CN116058324A - Mouse model for LPS-induced mammary gland fibrosis and construction method thereof - Google Patents
Mouse model for LPS-induced mammary gland fibrosis and construction method thereof Download PDFInfo
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Abstract
The invention discloses a mouse model for inducing mammary gland fibrosis by LPS and a construction method thereof, which are used for inducing mammary gland fibrosis by continuously pouring LPS into a milk duct of a female mouse in stages. The mammary gland fibrosis mouse model constructed by the method disclosed by the invention is prepared by selecting LPS to repeatedly intervene in a mouse for a plurality of times at low dose, and finally the mammary gland fibrosis model of the mouse is successfully built, which shows that the mammary gland fibrosis of the mouse can be successfully induced by repeatedly stimulating the mammary gland of the mouse by the LPS for a plurality of times, and provides experimental basis for further researching the pathological mechanism of the mammary gland fibrosis and clinical prevention and treatment researches.
Description
Technical Field
The invention relates to a mouse model for LPS-induced mammary gland fibrosis and a construction method thereof.
Background
The mammary gland fibrosis is a pathological phenomenon which occurs after the occurrence of mastitis, and is usually characterized by mastosclerosis, the lactation yield is greatly reduced, the dairy quality is reduced, and the mammary gland fibrosis can be developed into mastosclerosis when serious, and the lactation function is lost. Milk cow mammary gland fibrosis is one of the main reasons that milk cows are eliminated, and causes serious loss to the dairy cow industry.
A large amount of experimental data show that the research on diseases by using animal models is proved to be a more effective method, and the cost of adopting cows, dairy goats or pigs as research objects of the mammary gland fibrosis is too high and the operation is inconvenient, so that the establishment of the mammary gland fibrosis animal model which accords with clinical pathogenesis has important significance for researching the pathogenesis of the mammary gland fibrosis and preventing and treating the mammary gland fibrosis.
Diseases caused by gram-negative bacteria are very common, such as: colibacillosis (core and liver) of chickens, yellow-white diarrhea of piglets, mastitis of cows, edema disease of pigs and the like, and cause great economic loss for animal husbandry. Enterobacteria are the main causative agents of these diseases, but the process of directly constructing a mammary gland cell fibrosis model by using enterobacteria is slow and limited by the bioactivity of the enterobacteria, and the prepared biological models have poor identity, so that the method is unfavorable for developing large-scale control experiments.
Enterobacteria are the main causative agents of these diseases, and it was found by research that: the Lipopolysaccharide (LPS) of the escherichia coli is one of the most main pathogenic factors causing host damage by pathogens, and the research of the mouse mammary gland fibrosis disease model induced by the LPS at present is not reported at home and abroad.
Disclosure of Invention
Aiming at the problems of slower process and difficult control of model identity in the preparation of a mammary gland fibrosis biological model, the invention provides a mouse model for inducing mammary gland fibrosis by LPS and a construction method thereof.
The invention is realized by adopting the following technical scheme:
a construction method of a mouse model for inducing mammary gland fibrosis by LPS is provided, which is to continuously perfuse LPS into a mammary duct of a female mouse in stages for inducing mammary gland fibrosis.
In the method of constructing a mouse model of LPS-induced mammary fibrosis of the present invention, preferably, the female mouse is selected from BALB/C female mice which are delivered for 8 to 10 days.
In the method of constructing a mouse model of LPS-induced breast fibrosis of the present invention, it is preferable that each time LPS is infused, the concentration is 0.075-0.3mg/kg, preferably 0.1mg/kg.
In the method for constructing a mouse model of LPS-induced breast fibrosis of the present invention, the perfusion site is preferably a fourth pair of left and right breast ducts.
In the construction method of the LPS-induced mammary gland fibrosis mouse model, the specific steps are as follows:
(1) Selecting two groups of female mice for a control experiment, and marking the mice as a blank control group and an LPS dosage group respectively;
(2) Performing staged continuous infusion on the breast catheter at the same breast position by using normal saline for the blank group of mice, performing staged continuous infusion on the breast catheter at the same breast position by using equivalent LPS for the LPS dose group of mice, completing an induction process, performing normal saline infusion on the blank control group, and forming a control with a breast tissue fibrosis model finally constructed by the LPS dose group;
(3) After the induction is finished, the mice are sacrificed, mammary gland tissues of the perfusion part are selected to be respectively subjected to pathological section staining and extraction of RNA and protein, and the success of the construction of the mammary gland fibrosis model of the mice is verified from the results of pathological examination and RNA expression and protein expression.
In the method for constructing a mouse model of LPS-induced mammary gland fibrosis of the present invention, it is preferable that the blank group and the LPS dose group are each provided with at least two, preferably six, female mice.
In the method for constructing a mouse model of LPS-induced mammary gland fibrosis of the present invention, preferably, the mice in the blank group and the LPS-dose group are continuously perfused with the corresponding agent in stages on days 0, 3, 6, 9, 12 of the feed group, respectively, and induction is completed and the mice are sacrificed on day 21. The injection in the perfusion process can induce the damage of the mammary epithelial cells, the mammary epithelial cells are repeatedly damaged at different stages, the continuous micro damage of the mammary epithelial cells can cause the abnormal damage repair of the mammary tissue, the activation of the fibroblast and the excessive production of extracellular matrix are caused, and further, the occurrence and the development of the mammary fibrosis are promoted, and the successful modeling is further facilitated.
In the method for constructing a mouse model of LPS-induced mammary gland fibrosis of the present invention, preferably, the pathological section staining comprises the steps of:
(1) Taking mammary tissue perfused by mice, fixing with 10% neutral formalin solution, fixing for 48 hr, and repairing the mammary tissue into 1×2×3cm 3 Placing the small blocks in an embedding box, and replacing a new formalin solution for secondary fixation;
(2) Washing tissue blocks with running water overnight, dehydrating, waxing and embedding, slicing the tissue blocks with 3-5 mu m in a slicing machine, fully expanding the tissue blocks in a 42 ℃ sheet expanding machine, taking out the tissue blocks with a glass slide, and baking the tissue blocks on the sheet baking machine at 40 ℃ for about 2-4 hours; the slide was transferred to a staining rack for eosin-hematoxylin staining and Masson staining, the slides were slightly air-dried, and after blow-dried by a blower, the slides were observed by a microscope and recorded by photographing.
In the method for constructing a mouse model of LPS-induced mammary gland fibrosis of the present invention, preferably, the RNA and protein extraction comprises the steps of:
(1) Extracting total RNA from mammary tissue to synthesize cDNA, and detecting the expression of Collagen I and Collagen III mRNA in the mammary tissue;
(2) Homogenizing mammary tissue, ice-bathing for 30min, centrifuging for 15min to extract protein, adding loading buffer, denaturing at 100deg.C for 15min, performing SDS-PAGE electrophoresis on protein, transferring film, sealing 5% skimmed milk powder at room temperature for 2h, rinsing with TBST for 3 times, adding rabbit anti-vimentin primary antibody, incubating overnight at 4deg.C, rinsing with TBST for 3 times, adding HRP-labeled goat anti-rabbit secondary antibody, incubating at room temperature for 2h, rinsing with TBST for 3 times, adding ECL luminescent solution, and exposing in darkroom.
It is another object of the present invention to provide a mammary gland fibrosis mouse model constructed by the above method.
The mammary gland fibrosis mouse model constructed by the method disclosed by the invention is prepared by selecting LPS to intervene in a mouse repeatedly for a plurality of times at a low dose, and finally, the mammary gland fibrosis model of the mouse is successfully built, which shows that the LPS can stimulate the mammary gland of the mouse repeatedly for a plurality of times to successfully induce the mammary gland fibrosis of the mouse, PTCH1 is a membrane receptor of a Hedgehog (HH) signal path, when an HH ligand exists, the protein receptor is combined with the PTCH to release SMO activity inhibition, GLI is released from an SMO protein complex and is transferred into a nucleus, so that transcriptional activation of related genes downstream of the Hedgehog path is caused, and the condition that the level of the PTCH1 protein is obviously increased in the process of LPS-induced mammary gland fibrosis of the mouse is proved, so that the Hedgehog signal path possibly participates in the occurrence and development of mammary gland fibrosis is suggested, and experimental basis is provided for further research on pathological mechanism and clinical prevention and control research of the mammary gland fibrosis.
Drawings
FIG. 1 is a schematic diagram showing comparison of histopathological observations of breast fibrosis in LPS-induced mice in examples 1 and 2.
FIG. 2 is a schematic diagram showing comparison of LPS-induced mammary fibrosis histopathological observations of mice in example 4.
FIG. 3 is a graph showing the effect of LPS on the expression of type I collagen mRNA in mammary fibrotic tissue of mice in example 4.
FIG. 4 is a graph showing the effect of LPS on expression of type III collagen mRNA in mammary fibrotic tissue of mice in example 4.
FIG. 5 is a graph showing the effect of LPS on the expression of PTCH1 protein in mouse mammary fibrosis tissue in example 4.
Detailed Description
The following examples will assist one of ordinary skill in the art in further understanding the present invention. All embodiments obtained by a person of ordinary skill in the art without making any inventive effort based on the embodiments of the present invention are within the scope of the present invention.
Example 1
BALB/C master mice were used for 4-5 days of delivery of the SPF fraction and purchased from Hunan Stokes Levoda laboratory animals Co. The purchased mice were kept in a room with a temperature of 25℃and a light/dark cycle of 12/12, food and water were freely available, and modeling was started 4-5 days after the mice had been acclimatized.
The experiment was divided into two groups, namely a blank Control group (i.e., control group in the drawing), an LPS dose group (i.e., LPS group in the drawing), each group was provided with 5 mice, and the mice of the blank Control group were continuously perfused with 50 μl of physiological saline to the fourth pair of milk ducts of the mice on days 0, 3, 6, 9, 12 of the feeding group, and were continuously perfused with 50 μl of LPS to the fourth pair of milk ducts of the mice on days 0, 3, 6, 9, 12 of the feeding group, respectively, and the LPS concentration in this example was 0.075mg/kg, to complete the induction process.
After induction was completed, all mice survived.
Mice were sacrificed manually on day 21 of induction, and the mice were taken for a fourth pair of mammary tissue for pathological section staining by staged continuous perfusion.
The fourth pair of left mammary gland tissues of the mice are used10% neutral formalin solution was fixed. After 48h fixation, breast tissue was trimmed to 1X 2X 3cm 3 The small blocks are placed in an embedding box, and a new formalin solution is replaced for secondary fixation; washing tissue blocks with running water overnight, dehydrating, waxing and embedding, slicing the tissue blocks with 3-5 mu m in a slicing machine, fully expanding the tissue blocks in an expanding machine under the environment of 42 ℃, taking out the tissue blocks by a glass slide, and baking the tissue blocks on a baking machine at 40 ℃ for about 2-4 hours; the slides were transferred to a staining rack for eosin-Hematoxylin (HE) staining and Masson staining, the slides were slightly air dried, and after blow-dried with a blower, the microscope was observed and recorded with photographs, and the microscopic pictures of the blank control group and the LPS dose group are shown in the left and middle panels of fig. 1.
Example 2
In this example, a second LPS dose group was set on the basis of examples 1 and 2, and mice were continuously perfused in stages with LPS solution at a concentration of 0.15mg/kg, and the group setting and the perfusion were the same as examples 1 and 2.
Unlike example 1 and example 2, 3 mice died during induction with 0.15mg/kg of LPS in this example, with a mortality rate of 60%.
Surviving mice were artificially sacrificed after completion of the induction process of mice in the second LPS dose group, and the fourth perfused mammary tissue was used for pathological section staining.
A fourth pair of left mammary tissue from the second LPS dose group mice was fixed with 10% neutral formalin solution. After 48h fixation, breast tissue was trimmed to 1X 2X 3cm 3 The small blocks are placed in an embedding box, and a new formalin solution is replaced for secondary fixation; washing tissue blocks with running water overnight, dehydrating, waxing and embedding, slicing the tissue blocks with 3-5 mu m in a slicing machine, fully expanding the tissue blocks in an expanding machine under the environment of 42 ℃, taking out the tissue blocks by a glass slide, and baking the tissue blocks on a baking machine at 40 ℃ for about 2-4 hours; the slides were transferred to a staining rack for eosin-Hematoxylin (HE) staining and Masson staining, the slides were slightly air dried, blow dried with a blower, observed with a microscope and recorded with photographs, and the microscopic pictures of the second LPS dose group are shown in the right panel of fig. 1.
Example 3
In this example, a third LPS dose group was set on the basis of examples 1 and 2, and mice were continuously perfused in stages with LPS solution at a concentration of 0.3mg/kg, and the group setting and the perfusion were the same as examples 1 and 2.
In the LPS induction process of this example, all mice died and the model construction failed.
As a result, as shown in FIG. 1, the mice in the third LPS dose group (0.3 mg/kg) all died, the mortality rate was 100%, the mortality rate in the second LPS dose group (0.15 mg/kg) was 60%, and the first LPS dose group (0.075 mg/kg) did not die. From the histopathological results in FIG. 1, it was shown that the mammary gland acinar epithelium of the mice in the blank Control group (i.e., control group in the drawing) had complete structure, the lesions of the first LPS dose group (0.075 mg/kg) were mainly inflammatory exudation, the mammary gland acinar stroma had little fibroblast proliferation, and the second LPS dose group (0.15 mg/kg) was visibly evident stroma fibroblast proliferation, but the mortality was too high. Based on the mortality and fibrosis degree of mice, the LPS dose was adjusted to 0.1mg/kg, and a formal experiment was performed.
Example 4
BALB/C master mice were used for 4-5 days of delivery of the SPF fraction and purchased from Hunan Stokes Levoda laboratory animals Co. The purchased mice were kept in a room with a temperature of 25℃and a light/dark cycle of 12/12, food and water were freely available, and modeling was started 4-5 days after the mice had been acclimatized.
The experiment was divided into two groups, namely a blank Control group (i.e., control group in the drawing), an LPS dose group (i.e., LPS group in the drawing), each group was provided with 6 mice, the mice of the blank Control group were continuously perfused with 50 μl of physiological saline to the fourth pair of milk ducts of the mice on days 0, 3, 6, 9, 12 of the feeding group, and the mice of the LPS dose group were continuously perfused with 50 μl of LPS to the fourth pair of milk ducts of the mice on days 0, 3, 6, 9, 12 of the feeding group, and the LPS concentration of this example was 0.1mg/kg, to complete the induction process.
After induction was completed, all mice survived.
Mice were sacrificed manually on day 21 of induction, the mice were stained for pathological sections of the fourth pair of left mammary tissue by staged continuous perfusion, and the fourth pair of right mammary tissue was frozen in-80 ℃ refrigerator for subsequent RNA and protein extraction.
The fourth pair of left mammary tissue of the mice was fixed with 10% neutral formalin solution. After 48h fixation, breast tissue was trimmed to 1X 2X 3cm 3 The small blocks are placed in an embedding box, and a new formalin solution is replaced for secondary fixation; washing tissue blocks with running water overnight, dehydrating, waxing and embedding, slicing the tissue blocks with 3-5 mu m in a slicing machine, fully expanding the tissue blocks in an expanding machine under the environment of 42 ℃, taking out the tissue blocks by a glass slide, and baking the tissue blocks on a baking machine at 40 ℃ for about 2-4 hours; the slides were transferred to a staining rack for eosin-Hematoxylin (HE) staining and Masson staining, the slides were slightly air dried, and after blow-dried with a blower, the microscopic images of the blank and LPS dose groups were recorded as shown in the left and right panels of fig. 2.
As shown in fig. 2, the pathological section staining results of the present embodiment show that (i.e., the Control group in the drawing) the mammary tissue structure is fresh, no inflammatory cell infiltration is seen, the mammary gland acinar wall structure is complete, the acinar interval is not thickened, and no fibroblast proliferation is seen; compared with the control group, the mice of the LPS dosage group in the example have disordered mammary tissue structure, and the parenchymal mammary gland acinus is atrophic, and the mammary gland interstitial fibroblast is proliferated to form a fibrotic focus. Masson staining showed that most areas in the visual field of breast tissue were blue stained with aniline blue, indicating that there was a large amount of collagen deposition in the breast tissue and that breast fibrosis was evident.
Total RNA was extracted from the excised fourth pair of right breast tissues using RNAiso Plus (TaKaRa, japan) and cDNA synthesis was performed using First Strand cDNA synthesis Kit. And detecting the mRNA expression of the Collagen I and the Collagen III in the mammary tissue by adopting a SYBR Green and Bio-Rad CFX96 real-time PCR detection system, and finally obtaining the influence of LPS on the mRNA expression of the type I and III Collagen of the mammary fibrosis tissue of the mouse, wherein the influence is shown in figures 3 and 4 respectively.
In FIGS. 3 and 4, the RT-PCR detection results are shown in FIG. 3. After repeated stimulation of the mammary glands by LPS, the LPS group Collagen I mRNA level was significantly different from the control group (p < 0.05), and the Collagen III mRNA level was significantly different from the control group (p < 0.01).
Then homogenizing the mammary tissue for 30min in ice bath, centrifuging for 15min under 14000r/min to extract protein, proportionally adding a loading buffer solution, and denaturing for 15min at 100deg.C; secondly, transferring the protein after SDS-PAGE electrophoresis, sealing the protein for 2 hours at room temperature by using skimmed milk powder with the concentration of 5% after TBST dissolution, rinsing the protein for 3 times by using TBST, and adding rabbit anti-vimentin primary antibody for incubation at 4 ℃ overnight; rinsing for 3 times by TBST, adding HRP-marked goat anti-rabbit secondary antibody, and incubating for 2 hours at room temperature; finally, after TBST is rinsed for 3 times, ECL luminous solution is added, darkroom exposure is carried out, and Western blot is adopted to detect the expression condition of the PTCH1 protein of the mammary tissue, so that the detection of the PTCH1 protein expression of the mammary fibrosis tissue of the mouse induced by LPS is shown in a figure 5, and the result shows that the expression level of the PTCH1 protein of the LPS group is extremely obvious compared with that of a control group (p < 0.01).
Taken together, the results show that the repeated stimulation of the mammary gland of the mice with LPS with the concentration of 0.1mg/kg can successfully induce the mammary gland fibrosis model of the mice. PTCH1 is a membrane receptor for the Hedgehog (HH) signaling pathway, and when the HH ligand is present, through its binding to PTCH, the inhibition of SMO activity is released, GLI is released from the SMO protein complex and transferred into the nucleus, resulting in transcriptional activation of genes associated downstream of the Hedgehog pathway. The results of example 4 have preliminarily demonstrated that PTCH1 protein levels are significantly increased during LPS-induced mammary fibrosis in mice, suggesting that Hedgehog signaling pathways may be involved in the development of mammary fibrosis, providing experimental basis for further studies of the pathological mechanisms of mammary fibrosis and clinical control studies.
Claims (10)
- The construction method of the mouse model of LPS induced mammary gland fibrosis is characterized by comprising the following steps: the mammary duct of the female mouse is continuously infused with LPS in stages for inducing mammary cell fibrosis.
- 2. The method of constructing a mouse model of LPS-induced mammary fibrosis according to claim 1, wherein the female mouse is a BALB/C female mouse which has been delivered for 8-10 days.
- 3. The method of constructing a mouse model of LPS-induced mammary gland fibrosis according to claim 1, wherein each time the LPS is infused, the concentration is between 0.075 and 0.3mg/kg, preferably 0.1mg/kg.
- 4. The method of constructing a mouse model of LPS-induced mammary gland fibrosis according to claim 1, wherein the perfusion site is a fourth pair of left and right mammary ducts.
- 5. The method for constructing a mouse model of LPS-induced mammary gland fibrosis according to any one of claims 1 to 4, comprising the following specific steps:(1) Selecting two groups of female mice for a control experiment, and marking the mice as a blank control group and an LPS dosage group respectively;(2) Performing staged continuous perfusion on the breast catheter at the same breast position by using normal saline for the blank group of mice, and performing staged continuous perfusion on the breast catheter at the same breast position by using equivalent LPS for the LPS dose group of mice to complete the induction process;(3) After induction is completed, mice are sacrificed, and mammary tissue at the perfusion site is selected for pathological section staining and extraction of RNA and protein, respectively.
- 6. The method of constructing a mouse model of LPS-induced mammary gland fibrosis according to claim 5, wherein the blank group and the LPS dose group are each provided with at least two female mice.
- 7. The method of constructing a mouse model of LPS-induced mammary gland fibrosis according to claim 5, wherein the mice in the blank group and the LPS-dose group are continuously perfused with the corresponding agent in stages on days 0, 3, 6, 9, 12 of the feed group, respectively, and the induction is completed and the mice are sacrificed on day 21.
- 8. The method of constructing a mouse model of LPS-induced mammary gland fibrosis according to claim 5, wherein the pathological section staining comprises the steps of:(1) Taking mammary tissue perfused by mice, fixing with 10% neutral formalin solution, fixing for 48 hr, and repairing the mammary tissue into 1×2×3cm 3 Placing the small blocks in an embedding box, and replacing a new formalin solution for secondary fixation;(2) Washing tissue blocks with running water overnight, dehydrating, waxing and embedding, slicing the tissue blocks with 3-5 mu m in a slicing machine, fully expanding the tissue blocks in a 42 ℃ sheet expanding machine, taking out the tissue blocks with a glass slide, and baking the tissue blocks on the sheet baking machine at 40 ℃ for about 2-4 hours; the slide was transferred to a staining rack for eosin-hematoxylin staining and Masson staining, the slides were slightly air-dried, and after blow-dried by a blower, the slides were observed by a microscope and recorded by photographing.
- 9. The method of constructing a mouse model of LPS-induced breast fibrosis according to claim 5, wherein the extraction of RNA and protein comprises the steps of:(1) Extracting total RNA from mammary tissue to synthesize cDNA, and detecting the expression of Collagen I and Collagen III mRNA in the mammary tissue;(2) Homogenizing mammary tissue, ice-bathing for 30min, centrifuging for 15min to extract protein, adding loading buffer, denaturing at 100deg.C for 15min, performing SDS-PAGE electrophoresis on protein, transferring film, sealing 5% skimmed milk powder at room temperature for 2h, rinsing with TBST for 3 times, adding rabbit anti-vimentin primary antibody, incubating overnight at 4deg.C, rinsing with TBST for 3 times, adding HRP-labeled goat anti-rabbit secondary antibody, incubating at room temperature for 2h, rinsing with TBST for 3 times, adding ECL luminescent solution, and exposing in darkroom.
- 10. A mammary gland fibrosis mouse model constructed by the method of claims 1-9.
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