CN110537992A - Construction method for improving human nasopharyngeal carcinoma in-situ transplantation tumor model - Google Patents

Abstract

The invention relates to a nasopharyngeal carcinoma transplanted tumor model used in the technical field of medical models, which is improved on the basis of a nude mouse orthotopic transplanted tumor model and aims to overcome the technical difficulties of high incidence rate of asphyxia death during transplantation, short survival time after transplantation, inapplicability to longer-time experiments and the like. The invention adopts inhalation anesthesia in the preparation of a tumor model, firstly, a nude mouse reaches the anesthesia depth through aeroanesthesia, a 1ml insulin needle is used for sucking 105 5-8F cell suspension of stably transfected Luc, the oral cavity passes through 5mm below a white line at the boundary of a hard palate and a soft palate, the suspension passes through a mucosa to the left or right pharyngeal crypt for inoculation, the inoculation amount is controlled within 25 microliters, the result shows that the incidence rate of nasopharyngeal carcinoma of the nude mouse model is 100 percent, and the nude mouse which is dead due to dyspnea after planting does not occur in the planting process. The model is an in-situ nasopharyngeal carcinoma model which can be used for researching in-situ nasopharyngeal carcinoma development and metastasis and is also suitable for long-term experimental treatment and diagnosis research of nasopharyngeal carcinoma.

Description

Construction method for improving human nasopharyngeal carcinoma in-situ transplantation tumor model

Technical Field

The invention relates to the field of medical models, in particular to a construction method of an improved human nasopharyngeal carcinoma in-situ transplanted tumor model.

Background

at present, animal models of nasopharyngeal carcinoma at home and abroad comprise: carcinogen induction, tumor cell transplantation and transgenic animal models. The three models have certain defects, wherein the carcinogen induction method has long period and low success rate and is rarely applied. The transgenic method has high cost and low success rate, and is not suitable for long-term large-scale experiments.

The tumor cell transplantation method is the most commonly used method at present, but the widely applied nasopharyngeal carcinoma animal model at present is mainly subcutaneous transplantation tumor, but the subcutaneous transplantation tumor is easy to form envelope and transfer and the center of the tumor is easy to necrotize, so that the method cannot be well matched with preclinical research. In the past, a preparation method of a visualized nude mouse nasopharyngeal carcinoma orthotopic transplantation tumor model has been reported. This method illustrates an in situ vaccination method via the oral passage through the soft palate mucosa to the nasopharynx of nude mice, but in the course of our studies, some disadvantages in making this model were found: the cases of immediate asphyxiation death of nude mice after inoculation are very common in the case of liquid anesthesia; the survival time of the nude mice after planting is short, about 6-14 days, and the short survival time results in a low acquisition and transfer rate.

Disclosure of Invention

the present invention aims to overcome the above-mentioned shortcomings and provide a technical solution to solve the above-mentioned problems.

A construction method of an improved human nasopharyngeal carcinoma in-situ transplantation tumor model comprises the construction of an animal model, the preparation of a tumor source and the model making; constructing an animal model by selecting a nude mouse and selecting a nasopharyngeal planting anatomical position of the nude mouse; the tumor source is prepared by carrying out passage and culture on 5-8F-luc human nasopharyngeal carcinoma cell strains, preparing single cell suspension after the tumor cells are in logarithmic growth phase, adjusting the cell concentration to 1.4 multiplied by 105/ml, and then placing the single cell suspension in an ice box to keep the cell activity to the maximum extent for standby;

The method for making the model specifically comprises the following steps:

S1, in an aseptic experimental environment, selecting 16-18g of male nude mice, placing the male nude mice in a closed gas anesthesia (isoflurane) device, taking out the nude mice to be placed on an open gas anesthesia device to start to maintain anesthesia in an operation when the righting reflex of the nude mice to be detected disappears, muscles are relaxed, limbs do not move, and beards do not touch the reaction;

S2, sucking 25 mu L of cell suspension obtained in the tumor source preparation step by using a 1mL insulin fine needle, wherein the concentration is 1.4 multiplied by 105/mL, selecting a place 5mm below a white line at the junction of a soft palate and a hard palate, penetrating the fine needle through the soft palate to enter nasopharyngeal mucosa, slowly injecting cells after the fine needle slightly deviates from the left side or the right side to enter a pharyngeal crypt, and keeping the temperature of a nude mouse by using a warm water bag after operation while closely observing the vital signs of the nude mouse;

S3, placing the nude mice with tumor in a clean EVC mouse cage after the nude mice are awake, adding the sterilized feed and replacing the sterilized water;

S4, conveying the EVC mouse cage to an animal room through a fume hood; the sterilized padding, the feed and the sterilized water were changed every 3 days, and the vitality and the growth state of the nude mice were observed.

the invention has the beneficial effects that:

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1A is a schematic representation of the nasopharynx of a nude mouse;

FIG. 1B is a schematic view of the planting principle;

FIG. 2A is a graph of the intensity of bioluminescent tumor signals in vivo imaging of tumor-bearing nude mice with the coronal position of magnetic resonance T2WI as a reference for tumor location;

FIG. 2B is a graph showing the change in the intensity of bioluminescent tumor signals in vivo imaging of tumor-bearing nude mice;

FIG. 2C tumor locations reconstructed after live imaging 3D module capture;

FIG. 2D is a HE staining diagram of a tumor cross section tissue of a head of a tumor-bearing nude mouse;

FIG. 3A shows primary tumor and metastatic lymph nodes in supine position of nude mice bearing tumor in vivo;

FIG. 3B HE staining pattern of metastatic lymph node tissue of tumor-bearing nude mice.

FIG. 4A is a fluorescence profile 8h after probe injection in the advanced nasopharyngeal carcinoma model.

fig. 4B shows a corresponding full scan pathological section.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

1 materials and methods

1.1 cell strain and cell digestive juice (0.25% trypsin-EDTA, Gibco), DMEM (containing 10% fetal bovine serum and 1% double antibody solution), PBS buffer solution, cell incubator, inverted microscope, cell counting plate.

1.2BALB/C male nude mice 20, SPF grade, purchased from Beijing Wittingle laboratory animal technology Co., Ltd, aged 5-6 weeks, and weighing 16-18 g. The groups were randomly divided into 10 control groups and 10 model groups.

2 construction of animal models

the nasopharyngeal position of the nude mice is shown in figure 1A and the anatomical position of the nude mice is shown in figure 1B.

2.1 preparation of tumor Source

After 5-8F-luc culture and passage of the human nasopharyngeal carcinoma cell strain transfected with bioluminescent protein, tumor cells in logarithmic growth phase are prepared into single cell suspension, the cell concentration is adjusted to 1.4 multiplied by 105/ml, and the single cell suspension is placed in an ice box to keep the cell activity to the maximum extent.

2.2 method of making model

the whole experiment process is carried out on a sterile operating table;

s1, selecting 16-18g of male nude mice, placing the male nude mice in a sealed gas anesthesia (isoflurane) device, taking out the nude mice to be examined, placing the nude mice on an open gas anesthesia device, and starting to maintain anesthesia in an operation when the righting reflex of the nude mice disappears, muscles are relaxed, limbs do not move, and beards do not touch the reaction;

S2, sucking 25 mu L of cell suspension with 1mL of insulin fine needle, wherein the concentration is 1.4 multiplied by 105/mL, selecting 5mm below a white line at the junction of a soft palate and a hard palate, and making the fine needle penetrate through the soft palate to enter a position of a left or right pharyngeal recess, so that suffocation death in operation caused by injection at the position of the middle line can be better avoided, meanwhile, a transplanted tumor obtains a larger growth space and is closer to the growth environment of clinical nasopharyngeal carcinoma, the life cycle of a nude mouse with in-situ transplanted tumor is prolonged, the nude mouse is suitable for long-cycle experiments and establishment of a lymph node spontaneous metastasis model, a warm water bag is used for preserving heat of the nude mouse after the operation, and simultaneously, the vital signs of the nude mouse are closely observed;

S3, placing the nude mice with tumor in a clean EVC mouse cage after the nude mice are awake, adding the sterilized feed and replacing the sterilized water;

s4, EVC cages are delivered to the animal room through the fume hood. Changing the sterilizing padding, the feed and the sterilizing water every 3 days, and observing the vitality and the growth state of the nude mice;

The control group adopts liquid anesthesia before operation

The specific liquid anesthesia step comprises:

D1, preparation of liquid anesthetic: weighing 2.5g of tribromoethanol, sucking 5ml of tert-amyl alcohol by using a 5ml liquid transfer gun, adding the tribromoethanol and the tert-amyl alcohol into 200ml of deionized water, placing the mixture on a stirrer, setting the stirring speed to be 300r/min, stirring for 20min, filtering the reaction solution by using a 0.45-micrometer filter to remove unreacted substances and bacteria, and subpackaging the filtrate for later use;

D2, injecting 300 mu L liquid anesthetic into the abdominal cavity of the nude mouse, and allowing the nude mouse to enter deep anesthesia after ten minutes.

the model group adopts inhalation gas anesthesia before and during the operation

The specific inhalation gas anesthesia step comprises the following steps: the method comprises the steps of inhaling isoflurane, selecting a special inhalation anesthesia machine for small animals, mixing pure oxygen for anesthesia, volatilizing isoflurane through oxygen airflow, inducing the concentration of 2-3% for anesthesia, and maintaining the concentration for 1.5-2%. The anesthesia time is 2-3 min.

As shown in Table 1, the mortality rate of the model animals in the operative hours and the postoperative hours is obviously higher than that of the control group, and the statistical significance is achieved (P is less than 0.05).

Example 2

1 materials and methods

1.1 cell strain and cell digestive juice (0.25% trypsin-EDTA, Gibco), DMEM (containing 10% fetal bovine serum and 1% double antibody solution), PBS buffer solution, cell incubator, inverted microscope, cell counting plate.

1.2BALB/C male nude mice 20, SPF grade, purchased from Beijing Wittingle laboratory animal technology Co., Ltd, aged 5-6 weeks, and weighing 16-18 g. The groups were randomly divided into 10 control groups and 10 model groups.

2. construction of animal models

2.1 preparation of tumor Source

After 5-8F-luc culture passage of the human nasopharyngeal carcinoma cell line transfected with bioluminescent protein, tumor cells in logarithmic growth phase are prepared into single cell suspension, and the specific method is the same as that in example 1.

2.2 method of making model

the specific construction method of the model group is the same as that of example 1.

The control group was implanted under the mucosa of the right posterior nasopharynx after penetrating the soft palate and the hard palate with an injection needle as reported in the past literature, and the rest steps were identical to those of the model group.

as shown in Table 2, the mortality rate and survival time after operation of the model animals are significantly longer than those of the control group, and the statistical significance is achieved.

	Death in operation (/ only)	postoperative survival time (/ day)
			Model set (n is 10)	0/10	44±9
Control group (n ═ 10)	5/10	34±12
			P value	0.007	0.001

Example 3

1. Materials and methods

1.1 cell strain and cell digestive juice (0.25% trypsin-EDTA, Gibco), DMEM (containing 10% fetal bovine serum and 1% double antibody solution), PBS buffer solution, cell incubator, inverted microscope, cell counting plate.

1.2BALB/C male nude mice 20, SPF grade, purchased from Beijing Wittingle laboratory animal technology Co., Ltd, aged 5-6 weeks, and weighing 16-18 g. The groups were randomly divided into 20 control groups and 20 model groups.

2. Construction of animal models

2.1 preparation of tumor Source

after 5-8F-luc of the human nasopharyngeal carcinoma cell strain transfected with bioluminescent protein is cultured and passaged, tumor cells in logarithmic growth phase are prepared into single cell suspension, and the specific method is described in the invention.

2.2 method of making model

The specific construction method of the model group is expressed in the same way as the invention.

The control group was implanted under the mucosa of the right posterior nasopharynx after penetrating the soft palate and the hard palate with an injection needle as reported in the past literature, and the rest steps were identical to those of the model group. Both observation periods were 8 weeks.

As shown in Table 2, the model group animals showed cervical and pulmonary metastasis within 8 weeks after surgery, while the control group showed no metastasis.

	Neck metastasis	metastasis of lung
			Model set (n 20)	5/20	2/20
Control group (n ═ 20)	0/20	0/20
			P value	0.016	0.154

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Claims (2)

1. A construction method of an improved human nasopharyngeal carcinoma in-situ transplantation tumor model is characterized by comprising the steps of construction of an animal model, preparation of a tumor source and model making; the tumor source is prepared by carrying out passage and culture on 5-8F-luc human nasopharyngeal carcinoma cell strains, preparing single cell suspension after the tumor cells are in logarithmic growth phase, adjusting the cell concentration to 1.4 multiplied by 105/ml, and then placing the single cell suspension in an ice box to keep the cell activity to the maximum extent for standby;

the method for making the model specifically comprises the following steps:

S1, in an aseptic experimental environment, selecting 16-18g of male nude mice, placing the male nude mice in a closed gas anesthesia (isoflurane) device, taking out the nude mice to be placed on an open gas anesthesia device to start to maintain anesthesia in an operation when the righting reflex of the nude mice to be detected disappears, muscles are relaxed, limbs do not move, and beards do not touch the reaction;

s2, sucking 25 mu L of cell suspension obtained in the tumor source preparation step by using a 1mL insulin fine needle, wherein the concentration is 1.4 multiplied by 105/mL, selecting a place 5mm below a white line at the junction of a soft palate and a hard palate, penetrating the fine needle through the soft palate to enter nasopharyngeal mucosa, slowly injecting cells after the fine needle slightly deviates from the left side or the right side to enter a pharyngeal crypt, and keeping the temperature of a nude mouse by using a warm water bag after operation while closely observing the vital signs of the nude mouse;

S3, placing the nude mice with tumor in a clean EVC mouse cage after the nude mice are awake, adding the sterilized feed and replacing the sterilized water;

s4, conveying the EVC mouse cage to an animal room through a fume hood; the sterilized padding, the feed and the sterilized water were changed every 3 days, and the vitality and the growth state of the nude mice were observed.

2. The method for constructing an improved human nasopharyngeal carcinoma orthotopic transplantation tumor model according to claim 1, wherein the construction of the animal model comprises selecting a nude mouse, implanting the nasopharynx of the nude mouse with anatomical details, and performing gas anesthesia before surgery.