CN101919747A - Method for building improved orthotopic transplantation tumor model of colorectal tumor in nude mice - Google Patents

Abstract

The invention discloses a transplantation tumor model of colorectal tumor for medical scientific research, which is produced by improving the orthotopic transplantation tumor model of colorectal tumor in nude mice. The invention aims to overcome the defect that the orthotopic transplantation tumor model of colorectal tumor has high intestinal obstruction incidence rate and short model animal survival time and is not suitable for long-time experiments. In the invention, the prepared tumor is transplanted close to the blood vessel in mesocolon. The method comprises the following steps: (1) preparation of tumor: injecting single cell suspension of the tumor cell to subcutis of nude mice and preparing the source of the transplantation tumor; (2) transplanting the prepared tumor close to the blood vessel in mesenterium of nude mice but not the serosa of the intestinal tube; and (3) raising the nude mice in an aseptic laminar flow room, controlling the room temperature to be (25+/-1) DEG C and relative humidity to be 40-50% and continuously observing the nude mice. The animal model built by the invention can reproduce the natural clinical pathology process of human colorectal tumor, can not suffer from obstruction of the lumen in the digestive tract in the short term and has transfer mode similar to that of the patients.

Description

The construction method of improvement orthotopic transplantation tumor model of colorectal tumor in nude mice

Technical field

The present invention relates to a kind of new colon cancer transplanted tumor model that medical research is used, is improvement on the basis of orthotopic transplantation tumor model of colorectal tumor in nude mice.

Background technology

The animal model of intestinal canal tumour has both at home and abroad at present: chemical carcinogen revulsion, tumour transplatation method and transgenic animal model.

The chemical carcinogen revulsion modeling cycle is long, and success rate is low, less at present use.

Transgenic mouse cost height, strain is few, and using in long-term large-scale experiment has certain difficulty.

The tumour transplatation method is present the most frequently used method.Its advantage is: make a rout inoculate the oncocyte of same amount simultaneously, growth rate is relatively more consistent, individual variation is less, inoculation survival rate is nearly 100%, and is similar to host's influence, is easy to objective judgement curative effect, can of the same race or with the strain animal in transplant continuously, the long-term reservation, be for experiment, and the test period is generally all shorter, and experimental condition is easy to control etc.The tumour transplatation method comprises subcutaneous transplantation tumor method, ascites tumor method, orthotopic transplantation method etc. again.Orthotopic transplantation is because can offer the microenvironment that transplanted thing is similar to human body, so be used widely in recent years.As seen orthotopic transplantation tumor model of colorectal tumor in nude mice is all reported both at home and abroad at present.But in our research process, found some shortcomings of this model: the tumor bulk-growth is too fast, forms gastral blocking easily, causes experimental animal death, the too high (see figure 2) of mortality rate of experimental animal in the test period.

The present invention has used a kind of new method dexterously in experimentation, the transplanted tumor piece is transplanted in the mesocolon by the blood vessel, has successfully set up the improvement orthotopic transplantation tumor model of colorectal tumor in nude mice.

Summary of the invention

At the above-mentioned deficiency of prior art, technical problem to be solved by this invention provide a kind of method simple, can offer transplanted thing be similar to the microenvironment of human body, do not influence tumor route of metastasis, be difficult for causing the colon cancer transplanted tumor model that digestive tract blocks, host's survival rate is high.

In order to solve the problems of the technologies described above, the structure of colon cancer transplanted tumor model provided by the invention comprises the steps:

(1) preparation in tumor source

After colon tumor cell cultivated, goes down to posterity, be prepared into single cell suspension, adjustment cell concentration to 2 * 10 with the tumor cell of exponential phase ⁷/ ml; With the sterilization of iodine tincture local skin, with 0.1ml/ nude mice, it is subcutaneous to inject oxter, nude mice right side with aseptic disposable syringe before the injection; Nude mice was taken food naturally after injection finished; When treating that tumor body diameter reaches the 1cm left and right sides, put to death mice with tumor, sterilization skin downcuts the tumor piece, removes downright bad part, selects for use faint yellow or be flesh of fish shape tumor tissue to place normal saline, and the tumor piece that is cut into diameter 2mm is standby;

(2) model production method

Modeling nude mice fasting 4h is with 0.5% pentobarbital solution, 35～50mg/kg body weight intraperitoneal injection of anesthesia; With iodine tincture sterilization, take off the abdomen center or through the about 2cm of right side rectus incision, successively each layer of open abdomen retracts peritoneum with vascular forceps, recognize ascending colon; Colon intestinal axle parallel direction is done one 0.3～0.4cm otch in the ascending mesocolon upper edge of distance ileocecus 2cm, 2 of tumor pieces that prepared are placed in the mesocolon near the blood vessel, and with 3-0 silk suture mesentery otch; The conventional incision of abdominal wall of sewing up, the feed naturally of the clear-headed back of anesthesia; Add erythromycin ethylsuccinate 1g/100ml in the drinking-water; Nude mice is raised in aseptic Laminar Flow Room, and room temperature is controlled at (25 ± 1) ℃, and relative humidity 40%～50% observed for 3 weeks continuously.

The present invention transplants the tumor piece for preparing in mesocolon near the blood vessel, but not the placenta percreta of intestinal tube.This method has been set up and can have been reappeared human colon carcinoma nature clinical pathology process, the digestive tract tube chamber can not take place in a short time block, and the transfer mode animal model similar to clinical patients, and the life span after the animal pattern modeling is obviously prolonged.This is for the research and development of antitumor drug, especially screening and the Its Mechanisms to the Chinese medicine antitumor drug has important practical significance.

The present invention can realize being similar to the purpose of orthotopic transplantation tumor model of colorectal tumor, and the host's mortality rate that blocks generation because of the digestive tract tube chamber is obviously reduced.The inventive method is simple, and modeling success rate height is practical, has certain economic, practical value.

Description of drawings

Fig. 1 improves the structure flow chart of orthotopic transplantation tumor model of colorectal tumor in nude mice for the present invention.

Fig. 2 is the animal model sketch map (arrow is depicted as the transplanted tumor of compressing intestinal among the figure, and visible intestinal blocks, and blocks near-end intestinal tube inflatable dilatation) that tumor colon orthotopic transplantation method makes up.

Fig. 3 is transplanted tumor piece plantation position sketch map in the construction method of the present invention.

Among Fig. 3: (1) tumor piece plantation position; (2) colon; (3) arteriovenous branch before the mesentery; (4) anterior mesenteric artery; (5) caecum.

The specific embodiment

Below in conjunction with the drawings and specific embodiments, further set forth the present invention.These embodiment are interpreted as only being used to the present invention is described and are not used in restriction protection scope of the present invention.After the content of having read the present invention's record, those skilled in the art can make various changes or modifications the present invention, and these equivalences change and modify and fall into claim of the present invention institute restricted portion equally.

Embodiment 1

1. material and method

1.1 cell strain and reagent Digestive system (0.25% pancreatin solution+1mM EDTA), contain the two anti-culture fluid of RPMI1640+, PBS liquid, CO2 incubator, inverted microscope, the erythromycin ethylsuccinate sheet of 10% hyclone

1.2 20 of animal BALB/C nude mices, the SPF level, available from the Chinese Academy of Sciences, Mus 6～8 weeks of age, body weight 18～20g.Be divided into 10 of matched groups, 10 of model group at random.

2. the structure of animal model

Make up flow process as shown in Figure 1.

2.1 the preparation in tumor source

After human colon cancer cell SW480 cultivated, goes down to posterity, be prepared into single cell suspension with the tumor cell of exponential phase, it is subcutaneous to inject oxter, nude mice right side, prepares the tumor source.Concrete grammar is with describing in the summary of the invention.

2.2 model production method

Specifically with describing in the summary of the invention.

Matched group migrates to ascending colon serous coat apart from ileocecus 2cm with the tumor piece.

Model group migrates to the tumor piece apart from the ascending mesocolon blood vessel of ileocecus 2cm other (as shown in Figure 3).

As shown in table 1, the existence natural law of model group animal obviously is longer than matched group, has statistical significance (P＜0.05).

The existence situation of table 1. lotus tumor nude mouse (SW480)

Embodiment 2

1. material and method

1.1 cell strain and reagent Digestive system (0.25% pancreatin solution+1mM EDTA), contain the two anti-culture fluid of RPMI1640+, PBS liquid, CO2 incubator, inverted microscope, the erythromycin ethylsuccinate sheet of 10% hyclone

1.2 20 of animal BALB/C nude mices, the SPF level, available from the Chinese Academy of Sciences, Mus 6～8 weeks of age, body weight 18～20g.Be divided into 10 of matched groups, 10 of model group at random.

2. the structure of animal model

Make up flow process as shown in Figure 1.

2.1 after the preparation in tumor source is cultivated human colon cancer cell LOVO, go down to posterity, be prepared into single cell suspension with the tumor cell of exponential phase, it is subcutaneous to inject oxter, nude mice right side, prepares the tumor source.Concrete grammar is with describing in the summary of the invention.

2.2 model production method

Specifically with describing in the summary of the invention.

Matched group migrates to ascending colon serous coat apart from ileocecus 2cm with the tumor piece.

Model group migrates to the tumor piece apart from the ascending mesocolon blood vessel of ileocecus 2cm other (as shown in Figure 3).

As shown in table 2, the existence natural law of model group animal obviously is longer than matched group, has statistical significance (P＜0.05).

The existence situation of table 2. lotus tumor nude mouse (LOVO)

Embodiment 3

1. material and method

1.1 cell strain and reagent Digestive system (0.25% pancreatin solution+1mM EDTA), contain the two anti-culture fluid of RPMI1640+, PBS liquid, CO2 incubator, inverted microscope, the erythromycin ethylsuccinate sheet of 10% hyclone

1.2 20 of animal BALB/C nude mices, the SPF level, available from the Chinese Academy of Sciences, Mus 6～8 weeks of age, body weight 18～20g.Be divided into 10 of matched groups, 10 of model group at random.

2. the structure of animal model

Make up flow process as shown in Figure 1.

2.1 after the preparation in tumor source is cultivated gastric carcinoma cells MKN-45, go down to posterity, be prepared into single cell suspension with the tumor cell of exponential phase, it is subcutaneous to inject oxter, nude mice right side, prepares the tumor source.Concrete grammar is with describing in the summary of the invention.

2.2 model production method

Specifically with describing in the summary of the invention.

Matched group migrates to ascending colon serous coat apart from ileocecus 2cm with the tumor piece.

Model group migrates to the tumor piece apart from the ascending mesocolon blood vessel of ileocecus 2cm other (as shown in Figure 3).

As shown in table 3, the existence natural law of model group animal obviously is longer than matched group, has statistical significance (P＜0.05).

The existence situation of table 3. lotus tumor nude mouse (MKN45)

Above embodiment illustrates that all the existence natural law of model group animal obviously is longer than matched group.Presentation of results migrates near the existence natural law that can obviously prolong mice the nude mouse ascending mesocolon blood vessel with the tumor piece, delays to block the animal pattern death that causes because of the intestinal due to the tumor.

Claims (2)

1. a construction method of improveing orthotopic transplantation tumor model of colorectal tumor in nude mice is characterized in that, comprises the steps:

(1) preparation in tumor source

After colon tumor cell cultivated, goes down to posterity, be prepared into single cell suspension, adjustment cell concentration to 2 * 10 with the tumor cell of exponential phase ⁷/ ml; With the sterilization of iodine tincture local skin, with 0.1ml/ nude mice, it is subcutaneous to inject oxter, nude mice right side with aseptic disposable syringe before the injection; Nude mice was taken food naturally after injection finished; When treating that tumor body diameter reaches the 1cm left and right sides, put to death mice with tumor, sterilization skin downcuts the tumor piece, removes downright bad part, selects for use faint yellow or be flesh of fish shape tumor tissue to place normal saline, and the tumor piece that is cut into diameter 2mm is standby;

(2) model production method

Modeling nude mice fasting 4h is with 0.5% pentobarbital solution, 35～50mg/kg body weight intraperitoneal injection of anesthesia; With iodine tincture sterilization, take off the abdomen center or through the about 2cm of right side rectus incision, successively each layer of open abdomen retracts peritoneum with vascular forceps, recognize ascending colon; Colon intestinal axle parallel direction is done one 0.3～0.4cm otch in the ascending mesocolon upper edge of distance ileocecus 2cm, 2 of tumor pieces that prepared are placed in the mesocolon near the blood vessel, and with 3-0 silk suture mesentery otch; The conventional incision of abdominal wall of sewing up, the feed naturally of the clear-headed back of anesthesia; Add erythromycin ethylsuccinate 1g/100ml in the drinking-water; Nude mice is raised in aseptic Laminar Flow Room, and room temperature is controlled at (25 ± 1) ℃, and relative humidity 40%～50% observed for 3 weeks continuously.

2. the construction method of improvement orthotopic transplantation tumor model of colorectal tumor in nude mice according to claim 1 is characterized in that, in the preparation process in tumor source, sterilization and injection process are finished in 1 hour after being prepared into single cell suspension.

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