Rat uterus scar diverticulum model preparation method, identification method and histopathologic analysis method

Abstract

The invention relates to the technical field of biology and new medicine, in particular to a preparation method, an identification method and a histopathologic analysis method of a rat uterus scar diverticulum model. The preparation method of the rat uterus scar diverticulum model comprises the following steps: sequentially simulating rat pregnancy by using female progestational hormone, and infecting a uterus incision by using group B streptococcus (GBS) to obtain the rat uterus scar diverticulum model. The preparation method comprises the following steps: constructing a pseudopregnant rat model; preparing a GBS bacterial solution with a certain concentration, and immersing 3-0 silk threads with the length of about 1cm into the bacterial solution for 24 hours for later use; an incision with the length of about 2 cm is formed in the uterus on the two sides of the rat, and the incision is sutured discontinuously by an absorbable line. The simulated pregnant mouse is used, the influence of uterine hyperemia during human delivery and hormone change after female progestational hormone withdrawal on the uterine microenvironment is met, the formation of the human uterine scar diverticulum simulated by using the group B streptococcus infection incision is related to cesarean delivery incision infection, and therefore the success rate of the obtained model is high.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology and new medicine technologies. Specifically, it relates to a method for preparing a rat uterine scar diverticulum model, an identification method, and a histopathological analysis method. Background Art

[0002] For cesarean scar diverticulum (CSD) after cesarean section, also known as previous cesarean scar defect (PCSD), due to the lack of a stable and reliable animal model, both clinical and basic research on uterine scar diverticulum have been greatly restricted. In recent years, relevant personnel have made certain efforts in CSD research. However, most of them construct uterine scar models by means of full-thickness uterine injury. In the prior art, a PCSD model was constructed by injecting lipopolysaccharide (LPS) into the uterine myometrium of rats after cesarean section and electrocoagulating (with a power of 10 W) the uterine incision margin. It was found that compared with the scar group, the muscular layer of PCSD was thinner, and the endometrial defect rate and collagen fiber deposition were higher. However, there are still the following disadvantages: 1. The technical indicators of model establishment are not quantified, and the model establishment method lacks repeatability; 2. The principle of electro-injury is different from that of surgical injury, and the scar diverticulum caused by electro-injury fails to accurately simulate the scar diverticulum after cesarean section; 3. In the animal model after cesarean section, due to uterine contraction and repair, the sizes of the diverticula formed directly after cesarean section are different, and the uniformity is poor; 4. The method for preparing a rat uterine scar diverticulum model, an identification method, and a histopathological analysis method disclosed in the prior art with the publication number CN116269896A constructs a diverticulum in normal rats, which does not conform to the influence of the withdrawal of estrogen and progesterone on the uterine microenvironment after pregnancy and parturition in pregnant rats. The incision healing process and the sensitivity to external factors are different from those of non-pregnant rats, and it does not fit the clinical actual situation.

[0003] Therefore, aiming at the deficiencies of the prior art, it is very important to provide a method for preparing a rat uterine scar diverticulum model to solve the deficiencies of the prior art. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing a rat uterine scar diverticulum model, an identification method, and a histopathological analysis method to solve the problems raised in the above background art.

[0005] In the first aspect, to solve the above technical problems, an embodiment of the present invention provides a method for preparing a rat uterine scar diverticulum model, which uses sequential estrogen and progesterone to simulate pregnancy in rats and infects the wound with group B streptococcus liquid to obtain a rat uterine scar diverticulum model.

[0006] In an alternative embodiment, the preparation method comprises the following steps: S1. Construct a pseudopregnant rat model; S2. Prepare a Streptococcus group B standard strain bacterial solution at 1×10 7 CFU / mL, and immerse a 3-0 silk thread about 1 cm in length in the bacterial solution for 24 hours for standby; S3. Make an incision about 2 cm in length on both sides of the uterus of the rat, place the silk thread in step S2 into the uterine cavity, and intermittently suture the uterine serosa and muscular layer with a 4-0 absorbable suture; S4. After 3 weeks, observe the uterine incision of the rat by pathological section and diagnose whether a diverticulum is formed. If it is formed, a rat uterine scar diverticulum model is obtained.

[0007] In an alternative embodiment, the specific operating steps of step S1 are as follows: S1.1. Construct pseudopregnant rats by sequential subcutaneous injection of estrogen and progesterone; S1.2. Prepare a 3-0 silk thread with a bacterial solution concentration of 1×10 7 CFU / mL; S1.3. Shave the hair on the lower abdomen of the rat after successful anesthesia; S1.4. Place the rat supine and perform laparotomy under sterile conditions, layer by layer open the skin and muscle, and expose both uteruses; S1.5. Make an incision about 2 cm in length on both sides of the uterus of the rat, and place the silk thread in step S2 into the uterine cavity.

[0008] In an alternative embodiment, the specific operating steps of step S11 are as follows: Female rats are subcutaneously injected with 2.5 μg estradiol and 4 mg progesterone from day 1 to day 16, and 50 μg estradiol from day 17 to day 23.

[0009] In an alternative embodiment, the specific operating steps of step S12 are as follows: Take a single colony of the standard strain of Streptococcus group B and place it in Todd-Hewitt broth, and culture it in a constant temperature incubator at 37.5°C and 5% CO2 for 24 hours. Dilute the colony to a concentration of 1×10 7 CFU / mL using a microplate reader and sterile PBS, and at the same time place a 3-0 sterile silk thread about 1 cm in length into the above-mentioned bacterial solution for 24 hours.

[0010] In an alternative embodiment, the specific operating steps of step S1.5 are as follows: Make an incision with a length of 2 cm at two places near the junction of both uteruses, place a 3-0 silk thread about 1 cm in length with a bacterial solution concentration of 1×10 7 CFU / mL on one side, and place a 3-0 sterile silk thread about 1 cm in length on the other side (as a control).

[0011] In an alternative embodiment, the rat is a non-pregnant SD female rat.

[0012] In a second aspect, an embodiment of the present invention further provides a method for identifying a rat uterine scar diverticulum model. For a rat with uterine scar diverticulum prepared by using the above-mentioned method for preparing a rat uterine scar diverticulum model, identification is performed by embedding and sectioning the rat uterus and performing H&E staining.

[0013] In a third aspect, an embodiment of the present invention further provides a method for histopathological analysis of a rat uterine scar diverticulum model. A rat with uterine scar diverticulum prepared by using the above-mentioned method for preparing a rat uterine scar diverticulum model is sacrificed, the tissue of the uterine scar diverticulum is embedded and sectioned, then H&E staining is performed, and finally the remaining myometrial thickness of each incision, the depth of the diverticulum, and the ratio of the diverticulum depth to the remaining myometrial thickness are measured.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] The present invention uses simulated pregnant rats, which conform to the hyperemia of the uterus during human childbirth and the hormonal changes and physiological changes of the uterus after hormonal withdrawal. The wound healing is inconsistent with that of non-pregnant rats. Using Streptococcus group B to infect the incision to simulate the formation of human uterine diverticulum is related to incision infection. Therefore, the obtained model has a high success rate and is close to the actual clinical situation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a uterine diagram of a pseudopregnant rat constructed by sequential estrogen and progesterone administration.

[0017] Figure 2 It is a diagram of the remaining myometrial thickness of the rat uterus of the present invention.

[0018] Figure 3 It is a uterine diverticulum diagram obtained by the histopathological analysis method of the present invention 21 days after the method for preparing a rat uterine scar diverticulum model of Case 1 of the present invention.

[0019] Figure 4 It is a uterine diverticulum diagram obtained by the histopathological analysis method of the present invention 21 days after the method for preparing a rat uterine scar diverticulum model of Case 2 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods. The raw materials, reagent materials, etc. used in the following examples can be obtained from conventional biochemical reagent stores or pharmaceutical enterprises without special instructions.

[0022] As Figure 1 shown, Figure 1 in which (A) is the uterus of a healthy rat; (B-C) are the uteri of pseudopregnant rats in Case 1 and 2; (D) is the uterus after surgery; Figure 2 It is a pathological analysis diagram. (A) is the healthy side uterus of Case 1; (B) is the healthy side uterus of Case 2; RMT: remaining myometrial thickness; Figure 3 It is the postoperative pathological diagram of Case 1. Uterine diverticulum of Case 1 (A) and enlarged view (B), diverticulum: Niche; remaining myometrial thickness: RMT; adjacent myometrial thickness: AMT; diverticulum depth: Depth; Figure 4 It is the pathological analysis diagram of Case 2. Uterine diverticulum (A) and enlarged view (B), diverticulum: Niche; remaining myometrial thickness: RMT; adjacent myometrial thickness: AMT; diverticulum depth: Depth. The above figures are clear representative figures selected through multiple experiments.

[0023] Example 1:

[0024] As Figures 1-4 shown, the embodiment of the present invention provides a method for preparing a rat uterine scar diverticulum model, and a rat uterine scar diverticulum model is obtained.

[0025] A method for preparing a rat uterine scar diverticulum model uses sequential estrogen and progesterone to simulate rat pregnancy and infects the wound with Streptococcus agalactiae bacteria solution to obtain a rat uterine scar diverticulum model.

[0026] Furthermore, the preparation method includes the following steps: S1. Construct a pseudopregnant rat model; S2. Prepare a certain concentration of Streptococcus agalactiae bacteria solution, and immerse a 3-0 silk thread with a length of 1 cm in the bacteria solution for 24 hours for standby; S3. Make a 2-cm incision on both sides of the rat uterus, place the silk thread in step S2 into the uterine cavity, and intermittently suture the uterine serosa layer and myometrium with a 4-0 absorbable suture; S4. Three weeks later, perform H&E staining on the paraffin sections of the rat uterine tissue to confirm the formation of uterine incision diverticulum.

[0027] The specific operation steps of step S1 are as follows: S1.1. Use sequential estrogen and progesterone subcutaneous injection to construct pseudopregnant rats, and stop the drug one day before surgery; S1.2. Prepare a 3-0 silk thread dipped in a bacteria solution with a concentration of 1*10 7 CFU / mL; S1.3. Shave the lower abdomen of the rats after successful anesthesia. S1.4. Place the rats in the supine position and perform laparotomy under sterile conditions. Open the skin and muscles layer by layer to expose the bilateral uteruses. S1.5. Make a 2-cm incision on the bilateral uteruses of the rats and place the silk thread in step S2 into the uterine cavity.

[0028] The specific operation steps of step S1.1 are as follows: Subcutaneously inject 2.5 μg of estradiol and 4 mg of progesterone into female rats from the 1st day to the 16th day, and subcutaneously inject 50 μg of estradiol from the 17th day to the 23rd day.

[0029] The specific operation steps of step S12 are as follows: Pick a single colony of group B streptococcus and put it into group B streptococcus enrichment broth, and culture it in a constant temperature incubator at 37.5 °C and 5% CO2 for 24 h. Dilute the colony to a concentration of 1×10 7 CFU / mL using an enzyme-linked immunosorbent assay (ELISA) reader and sterile PBS. At the same time, put several 3-0 sterile silk threads about 1 cm long into the above-mentioned bacterial solution overnight.

[0030] The specific operation steps of step S1.5 are as follows: Make a 2-cm incision at a distance of 2 cm from the junction of the bilateral uteruses. Place a 3-0 silk thread about 1 cm long with a bacterial solution concentration of 1×10 7 CFU / mL on one side, and place a 3-0 sterile silk thread about 1 cm long on the other side.

[0031] The rats are non-pregnant SD female rats.

[0032] Example 2:

[0033] The present invention also provides a method for identifying a rat uterine scar diverticulum model. For rats with uterine scar diverticulum prepared by using the method for preparing a rat uterine scar diverticulum model described in any item of Example 1, identify them by embedding and sectioning the rat uterus and performing H&E staining.

[0034] Furthermore, select to sacrifice or anesthetize and excise the uterine tissue on the modeling side to make tissue paraffin sections. For rats with uterine scar diverticulum prepared by using the method for preparing a rat uterine scar diverticulum model described in any item of Example 1, embed and section the tissue of the uterine scar diverticulum, then perform H&E staining, and finally measure the remaining myometrial thickness of the incision, the depth of the diverticulum, and calculate the ratio of the depth of the diverticulum to the remaining myometrial thickness.

[0035] It should be noted that the bacterial solution described in the present invention is a purchased standard strain, or it can also be separated and cultured by oneself. The bacterial solution is prior art and will not be elaborated here.

[0036] On pathological sections, the depth of the diverticulum is described by depth, the remaining myometrial thickness around the incision is described by RMT, and the myometrial thickness adjacent to the incision is described by AMT. Similar to the clinical evaluation of the definition of a large diverticulum, these three indicators are also measured under transvaginal ultrasound. The diagnosis of uterine scar diverticulum according to the diverticulum definition in the guidelines is similar. However, the mouse uterus is relatively small and it is difficult to accurately evaluate these three indicators by ultrasound. Therefore, the above three indicators are measured through uterine coronal tissue pathological sections to evaluate the size of the diverticulum, which is objectively reproducible.

[0037] The present invention uses simulated pregnant mice, which conform to the hormonal changes and physiological changes of the uterus after uterine congestion and hormonal withdrawal during human childbirth. The wound healing is inconsistent with that of non-pregnant mice. The formation of human uterine diverticulum is simulated by infecting the incision with group B streptococcus, which is related to incision infection. Therefore, the success rate of the obtained model is high.

[0038] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification are only preferred examples of the present invention and are not used to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a rat uterine scar diverticulum model, characterized in that, Using sequential estrogen and progesterone to simulate pregnancy in rats, and infecting the wound with Streptococcus group B bacterial solution to obtain a rat uterine scar diverticulum model.

2. The method for preparing a rat uterine scar diverticulum model according to claim 1, wherein The preparation method includes the following steps: S1. Construct a pseudopregnant rat model; S2. Prepare Streptococcus B bacterium solution at 1×10 7 CFU / mL. Immerse a 3-0 silk thread with a length of 1 cm into the bacterium solution for 24 hours for standby; S3. Make a 2-cm incision on both sides of the rat uterus, place the silk thread in step S2 into the uterine cavity, and intermittently suture the uterine serosa and muscular layer with 4-0 absorbable sutures. S4. Three weeks later, take the rat uterine tissue, embed it in paraffin, section it, and stain it with H&E to confirm the formation of uterine incision diverticulum.

3. The method for preparing a rat uterine scar diverticulum model according to claim 2, wherein, The specific operation steps of step S1 are as follows: S1.

1. Use sequential estrogen and progesterone subcutaneous injection to construct pseudopregnant rats, and stop the drug one day before the operation. S1.

2. Prepare 3-0 silk threads dipped in a bacterial solution with a concentration of 1*10 7 CFU / mL; S1.

3. Shave the hair on the lower abdomen of the rats after successful anesthesia. S1.

4. Place the rats supine and perform laparotomy under sterile conditions, layer by layer open the skin and muscles to expose both uteri. S1.

5. Make a 2-cm incision on both sides of the rat uterus, and place the silk thread in step S2 into the uterine cavity.

4. The method for preparing a rat uterine scar diverticulum model according to claim 3, wherein, The specific operation steps of step S1.1 are as follows: Subcutaneously inject 2.5 μg estradiol and 4 mg progesterone into female rats from day 1 to day 16, and subcutaneously inject 50 μg estradiol from day 17 to day 23.

5. The method for preparing a rat uterine scar diverticulum model according to claim 3, wherein The specific operation steps of the step S1.2 are as follows: Take a single colony of group B streptococcus and place it in Todd-Hewitt broth, and culture it in a constant temperature incubator at 37.5°C and 5% CO2 for 24 hours. Use an enzyme-linked immunosorbent assay (ELISA) reader and sterile PBS to dilute the colony to a concentration of 1*10 7 CFU / mL. At the same time, place several 3-0 sterile silk threads about 1 cm long into the above-mentioned bacterial solution for 24 hours.

6. The method for preparing a rat uterine scar diverticulum model according to claim 3, characterized in that, The specific operation steps of step S1.5 are as follows: make an incision with a length of 2 cm at a position 2 cm away from the junction of the bilateral uterus, place a 3-0 silk thread with a length of about 1 cm and a bacterial liquid concentration of 1*10 7 CFU / mL on one side, and place a 3-0 sterile silk thread with a length of about 1 cm on the other side.

7. The method for preparing a rat uterine scar diverticulum model according to claim 3, characterized in that, The rats are non-pregnant SD female rats.

8. A method for identifying a rat uterine scar diverticulum model, characterized in that, For the rats with uterine scar diverticulum prepared by using the preparation method of the rat uterine scar diverticulum model described in any one of claims 1 to 7, identify them by paraffin embedding section and H&E staining of the rat uterus.

9. A histopathological analysis method for a rat uterine scar diverticulum model, characterized in that, Select to sacrifice, or anesthetize and excise the uterus on the modeling side to make tissue paraffin sections. For the rats with uterine scar diverticulum prepared by the preparation method of the rat uterine scar diverticulum model described in any one of claims 1 to 7, embed and section the tissue of the uterine scar diverticulum, then perform H&E staining, and finally measure the remaining uterine muscle layer thickness at the incision, the diverticulum depth, and calculate the ratio of the diverticulum depth / remaining muscle layer thickness.

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