ray irradiation in step (3) is performed 7 days after the first subcutaneous injection of the rats.

[0019] Preferably, the cumulative irradiation dose of the X-ray irradiation in step (3) is 28-50 Gy.

[0020] Further, the cumulative irradiation dose of the X-ray irradiation in step (3) is 50 Gy, 10 Gy / day, and the irradiation is performed continuously for 5 days.

[0021] In another aspect, the present application provides the use of the rat model obtained by the above model construction method in evaluating the therapeutic effect of a drug for treating nasopharyngeal carcinoma.

[0022] Preferably, the drug is a single traditional Chinese medicine, a traditional Chinese medicine composition, or a traditional Chinese medicine preparation.

[0023] Within the scope of the present application, the "single traditional Chinese medicine" refers to a single traditional Chinese medicine or an extract thereof. The "traditional Chinese medicine composition" refers to a composition of two or more traditional Chinese medicine materials or extracts thereof. The "traditional Chinese medicine preparation" refers to a preparation of the single traditional Chinese medicine or the traditional Chinese medicine composition and conventional pharmaceutical excipients. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Figure 2 shows the pathological picture of the nasopharyngeal carcinoma tumor in the rats in Example 2 (×400). DETAILED DESCRIPTION

[0025] The present application will be further described in detail below with reference to the specific embodiments, and the examples given are only for the purpose of illustrating the present application, but not for limiting the scope of the present application.

[0026] Example 1

[0027] 1. X-ray irradiation of the nasopharynx

[0028] Thirty rats were divided into five groups, i.e., the irradiation dose 1 group, the irradiation dose 2 group, the irradiation dose 3 group, the irradiation dose 4 group, and the irradiation dose 5 group, with 6 rats in each group. Different doses of X-ray irradiation were given to the nasopharynx of the rats in each group, and the specific irradiation doses were as follows:

[0029]

[0030] 2. Observation indexes

[0031] ① Animal mortality: the death of the animals was observed every day after the first X-ray irradiation of the nasopharynx of the rats.

[0032] ②Nasal discharge: The survival rate was 100% in the group of rats, after the first X-ray irradiation of the nasopharynx, the nasal mucosa of rats was observed at 1, 2, 3, 4 weeks, respectively, to observe the congestion of the nasal mucosa, and the nasal discharge was collected with a cotton ball, the cotton ball was washed with quantitative distilled water, and the amount of discharge was determined by turbidimetry.

[0033] 3. Experimental results

[0034] ① Animal mortality

[0035] No animal death occurred in groups 1-4, and the total irradiation dose of group 5 was 60 Gy, with a mortality rate of 50%.

[0036] Table 1-1 Mortality rate after local X-ray irradiation of rats (n = 6)

[0037]

[0038] ②Nasal discharge

[0039] In groups 1-4, no obvious changes were observed in the appearance of the rat nose at 1 week after the first X-ray irradiation of the nasopharynx, and at 2-4 weeks, the nasal cavity of rats in each group appeared to have visible secretions. The secretions were collected for detection, and the OD value of the nasal discharge of rats in the irradiation dose 5 group was the highest.

[0040] Table 1-2 Detection results of nasal discharge after local X-ray irradiation of rats (n = 6)

[0041]

[0042]

[0043] In summary, the irradiation dose of group 4 is the best, which can ensure the production of enough nasal discharge and will not cause the death of rats.

[0044] Example 2

[0045] 1. Preparation of FAT cell suspension

[0046] (1) Resuscitation culture

[0047] The frozen tube of murine nasopharyngeal carcinoma cells (FAT cells) was taken out from liquid nitrogen and directly put into a 37°C water bath, while being shaken to melt quickly. Fresh DMEM high-sugar culture solution containing 10% fetal bovine serum and 1% double antibody was added in advance into a centrifuge tube, and the thawed cell suspension was gently blown and transferred into the centrifuge tube. The cell suspension was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded. Fresh DMEM high-sugar culture solution was added to prepare a cell suspension. 5 ml of the cell suspension was taken and inoculated into a T25 cell culture bottle, which was cultured at 37°C, 5% CO2, and saturated humidity. The culture medium was high-sugar DMEM containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin. The cells were adherently grown, and the culture medium was replaced every other day. When the cells reached 90% confluence, they were digested with 0.25% trypsin containing 0.02% EDTA.

[0048] (2) Preparation of cell suspension

[0049] The culture solution after digestion in step (1) was centrifuged (1000 r / min) for 10 min, and the cells were collected to prepare a single cell suspension with PBS. The cell density was adjusted to 4 x 10 7 cells / mL for standby use.

[0050] 2. Cancer cell transplantation and X-ray irradiation

[0051] Ten SPF male F344 rats were taken as a model group to perform modeling. The fur on the hind limbs and armpits was removed with an electric shaver, and the rats were subcutaneously injected with the rat nasopharyngeal carcinoma FAT cell strain at a dose of 4 x 10 7 cells / rat. The injection was performed once every 5 days at the same position, and a total of 3 injections were performed. Seven days after the first injection of tumor cells, the rats were given X-ray irradiation on the nasopharynx, once a day, at a dose of 10 Gy, for 5 consecutive days. Another 10 F344 rats were not treated and served as a normal control group.

[0052] 3. Observation indexes

[0053] (1) Nasal secretion: After the first X-ray irradiation on the nasopharynx of the rats, the nasal mucosa congestion of the rats was observed at 1, 2, 3, and 4 weeks, respectively. The nasal secretion was collected with a cotton ball, the cotton ball was washed with a certain amount of distilled water, and the amount of secretion was determined by the turbidimetry method.

[0054] (2) Pain and itching reaction: The rat nose was touched to observe the rat's avoidance and screaming to evaluate the rat's pain, and the rat's scratching of the nose was observed to evaluate the itching symptoms.

[0055] Appendix: Criteria for pain and itching relief in rats

[0056]

[0057] (3) Tumor observation: After the first injection of tumor cells, the injection site was observed daily; on day 35, the rats were anesthetized, and the tumors were dissected and measured for volume, fixed with 4% formaldehyde, and observed for pathological changes by HE staining.

[0058] (4) Thymus observation: The rats were dissected, and the thoracic cavity was opened to observe the thymus.

[0059] 4. Experimental results

[0060] (1) Nasal secretions

[0061] In the first week after the first X-ray irradiation of the rat nasopharynx, no obvious changes were observed in the appearance of the rat nose; in the second to fourth weeks, the rat nose began to swell, and nasal secretions appeared subsequently. In the normal group without irradiation, no abnormalities were observed.

[0062] Nasal secretions were collected and measured by spectrophotometry. The results showed that, compared with the normal group, in the second to fourth weeks after the first X-ray irradiation of the rat nasopharynx, the OD value of the secretions in the model group increased significantly (P<0.01).

[0063] 0.01).

[0064] Table 2-1 Detection results of rat nasal secretions after irradiation (n=10)

[0065]

[0066] Note: Compared with the normal group, ** represents P<0.01.

[0067] (2) Itching reaction

[0068] In the first week after the first X-ray irradiation of the rat nasopharynx, no obvious changes were observed in the behavior of the rats when touching the rat nose; in the second to fourth weeks, when touching the rat nose, the rats began to show symptoms such as avoidance, screaming, and scratching the nose. In the normal group, no avoidance, screaming, and other pain reaction symptoms were observed when touching the rat nose; compared with the normal group, the pain reaction symptoms such as avoidance and screaming in the model group were very obvious (P<0.01).

[0069] No itching reaction symptoms such as scratching the nose were observed in the normal group; compared with the normal group, in the second to fourth weeks, the itching reaction symptoms such as scratching the nose in the model group were very obvious (P<0.01).

[0070] Table 2-2 Scoring results of nasal pain reaction symptoms in rats after irradiation (n=10)

[0071]

[0072] Note: Compared with the normal group, ** represents P<0.01.

[0073] Table 2-3 Scoring results of nasal itching reaction symptoms in rats after irradiation (n=10)

[0074]

[0075] Note: Compared with the normal group, ** represents P < 0.01.

[0076] (3) Tumor observation

[0077] Seven days after the first injection of tumor cells, small nodules were visible under the skin. After dissection on the 35th day, the size of the rat nasopharyngeal carcinoma tumor was 2.80±0.89cm. 3 Pathological examination showed that the nuclei were of different morphologies, with clear nucleoli, coarse chromatin, and nuclear division figures. The cells were highly malignant (see attached Figure 1 ).

[0078] (4) Thymus observation

[0079] The thymus was dissected and observed with the naked eye, and no abnormalities were found in the thymus.

[0080] In summary, the model construction method of the present invention adopts the nasopharyngeal carcinoma cell transfer inoculation method, which is simple to operate, small nodules can be seen subcutaneously in 7 days, and the modeling time is short; the obtained rat model did not show abnormal changes in the immune organ - thymus during dissection, the immune system was normal, nasal secretions increased, pain and itching symptoms were obvious, and significant nasopharyngeal damage and increased secretions, which are common symptoms after radiotherapy, were shown. It can be used for the study of the comprehensive effect of traditional Chinese medicine in treating nasopharyngeal carcinoma and reducing radiotherapy damage.

Claims

1. A method for constructing a nasopharyngeal carcinoma tumor-bearing rat model with increased nasal secretion, comprising the following steps: (1) The mouse nasopharyngeal carcinoma cells are cultured to prepare a cell suspension of FAT cells, and the density of the cell suspension is 4 x 10 7 cells / mL; (2) 2-4 times of subcutaneous injection of the F344 rats with the cell suspension prepared in step (1), the single injection dose of the subcutaneous injection being 2 x 10 7 4 x 10 7 cells per rat; (3) After the first subcutaneous injection for 7 days, the nasopharynx of the rat after subcutaneous injection in step (2) is locally irradiated using X-rays, and the cumulative irradiation dose of the local irradiation of the nasopharynx of the rat using X-rays is 28-50 Gy.

2. The construction method of claim 1, wherein, The single injection dose for the subcutaneous injection in step (2) is 4 x 10 7 cells / animal.

3. The construction method of claim 1, wherein, The subcutaneous injection in step (2) is performed once every 5 days at the same position, and a total of 3 times.

4. The construction method of claim 1, wherein, The cumulative irradiation dose of the local irradiation of the nasopharynx of the rat using X-rays in step (3) is 50 Gy, 10 Gy per day, and continuous irradiation for 5 days.

5. Use of the rat model obtained by the construction method of any one of claims 1-4 in screening drugs for treating nasopharyngeal carcinoma.

6. Use according to claim 5, characterized in that, The drug is a single traditional Chinese medicine, a traditional Chinese medicine composition, or a traditional Chinese medicine preparation.

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