A preparation method of a bladder cancer orthotopic tumor-bearing rat model with blood stasis and toxin

Abstract

The application discloses a preparation method of a bladder cancer orthotopic tumor-bearing rat model of blood stasis and toxin syndrome, and has the characteristics that 0.3ml / g of chloral hydrate is injected into the abdominal cavity of an SD rat to perform anesthesia, after the anesthesia takes effect, the rat is fixed in a supine position, the perineum of the rat is disinfected with iodophor, a catheter is slowly inserted into the bladder along the posterior wall of the urethral orifice of the rat to suck out urine, 0.2mL of MNU solution is injected into the bladder of the rat, after the injection is completed, the catheter is withdrawn, and the urethral orifice is gently clamped, the injection is repeated once every two weeks, and the injection is performed four times in total, 0.2ml of adrenaline hydrochloride is subcutaneously injected into the nape of the rat, the injection is performed once every two days, and the injection is continuously performed for eight weeks, the self-tumorigenic model of the bladder cancer model of the rat is constructed, compared with tumor-bearing and ectopic transplantation and other bladder cancer models, the model can better reflect the pathological process of the bladder cancer, the model construction simultaneously considers the syndrome characteristics of the blood stasis and toxin syndrome of traditional Chinese medicine, is beneficial to observation of the pathological process of the blood stasis and toxin type bladder cancer model in the later stage, and can be used for observation of curative effects and indexes after intervention of traditional Chinese medicines.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, specifically a method for preparing an orthotopic rat model of bladder cancer with blood stasis syndrome. Background Technology

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system, and it is prone to recurrence and has a poor prognosis. The etiology and pathogenesis of bladder cancer are not fully understood. It is currently known that bladder cancer is a multifactorial and complex pathological process, involving both external environmental factors and internal genetic factors. The more clearly defined risk factors are smoking and occupational factors such as long-term exposure to industrial chemicals. Currently, Western medicine primarily treats bladder cancer with surgery, followed by postoperative bladder instillation of medication to delay recurrence. Traditional Chinese medicine classifies bladder cancer as "hematuria" or "blood in urine," believing that toxins, blood stasis, and phlegm-dampness permeate the entire course of the disease. Traditional Chinese medicine treatment, based on syndrome differentiation and treatment, utilizes oral Chinese herbal medicine in addition to surgical treatment, achieving significant clinical efficacy in our previous clinical studies. To elucidate the specific mechanisms of action, suitable animal models are indispensable. Currently, there are many methods for preparing animal models of bladder cancer in pharmacological experimental research, but these methods are mostly guided by Western medicine, lacking traditional Chinese medicine syndrome models, which limits related research in traditional Chinese medicine. Therefore, it is necessary to establish more comprehensive animal models that combine "disease" and "symptom" for reference in new drug development and clinical trials.

[0003] Existing rat models of bladder cancer lack specific TCM syndrome classifications, making it impossible to reasonably interpret the mechanism of action of TCM under the "disease-syndrome-prescription" framework. Existing animal models of blood stasis and toxin syndrome are relatively simple, cover a limited number of diseases, have low tumor formation efficiency, short observation time, and lack animal models of bladder cancer. This results in a lack of reliable model basis for pharmacological and toxicological research on TCM treatment of bladder cancer. Furthermore, there is a lack of standard procedures and reference indicators for identifying "rat models of bladder cancer with blood stasis and toxin syndrome," and no standardized methods for creating rat models. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing an orthotopic rat model of bladder cancer with blood stasis syndrome, which can effectively solve the problems mentioned in the background art.

[0005] To address the aforementioned issues, our previous research revealed that postoperative oral administration of traditional Chinese medicine (TCM) with blood-activating and detoxifying properties combined with bladder instillation in bladder cancer patients with blood stasis and toxin syndrome effectively improved their quality of life and inhibited postoperative tumor recurrence. Furthermore, through related cell experiments, we discovered that TCM exerts a multi-pathway and multi-channel inhibitory effect on bladder cancer progression. However, the lack of a suitable animal model for bladder cancer with blood stasis and toxin syndrome hindered our further research as we could not simulate the metabolism and action of TCM. Therefore, we conducted extensive research and verification, and through numerous preliminary experiments and repeated model construction, we explored a method for stably culturing a rat model of bladder cancer with blood stasis and toxin syndrome. We also provided corresponding identification methods for the culturated rat model. This provides a research foundation for elucidating the possible mechanisms by which TCM exerts anti-tumor therapeutic effects in bladder cancer treatment and for the synergistic effect of TCM with chemotherapy in bladder cancer.

[0006] The technical solution adopted in this invention is as follows: A method for preparing an orthotopic rat model of bladder cancer with blood stasis syndrome. SD rats are anesthetized by intraperitoneal injection of 0.3 ml / g chloral hydrate. After the effect takes, the rats are fixed in a supine position. The perineum is disinfected with povidone-iodine. A urinary catheter is slowly inserted into the bladder through the external urethral orifice along the posterior wall and the urine is drained. 0.2 mL of MNU solution is instilled into the rat's bladder. After instillation, the catheter is removed, and the urethral orifice is gently clamped. Instillation is repeated once every 2 weeks for a total of 4 times. 0.2 ml of epinephrine hydrochloride is subcutaneously injected into the neck and back of the rats once every 2 days for 8 consecutive weeks.

[0007] As a further preferred embodiment of the present invention, the concentration of chloral hydrate is 10%.

[0008] As a further preferred embodiment of the present invention, the MNU solution is prepared by mixing N-methyl-N-nitrosourea and sodium citrate solution in a ratio of 1:5; the concentration of the MNU solution is 10 mg / ml.

[0009] As a further preferred embodiment of the present invention, the concentration of the epinephrine hydrochloride is 1%.

[0010] As a further preferred embodiment of the present invention, the SD rat is an SPF-grade male SD rat.

[0011] As a further preferred embodiment of the present invention, it also includes model identification: symptom assessment of TCM syndrome type, physicochemical index identification of TCM blood stasis syndrome type, and bladder cancer tissue identification of the model rats; the symptom assessment of the TCM syndrome type includes open field detection, tongue RGB detection, and mechanical pain detection; the physicochemical index identification of the TCM blood stasis syndrome type includes coagulation detection, and physicochemical index detection of vascular endothelial and platelet function.

[0012] As a further preferred embodiment of the present invention, the model rats exhibit greater anxiety symptoms than normal rats in the open field test; the R, G, and B values ​​in the tongue image of the model rats are simultaneously lower than those of normal rats in the tongue RGB test; and the pain threshold of the model rats is lower than that of normal rats in the mechanical pain test.

[0013] As a further preferred embodiment of the present invention, the coagulation test uses a coagulation method to detect the expression levels of thrombin time, prothrombin time, activated partial thromboplastin time, and fibrinogen (FIB); wherein the thrombin time, prothrombin time, and activated partial thromboplastin time of the model group rats are all shorter than those of normal rats; and the expression level of fibrinogen (FIB) of the model group rats is higher than that of normal rats.

[0014] As a further preferred embodiment of the present invention, the physicochemical indicators of vascular endothelial and platelet function are detected by ELISA to detect the expression levels of vascular endothelial growth factor, endothelin-1, nitric oxide, tissue plasminogen activator, plasminogen activator inhibitor-1, 6-ketoprostaglandin, and thromboxane B2; wherein the expression levels of vascular endothelial growth factor, nitric oxide, tissue plasminogen activator, and 6-ketoprostaglandin in the model rats are lower than those in normal rats; and the expression levels of endothelin-1, plasminogen activator inhibitor-1, and thromboxane B2 in the model rats are higher than those in normal rats.

[0015] Compared with the prior art, the present invention provides a method for preparing an orthotopic rat model of bladder cancer with blood stasis syndrome, which has the following beneficial effects:

[0016] This invention constructs a spontaneous tumor formation model of rat bladder cancer, which better reflects the pathogenesis and pathological process of bladder cancer compared to other bladder cancer models such as tumor-bearing and ectopic transplantation. The model construction also takes into account the symptom characteristics of "blood stasis and toxin syndrome" in traditional Chinese medicine, which is beneficial for later observation of the pathogenesis and pathological process of blood stasis and toxin type bladder cancer model, and can also be used to observe the efficacy and indicators after intervention with traditional Chinese medicine. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the model preparation and identification process of the present invention;

[0018] Figure 2 This is a schematic diagram of the infusion operation;

[0019] Figure 3 This is a diagram recording the results of an open field experiment.

[0020] Figure 4 This is a graph showing the mechanical pain threshold detection.

[0021] Figure 5 This is a diagram showing the growth of a rat bladder tumor.

[0022] Figure 6 Image of HE staining in rat bladder; Detailed Implementation

[0023] refer to Figure 1

[0024] 1. After rats were acclimatized for one week, a rat model of bladder cancer with blood stasis syndrome was obtained by bladder instillation of N-methyl-N-nitrosourea (MNU) sodium citrate solution combined with subcutaneous injection of adrenaline for eight consecutive weeks. (Continuous subcutaneous injection of adrenaline hydrochloride can induce blood stasis in rats, while MNU, as a chemical carcinogen for bladder cancer, combined with bladder instillation of sodium citrate, can spontaneously induce bladder cancer in rats. Furthermore, MNU, as a toxic substance, conforms to the pathological process of bladder cancer in rats with blood stasis syndrome.) The median survival time of this model rats was 24.7 weeks, which can fully cover the entire life cycle observation of tumor treatment.

[0025] 2. To validate the rat model of bladder cancer with blood stasis and toxin syndrome, we developed a systematic evaluation method: We used the open field test, RGB tongue test, and mechanical pain test to observe the physical characteristics and changes in the four diagnostic methods of traditional Chinese medicine (TCM) in the model rats, determining the TCM diagnostic features of the blood stasis and toxin syndrome in the model rats; and we used enzyme-linked immunosorbent assay (ELISA) to detect the levels of vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), nitric oxide (NO), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1) in rat serum. Platelet count, vascular endothelial cell function, and other indicators such as 6-keto-PEGF1a, thromboxane B2 (TXB2), were measured. Coagulation function indicators, including thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen (FIB), were assessed using the coagulation method to evaluate the microscopic physicochemical characterization of bladder cancer in rats with blood stasis syndrome. Rat MRI scans were used to detect in situ tumor formation in the bladder during the modeling period. Hematoxylin-eosin (HE) staining was used to observe the morphology of rat bladder cancer tissue.

[0026] As a specific embodiment of the present invention:

[0027] I. Steps in Model Preparation

[0028] (1) SPF-grade male SD rats were acclimatized for 1 week. During the experiment, they had free access to water and were fed solid food. The temperature was 22-24℃ and the humidity was 40%-60%.

[0029] (2) Preparation of MNU solution: Prepare a mixed solution (concentration: 10mg / ml) by mixing N-methyl-N-nitrosourea (MNU) and sodium citrate solution (pH = 6.0) in a water bath at 50℃.

[0030] (3) Spontaneous bladder tumor model: Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3 ml / g). After the anesthesia took effect, the rats were fixed in a supine position, and the perineum was disinfected with povidone-iodine. The external urethral orifice was identified and lifted. A self-made catheter dipped in paraffin oil was slowly inserted into the urethral orifice along the posterior wall. After inserting the self-made rat catheter about 3-4 cm, a significant resistance was felt, indicating that the catheter was in the bladder. Urine was drained, and each rat was injected with 0.2 ml of MNU solution for bladder irrigation. After irrigation, the catheter was removed, and the urethral orifice was gently clamped for 15-20 minutes. Irrigation was performed once every 2 weeks for 4 consecutive weeks, for a total of 8 weeks (e.g., if...). Figure 2 A capillary catheter was selected and marked at 4 cm. It was then connected to a skin test needle syringe to serve as a urinary catheter for rats. The catheter was inserted through the rat urethra and the solution was injected into it.

[0031] (4) Construction of the blood stasis and toxin syndrome model: 0.2 ml of 1% adrenaline hydrochloride (0.08 mg / kg body weight) was injected subcutaneously into the neck and back of rats, once every 2 days, for 8 consecutive weeks.

[0032] II. Model Authentication Methods

[0033] 1. Symptom assessment of TCM syndrome types:

[0034] (1) Open field test: A 100 cm × 100 cm open field box was prepared, and the time spent in the central area of ​​the rat within 3 minutes was recorded using VisuTrack video recording and analysis software to assess the rat's anxiety symptoms. The open field test was conducted once every 2 weeks for a total of 8 weeks (results are referenced). Figure 3 The model rats spent less time in the center, reflecting anxiety symptoms in the model rats (rats with bladder cancer due to blood stasis syndrome).

[0035] (2) RGB detection of tongue: A fixed camera was used to take photos of the tongue. Then, the "histogram" tool in Photoshop was used to calculate the red (R), green (G), and blue (B) component values ​​of the defined areas of the tongue (point 1 at the tip and point 2 at the root) and specific gray areas. The relative R, G, B values ​​were calculated as (128 / gray area R, G, B component values) × tongue R, G, B component values. The test was conducted once every 2 weeks for a total of 8 weeks. The statistical results are shown in the table below:

[0036]

[0037] The R, G, and B values ​​in the tongue appearance of the model rats were significantly lower than those of normal rats.

[0038] (3) Mechanical pain detection: The skin in the middle of the rat's foot was stimulated with the same force using a Von Frey nylon rope, and the rat's paw withdrawal response was observed. The above operation was repeated 3 times. Finally, the mean of 50% Von Frey response thresholds in the 3 times was calculated as the mechanical pain threshold (results are shown in the figure). Figure 4 Compared with normal rats, the pain threshold of the model rats was significantly reduced.

[0039] 2. Physicochemical indicators for identifying blood stasis syndrome in Traditional Chinese Medicine:

[0040] (1) Coagulation method for detecting four coagulation parameters: 2 mL of blood was collected from the jugular vein using a sodium citrate anticoagulation blood collection tube. Within 3 hours, the expression levels of thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen (FIB) were detected using the coagulation method (see table below for details).

[0041]

[0042] The results showed that the TT, PT, and APTT times of the model group rats were shorter than those of the normal rats, while the FIB time was higher than that of the normal rats.

[0043] (2) ELISA method for detecting relevant physicochemical indicators of vascular endothelial and platelet function: 3-5 mL of blood was collected from the jugular vein using a regular non-anticoagulated blood collection tube. The expression levels of vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), nitric oxide (NO), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), 6-keto-PEGF1a, and thromboxane B2 (TXB2) were detected by ELISA (see table below for details):

[0044]

[0045] The results showed that the expression levels of VEGF, NO, t-PA, and 6-keto-PEGF1a in the model rats were significantly lower than those in normal rats, while the levels of ET-1, TXB2, and PAI-1 were significantly higher than those in normal rats.

[0046] III. Bladder Cancer Tissue Identification

[0047] (1) MRI scan: A 3.0T magnetic resonance imaging system with a slice thickness of 1.0 mm was used. At the end of week 8 of the experiment, rats were anesthetized and abdominal MRI scans were performed to observe bladder tumor images (see details). Figure 5 The results showed that, using our construction method, bladder tumors in rats could be observed by MR examination at week 8.

[0048] (2) Bladder tissue morphology examination:

[0049] 1. Take rat bladder tissue and immerse fresh bladder tissue in 10% formalin solution for 72 hours. Rinse with running water to remove the fixative remaining in the tissue for 2 hours, and then dehydrate layer by layer with ethanol. Immerse the tissue block first in an equal volume mixture of anhydrous ethanol and xylene for 1 hour, and then immerse it in pure xylene for 1.5 hours. Place the specimen box containing the specimen in a 60°C incubator. Take small pieces of pure paraffin with a melting point of 52-56°C and gradually add them to the specimen box until the specimen is saturated with paraffin. Pour the molten paraffin and specimen into a pre-made small box while hot, adjust the position of the specimen, and allow the paraffin to gradually cool and solidify. Pour out the paraffin block and cut it into 4mm thick continuous paraffin strips. Place the cut strips on a glass slide and bake them. Dewax and rehydrate layer by layer with xylene and ethanol, and then stain with hematoxylin and eosin (HE staining).

[0050] 2. Place the prepared slides under a 400x optical microscope to observe the pathological changes in the rat bladder tissue structure and collect images. Assess the degree of bladder malignancy from three aspects: normal mucosa, atypical hyperplasia, and bladder cancer. Observe at least five fields of view for each slide (see details). Figure 6 ).

[0051] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention's specification and drawings under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A preparation method of a bladder cancer orthotopic tumor-bearing rat model with blood stasis and toxin, characterized in that, The SD rats are anesthetized by intraperitoneal injection of 0.3ml / g of 10% chloral hydrate, and after the anesthetic takes effect, the rats are fixed in a supine position, the perineum is disinfected with iodophor, and a catheter is slowly inserted into the bladder along the posterior wall of the urethral orifice to remove urine; 0.2mL of MNU solution is injected into the bladder of the rat, wherein the MNU solution is prepared by mixing N-methyl-N-nitroso urea and sodium citrate solution at a ratio of 1:5; the concentration of the MNU solution is 10mg / ml; After the perfusion is completed, the catheter is withdrawn, and the urethral orifice is gently clamped, and the perfusion is repeated once every 2 weeks, a total of 4 times; 0.2ml of 1% epinephrine hydrochloride is subcutaneously injected into the nape of the rat, and the injection is performed once every 2 days, and the injection is continuously performed for 8 weeks; the SD rats are SPF male SD rats.

2. The preparation method of the blood stasis and toxin accumulation syndrome bladder cancer orthotopic tumor-bearing rat model according to claim 1, characterized in that, Further comprising model identification: the model rats are subjected to syndrome evaluation of TCM syndrome type, physicochemical index identification of TCM stasis toxin syndrome type, and bladder cancer tissue identification; the syndrome evaluation of TCM syndrome type comprises open field test, tongue RGB detection, and mechanical pain test; the physicochemical index identification of TCM stasis toxin syndrome type comprises coagulation test, and physicochemical index detection of vascular endothelial and platelet function.

3. The preparation method of the blood stasis and toxin accumulation syndrome bladder cancer orthotopic tumor-bearing rat model according to claim 2, characterized in that, In the open field test, the anxiety symptoms of the model rats are greater than those of normal rats; in the tongue RGB detection, the R, G and B values in the tongue image of the model rats are simultaneously lower than those of normal rats; in the mechanical pain test, the pain threshold of the model rats is lower than that of normal rats.

4. The preparation method of the blood stasis and toxin accumulation syndrome bladder cancer orthotopic tumor-bearing rat model according to claim 2, characterized in that, In the coagulation test, the expression levels of thrombin time, prothrombin time, activated partial thromboplastin time and fibrinogen are detected; wherein the thrombin time, prothrombin time and activated partial thromboplastin time of the model rats are shorter than those of normal rats; the expression level of fibrinogen of the model rats is higher than that of normal rats.

5. The preparation method of the blood stasis and toxin accumulation syndrome bladder cancer orthotopic tumor-bearing rat model according to claim 2, characterized in that, In the physicochemical index detection of vascular endothelial and platelet function, the expression levels of vascular endothelial growth factor, endothelin-1, nitric oxide, tissue-type plasminogen activator, plasminogen activator inhibitor-1, 6-ketoprostaglandin and thromboxane B2 are detected by ELISA; wherein the expression levels of vascular endothelial growth factor, nitric oxide, tissue-type plasminogen activator and 6-ketoprostaglandin of the model rats are lower than those of normal rats; the expression levels of endothelin-1, plasminogen activator inhibitor-1 and thromboxane B2 of the model rats are higher than those of normal rats.

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