A method for constructing a mouse model of uterine atony

Abstract

The present invention provides a method for constructing a uterine atony mouse model, which belongs to the field of biomedicine. The present invention constructs a uterine atony mouse model by specifically knocking out the uterine smooth muscle CBS gene. The mouse model has characteristic symptoms of delayed labor initiation time, prolonged labor, and uterine muscle atony, which are consistent with the symptoms and pathological process of human uterine atony, and has good controllability and stability. The mouse model constructed by the method of the present invention can be used to study the occurrence and development mechanism and prevention and treatment measures of uterine atony, as well as for the development and research of new drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for constructing a uterine atony mouse model and an application thereof. Background Art

[0002] The contraction function of the uterine muscle plays a key role in the maintenance and initiation of pregnancy. The regulatory mechanism of the transition of the uterine muscle from the resting state to the contracted state, as well as the factors involved in the regulation of the uterine muscle contraction process, have not yet been thoroughly elucidated. Because the mechanism is not clear, there is still a relative lack of clinical treatment methods for diseases related to the contraction function of the uterine muscle, such as premature birth and uterine atony, making it difficult to achieve a stable and efficient treatment effect.

[0003] The uterine muscle during pregnancy can be divided into four stages: resting period, activation period before delivery, contraction period during delivery, and recovery period after delivery. Resting period: During the 36th to 38th week of pregnancy, the uterine muscle is in a relaxed state under the conditions of progesterone and relaxin. Activation period: During the last 2-4 weeks of pregnancy, the uterine muscle transitions from the resting period to the contraction period and is activated, and weak and irregular uterine contractions may occur. At this time, there will be obvious changes in the structure and function of the uterine muscle and cervix, such as an increase in gap junctions between uterine muscle cells, a large increase in contraction-related proteins such as oxytocin and prostaglandin receptors, an enhanced response of the uterine muscle to oxytocin and prostaglandins, and softening and maturation of the cervix and formation of the lower uterine segment. Contraction period: When labor begins, the sensitivity of the uterine muscle to oxytocin and prostaglandins is further enhanced, and it develops from irregular contractions to rhythmic and strong contractions. Recovery period: The contraction of the uterine muscle weakens and gradually changes to the state before pregnancy. During this approximately 40-week period, uterine muscle cells need to switch from a resting, proliferative, synthetic phenotype, etc., to a contractile phenotype during labor. If an abnormality occurs during the phenotypic conversion process, it may result in the inability of the uterine muscle to maintain its proper contractile function during the final labor stage, such as premature contraction of the uterine muscle, which manifests as premature birth, or insufficient contraction of the uterine muscle, which manifests as prolonged labor and uterine atony.

[0004] Uterine atony is the underlying cause of postpartum hemorrhage. Data from a large global randomized controlled trial suggest that uterine atony accounts for 30-80% of all cases of postpartum hemorrhage. Currently, the treatments for uterine atony include oxytocin, ergonovine, misoprostol, etc. The effects of these drugs are sometimes not ideal and may also bring a series of cardiovascular effects, including hypotension, tachycardia, ST segment depression, myocardial ischemia and arrhythmia. Further treatments include uterine massage, uterine packing, and uterine compression suture, but they may lead to uterine necrosis and intrauterine adhesions. When the above methods fail, in order to save lives, bilateral uterine artery ligation, internal iliac artery ligation, or even hysterectomy can be selected, which will not be able to preserve normal reproductive function.

[0005] At present, for the study of uterine atony, wild-type mice can be used. After the mice become pregnant and before delivery, drugs are injected to intervene, so that the mice develop the phenotype of uterine atony. However, the method of modeling by drug administration alone still has limitations, and gene knockout modeling can better study uterine atony. In addition, due to the medical ethics constraints of human experiments, animal models have become an important tool for studying the occurrence and development of uterine atony and exploring prevention and control measures. However, there is currently no phenotypically stable and large-scale fertilized animal model of uterine atony. Therefore, there is a need in the art for a method for constructing an animal model of uterine atony. Summary of the invention

[0006] Therefore, the present invention provides a method for constructing a uterine atony mouse model, which comprises constructing a uterine atony mouse model by specifically knocking out the CBS gene of the uterine smooth muscle of the mouse.

[0007] In a specific embodiment, loxP sequences are inserted into both ends of the mouse CBS gene to mark and effectively knock out the CBS gene. The resulting mice with loxP sequences inserted into both ends of the CBS gene are named loxP-flox-CBS mice, or CBS f / f Then, the loxP-flox-CBS mice were mated with the tool mice carrying the Tagln-Cre enzyme and backcrossed with the loxP-flox-CBS mice to purify the uterine atony mouse model, i.e., CBS f / f / Tagln-Cre mice, or CBS d / d mice; the Tagln-Cre enzyme is a smooth muscle driven Cre.

[0008] In a specific embodiment, the uterine atony mouse model, namely CBS f / f / Tagln-Cre mice, the model mice were CBS f / f CBS of male mice of Tagln-Cre mice and control mice f / f The female mice were caged together to breed more CBS model mice. d / d CBS mice and control mice f / f Mouse.

[0009] In a specific embodiment, the male-female cage ratio is 3:1 or more.

[0010] In a specific embodiment, female CBS are selected at the beginning of establishing the mouse model. f / f The mice carry the flox sequence, while the male mice are tool mice carrying the Tagln-Cre enzyme.

[0011] In the present invention, the flox sequence is the loxP sequence.

[0012] In a specific embodiment, the mouse is an SPF grade C57BL / 6 mouse.

[0013] The present invention also provides an application of a uterine atony mouse model, including the uterine atony mouse model prepared by the construction method for use in the development of drugs for treating and / or preventing uterine atony in animals and humans.

[0014] The present invention also provides a method for screening drugs for treating and / or preventing uterine atony in animals and humans, the method comprising the following steps:

[0015] 1) administering a drug to be tested to the mouse model constructed by the construction method according to any one of claims 1 to 6;

[0016] 2) Analyze and evaluate the therapeutic effects of the drugs to be tested, and select test drugs that can significantly improve the pathological characteristics of model mice.

[0017] The present invention has the following beneficial effects:

[0018] 1. The present invention uses a mouse model to simulate the natural state of the human body to conduct experiments to study the pathogenesis and treatment, which reduces the necessity of conducting experiments on humans and avoids the ethical disputes of human experiments.

[0019] 2. Systemic knockout of CBS will lead to embryonic death of mice. The present invention constructs a uterine atony mouse model by specifically knocking out the CBS gene in uterine smooth muscle cells. The mouse survival rate is high and the knockout efficiency is high. After successful construction, the phenotype is stable, and the symptoms will not be alleviated or disappear with the increase of time and mating times.

[0020] 3. The pathological manifestations of the uterine atony model in the invention include delayed labor initiation, prolonged labor, uterine muscle atony, etc. It is consistent with the pathological changes of human uterine atony, can better simulate the natural disease state of the human body, and has good representativeness.

[0021] 4. The mouse model of the present invention has an obvious phenotype of uterine atony, which can fill the gap of the current lack of relevant animal models and is conducive to the observation and design of relevant experiments and the development of relevant drugs.

[0022] 5. The present invention can verify and observe gene knockout from multiple angles such as genes, proteins, tissues and organs, and the experimental results are real, credible and convincing.

[0023] In general, the present invention constructs a mouse model of uterine atony by conditionally knocking out the CBS gene of uterine smooth muscle cells. The mouse model has phenotypes such as delayed labor initiation, prolonged labor, and uterine muscle atony, which are consistent with the symptoms and pathological processes of human uterine atony, and has good controllability and stability. The uterine atony mouse model of the present invention can be used to study the occurrence and development mechanism and prevention and treatment measures of uterine atony, as well as for the development and research of new drugs. In addition, the mouse model constructed by conditional gene knockout can further systematically explore the molecular mechanism of uterine muscle transformation before pregnancy and in the early stages of pregnancy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure is a flowchart for constructing a mouse model of uterine smooth muscle cell-specific knockout of the CBS gene in the present invention.

[0025] Figure 2 Another flowchart for constructing a mouse model of uterine smooth muscle cell-specific CBS gene knockout in the present invention.

[0026] Figure 3 This is the nucleic acid electrophoresis verification diagram of the CBS gene.

[0027] Figure 4 This is the nucleic acid electrophoresis verification diagram of the Tagln-Cre gene.

[0028] Figure 5 CBS d / d and control mice CBS f / f Protein electrophoresis of CBS protein and internal reference protein β-actin in uterine smooth muscle tissue.

[0029] Figure 6 Yes Figure 5 Gray value statistics of protein electrophoresis results.

[0030] Figure 7 CBS d / d and control mice CBS f / f Immunohistochemical staining of CBS was performed on uterine sections to verify the knockout of CBS in the uterine smooth muscle area at the protein level.

[0031] Figure 8 CBS d / d and control mice CBS f / f Schematic diagram of the relative expression of mRNA in uterine smooth muscle tissue, which verifies the CBS model mouse at the transcriptional level. d / d The gene was specifically knocked out.

[0032] Fig. 9 CBS d / dCBS mice and control mice f / f Statistical chart of mouse gestation duration.

[0033] Fig.10 CBS d / d CBS mice and control mice f / f Statistical chart of mouse delivery duration.

[0034] Fig.11 CBS d / d CBS mice and control mice f / f Contraction tension detection curve of isolated uterine muscle strips of mice during delivery.

[0035] Fig.12 Yes Fig.11 The contraction tension curves shown were further analyzed to calculate the peak-to-peak value, mean contraction force, maximum contraction force, and area under the curve.

[0036] Fig.13 CBS f / f Contraction force detection curve of isolated uterine muscle strips of mice during labor before and after oxytocin stimulation.

[0037] Fig.14 CBS d / d Contraction force detection curve of isolated uterine muscle strips of mice during labor before and after oxytocin stimulation. DETAILED DESCRIPTION

[0038] The cystathionine beta synthase (CBS) gene is located in the subtelomeric region q.22.3 on human chromosome 21 and has 23 exons, 15 of which encode the enzyme. CBS has two major promoters -1a and -1b. Both promoters are GC-rich and contain many putative transcription factor binding sites and estrogen receptor binding sites. Human CBS is a tetramer composed of 551 amino acids with a subunit molecular weight of 63kDa. CBS is a key enzyme in the methionine and cysteine ​​metabolic pathways and is widely involved in various biochemical processes.

[0039] The inventors observed that the start of labor in mice was delayed, the labor process was prolonged, and the contraction force of isolated uterine muscle strips was weakened by knocking out CBS in the uterine smooth muscle of mice. These phenotypes are consistent with the symptoms of the disease of uterine atony. The inventors found that by specifically knocking out the CBS gene in the uterine smooth muscle of mice, by interfering with the phenotypic conversion process of the uterine muscle after pregnancy, the uterine muscle cells of pregnant mice during the labor period were not completely converted to a contractile phenotype, and ultimately showed uterine atony as a whole. Therefore, the present invention constructs an animal model of uterine atony with specific knockout of the CBS gene in uterine smooth muscle.

[0040] The present invention aims to provide a method for constructing a mouse model of uterine atony by specifically knocking out the CBS gene in mouse uterine smooth muscle cells. The mouse model of uterine atony can better restore the pathological manifestations of the disease, is consistent with the symptoms and pathological process of human uterine atony, has good controllability and stability, and can be used to study the occurrence and development mechanism of uterine atony, preventive and therapeutic measures, and for new drug research.

[0041] The invention provides a method for constructing a uterine atony mouse model.

[0042] First build CBS f / f Mouse, loxP sequences were inserted into both ends of the mouse CBS gene to mark and effectively knock out the CBS gene. The resulting mouse with loxP sequences inserted into both ends of the CBS gene was named loxP-flox-CBS mouse, or CBS f / f Mice. Specifically, the CBS gene was floxed by using the principle of homologous recombination and embryonic stem cell (ES cell) targeting. Exon exon2 in the transcript of the CBS gene Cbs-001 (ENSMUST00000067801) was floxed to construct an ES cell targeting vector. The vector contains a sequence homologous to the 5' end of the CBS gene, a flox gene sequence, a PGK-Neo-polyA positive screening marker, a sequence homologous to the 3' end of the CBS gene, and an MC1-TK-polyA negative screening marker. The vector was transcribed into ES cells, and after G418 and Ganc drug screening and PCR identification, positive cloned ES cells with correct homologous recombination were obtained. After amplification, the positive ES cell clones were injected into the blastocysts of C57BL / 6J mice to obtain chimeric mice. The male mice born after targeting are mated with normal female mice that provided the blastocysts. Some of the offspring mice born are heterozygous mice (- / +). The heterozygous mice are then intercrossed, and homozygous gene-targeted mice (+ / +) are identified and screened out from the offspring. The loxP sequences are inserted at both ends of the CBS gene of these mice, and transgenic mice (i.e., loxP-flox-CBS mice) strains can be established through breeding.

[0043] The process of customizing the loxP-flox-CBS mice is generally completed in a genetic company, and the method for preparing the loxP-flox-CBS mice is a routine technology that can be obtained by genetic companies.

[0044] Then, construct a tool mouse of Tagln-Cre enzyme, and this transgenic mouse can express Cre recombinase in smooth muscle tissue. Since the tool mouse of Tagln-Cre enzyme is available for direct purchase, the construction method of the mouse is not described here.

[0045] Afterwards, loxP-flox-CBS mice were mated with tool mice carrying Tagln-Cre enzyme and backcrossed to purify, thereby obtaining the uterine atony mouse model, namely CBS f / f / Tagln-Cre mice.

[0046] The genotype of mice is usually determined by testing the toes or tails of 3-week-old mice. f / f The CBS protein and mRNA expression levels in the uterine smooth muscle tissue of / Tagln-Cre mice were measured to verify the knockout of the CBS gene in mouse endometrial cells and to further determine whether the uterine atony mouse model was successfully constructed. f / f CBS / Tagln-Cre mice d / d CBS f / f Female mice were caged together to breed more model mice and control mice.

[0047] Specifically: Select CBS f / f / Tagln-Cre model mouse CBS d / d CBS f / f Female mice are mated to produce more model mice and control mice. The genotype of the offspring mice can be verified at about 3 weeks after birth, and the CBS gene can be identified as wild type (no flox sequence is inserted in the two paired DNA chains), heterozygous (only one of the two paired DNA chains has a flox sequence inserted at both ends of its CBS gene), or f / f type (both paired DNA chains have flox sequences inserted at both ends of its CBS gene), and f / f type mice are selected to analyze their Tagln-Cre genotype to identify whether they are model mice or control mice. The CBS gene of the control mouse is f / f type, and the Tagln-cre genotype is negative, so the CBS gene is not knocked out in this mouse. Controlling the ratio of male to female cages to 3:1 can maximize the mating success rate, thereby ensuring the orderly progress of the model construction experiment and avoiding the problem of prolonged experimental period caused by long-term mating failure during the experimental process.

[0048] Example 1

[0049] This example describes the construction process of genetic mice.

[0050] Figure 1 and Figure 2The flowchart of the construction of the mouse model of uterine smooth muscle cell-specific knockout of CBS gene in the present invention is as follows. SPF-grade C57BL / 6 background mice were selected, loxP sequences were first inserted at both ends of the CBS gene, and the target gene with loxP fragments was integrated into the original genome to mark and knock out the CBS gene, thereby obtaining loxP-flox-CBS mice, namely CBS f / f Then, loxP-flox-CBS mice were mated with tool mice carrying Tagln-Cre enzyme and backcrossed to purify, thus obtaining a mouse model with uterine smooth muscle cell-specific knockout of CBS gene, namely CBS f / f / Tagln-Cre mice, also known as CBS d / d Mouse.

[0051] Figure 1 In the control mouse CBS customized by Gene Company f / f The CBS gene of mice is homozygous with loxP sequences inserted in both nucleotides. The commercialized tool mice carrying Tagln-Cre enzyme purchased from a gene company have both nucleotides of the Cre gene positive and homozygous; their offspring mice CBS f / -, The CBS gene of Tagln-Cre mice is a heterozygote with only one nucleotide inserted into the loxP sequence, and its Cre gene is also a heterozygote with only one nucleotide positive. However, the Cre enzyme only needs one nucleotide positive in the Cre gene to be effective, and the heterozygote of the Cre gene is as effective as the homozygote. f / -, Tagln-Cre mice were then crossed with control mice CBS f / f Mice were backcrossed; half of the resulting offspring mice were homozygous with loxP sequences inserted into both nucleotides of the CBS gene, and the other half were heterozygous with only one nucleotide of the CBS gene inserted into the loxP sequence. The heterozygous mice were discarded, and half of the homozygous mice were Cre enzyme positive, i.e., model mice CBS d / d The other half of the homozygous mice are Cre enzyme negative, that is, the control mice CBS f / f mice. Therefore, in general, CBS f / -, Tagln-Cre mice and control mice CBS f / f Among the offspring mice obtained after backcrossing, 1 / 4 are model mice, which is the total number of female and male model mice. Therefore, the probability of female and male model mice is 1 / 8 each.

[0052] In addition, although male mice do not have a uterus and therefore no uterine smooth muscle, because the effect of Tagln-Cre is to knock out the gene in the smooth muscle part of the mouse, the model mouse CBS d / dMale mice can be generated in which the CBS gene is selectively knocked out in the smooth muscle area.

[0053] In actual operation, the inventors found that if the model mouse CBS d / d Male mice and model mice CBS d / d The female mice tried to breed more model mice, but the female mice were not easy to get pregnant, and the model mice CBS d / d The delivery time of female mice was prolonged and the survival rate of offspring was reduced.

[0054] Therefore, the model mouse CBS was selected in the present invention. d / d CBS f / f When female mice mate, half of their offspring are model mice and the other half are control mice; specifically, one quarter of the offspring are model mice CBS. d / d Female mice, one quarter of the control mice CBS f / f Female mice.

[0055] Example 2

[0056] This example verifies CBS gene knockout.

[0057] Figure 3 This is the CBS gene verification diagram. Figure 4 This is the Tagln-Cre gene validation diagram. Figure 3 The nucleic acid gel electrophoresis shows the genotype of the mouse CBS gene. The leftmost column is the bright band of the standard reagent, which is used to indicate the size of the nucleic acid. The second column is the blank control, and the third column is the wild type, that is, the CBS gene of the two DNA chains has no loxP sequence inserted at both ends. The nucleic acid size is 355bp. Columns 4-6 represent three different mice, but all three are homozygous f / f, that is, the CBS gene of the two DNA chains has loxP sequences inserted at both ends, and the nucleic acid size is 437bp. Finally, the combination Figure 3 and Figure 4 It can be seen that the mice in columns 4-6 are Figure 1 CBS d / d The genotype of the Tagln-Cre gene was verified by nucleic acid electrophoresis. The verification results are shown in Figure 4, the leftmost column in the figure is the bright band of the standard reagent, which is used to indicate the size of the nucleic acid. The 2nd column is the blank control, and the 3rd column is the wild type. No nucleic acid band is seen at the 100bp position. Columns 4-6 represent three different mice, but all three are Tagln-Cre genotype positive, and nucleic acid bands can be seen at the 100bp position. Mice that are homozygous for the CBS gene (i.e. f / f type) and negative for the Tagln-Cre genotype (the CBS gene on uterine smooth muscle cells has not been knocked out) can be used as control mice for the model mice in the present invention. Figure 3 and Figure 4 It can be seen that the mouse model CBS obtained by the present invention d / d The genotype of the mice was homozygous for the CBS gene and carried the Tagln-Cre gene.

[0058] Figure 5 CBS d / d and control mice CBS f / f The protein electrophoresis diagram of CBS protein and internal reference protein β-actin in uterine smooth muscle tissue of mice with homozygous CBS gene (i.e. f / f type), with a molecular weight of about 63kDa, and the second row is a schematic diagram of β-actin, the internal reference protein, with a molecular weight of about 43kDa. The three bands on the left in the first row are all from control mice with homozygous CBS gene (i.e. f / f type) and negative Tagln-Cre genotype (i.e. CBS f / f The protein electrophoresis of the mouse) showed that the protein expression level was high; this indicated that the CBS protein in the mouse uterine smooth muscle tissue was higher than that in the control mouse CBS f / f The three bands on the right in the first row are all model mice with homozygous CBS gene (i.e. f / f type) and positive Tagln-Cre genotype (i.e. CBS d / d The protein electrophoresis of the mouse (Figure 2A) shows that the expression of CBS protein in the uterine smooth muscle tissue of the mouse is reduced or even disappeared.

[0059] Figure 6 Yes Figure 5 Quantitative analysis of the results. Figure 6 CBS d / d and control mice CBS f / f Schematic diagram of the protein expression ratio of CBS protein in uterine smooth muscle tissue and the internal reference protein β-actin. Figure 6 Each data point represents a mouse. As can be seen in the figure, the expression level of the protein in the endometrial tissue of the control mouse is significantly higher than that of the model mouse, and there is a statistical difference between the two, and the *** in the figure represents p<0.001.

[0060] Figure 7 CBSd / d and control mice CBS f / f The microscope image of the uterus after immunohistochemical staining, i.e. the verification image at the protein level. CBS, 1:500, Proteintech, 67861-1-Ig, where the left side is the control mouse (i.e. CBS f / f mice), and the right side is the model mouse (i.e., CBS d / d mice), as shown, with CBS f / f Compared with mice, CBS d / d The expression of CBS protein in the smooth muscle of mouse uterus was significantly reduced.

[0061] Figure 8 CBS d / d and control mice CBS f / f Schematic diagram of the relative expression of mRNA in uterine smooth muscle tissue, which verifies the CBS model mouse at the transcriptional level. d / d The gene was specifically knocked out. As can be seen from the figure, the relative expression of CBS mRNA in the uterine smooth muscle tissue of the control mice was significantly higher than that of the model mice, and there was a statistical difference between the two, and p<0.001 (*** represents p<0.001).

[0062] from Figures 5 to 8 It can be seen that protein immunoblotting, mRNA expression and immunohistochemical staining showed that compared with the control mouse CBS f / f CBS d / d The CBS expression level in mouse uterine smooth muscle tissue was significantly decreased at the protein and mRNA levels.

[0063] Example 3

[0064] This example is the observation of the uterine atony phenotype in mice.

[0065] Fig. 9 CBS d / d CBS mice and control mice f / f Mouse pregnancy duration statistics, each data point in the figure represents a mouse. As shown in the figure, control mice (i.e. CBS f / f The average gestational length of mice is 19.46 days, and the model mice (i.e., CBS d / d The average gestational length of CBS is 20.05 days. f / f Compared with mice, CBS d / d The gestational duration of mice was significantly increased, and there was a statistical difference between the two, and p<0.01 (** represents p<0.01).

[0066] Fig.10 CBS d / dCBS mice and control mice f / f Statistical graph of mouse delivery time, each data point in the graph represents one mouse. As shown in the figure, control mice (i.e. CBS f / f The average delivery time of the model mice (CBS d / d The average delivery time of mice was 114.2 minutes. f / f Compared with mice, CBS d / d The delivery time of mice was significantly increased, and there was a statistical difference between the two, and p<0.001 (*** represents p<0.001).

[0067] Fig.11 CBS d / d CBS mice and control mice f / f The contraction tension detection curve of isolated uterine muscle strips of mice during delivery. The first column corresponds to 5 control mice (i.e. CBS f / f The second column corresponds to the curve of 5 model mice (i.e. CBS d / d As shown in the figure, compared with the control mice, the model mice, namely CBS d / d The contraction curve of the mouse uterus was significantly gentler. Fig.12 Yes Fig.11 The contraction tension curve shown was further analyzed to calculate the peak-to-peak value, average contraction force, maximum contraction force and area under the curve. As shown in the figure, compared with the control mice, the model mice, namely CBS d / d The peak-to-peak value, average contraction force, maximum contraction force, and area under the curve of isolated mouse uterine muscle strips were significantly decreased (** represents p<0.01, *** represents p<0.001). Fig.11 and Fig.12 It can be seen that compared with the control mice CBS f / f CBS d / d The contraction ability of the uterine muscles of mice was significantly weakened during delivery.

[0068] In summary, the present invention first constructs the CBS gene smooth muscle specific knockout mouse of the present invention by using Cre enzyme and Loxp system, that is, Figure 1 and Figure 2 The contents shown in the figure were verified by nucleic acid gel electrophoresis, namely, the CBS gene and Tagln-Cre gene Figure 3 and Figure 4 The results show that the model mice (i.e. CBS d / d mice) and control mice (i.e., CBS f / f mice).

[0069] Then we need to verify the knockout of CBS on uterine smooth muscle. We obtain the uterine muscle tissue of mice, remove the non-uterine muscle components such as the endometrium, extract the protein, and detect the protein expression of CBS by western blotting. The expression of CBS protein in the model mice is significantly reduced, while the expression of CBS protein in the control mice is normal. Figure 5 and Figure 6 The uterine tissue of mice was obtained, embedded and sliced, and then immunohistochemical staining was performed. The expression of CBS protein in the uterine smooth muscle of model mice was significantly reduced, while the expression of CBS protein in the uterine smooth muscle of control mice was normal. Figure 7 The mRNA was extracted and reverse transcribed before using qPCR to detect the relative expression of CBS mRNA. The CBS mRNA expression in the model mice was significantly reduced, while the CBS mRNA expression in the control mice was normal. Figure 8 Content shown.

[0070] Next, the birth phenotype of the genetic mice of the present invention needs to be observed. Female genetic mice (including model mice CBS d / d CBS mice and control mice f / f The mice were mated with wild-type C57 male mice. The presence of a thrombus at 8am the next day was recorded as 0.5 days of pregnancy. The duration of pregnancy and delivery of the mice were observed and recorded. The average duration of pregnancy of the model mice was 20.05 days, which was longer than the average duration of pregnancy of the control mice, which was 19.46 days. Fig. 9 The average labor time of the model mice was 114.2 minutes, which was longer than the average labor time of the control mice, which was 68.33 minutes. Fig.10 Content shown.

[0071] Next, it is necessary to further test the gene mice (including the model mice CBS d / d CBS mice and control mice f / f The contraction of uterine muscle of model mice was investigated by obtaining uterine muscle strips during pregnancy and testing the contraction tension in an in vitro constant temperature perfusion system. The contraction curve showed that the waveform of the contraction curve of the uterus of the model mice was significantly flat, while the waveform of the contraction curve of the control mice was significantly undulating, i.e. Fig.11 Further analysis of the contraction tension curve showed that the peak-to-peak value, average contraction force, maximum contraction force, and area under the curve of the isolated uterine muscle strips of the model mice were significantly decreased compared with those of the control mice. Fig.12 Content shown.

[0072] In summary, the present invention constructs a mouse model of uterine atony by specifically knocking out the CBS gene of mouse uterine smooth muscle.

[0073] In addition, the inventors further explored the CBS model mouse constructed by the present invention. d / d CBS mice and control mice f / f Uterine atony phenotype in mice. Oxytocin is a commonly used drug for the clinical treatment of uterine atony. The inventors obtained uterine muscle strips from mice during labor, gave them oxytocin stimulation, and observed its effect on the contraction tension of the uterine strips.

[0074] Fig.13 CBS f / f The in vitro uterine muscle strip contraction force detection curve of mice during delivery. The first column corresponds to the curve of tension detection of 3 control mice without drug stimulation, and the second column corresponds to the curve of tension detection of 3 control mice after 0.01U / ml oxytocin stimulation. As shown in the figure, when no drug stimulation was given, the waveform of the contraction curve of the uterus of the control mice fluctuated significantly. After oxytocin stimulation, the waveform of the contraction curve of the uterus fluctuated more violently, indicating that oxytocin stimulation had an important effect on the CBS of the control mice. f / f It has a significant effect in promoting uterine muscle contraction in mice.

[0075] Fig.14 CBS d / d The in vitro uterine muscle strip contraction force detection curve of mice during delivery. The first column corresponds to the curve of tension detection of 3 model mice without drug stimulation, and the second column corresponds to the curve of tension detection of 3 model mice after 0.01U / ml oxytocin stimulation. When no drug stimulation was given, the waveform of the uterine contraction curve of the model mice was gentle. After oxytocin stimulation, the waveform of the uterine contraction curve was still gentle without obvious fluctuations, indicating that oxytocin stimulation had a positive effect on the CBS of model mice. d / d The effect of promoting uterine muscle contraction in mice was poor.

[0076] In general, the present invention provides a method for constructing a mouse model of uterine atony. The present invention constructs a mouse model of uterine atony by specifically knocking out the CBS gene of uterine smooth muscle. The mouse model has characteristic symptoms of delayed labor initiation, prolonged labor, and uterine muscle atony, which are consistent with the symptoms and pathological process of human uterine atony, and has good controllability and stability. The mouse model constructed by the method of the present invention can be used to study the occurrence and development mechanism and prevention and treatment measures of uterine atony, as well as for the development and research of new drugs.

[0077] After the mouse model of the present invention is successfully established, the key phenotypes of human uterine atony may appear partially or completely. The animal model provides a guarantee for the disease characteristics, pathogenesis, disease outcome, research and development and screening of intervention strategies and new therapeutic drugs for uterine atony.

[0078] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions and substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope of the present invention.

Claims

1. A method for constructing a uterine atony mouse model, characterized in that: The method comprises constructing a uterine atony mouse model by specifically knocking out the CBS gene of the uterine smooth muscle of the mouse; and specifically comprises inserting loxP sequences into both ends of the mouse CBS gene to mark and effectively knock out the CBS gene, and the obtained mouse having loxP sequences inserted into both ends of the CBS gene is named loxP-flox-CBS mouse, or CBS f / f mice; then, the loxP-flox-CBS mice were mated with tool mice carrying Tagln-Cre enzyme and backcrossed with the loxP-flox-CBS mice for purification to obtain the uterine atony mouse model, i.e., CBS f / f / Tagln-Cre mice, or CBS d / d mice; the Tagln-Cre enzyme is smooth muscle driven Cre.

2. The method for constructing a uterine atony mouse model according to claim 1, characterized in that: The uterine atony mouse model, namely CBS f / f / Tagln-Cre mice, the model mice were CBS f / f CBS of male mice of Tagln-Cre mice and control mice f / f The female mice were caged together to breed more CBS model mice. d / d CBS mice and control mice f / f Mouse.

3. The method for constructing a uterine atony mouse model according to claim 2, characterized in that: The ratio of male to female in the cage should be 3:1 or above.

4. The method for constructing a uterine atony mouse model according to any one of claims 1 to 3, characterized in that: Select female CBS at the very beginning of mouse model establishment f / f The mice carry the flox sequence, while the male mice are tool mice carrying the Tagln-Cre enzyme.

5. The method for constructing a uterine atony mouse model according to any one of claims 1 to 3, characterized in that: The mice are SPF grade C57BL / 6 mice.

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