Construction method and application of CRHPVN neuron activated liver depression type breast cancer mouse model

Abstract

The invention discloses a construction method and application of a CRHPVN neuron activated liver depression type breast cancer mouse model, and belongs to the technical field of animal model construction. The construction method comprises the following steps: (1) injecting 140 to 160 nl of liquid containing rAAV-CRH-CRE-WPRE-hGH polyA and a chemically genetically activated virus rAAV-hM3D (Gq)-mCherry at the same time into a PVN brain region of a CRH-IRES-Cre mouse, wherein the titers of the rAAV-CRH-CRE-WPRE-hGH polyA and the chemically genetically activated virus rAAV-hM3D (Gq)-mCherry are both 2.4 to 2.6 * 10 < 12 > pfu; and (2) 25-30 days after virus injection, performing breast cancer cell planting on all mice, and 38-42 days after breast cancer cell inoculation, obtaining the CRHPVN neuron activated liver depression type breast cancer mouse model. The construction method provided by the invention is simple to operate and good in repeatability, and can overcome the defect of large animal individual difference.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal model construction, and particularly relates to a CRH PVN Construction method and application of a neuron-activated liver-depression type breast cancer mouse model. Background Art

[0002] Breast cancer is the most common malignant tumor in women and one of the three most common cancers in the world. The probability of women developing breast cancer in their lifetime is as high as 8%-10%, which seriously threatens the health of modern women. Mental and psychological factors are one of the important causes of the occurrence and development of breast cancer, and the deterioration of breast cancer can increase the mental and psychological burden of patients, which in turn further aggravates tumor growth and forms a vicious cycle. The growth of breast cancer caused by mental stress factors is very similar to liver depression type breast cancer in traditional Chinese medicine, suggesting that the treatment of breast cancer should focus on soothing the liver and relieving depression.

[0003] In order to further explore the essence of liver depression promoting breast cancer growth and explore its effective Chinese medicine for treatment, it is necessary to study the replication of animal models of liver depression breast cancer. The ideal replication method of animal models should be simple, objective, stable and reproducible, and the etiology, manifestations, and efficacy should be in line with the guidance of traditional Chinese medicine theory.

[0004] However, due to various technical difficulties, there is currently a lack of an animal model that can simulate liver depression syndrome breast cancer. At the same time, due to the shortcomings of complex operation, poor repeatability, large individual differences, long modeling time, and high animal mortality rate in the animal model currently used in liver depression syndrome research, it is impossible to directly apply it to the animal model of simulating induced breast cancer. Therefore, there is an urgent need to develop a stable, repeatable, easy-to-operate animal model that can simulate the pathological occurrence and development process of breast cancer in humans with liver depression. At present, there is no report on a mouse model of liver depression syndrome breast cancer.

[0005] Due to the restrictions of medical ethics, many clinical studies on liver depression syndrome are difficult to carry out, and sample tissues of patients with liver depression syndrome are even more difficult to obtain. The patient's non-cooperation also adds a lot of difficulties to the clinical research of liver depression syndrome. Therefore, it is necessary to find a substitute for human clinical research. The animal model of TCM syndrome provides a method to solve this problem because of the sufficient number of animals, strong operability, and reliable model. It has been widely used in modern research in TCM. Because the emotional changes such as depression and low mood manifested by liver depression syndrome are very similar to the main symptoms of depression (low mood, loss of interest), TCM researchers borrowed the animal model of depression in Western medicine and combined it with the etiology and pathogenesis theory of liver depression syndrome in TCM to produce an animal model of liver depression syndrome with TCM characteristics. At present, the more mature animal models of liver depression syndrome in TCM syndrome mainly include the following 5 types:

[0006] (1) Chronic stress modeling

[0007] The modeling procedure of the chronic unpredictable stress model (CUS) is to model through a variety of chronic stimulations. Although the animals show symptoms of liver depression syndrome such as anhedonia and reduced desire for survival after modeling, the high mortality rate of animals is not conducive to animal research on liver depression syndrome due to the large number of stimulation factors or excessive stimulation intensity. Therefore, Wilmer and other scholars improved on this basis and proposed the chronic unpredictable mild stress model (CUMS), which is modeled through a variety of chronic, unpredictable, and low-intensity stress events, including fasting, 3-day water deprivation, empty bottle hanging, restraint, ice water swimming, hot environment, horizontal shock, foreign body stimulation, tail clamping stimulation, lighting overnight, etc. Rodents are exposed to a series of repeated unpredictable mild stimulations for a certain period of time (3 weeks to 3 months), causing anhedonia and inducing depression-related behaviors, such as weight loss, reduced sugar water consumption, and reduced desire for survival.

[0008] This model has many stress factors and low stress intensity, and the stimulation effect is highly effective and lasts for a long time. It better simulates the situation in which people suffer from various adverse physical and mental stimulations in daily life for a long time, which leads to liver depression. Antidepressant treatment is effective and similar to clinical treatment. It has certain value in studying the etiology, pathogenesis and treatment mechanism of depression, and is one of the most widely used models. However, the operation is complicated and there are large individual differences.

[0009] (2) Drug modeling method

[0010] ① Carbon tetrachloride (CCl) acute poisoning method

[0011] Due to emotional frustration, anger and depression that damage the liver, liver dysfunction, and spleen and earth; or improper diet and excessive fatigue that damage the spleen, spleen dysfunction, dampness and wood depression, and liver dysfunction, thus forming "liver depression and spleen deficiency syndrome", which is mainly manifested as: depression, poor appetite, abdominal distension, limb fatigue, intestinal rumbling and loss of breath, and flank pain. CC4 is a recognized liver poison. Acute CCl poisoning can cause neurological symptoms (such as headache, dizziness, mental confusion, convulsions, hyperreflexia, drowsiness, coma, respiratory muscle paralysis, etc.) and digestive system symptoms (hepatomegaly, liver damage, nausea, vomiting, abdominal pain, diarrhea) and other symptoms. In 1979, Hunan Medical College first used CC4 acute poisoning method to establish the animal model of "liver depression and spleen deficiency syndrome" in mice and rats. A study selected rats and gave them subcutaneous injections of CC peanut oil solution (0.005mlg) once on the 1st day and the 4th day respectively. The rats showed symptoms of "liver depression and spleen deficiency": dry hair, lack of strength to struggle, decreased appetite, reduced body weight, soft and light-colored stools; accompanied by liver lesions, such as significant liver enlargement, hepatocyte necrosis, and abnormal liver function. This model has opened up a new example for the study of animal models of liver depression.

[0012] ② Artemisia argyi injection method

[0013] Artemisia argyi, pungent, warm, bitter, belongs to the liver, spleen, and kidney meridians, and has the effects of warming the meridians to stop bleeding, dispelling cold and relieving pain; external use to remove dampness and relieve itching. Artemisia argyi oil is the volatile oil contained in its dry or fresh leaves, and its main active ingredients are terpinenol-4 and a-terpineol. Studies have shown that artemisia argyi oil has an adrenaline-like effect and has an excitatory effect on the central nervous system (CNS). Taking a large amount of it can cause toxic hepatitis. In 1979, Hunan Medical College selected wild-type mice for intraperitoneal injection of artemisia argyi oil (6.4g / kg, 2 days / time). After continuous injection for 2-3 months, the mice showed manic manifestations of liver depression: the mice increased their activity, fought continuously, and were not easy to scratch. Mice were given intraperitoneal injection of mugwort leaf injection (0.6 ml per time, once a day), and rats were given intraperitoneal injection of mugwort leaf injection (2 ml per time, twice a day). After continuous medication for 40 days, the mice showed symptoms of liver depression18, and pathological changes in the liver occurred, such as increased CAMP and CAMP / CGMP in the liver, cerebral cortex and thalamus.

[0014] ③Adrenal subcutaneous injection

[0015] The liver is responsible for regulating qi and maintaining the ascending and descending movement of the body's qi. If various causes lead to liver failure, qi will be unfavorable. Qi is the leader of blood. When qi moves, blood moves. When qi stagnates, blood stagnates, forming the syndrome of "qi stagnation and blood stasis". The subcutaneous adrenal injection model is a representative model. Xiao Chun et al. used surgical transplantation plus 1% adrenal subcutaneous injection (0.15m, once a week, for 4 consecutive weeks) to replicate the rat model of endometriosis with liver depression. This animal model has clinical symptoms related to liver depression and pathological changes of the adrenal glands. The cycle is short, but it requires high surgical skills and the animal survival rate is low.

[0016] ④ Polyinosinic-polycytidyl acid (Poly I: C) intraperitoneal injection

[0017] Clinical studies have found that after high-dose interferon alpha (IFN-a) is used to treat liver infection or some malignant tumors, up to 40%-50% of patients will experience symptoms of liver depression, such as depression or anxiety, silent loss of appetite, and abnormal behavior. PolyI:C is a double-stranded RNA polymerized by inosinic acid and cytidylic acid, which can induce human cells and animal cells to produce tumor necrosis factor-a (TNF-a). Huang Weiliang et al. used a high dose of PolyIC (3 mg / kg) to induce a mouse liver depression model. The results of behavioral tests showed that the activity of mice in the open field test (OFT) was significantly reduced, the sucrose consumption rate in the sucrose preference test (SPT) was significantly reduced, and the immobility time in the tail suspended test (TST) and forced swimming test (FST) was prolonged.

[0018] The above drug modeling methods simulate the manifestations of liver depression syndrome to a certain extent, but because the drugs have great side effects, affect more organs, and are quite different from the disease patterns of clinical liver depression patients, they are not the preferred modeling method for the animal model of liver depression syndrome in traditional Chinese medicine.

[0019] (3) Emotional stimulation modeling

[0020] The main cause of liver depression is emotional disorder, emotional injury, and emotional failure, which leads to liver failure to release qi and liver qi stagnation, resulting in liver depression. Therefore, many researchers have created the following animal models of liver depression syndrome, including tail clamping, anger fighting, neck shackle, and single cage feeding, and binding. Xu Huiren et al. used tail clamping stimulation to induce rats to fight, making the rats angry. After three days of continuous stimulation, the rats showed symptoms of liver depression, namely, weakened biting, body fatigue, and even loss of appetite, weight loss, and body mass. Chen Xiaoye et al. put a homemade neck shackle on the neck of the rat to restrict the rat's daily behaviors such as grooming and itching. After 7-10 days of modeling, the rats showed slow movements, dull and yellow fur, fatigue and less food, and even dark purple claws and dark scales on the tail. Qiao Mingqi and Lv Zhiping et al. used medical bandages to tie up the limbs of rats, restricting their movements and making it difficult for them to walk. After one week of restraint, the rats showed symptoms of liver qi stagnation: reduced activity, dull eyes, reduced diet, and avoidance of irritation. Peng Cheng et al. used the activity restriction and water immersion method to immerse the rats below the xiphoid process in water and restrict their movements so that they had to stand in the water, achieving the effect of modeling liver qi stagnation.

[0021] This modeling method is consistent with the pathological process of emotional disorder leading to liver failure to release, which makes the modeled animals angry. However, the current research is more about the liver qi stagnation syndrome, which is mainly manifested as emotional inhibition and depression rather than anger; and it is inconsistent with the clinical pathogenic mechanism of liver failure to release due to unsatisfactory wishes and unfulfilled desires.

[0022] (4) Orphanage or maternal separation (MS)

[0023] Animals that are used to being raised in groups are separated and raised alone. Animals that are raised alone for a long time will show symptoms of liver qi stagnation, such as depression, silence and unwillingness to eat. The MS model is a kind of early life stress. By repeatedly separating the young mice from their mothers, the physiological functions and behaviors of the young mice will undergo long-term changes. A few months after modeling, the young mice will show symptoms of liver qi stagnation, such as depression and melancholy. This model has a single method of prediction and is simple to operate. It is mainly used to study the effects of early stress on the physiological, pathological and behavioral changes of young mice in adulthood. However, there are certain shortcomings because there are few children with liver qi stagnation in clinical practice and solitary raising occupies a large number of places.

[0024] (5) Combining multiple modeling methods

[0025] In order to better achieve the liver depression model, two or more of the above modeling methods can be combined. Cui Xiangyang et al. used a liver depression rat model induced by tail clamping and adrenaline injection, and found that the blood estradiol (E2) and progesterone (P) of the model rats were significantly reduced, and prolactin (PRL) was significantly increased. Zhu Xiaoxia et al. used the CUMS combined with solitary raising method to replicate the liver depression rat model and explore the mechanism of action of Xiaoyaosan in improving liver depression. This modeling method is based on many years of research on liver depression. The drugs used for modeling are also some hormones that have a significant correlation with liver depression selected after multiple screenings and combined with some more mature modeling methods. However, whether the increase in these indicators can represent the essence of liver depression still requires a lot of experimental research. Summary of the invention

[0026] One of the purposes of the present invention is to provide a CRH PVN A method for constructing a neuron-activated liver depression type breast cancer mouse model, the method comprising the following steps:

[0027] (1) 140-160 nl of liquid containing rAAV-CRH-CRE-WPRE-hGH polyA and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry was injected into the PVN brain region of CRH-IRES-Cre mice, where the virus titers of rAAV-CRH-CRE-WPRE-hGH polyA and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry were both 2.4-2.6×10 12 pfu;

[0028] (2) 25-30 days after virus injection, all mice were implanted with breast cancer cells. CRH was obtained 38-42 days after breast cancer cell inoculation. PVN A mouse model of liver-depression breast cancer with neuronal activation.

[0029] Preferably, in step (1), the PVN brain region of mice is injected with 150 nl of liquid containing both rAAV-CRH-CRE-WPRE-hGHpolyA and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry.

[0030] More preferably, in step (1), the virus titers of rAAV-CRH-CRE-WPRE-hGHpolyA and chemical genetically activated virus rAAV-hM3D(Gq)-mCherry are both 2.5×10 12 pfu.

[0031] More preferably, 28 days after the virus injection in step (2), all mice are implanted with breast cancer cells.

[0032] More preferably, the specific process of implanting breast cancer cells in step (2) is as follows:

[0033] The breast cancer cell Py230 precipitate was suspended in PBS solution and then mixed with Matrigel to make a density of 6-8×10 6 The cell suspension of 100 cells / mL was inoculated in situ into the 4th papillary fat pad on the right side of the mouse, with 0.08-0.12 mL injected into each mouse.

[0034] More preferably, the density of breast cancer cells Py230 in the cell suspension in step (2) is 7×10 6 Pieces / mL.

[0035] More preferably, in step (2), each mouse is injected with 0.1 ml.

[0036] More preferably, in step (2), CRH can be obtained 40 days after breast cancer cells are inoculated. PVN A mouse model of liver-depression breast cancer with neuronal activation.

[0037] The second purpose of the present invention is to provide an application of the mouse model constructed by the above construction method in drug screening for liver depression type breast cancer.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The construction method of the present invention is simple to operate and has good repeatability, and can overcome the disadvantage of large individual differences in animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the central CRH in Example 1 PVN Liver depression phenotypes of neuron activation, including A. Experimental design diagram; B. Sugar water preference test results of each group of mice; C. Open field test results of each group of mice; D. Tail suspension test results of each group of mice, n=6 / group; Ctrl: control group; CRH+: central CRH PVN Neuron activation group; CRH+XYS: central CRH PVN Neuronal activation+Xiaoyaosan group; ** indicates P < 0.01.

[0041] Figure 2 is the central CRH in Example 1 PVNNeuron activation-induced breast cancer with liver depression, where A. tumor representative image; B. tumor growth curve of mice in each group; C. comparison of tumor weight of mice in each group; D. comparison of Ki67 expression in tumor tissue of mice in each group, n=6 / group; Ctrl: control group; CRH+: central CRH PVN Neuron activation group; CRH+XYS: central CRH PVN Neuron activation + Xiaoyaosan group; * indicates P < 0.05, ** indicates P < 0.01.

[0042] Figure 3 is the central CRH in Example 2 PVN Comparison of the effects of neurons and CUMS on the induction of liver depression phenotype, including A. Sugar water preference test of mice in each group; B. Open field test of mice in each group; C. Tail suspension test of mice in each group. n=6 / group; Ctrl: control group; CRH+: central CRH PVN Neuronal activation group; CUMS: emotional stress group; CUMS+CRH-: emotional stress + central CRH PVN Neuron inhibition group; * indicates P < 0.05, ** indicates P < 0.01.

[0043] Figure 4 is the central CRH in Example 2 PVN Results of tumor growth detection in mice regulated by neurons, including A. tumor representative image; B. tumor growth curve of mice in each group; C. comparison of tumor weight of mice in each group; D. comparison of Ki67 expression in tumor tissue of mice in each group, n=6 / group; Ctrl: control group; CRH+: central CRH PVN Neuronal activation group; CUMS: emotional stress group; CUMS+CRH-: emotional stress + central CRH PVN Neuron inhibition group; * indicates P < 0.05, ** indicates P < 0.01. DETAILED DESCRIPTION

[0044] Example 1

[0045] SPF clean grade CRH-IRES-Cre mice, female, 8 weeks old. After 1 week of adaptive feeding, the control group (Ctrl), central CRH PVN Neuronal activation group (CRH+) and central CRH PVN Neuronal activation + Xiaoyaosan group (CRH + XYS). Among them, 150nl of liquid containing a virus titer of 2.5×10 12pfu of rAAV-CRH-CRE-WPRE-hGH polyA and chemical genetics blank vector virus rAAV-DIO-mCherry; 150 nl of liquid was injected into the PVN brain area of ​​mice in the CRH+ group and CRH+XYS group, and the virus titer in the liquid was 2.5×10 12 pfu of rAAV-CRH-CRE-WPRE-hGH polyA and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry. Four weeks after virus injection, all mice were implanted with breast cancer cells, namely: breast cancer Py230 cells in the logarithmic growth phase were taken, and the cells were digested and centrifuged when the cells were in good adhesion and growth state. The cell pellet was suspended in PBS solution and mixed with Matrigel 1:1 to make a density of 7×10 6 A cell suspension of 500 g / mL was inoculated in situ into the fourth papillary fat pad on the right side of all mice, with 0.1 mL injected per mouse. After tumor implantation, all mice were treated with clozapine N-oxide (CNO). In addition, the mice in the CRH+XYS group were treated with Xiaoyaosan (6.5 g / kg), and the remaining mice were treated with the same volume of normal saline. The tumor diameter of the mice was measured every 5 days, and the tumor volume of the mice was calculated. Behavioral tests were performed 40 days after tumor implantation to evaluate the depressive state of mice in each group. Mouse breast cancer cells Py230 were purchased from the American Type Culture Collection (ATCC), and the specific scheme is as follows:

[0046] (1) Animal grouping:

[0047] SPF clean grade CRH-IRES-Cre mice, female, 8 weeks old. After 1 week of adaptive feeding, they were divided into groups using a simple random allocation method, with 6 mice in each group. They were control group (Ctrl), central CRH PVN Neuronal activation group (CRH+) and central CRH PVN Neuronal activation + Xiaoyaosan group (CRH+XYS).

[0048] (2) Stereotactic brain injection:

[0049] After anesthetizing the mouse with isoflurane, shave the hair on the head of the mouse. Fix the head of the mouse on a stereotaxic instrument, observe the position of the mouse's head from front to back and left to right to be horizontal, and observe that the mouse is in good respiratory condition. After the head is fixed, apply erythromycin eye ointment to the mouse's eyes, disinfect the head with iodine, cut the mouse's scalp, and then wipe the surface tissue of the skull with a cotton swab dipped in saline to fully expose the Bregma point. Level the head according to the Bregma point and Lambda point to ensure that the two points are arranged in a straight line and the depth difference between the two points does not exceed 0.05mm. Then measure the depth of the left (AP 0, ML-2.0) and right (AP 0, ML+2.0) points with the Bregma point as the center to ensure that the depth difference between the two points does not exceed 0.05mm. After the head is leveled, the microelectrode is moved above the target brain area, and the skull is gently ground at the PVN brain area (ML: ±0.3; AP: -0.6; DV: -4.5) using a skull drill. When the dura mater appears, the dura mater is carefully punctured with a medical syringe needle. 50nl of virus is sucked up using a microelectrode and injected into the PVN brain area at a rate of 10nl / min. After the virus is injected, the microelectrode is left in the PVN brain area for 15 minutes before being slowly pulled out to prevent the virus from leaking out of the brain area as the needle is pulled out. After the microelectrode is pulled out, the skull is wiped with saline, and the skin of the mouse head is sutured with 6-0 sutures and disinfected with iodine. After the operation, the mouse is placed on a heating pad and moved back to the cage after the mouse wakes up.

[0050] (3) Specific virus injection:

[0051] The PVN of mice in the Ctrl group was injected with 150 nl of sterile PBS containing a virus titer of 2.5 × 10 12 pfu of rAAV-CRH-CRE-WPRE-hGH polyA (BrainVTA, PT-0588) and chemical genetics blank vector virus rAAV-DIO-mCherry (BrainVTA, PT-0115); 150 nl of liquid was injected into the PVN brain region of mice in the CRH+ group and CRH+XYS group, and the virus titer was 2.5×10 12 pfu of rAAV-CRH-CRE-WPRE-hGH polyA (BrainVTA, PT-0588) and chemical genetically activated virus rAAV-hM3D(Gq)-mCherry (BrainVTA, PT-0019).

[0052] (4) Tumor cell implantation and subsequent intervention:

[0053] Four weeks after virus injection, all mice were implanted with breast cancer cells. Py230 breast cancer cells in the logarithmic growth phase were taken. When the cells were well attached to the wall and growing, the cells were digested and centrifuged. The cell pellet was suspended in PBS solution and mixed with Matrigel 1:1 to make a density of 7×10 6 / mL of cell suspension was inoculated in situ in the fourth papillary fat pad on the right side of all mice, with 0.1mL injected per mouse. After tumor implantation, all mice were treated with clozapine N-oxide (CNO), specifically 1.0mg / kg CNO was injected intraperitoneally every day, and 1.25mg / kg CNO was added to the drinking water of the mice. The use period was until the day of sampling (42 days after tumor inoculation). In addition, the mice in the CRH+XYS group were given Xiaoyaosan (6.5g / kg) for treatment, specifically gavage, once a day, and the use period was until the day of sampling (42 days after tumor inoculation). The remaining mice were treated with the same volume of normal saline. The tumor diameter of the mice was measured every 5 days, and the tumor volume of the mice was calculated. After 40 days, behavioral tests were performed to evaluate the depressive state of mice in each group. Mouse breast cancer cells Py230 were purchased from the American Type Culture Collection (ATCC).

[0054] (5) Assessment of Liver Depression Syndrome:

[0055] ① Sugar water preference experiment: 40 days after tumor implantation, the mice were first housed individually and trained to drink sucrose water within the next 48 hours. During this training phase, we provided two drinking options: one bottle contained 2% sucrose solution, and the other bottle was pure water. To ensure the fairness of the results, we exchanged the positions of the two bottles of liquid 24 hours after the start of training. After the training was completed, a 16-hour water deprivation treatment (but not fasting) was implemented, followed by a 4-hour test. After the first 2 hours of the test, the positions of the two bottles were exchanged again. At the end of the test, we recorded the consumption of sucrose water and pure water. Sugar water preference (%) = sugar water consumption ÷ (distilled water consumption + sugar water consumption) × 100%.

[0056] ② Open field experiment: 41 days after tumor implantation, the animals were placed in the center of the box, the experimental box specifications were 50cm×50cm×45cm. After 10 minutes of adaptation, the camera system was used to record the movement distance of the mice within 5 minutes. The behavior video was analyzed using Smart3.0 behavioral software.

[0057] ③ Tail suspension test: 42 days after tumor implantation, the mouse tail was fixed with tape and hung upside down for 6 minutes. The height between the tip of the mouse tail and the ground was about 30 cm. The total immobility time of the mouse within 6 minutes was recorded by a camera system (the mouse gave up struggling and was completely immobile). The behavior video was analyzed using Smart3.0 behavioral software.

[0058] The mice had liver depression, which was one or more of the following phenotypes:

[0059] ① The preference for sugar water (%) was significantly lower than that of the control group, P<0.05; the preference for sugar water increased after Xiaoyaosan intervention, P<0.05;

[0060] ② The movement distance in the open field test decreased, P<0.05; the movement distance increased after Xiaoyaosan intervention, P<0.05;

[0061] ③ The immobility time in the tail suspension test was prolonged, P<0.05; the immobility time was shortened after Xiaoyaosan intervention, P<0.05.

[0062] result:

[0063] (1) Central CRH PVN Neuronal induction of liver depression phenotype

[0064] In order to clarify the central CRH PVN To investigate the causal relationship between neurons and liver depression in mice, we used chemical genetics to activate central CRH in breast cancer mice. PVN Neurons were treated with Xiaoyaosan to observe the central CRH PVN Can neuronal activation induce liver depression phenotype? Figure 1 A). The results of the sucrose preference experiment showed that the preference for sucrose in the CRH+XYS group was significantly higher than that in the CRH+ group (P<0.01, Figure 1 B), and there was no significant difference with the Ctrl group ( Figure 1 B). The results of the open field test showed that the exploration distance of the mice in the CRH+XYS group to the surrounding environment was significantly higher than that in the CRH+ group (P<0.01, Figure 1 C), and there was no significant difference with the Ctrl group ( Figure 1 C). The results of the tail suspension test showed that the proportion of mice in the CRH+XYS group showing a state of "behavioral despair" when facing stress was significantly lower than that in the CRH+ group (P<0.01, Figure 1 D), and there was no significant difference with the Ctrl group ( Figure 1 D).

[0065] (2) Central CRH PVN Neuron-induced liver depression in breast cancer

[0066] By observing the growth volume of breast cancer in each group of mice, we found that the tumor volume of mice in the CRH+XYS group was significantly lower than that in the CRH+ group (P<0.05, Figure 2 AB), and there was no significant difference between the mice in the Ctrl group ( Figure 2 AB). After 40 days, the tumor-bearing mice were sampled and the tumor weight was measured. The results also showed that the tumor weight of the CRH+XYS group mice was significantly smaller than that of the CRH+ group mice (P<0.01, Figure 2 C), and there was no significant difference between the mice in the Ctrl group. Immunohistochemistry results also showed that the level of Ki-67 in tumor tissues of mice in the CRH+XYS group was significantly lower than that in the CRH+ group (P < 0.05, Figure 2 D), and there was no significant difference between the mice in the Ctrl group ( Figure 2 D) The above results show that Xiaoyaosan can relieve the central CRH PVN Neuronal activation induces the progression of tumor malignant growth.

[0067] Example 2

[0068] The process of CUMS liver depression syndrome modeling method is as follows:

[0069] SPF clean grade CRH-IRES-Cre mice, female, 8 weeks old. After 1 week of adaptive feeding, they were divided into groups using a simple random allocation method, with 6 mice in each group. They were control group (Ctrl), central CRH PVN Neuronal activation group (CRH+), emotional stress group (CUMS) and emotional stress + central CRH PVN Neuron inhibition group (CUMS+CRH-). 150 nl of liquid containing virus with a titer of 2.5×10 12 pfu of rAAV-CRH-CRE-WPRE-hGH polyA (BrainVTA, PT-0588) and chemical genetics blank vector virus rAAV-DIO-mCherry (BrainVTA, PT-0115); 150 nl of liquid containing both viruses with a titer of 2.5×10 12 pfu of rAAV-CRH-CRE-WPRE-hGH polyA (BrainVTA, PT-0588) and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry (BrainVTA, PT-0019); 150 nl of liquid containing both viruses with a titer of 2.5×10 12pfu of rAAV-CRH-CRE-WPRE-hGHpolyA (BrainVTA, PT-0588) and chemical genetics inhibition virus rAAV-hM4D(Gi)-mCherry (BrainVTA, PT-0020). Five days after virus injection, mice in the CUMS group and CUMS+CRH- group were intervened with the CUMS regimen. After 4 weeks of CUMS intervention, all mice were implanted with breast cancer cells, that is, breast cancer Py230 cells in the logarithmic growth phase were taken, and the cells were digested and centrifuged when the cells were in good adhesion and growth state. The cell pellet was suspended in PBS solution and mixed with Matrigel 1:1 to make a density of 7×10 6 A cell suspension of 100 / mL was inoculated in situ into the 4th nipple fat pad on the right side of all mice, with 0.1 mL injected per mouse. After tumor implantation, CUMS was continued to be used to intervene in the CUMS group and CUMS+CRH-group mice. At the same time, all mice were given CNO treatment. The tumor diameter of the mice was measured every 5 days, and the tumor volume of the mice was calculated. Behavioral tests were performed 40 days after tumor implantation to evaluate the depressive state of each group of mice. Mouse breast cancer cells Py230 were purchased from the American Type Culture Collection (ATCC).

[0070] The above-mentioned chronic unpredictable mild stress (CUMS) intervention process is as follows:

[0071] Each day, the rats were randomly given two different chronic unpredictable mild stress (CUMS) stimuli, including restraint, cage tilt, bedding removal, wet bedding, food and water deprivation, ice water swimming, stroboscopic light, and day and night reversal. The same stimulation could not be given on two consecutive days (see Table 3.1-1 for specific operating methods).

[0072] Table 3.1-1 CUMS specific operation methods

[0073]

[0074] (1) Central CRH PVN Comparison of the effects of neurons and CUMS on inducing liver depression phenotype

[0075] Chemical genetics to regulate central CRH PVN The mice with neuronal activity underwent depressive-like behavioral tests (including sugar preference test, open field test and tail suspension test 40 days after tumor implantation). The results showed that compared with the Ctrl group, the CRH+ group mice showed a reduced intake of sucrose (P < 0.01, Figure 3 A), exploration of the surrounding environment decreased (P < 0.01, Figure 3 B) and showed a state of "behavioral despair" when facing stress (P < 0.01, Figure 3 C). Compared with the CUMS group, there were no significant differences in the behavioral indicators of mice in the CRH+ group ( Figure 3 AC). On the other hand, compared with the CUMS group, the sucrose intake ratio of mice in the CUMS+CRH- group was higher (P<0.01, Figure 3 A) Exploration of the surrounding environment (P<0.05, Figure 3 B) increased, and the proportion of those who showed "behavioral despair" when facing pressure decreased (P < 0.01, Figure 3 C). Compared with the Ctrl group, there were no significant differences in the behavioral indicators of mice in the CUMS+CRH- group ( Figure 3 AC). The above results show that central CRH PVN Neuronal activation can induce liver depression phenotype in mice, and there is no significant difference between the CUMS group and the CUMS group; however, inhibition of central CRH PVN Neuronal function can alleviate the liver depression induced by CUMS. PVN The neuron is one of the key neurons that induce liver depression in mice.

[0076] (2) Central CRH PVN Comparison of the effects of neurons and CUMS in inducing breast cancer phenotypes with liver depression syndrome

[0077] Subsequently, we dynamically observed the growth of breast cancer in each group of mice. Around the 5th day after the inoculation of mouse-derived breast cancer cells, tumor nodules could be measured under the skin of each group of tumor-bearing mice. Tumor volume was measured every 5 days, and tumor growth curves were drawn based on the tumor volume of each breast cancer mouse at different times ( Figure 4 AB). The results showed that around 30 days after inoculation, the tumor volume of mice in the CRH+ group began to be significantly larger than that of the Ctrl group (P < 0.01, Table 1); on the contrary, around 30 days after inoculation, the tumor volume of mice in the CUMS+CRH- group began to be significantly smaller than that of the CUMS group (P < 0.05, Table 1). After 40 days, the tumor-bearing mice were sampled and the tumor weight was weighed. The results also showed that the tumor weight of mice in the CRH+ group was significantly larger than that of mice in the Ctrl group (P < 0.01, Figure 4 C), while the tumor weight of mice in the CUMS+CRH- group was significantly lower than that in the CUMS group (P < 0.01, Figure 4 C). In addition, immunohistochemistry results showed that the level of Ki-67 in tumor tissues of mice in the CRH+ group was significantly higher than that in the Ctrl group (P < 0.05, Figure 4 D), while the Ki-67 level in tumor tissues of mice in the CUMS+CRH- group was significantly lower than that in the CUMS group (P<0.05, Figure 4D) The above results show that central CRH PVN Neuronal activation can not only induce emotional stress in mice, but also promote the malignant growth of breast cancer; central CRH PVN Neuronal inhibition can mitigate the effects of emotional stress on breast cancer growth. PVN This provides strong evidence that neurons play an important role in the malignant growth of emotional stress-induced breast cancer.

[0078] Table 1 Central CRH PVN Effects of neurons on tumor volume in mice

[0079]

[0080]

[0081] Note: Shapiro-Wilk test, P < 0.05 on the 5th, 10th and 35th days, did not meet the normal distribution, and M (P 25 , P 75 ). Kruskal-Wallis test was used, and the difference between the groups on day 5 (H=2.397, P=0.494>0.05) was not statistically significant; the difference between the groups on day 10 (H=15.614, P=0.001<0.01) and day 35 (H=16.267, P=0.001<0.01) was statistically significant, and Bonferroni test was used for multiple comparisons. On days 15, 20, 25, 30, and 40, P>0.05, which met the normal distribution, and x±s was used for reporting. Variance homogeneity test, on days 15, 20, 30, and 40, P>0.05, which met the variance homogeneity; one-way analysis of variance was used, and Tukey test was used for multiple comparisons. Variance homogeneity test, on day 25, P < 0.05, did not meet the variance homogeneity; Welch's ANOVA analysis was used, and Dunnett T3 test was used for multiple comparisons. P1 represents comparison with the Ctrl group; P2 represents comparison with the CUMS group.

[0082] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A CRH PVN The method for constructing a neuron-activated liver depression type breast cancer mouse model is characterized by: The construction method comprises the following steps: (1) 140-160 nl of liquid containing rAAV-CRH-CRE-WPRE-hGHpolyA and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry was injected into the PVN brain region of CRH-IRES-Cre mice, where the virus titers of rAAV-CRH-CRE-WPRE-hGHpolyA and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry were both 2.4-2.6×10 12 pfu; (2) 25-30 days after virus injection, all mice were implanted with breast cancer cells. CRH was obtained 38-42 days after breast cancer cell inoculation. PVN A mouse model of liver-depression breast cancer with neuronal activation.

2. The construction method according to claim 1, characterized in that: In the step (1), 150 nl of liquid containing both rAAV-CRH-CRE-WPRE-hGHpolyA and chemical genetic activation virus rAAV-hM3D(Gq)-mCherry was injected into the PVN brain region of mice.

3. The construction method according to claim 2, characterized in that: The virus titers of rAAV-CRH-CRE-WPRE-hGHpolyA and chemical genetically activated virus rAAV-hM3D(Gq)-mCherry in step (1) were both 2.5×10 12 pfu.

4. The construction method according to claim 3, characterized in that: 28 days after the virus injection in step (2), all mice were implanted with breast cancer cells.

5. The construction method according to claim 4, characterized in that: The specific process of breast cancer cell implantation in step (2) is as follows: The breast cancer cell Py230 precipitate was suspended in PBS solution and then mixed with Matrigel to make a density of 6-8×10 6 The cell suspension of 100 cells / mL was inoculated in situ into the 4th papillary fat pad on the right side of the mouse, with 0.08-0.12 mL injected into each mouse.

6. The construction method according to claim 5, characterized in that: The density of breast cancer cells Py230 in the cell suspension in step (2) is 7×10 6 Pieces / mL.

7. The construction method according to claim 6, characterized in that: In the step (2), each mouse was injected with 0.1 ml.

8. The construction method according to claim 7, characterized in that: In step (2), CRH can be obtained 40 days after breast cancer cells are inoculated. PVN A mouse model of liver-depression breast cancer with neuronal activation.

9. Use of the mouse model constructed by the construction method according to any one of claims 1 to 8 in drug screening for liver depression type breast cancer.

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