CN114342878B - Establishment method of slow transmission type constipation animal model - Google Patents
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Abstract
The invention discloses a method for establishing a slow transmission type constipation animal model, which is characterized in that: injecting mixed reagents of two viruses pAAV9-CAG-EGFP-P2A-Cre-WPRE and pAAV9-EF1a-DIO-hM4D (Gi) -mCherry at multiple sites on the colon wall of a mouse; and then the slow transit constipation animal model is obtained by administering the dechlorazepine intraperitoneal injection after one month. The invention adopts a chemical genetic method, the virus expressing hM4D is transfected to colon enteric neurons, and the slow-transit constipation animal model is successfully established by injecting dechloropressin to activate a receptor in an abdominal cavity, is stable, simulates a clinical pathophysiological mechanism, and has the characteristics of high efficiency, accuracy, controllability and reversibility.
Description
Technical Field
The invention belongs to the field of biomedicine, relates to an animal model establishing method, and particularly relates to a slow-transmission constipation animal model establishing method.
Background
Slow Transit Constipation (STC) is a common gastrointestinal motility disorder disease, the prevalence rate of adult chronic constipation is 4% -10%, more common in the elderly, the prevalence rate of people over 70 years old reaches 23%, and the prevalence rate of women is higher than that of men. The patients mainly have the symptoms of difficult defecation and (or) reduced defecation frequency, dry and hard feces, abdominal distension, abdominal pain and reduced appetite, and the life quality of the patients is seriously influenced. At present, laxatives or prokinetic agents are usually adopted to treat slow transit constipation clinically, and severe patients need to be subjected to colon resection by an operation, but the clinical treatment effect is poor, so that the difficulty in clinical practice is caused. By establishing an animal model similar to the clinical pathogenesis of STC, the pathophysiological mechanism of STC is defined, and the method has important guiding significance for the treatment of STC.
The pathophysiological characteristics of STC are that the colon transmission time is obviously prolonged, the motor ability is reduced, and the abnormality of the colon peristalsis loop caused by the disturbance of the intestinal nerve axis regulation is an important pathological basis, including the morphologic change of the intermuscular and submucosal intestinal plexus of STC colon tissues, the abnormality of the number of the intermuscular neurons, and the concomitant up-regulation of excitatory neurotransmitters such as substance P down-regulation (SP), inhibitory neurotransmitters such as Vasoactive Intestinal Peptide (VIP) or Nitric Oxide (NO). At present, the method for establishing the slow-transmission constipation animal model mainly adopts compound diphenoxylate and loperamide for stomach filling and molding, but the existing construction method has no unified standard on the medicament dosage and the medicament administration time, the molding success rate is difficult to ensure, the molding stability is poor, and more importantly, the method does not accord with the pathogenesis of clinical patients.
Disclosure of Invention
The invention provides a novel construction method of a slow transmission type constipation model, which is a novel method for constructing a reversible slow transmission type constipation animal model with high success rate, stable model, simulated clinical pathophysiological mechanism, accurate regulation and control and high efficiency.
In order to achieve the above object, the present invention provides a method for establishing a slow transit constipation animal model, which has the following characteristics: injecting mixed reagents of two viruses pAAV9-CAG-EGFP-P2A-Cre-WPRE and pAAV9-EF1a-DIO-hM4D (Gi) -mCherry at multiple sites of colon wall of a mouse; and then the slow transit constipation animal model is obtained by administering the dechlorazepine intraperitoneal injection after one month.
Further, the present invention provides a method for establishing a slow transit constipation animal model, which may further have the following characteristics: wherein, the volume ratio of the two viruses pAAV9-CAG-EGFP-P2A-Cre-WPRE and pAAV9-EF1a-DIO-hM4D (Gi) -mCherry is 1: 1.
Further, the present invention provides a method for establishing a slow transit constipation animal model, which may further have the following characteristics: wherein, viruses pAAV9-CAG-EGFP-P2A-Cre-WPRE and pAAV9-EF1a-DIO-hM4D (Gi) -mCherry are injected into each site for 1 ul.
Further, the present invention provides a method for establishing a slow transit constipation animal model, which may further have the following characteristics: wherein the injection dosage of the clozapine is 100 ug/kg.
Further, the invention provides a method for establishing a slow transit constipation animal model, which can also have the following characteristics: wherein the mice are SPF male C57BL/6J mice, and the selection condition is that the mice with the body weight of 15-20g and the age of 8 weeks are selected.
Further, the present invention provides a method for establishing a slow transit constipation animal model, which may further have the following characteristics: wherein the breeding conditions of the mice are as follows: the temperature is 24 +/-2 ℃, the humidity is 50-70%, and 12h of intermittent illumination is carried out.
Further, the present invention provides a method for establishing a slow transit constipation animal model, which may further have the following characteristics: wherein, before injecting virus into mice, fasting for 24 hours, anaesthetizing, preparing skin, sterilizing, performing abdominal median incision, exposing colon, performing blunt separation, injecting at multiple sites of colonic muscle layer by using 5ul Hamilton injection needle with needle D of 0.47mm parallel to colon wall.
Further, the invention provides a method for establishing a slow transit constipation animal model, which can also have the following characteristics: wherein, the colonic muscularis multidot injection method of the virus comprises the following steps: pAAV9-CAG-EGFP-P2A-Cre-WPRE and pAAV9-EF1a-DIO-hM4D (Gi) -mCherry were mixed as follows: 1, mixing, namely, uniformly distributing six sites from the caecum colon junction to the rectum part according to the distance, injecting a sample injection needle in the ileum muscular layer in a direction parallel to the intestinal canal, and injecting 2ul of mixed solution at each site.
Further, the present invention provides a method for establishing a slow transit constipation animal model, which may further have the following characteristics: wherein the injection method of the dechlorazepine comprises the following steps: preparing clozapine into a clozapine solution, and injecting 100ug/kg of clozapine intraperitoneally into each mouse one month after viral infection; the preparation method of the clozapine solution comprises the following steps: dissolving clozapine in dimethyl sulfoxide to prepare a clozapine mother solution, and mixing the clozapine mother solution, polyethylene glycol 300, an emulsifier T-80 and normal saline to obtain a clozapine solution.
Further, the present invention provides a method for establishing a slow transit constipation animal model, which may further have the following characteristics: in the preparation of the clozapine solution, the concentration of the clozapine mother liquor is 1mmol/L, and the volume ratio of the clozapine mother liquor, the polyethylene glycol 300, the emulsifier T-80 and the physiological saline is 2: 8: 1: 9.
the invention has the beneficial effects that:
the invention provides a novel construction method of a slow transmission type constipation animal model, which adopts a virus transfection method to deliver inhibitory chemical genetic receptors to enteric nerves and express the inhibitory chemical genetic receptors, and injects a chemical genetic ligand, namely clozapine, into an abdominal cavity to activate the receptors, so as to provide a novel method for establishing the reversible slow transmission type constipation animal model with high success rate, stable model, simulation of clinical pathophysiology mechanism, accurate regulation and control.
The chemical genetic technology has the function of selectively regulating and controlling the excitability of neurons by cells and accurately acts on enteric neurons, thereby playing the role of promoting or inhibiting the intestinal motility. The invention transduces the chemogenetic inhibitory receptor to the enteric neuron through virus and expresses the chemogenetic inhibitory receptor, and then the chemogenetic ligand DCZ is injected into the abdominal cavity to activate the receptor, so that the receptor can effectively control the activity of the enteric neuron for a long time, and the chemogenetic ligand DCZ has the function of efficiently, reversibly and specifically inhibiting the enteric neuron, thereby realizing the inhibition of the colonic movement.
Drawings
FIG. 1 is a stool quality and stool water content determination for two groups of mice 24h before surgery, after virus injection, one week after DCZ injection, and after DCZ injection discontinuation;
FIG. 2 is a graph of the contraction of the muscle bars of two groups of mice after modeling for ex vivo colonic smooth muscle bar tonometry;
figure 3 is a graph of enteric nerve staining after modeling for two groups of mice.
Detailed Description
The present invention is further illustrated by the following specific examples.
The invention provides a method for establishing a slow-transmission constipation animal model, which selects an SPF male C57BL/6J mouse under the specific conditions of 8-week-old mouse weight of 15-20 g. Feeding under appropriate conditions: the temperature is 24 +/-2 ℃, the humidity is 50-70 percent, and the illumination is interrupted for 12 hours. Randomly dividing the mice into 2 groups, wherein the first group is a sham operation group (control group), and the mice in the sham operation group are injected with physiological saline at multiple sites on the colon intestinal wall; the second group is an experimental group, and the mixed reagent of two viruses pAAV9-CAG-EGFP-P2A-Cre-WPRE and pAAV9-EF1a-DIO-hM4D (Gi) -mCherry is injected at a plurality of sites on the colon and intestinal wall of a mouse. Both groups were injected intraperitoneally one month later with clozapine (deschloloclozapine, DCZ).
In this example, pAAV9-CAG-EGFP-P2A-Cre-WPRE was purchased from Heyuanbio, Inc., and the batch was CN 396; pAAV9-EF1a-DIO-hM4D (Gi) -mCherry was purchased from Virgilla, having the English name wzbio and a batch of 20210730018.
The specific method for establishing the slow transmission constipation model of the experimental group comprises the following steps: mice were fasted for 24 hours, anesthetized, prepped, sterilized, passed through a median abdominal incision, with colon exposed and blunt dissection, injected at multiple sites in the colonic muscularis using a 5ul Hamilton injection needle, with needle D ═ 0.47mm parallel to the colon wall, and injected at each site with 1ul each of the viruses pAAV9-CAG-EGFP-P2A-Cre-WPRE and pAAV9-EF1a-DIO-hM4D (Gi) -mCherry. One month later, the DCZ was given as an intraperitoneal injection at a dose of 100 ug/kg.
The colon muscularis multisite injection method of the virus comprises the following steps: pAAV9-CAG-EGFP-P2A-Cre-WPRE and pAAV9-EF1a-DIO-hM4D (Gi) -mCherry were mixed as follows: 1 and mixing. Six sites are uniformly distributed from the caecum colon junction to the rectum part according to the distance, the sample injection needle is parallel to the direction of the intestinal canal and injects in the ileum muscular layer, and each site injects 2ul of mixed solution.
The intraperitoneal injection method of DCZ comprises the following steps: DCZ was formulated as a DCZ solution and each mouse was injected intraperitoneally with DCZ at a dose of 100ug/kg one month after viral infection.
The preparation method of the DCZ solution comprises the following steps: after 10mg of clozapine powder is dissolved in 34.2021ml of dimethyl sulfoxide solution to prepare 1mM clozapine mother liquor, the clozapine mother liquor, polyethylene glycol 300, emulsifier T-80 and physiological saline are mixed according to the volume ratio of 2: 8: 1: 9 to a final solution of clozapine. In this example, DCZ was purchased from Haoyuan biomedical science and technology, Inc., Shanghai under the trademark MCE and under the trademark HY-42110.
In the experimental process, the quality and the water content of the excrement are monitored before the operation of the mouse, after the virus injection, one week after the DCZ injection and 24 hours after the DCZ injection is stopped; post-surgery the efficacy of the chemogenetic virus infection of enteric nerves was assessed by enteric nerve staining by ex vivo colonic smooth muscle strip tonometry to assess whether the group of mice progressed to chemogenetic slow transit constipation.
The method for measuring the quality and the water content of the excrement in 24 hours comprises the following steps: the method comprises the steps of respectively selecting four time points before operation, after virus injection, one week after DCZ injection and after DCZ injection to collect excrement, placing a mouse in a metabolism cage, giving corresponding food and water, immediately taking the excrement by using clean forceps after the mouse defecates, enabling the excrement to be convenient for an aseptic freezing tube, temporarily storing the freezing tube in liquid nitrogen in the process, weighing the collected excrement, drying the weighed excrement in a 38-DEG C oven for 24 hours, and recording the water content of the excrement.
The method for measuring the tension of the isolated colon smooth muscle strip comprises the following steps: taking fresh colon tissue, making 10mm intestinal canal muscle strips, fixing in an organ bath containing krebs solution, circularly heating at 37 deg.C, stabilizing for 10min, adding DCZ into the organ bath, sequentially adding 0.1nM, 1nM, 10nMDCZ and 20nMDCZ into the organ bath, converting with a tension transducer, and analyzing tension change curve with a multichannel physiological collector.
The enteric nerve staining method comprises the following steps: stripping the mucosa layer, the submucosa layer and the annular muscle layer of the colon intestine section, then paving the stripped mucosa layer, the submucosa layer and the annular muscle layer on a glass slide to prepare a colon white-mount patch, dripping 4% formaldehyde, fixing for 10 minutes at room temperature, sealing for 1 hour, dripping mCherry primary antibody on the patch, placing the patch for incubation for 24 hours in a refrigerator at 4 ℃, then incubating anti-rabbitt red secondary antibody for 1 hour in a dark place, finally carrying out DAPI staining, and observing and analyzing by using a microscope.
As shown in fig. 1, there is no significant difference in stool quality and stool water content between the model group and the sham operation group after the operation, after the virus injection and after the stop of DCZ injection (p >0.05), and stool quality of the model group 24h after DCZ injection is significantly reduced (p <0.05) compared with the sham operation group; in the model group, the quality and water content of feces 24h after DCZ injection are obviously reduced (p is less than 0.05) compared with those before DCZ injection, while in the sham operation group, no statistical difference exists before and after administration (p is more than 0.05), which indicates that constipation symptoms appear when the chemogenetic mice are injected with DCZ.
As shown in FIG. 2, compared with 0 nMDZ environment, the frequency and amplitude of contraction of the colon smooth muscle strips of the model mouse under the action of 10 nMDZ and 20 nMDZ are obviously reduced (p is less than 0.05), while the frequency and amplitude of contraction of the colon smooth muscle strips of the model mouse are not obviously changed in the sham operation group (p is greater than 0.05), which indicates that the contraction force of the isolated colon smooth muscle of the chemogenetic mouse is obviously reduced in the DCZ environment.
As shown in FIG. 3, the virus staining of the model-made mice was distributed in the enteric nerve, the red part was hM4D virus staining, and the blue part was DAPI, indicating that the virus was specifically distributed in the colonic nerve.
By combining the results, the virus expressing hM4D is transfected into colon enteric neurons by a chemical genetic method, and the receptor is activated by injecting clozapine into the abdominal cavity, so that the establishment of a slow-transmission constipation animal model is successfully induced, the model is stable and accurate in regulation and control, and the success rate is high.
The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention in any way, and any person skilled in the art can make any simple modification, equivalent replacement, and improvement on the above embodiment without departing from the technical spirit of the present invention, and still fall within the protection scope of the technical solution of the present invention.
Claims (6)
1. An agent for establishing a slow-transit constipation animal model, characterized in that:
comprises a viral agent and a solution of clozapine;
the virus reagent comprises a mixed reagent of two viruses pAAV9-CAG-EGFP-P2A-Cre-WPRE and pAAV9-EF1a-DIO-hM4D (Gi) -mCherry injected at multiple sites of the colon wall of a mouse.
2. The agent for establishing a slow transit constipation animal model according to claim 1, characterized in that:
wherein, the volume ratio of the two viruses pAAV9-CAG-EGFP-P2A-Cre-WPRE and pAAV9-EF1a-DIO-hM4D (Gi) -mCherry is 1: 1.
3. The agent for establishing a slow transit constipation animal model according to claim 1, characterized in that:
wherein, viruses pAAV9-CAG-EGFP-P2A-Cre-WPRE and pAAV9-EF1a-DIO-hM4D (Gi) -mCherry are injected into each site for 1 ul.
4. The agent for establishing a slow transit constipation animal model according to claim 1, characterized in that:
wherein the injection dosage of the clozapine is 100 ug/kg.
5. The agent for establishing a slow transit constipation animal model according to claim 1, characterized in that:
the preparation method of the clozapine solution comprises the following steps: dissolving clozapine in dimethyl sulfoxide to prepare a clozapine mother solution, and mixing the clozapine mother solution, polyethylene glycol 300, an emulsifier T-80 and normal saline to obtain a clozapine solution.
6. The agent for establishing a slow transit constipation animal model according to claim 5, characterized in that:
wherein in the preparation of the clozapine solution, the concentration of the clozapine mother liquor is 1mmol/L, and the volume ratio of the clozapine mother liquor, the polyethylene glycol 300, the emulsifier T-80 and the normal saline is 2: 8: 1: 9.
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| CN110904154A (en) * | 2019-11-28 | 2020-03-24 | 东南大学 | AAV vector construction method and application for mouse hippocampal CA2 region specific expression CRE |
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