CN109984857B - Method for establishing accurate peritoneal adhesion animal model and application - Google Patents
Method for establishing accurate peritoneal adhesion animal model and application Download PDFInfo
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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Abstract
The invention relates to a method for establishing an accurate peritoneal adhesion animal model, which comprises the following steps: 1) anaesthetizing SD rats, disinfecting abdominal areas and paving towels; 2) opening the center of the abdomen of the SD rat, exposing the cecum, scraping the cecum with surgical sterile gauze until punctiform bleeding occurs, and counting the number of bleeding points; scraping cecum with a surgical blade on the abdominal wall side of the SD rat; 3) placing the cecum back into the abdominal cavity, and fixing at a position 0.5cm above the injury of the cecum and the abdominal wall; 4) closing abdomen layer by layer, and sterilizing. A method for screening materials for preventing postoperative peritoneal adhesion comprises accurately establishing a rat peritoneal adhesion model according to the requirements, and respectively injecting 1-5 mL of screening material solution or negative control solution to the joint of the cecum and the abdominal wall before closing the abdomen. After 7 days of operation, the abdomen is opened, and the effect of the screening material for preventing the peritoneal adhesion is evaluated by counting the ratio of the peritoneal adhesion, inflammatory reaction and pathological conditions of adhesion tissues.
Description
Technical Field
The invention relates to an animal model establishing method, in particular to an establishing method of an accurate peritoneal adhesion animal model and application of the animal model in screening and preventing postoperative peritoneal adhesion materials.
Background
Peritoneal adhesions are the products of the body's own defenses after peritoneal injury, often occur between the visceral organs and abdominal wall in the abdominal cavity, and are the common serious complications after abdominal surgery in clinical practice. Clinical data show that the incidence of intra-abdominal adhesions in patients with laparotomy is as high as 90%, and that peritoneal adhesions can occur again in about ten years after surgery in 1/3 patients. Peritoneal adhesions can lead to chronic pain, female infertility and chronic pelvic pain, intestinal obstruction, and even death. At present, the clinical preventive measures are mainly precise operation so as to reduce peritoneal trauma, reduce inflammatory reaction by using medicaments, inhibit agglutination reaction, promote fibrinolysis and reduce the probability of adhesion by isolating peritoneum by using biological materials such as chitosan and the like. Although the literature reports that the causes of postoperative peritoneal adhesion are mainly trauma, ischemia, infection and foreign body, the specific pathogenesis of the postoperative peritoneal adhesion is not clear yet.
Research on the pathogenesis of peritoneal adhesions and development of novel drugs for preventing peritoneal adhesions are one of the hot spots in clinical research at present. Clinically, the peritoneal adhesion condition can be observed intuitively only by re-operation, so that the establishment of a peritoneal adhesion animal model is very important for the research and development of pathogenesis and preventive medicaments of the peritoneal adhesion animal model, but the establishment of an accurate peritoneal adhesion animal model troubles the research and development of the preventive medicaments of the peritoneal adhesion. At present, the method of inducing the experimental animals to generate the adhesions by adopting abdominal wall-cecum defect, abdominal wall defect, uterine horn-abdominal wall defect and the like is a main method for establishing the peritoneal adhesion animal model, however, the damage degree of each tissue is not determined in the molding process, so that the individual difference is too large, and the molding stability is influenced. Meanwhile, the evaluation method for establishing the model is mainly a scoring method, but the scoring system has high subjectivity and cannot completely and objectively reflect the establishment of the animal model. Therefore, the accurate modeling and evaluation method with good repeatability and simple operation is applied to the establishment of the peritoneal adhesion animal model, can not only provide help for researching the pathogenesis of the peritoneal adhesion, but also provide a suitable platform for the research and development of the medicament for preventing the peritoneal adhesion.
The invention provides a method for establishing and evaluating an accurate postoperative peritoneal adhesion animal model, which quantifies the damage degree of different tissues in the process of establishing the model and avoids individual difference caused by subjectivity of a modeling method. In the model evaluation process, the adhesion ratio is used as an accurate evaluation index, the degree of peritoneal adhesion is quantified, and the degree of peritoneal adhesion is objectively reflected. Meanwhile, the invention applies the refined rat peritoneal adhesion model to screening of novel postoperative peritoneal adhesion prevention materials, and research results show that compared with clinical commercialized peritoneal adhesion prevention medicine, namely chitosan, sericin extracted by an alkaline water extraction method has a more remarkable effect of preventing peritoneal adhesion. The establishment of the accurate peritoneal adhesion animal model provides a new research platform for the research and development of novel materials for preventing postoperative peritoneal adhesion.
Disclosure of Invention
The invention aims to provide a method for establishing a precise postoperative peritoneal adhesion animal model and application thereof. Meanwhile, the invention aims to apply the accurate peritoneal adhesion animal model to the research of preventing the peritoneal adhesion by using the novel sericin material.
The invention provides a method for establishing an accurate peritoneal adhesion animal model, which is characterized in that surgical sterile gauze is used for causing punctate bleeding at the cecum, and the number of bleeding points is accurately counted so as to reduce individual difference in the modeling process. Meanwhile, the surgical blade is used for causing injury of a corresponding area at the abdominal wall of a rat, then the cecum is fixed, and the degree of peritoneal adhesion is evaluated through adhesion occupation ratio and histochemical staining at a corresponding time point.
The invention also provides application of the rat peritoneal adhesion model in screening of novel biological materials for preventing peritoneal adhesion.
The invention provides a method for establishing an accurate peritoneal adhesion animal model, which comprises the following steps:
1) anaesthetizing SD rats, disinfecting abdominal areas and paving towels;
2) opening the center of the abdomen of the SD rat, exposing the cecum, scraping the cecum with surgical sterile gauze until punctiform bleeding occurs, and counting the number of bleeding points;
3) scraping cecum with a surgical blade on the abdominal wall side of the SD rat;
4) placing the cecum back into the abdominal cavity, and fixing at a position 0.5cm above the injury of the cecum and the abdominal wall;
5) closing abdomen layer by layer, and sterilizing.
Further, in the protocol of the present application, the SD rat weighs 200-250 g.
Further, in the scheme of the application, the anesthesia is performed by intraperitoneal injection of 0.3mL/100g with 8-10 g/100 mL chloral hydrate solution.
Furthermore, in the scheme of this application, the belly median opening length is 4~5 cm.
Further, in the context of the present application, the sterile surgical gauze has a density of 21X 31/in.2。
Furthermore, in the scheme of the application, the area of the gauze scraping cecum is 2.5-3.5 cm above the root of the cecum.
Further, in the protocol of the present application, the gauze scraped cecum area was 1X 2 cm2 。
Furthermore, in the scheme of the application, the number of times of scraping the cecum by the gauze is 30-50.
Furthermore, in the scheme of the application, the frequency of scraping the cecum by the gauze is 90-120 times/min.
Further, in the embodiments of the present application, the blade scrapes the abdominal wall area corresponding to the cecum.
Further, in the protocol of the present application, the blade scraped abdominal wall area was 1X 2 cm2 。
Further, in the scheme of the application, the scraping abdominal wall thickness of the blade is 0.1-0.15 cm.
Furthermore, in the protocol of the present application, 3-0 silk thread is used for cecal and abdominal wall fixation.
The invention also provides a method for applying the molding method to screening of a novel material for preventing postoperative peritoneal adhesion, which comprises the following steps:
respectively establishing two groups of models A and B according to the following steps 1) to 4):
1) anaesthetizing SD rats, disinfecting abdominal areas and paving towels;
2) opening the center of the abdomen of the SD rat, exposing the cecum, scraping the cecum with surgical sterile gauze until punctiform bleeding occurs, and counting the number of bleeding points;
3) scraping cecum with a surgical blade on the abdominal wall side of the SD rat;
4) placing the cecum back into the abdominal cavity, and fixing at a position 0.5cm above the injury of the cecum and the abdominal wall;
5) respectively injecting 1-5 mL of screening material solution and negative control solution to the joint of the cecum and the abdominal wall of the A and B group models; wherein the negative control solution is 0.25-5% medical chitosan solution by mass ratio;
6) closing abdomen layer by layer and sterilizing;
7) after 7 days of operation, the abdomen is opened, and the effect of the screening material for preventing the peritoneal adhesion is evaluated by counting the ratio of the peritoneal adhesion, inflammatory reaction and pathological conditions of adhesion tissues.
The screening material solution is a sericin solution prepared by the following method:
1) extracting sericin: weighing 1-5 g of silkworm cocoon, cutting, washing with deionized water, removing water, and placing in 20-1000 mL Na with concentration of 0.01-0.02 mol/L2CO3In the solution, stirring and reacting for 0.5-1 hour at the temperature of 80-100 ℃ to completely dissolve sericin; centrifuging to remove insoluble precipitate in the solution, collecting supernatant, and dialyzing for 2-3 days to obtain clarified sericin solution; freeze-drying to obtain sericin powder;
2) adding the sericin powder obtained in the step 1) into a PBS (phosphate buffer solution) to obtain a sericin solution with the mass ratio of 0.25-5%.
Further, in the scheme of the application, the ratio of the peritoneal adhesions is as follows: peritoneal adhesion area/injury area.
The invention also provides application of the sericin in preparation of a material for preventing postoperative peritoneal adhesion.
The scheme of the invention has the following advantages:
1) according to the modeling method of the postoperative peritoneal adhesion animal model, the degree of cecal injury is quantified through the statistics of bleeding points in unit area for the first time in the modeling process;
2) in the modeling process, the abdominal wall injury degree is quantified by counting the abdominal wall injury area for the first time, and the individual difference caused by the subjectivity of the modeling method is reduced;
3) more quantitative evaluation indexes such as adhesion ratio and the like are adopted in the evaluation of the model stability, and the degree of peritoneal adhesion is objectively reflected;
4) the modeling method of the postoperative peritoneal adhesion animal model provided by the invention can be applied to screening of novel materials for preventing postoperative peritoneal adhesion.
5) Compared with the commercialized medical chitosan of the control group, the sericin has better effect of preventing peritoneal adhesion, the preparation method of the sericin is simple and easy to implement, the treatment effect evaluation is more accurate, and the obtained result is more credible.
Drawings
FIG. 1 is a model drawing (a) a drawing of a caecum hemorrhage; (b) a picture of an abdominal wall injury object; (c) picture of abdominal membrane adhesion real object after 1 week; (d) abdominal wall side adhesions.
Fig. 2 is statistics of the bleeding point of cecum at the time of molding.
FIG. 3 is a statistic of the percentage of peritoneal adhesions after molding.
Fig. 4 (a-f) are physical diagrams of physiological saline, medical chitosan, hyaluronic acid and sericin applied to treatment of peritoneal adhesion. (g) And (5) counting bleeding points. (h) And (5) counting the adhesion percentage.
FIG. 5 after 7 days of molding treatment, saline, medical chitosan and sericin were applied to H & E staining of the adhesion tissue for treatment of peritoneal adhesion.
FIG. 6 is a measurement of mRNA levels of PAI-1, TGF- β, TNF- α incubated with NIH3T3 cells in physiological saline, chitosan, hyaluronic acid, and sericin.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified.
Example 1 establishment of post-operative peritoneal adhesions animal model
First, the experimental process
SD rat weight 200g-250 g.
2. Weighed before modeling, and injected intraperitoneally with 0.3mL/100g 10% (g/mL) chloral hydrate. Rat abdomen was depilated, iodophor was sterilized, and sterile towel was laid.
3. Opening a 4-5 cm middle part of the abdomen, performing laparotomy layer by layer, fully exposing the cecum, scraping the cecum for 30-50 times to punctate bleeding of the cecum at a position 2.5-3.5 cm above the root of the cecum by using surgical sterile gauze, wherein the scraping frequency is 90-120 times/min, and the scraping area is 1 multiplied by 2 cm2And counting the number of bleeding points.
4. Scraping the abdominal wall with a surgical operation blade on the corresponding abdominal wall side of the cecum, wherein the scraping thickness is 0.1-0.15 cm, and the scraping area is 1 multiplied by 2 cm2 。
5. The cecum was placed back into the abdominal cavity and fixed by 3-0 silk thread 0.5cm above the cecum and abdominal wall lesions, respectively.
6. Closing abdomen layer by layer, and sterilizing with iodophor.
7. After the model is made, the rat lies flat, is naturally clear and is raised conventionally.
Second, experimental analysis
FIG. 1 is a model object diagram, (a) a caecum injury object diagram, and a black dotted line frame is a caecum bleeding point; (b) an abdominal wall injury object image, wherein a black dotted line frame is an abdominal wall injury area; (c) 7 days after the model is made, a real object diagram of the peritoneal adhesions is shown, and a black dotted line is a boundary of the intestinal tract and the abdominal wall adhesions; (d) the adhesion condition of the abdominal wall side of the adhesion tissue is separated 7 days after molding, and the black dotted line frame is the area of peritoneal adhesion.
Example 2 evaluation method for post-operative peritoneal adhesion animal model establishment
First, the experimental process
1. Evaluation of the damage degree of the caecum: the number of the cecal bleeding points in step 3) of example 1 was counted by manual counting and photograph taking.
2. Evaluation of degree of peritoneal adhesion:
the general view of peritoneal adhesions is as follows: SD rats were sacrificed by intraperitoneal injection of 1 mL/100g of 10% chloral hydrate on day 7 after modeling, abdominal skin was incised with a "U" shaped incision, and the area of the peritoneal adhesion region was counted to calculate the adhesion ratio (adhesion ratio = adhesion area/lesion area).
Adhesion tissue pathological section: injecting 1 mL/100g 10% chloral hydrate into abdominal cavity to kill SD rat on 7 days after model making, cutting abdominal skin with U-shaped incision, taking out adhesion tissue, soaking in 4% paraformaldehyde solution for fixation, embedding in paraffin, and performing HE staining to obtain pathological section.
Second, experimental analysis
1. As shown in FIG. 2, the number of bleeding points in the cecum was about 11, and the number of bleeding points was stable, indicating that the extent of cecum injury was consistent.
2. As shown in FIG. 3, the area of the peritoneal adhesion region of the rat was about 1.4 cm after 7 days of molding2 The adhesion ratio is about 0.7, and all experimental animals have peritoneal adhesion, so that the molding is successful.
Example 3 application of animal model of postoperative peritoneal adhesion to screening of novel materials for preventing postoperative peritoneal adhesion
First, the experimental process
The method for molding the post-operative peritoneal adhesion animal model in this example is the same as the molding method in example 1, except that step 1) is performed as follows: the weight of the SD rat is 200g-250g, the number is 36, the SD rat is randomly divided into 6 groups which are respectively a normal saline group, a medical chitosan group, a hyaluronic acid group, a 1.5% sericin group, a 0.5% sericin group and a 0.25% sericin group, and each group comprises 6 rats; except that step 5) was carried out as follows: the cecum was placed back into the abdominal cavity and fixed by 3-0 silk thread 0.5cm above the cecum and abdominal wall lesions, respectively. 1ml of physiological saline, medical chitosan, hyaluronic acid, 1.5% (w/v) sericin, 0.5% (w/v) sericin and 0.25% (w/v) sericin are respectively injected at the caecum and abdominal wall injury positions by a 1ml syringe.
Second, experimental analysis
1. As shown in fig. 4, 7 days after molding, the ratio of peritoneal adhesions was significantly reduced in the sericin-treated rats compared with the control group. The above results indicate that the medical sodium hyaluronate gel commonly used in clinic can reduce the postoperative peritoneal adhesion.
2. As shown in FIG. 5, after 7 days of molding, the adherent tissues were stained with H & E, and the results showed that the area of the adherent region was significantly smaller in the sericin solution group and the inflammatory cell infiltration was less in the medical chitosan group than in the physiological saline group.
EXAMPLE 4 treatment of post-operative peritoneal adhesions with sericin by modulating PAI-1, TGF-beta and TNF-alpha
First, the experimental process
1. DMEM high-sugar medium was first used at 37oC, at 5% CO2NIH3T3 cells were cultured for 24 hours at 100% humidity
2. Then adding physiological saline, medical chitosan and sericin into the cell culture holes respectively, and culturing for 24 hours to obtain parallel samples with three holes in each group;
3.24 hours later, cellular RNA was extracted and assayed for mRNA levels of PAI-1, TGF- β and TNF- α;
second, experimental analysis
1. As shown in FIG. 6, mRNA levels of PAI-1, TGF-. beta.and TNF-. alpha.were significantly lower in the sericin group than in the other control groups.
2. Experimental results prove that sericin can treat postoperative peritoneal adhesion by regulating PAI-1, TGF-beta and TNF-alpha.
Claims (1)
1. The application of sericin in preparing a material for preventing postoperative peritoneal adhesion is characterized in that the sericin is a sericin solution with the mass ratio of 0.25-5%; the sericin solution is prepared by the following method:
1) extracting sericin: weighing 1-5 g of silkworm cocoon, cutting, washing with deionized water, removing water, and placing in 20-1000 mL Na with concentration of 0.01-0.02 mol/L2CO3In the solution, stirring and reacting for 0.5-1 hour at the temperature of 80-100 ℃ to completely dissolve sericin; centrifuging to remove insoluble precipitate in the solution, collecting supernatant, and dialyzing for 2-3 days to obtain clarified sericin solution; freeze-drying to obtain sericin powder;
2) adding the sericin powder obtained in the step 1) into a PBS (phosphate buffer solution) to obtain a sericin solution with the mass ratio of 0.25-5%.
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