CN114027256B - Construction method and application of SD rat in-situ liver cancer model with high liver hardness background - Google Patents
Construction method and application of SD rat in-situ liver cancer model with high liver hardness background Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a construction method and application of an in-situ liver cancer model of a high liver hardness background SD rat, comprising the following steps: injecting carbon tetrachloride subcutaneously into the abdomen of SD rat to induce the formation of SD rat with high liver hardness, injecting low growth factor Matrigel mixed Buffalo liver cancer cell under the liver capsule, and combining short-term intramuscular injection of small dose glucocorticoid to form in-situ liver cancer model of SD rat with high liver hardness background after 12-14 days. The SD rat and the liver cancer cells of the rat adopted by the invention are easy to obtain and low in cost, have the advantages of high tumor formation rate, controllable tumor number and position, short tumor formation time, high metastasis and the like, effectively solve the technical difficulty and modeling cost problems of the establishment of the existing liver cancer animal model with the hardness background, better simulate the malignant pathological characteristics of a liver cancer patient with the high liver hardness background, and provide a practical and efficient novel liver cancer animal model for the mechanism analysis of gene functions, radiotherapy resistance, immunosuppression and the like under the hardness mechanical microenvironment.
Description
Technical Field
The invention relates to the technical field of animal models, in particular to a construction method and application of an in-situ liver cancer model of a high liver hardness background SD rat.
Background
Matrix hardening is mainly caused by excessive deposition and crosslinking of extracellular matrix proteins, and the mechanical hardness change often damages the balance of cell surface forces, promotes integrin aggregation and focal adhesion formation, and transmits exogenous hardness mechanical signals into intracellular to be converted into biochemical signals, thereby affecting and changing the cell biological phenotype and characteristics. Matrix hardening is a typical biomechanical phenotype of solid tumors, often accompanied by tumorigenesis and progression, and currently, matrix hardness evaluation (elastography) is clinically used as a method for predicting the development and prognosis of solid tumors such as breast cancer, hepatocellular carcinoma and the like, and research shows that matrix hardness increase in a tumor microenvironment can be involved in regulation and control of tumor invasion and metastasis as an initiating factor, and matrix hardness is an important biomechanical parameter of a cellular microenvironment, and can influence and participate in various pathophysiological processes in vivo such as tissue development, fibrosis, tumor metastasis and the like. However, the lack of an in-vivo and in-vitro experimental platform related to the hardness of an ideal matrix, particularly the lack of an animal model of an ideal tumor related to the hardness, still severely restricts the development of molecular mechanism researches related to the development of tumor development of matrix hardness regulation.
The Chinese patent application ZL201410025039.9 organically combines the adjustability of the hardness of the polyacrylamide gel with the matrix protein coating to prepare an in-vitro cell culture platform with different hardness substrates having biological interfaces and hardness changes, so as to better simulate the state of changing the hardness of normal liver tissues to cirrhosis; meanwhile, the Chinese patent invention ZL201310127532.7 establishes liver cancer models of rats with different liver matrix hardness backgrounds, realizes the combined modeling of the hardness backgrounds and the liver cancer, and better simulates and displays malignant pathological characteristics of the liver cancer under the influence of the liver matrix hardness, including histopathology, liver function, tumor proliferation, far-end lung metastasis, invasion metastasis associated genes and the like. The in vitro and in vivo research system is established, so that the problems that the liver matrix hardness background change participates in liver cancer occurrence progress research lacks an ideal experimental system, lacks an ideal animal model and the like are solved, a series of evidence that the matrix hardness change affects malignant biological characteristics of liver cancer cells is obtained, and the physical and mechanical characteristics participate in a liver cancer occurrence and development molecular mechanism are clarified. However, due to the purchase cost and source limitations of Buffalo rats, the creative ability to construct a practical and novel surrogate animal model is a fundamental problem that needs to be explored and solved.
Hepatocellular carcinoma (HCC) is characterized by other tumors, which usually undergoes chronic or acute liver injury (hepatitis) → liver self-repair→ liver fibrosis→ liver cirrhosis→ liver cancer, and liver cancer patients often have obvious necrotic inflammation, abnormal angiogenesis and extensive fibrosis of the liver and cirrhosis, and it is seen that changes in microenvironment during the course of hepatitis to cirrhosis play an important role in the development of liver cancer, and changes in liver perfusion, metabolic changes, etc. caused by the microenvironment changes during the course of hepatitis to cirrhosis also affect the effect of drug therapy. Clinical data shows that over 80% of liver cancer patients have liver fibrosis or cirrhosis background, and liver cancer patients with high cirrhosis background generally have poor prognosis. However, the mechanism of regulating liver cancer progression by the mechanical signals generated by liver fibrosis and cirrhosis is still poorly understood at present, and the lack of an ideal liver cancer animal model with liver cirrhosis background is an important cause. At present, an animal model of normal healthy liver in-situ transplanted tumor or subcutaneous tumor cannot simulate the influence of matrix hardness background change on malignant characteristics of liver cancer and the influence of matrix hardness microenvironment on drug reactivity. Therefore, whether a liver cancer animal model which is more practical and efficient and can simulate the liver matrix hardness microenvironment can be constructed is important to solving the research on the biomechanical signal action mechanism in the microenvironment.
At present, the common liver cancer animal model has the following advantages and disadvantages:
1) Model of induced liver cancer: refers to the induction of liver cancer by specific chemical inducers, including diethyl nitrosamine (DEN), aflatoxin (AF) and Dimethylaminoazobenzene (DAB), and the induction of liver fibrosis/liver cirrhosis of mice/rats by the chemical inducers, thereby inducing the liver cancer, which can induce HCC in immunocompetent mice/rats. The model is mainly used for researching etiology, genetics, pathogenesis and genetics of liver cancer and detecting cancer risks and chemotherapy of candidate medicaments. However, there are significant differences in the time of tumor induction and tumor size from mouse/rat strain to strain, and multiple tumor lesions often occur in this model, and the induction model is difficult to normalize. In addition, the model animal has low tumorigenesis rate and high mortality rate, and the animals have poor general conditions and short survival time after tumorigenesis, so that the model animal is difficult to withstand subsequent research.
2) Xenograft model: the model is a subcutaneous injection of human HCC cell lines in immunodeficient mice, such as athymic nude mice or Severe Combined Immunodeficiency (SCID) mice. The model is beneficial to research on treatment response and some research on malignant behaviors of cancer cells in the tumor progress process because the tumor is easy to obtain and can continuously monitor tumor growth. In addition, the human xenograft model (PDX) established by directly transplanting the tumor tissue of the patient to the immunodeficiency mouse reserves most of the characteristics of primary tumor in histopathology, molecular biology and gene level, has better clinical efficacy predictability, and is widely applied to new drug development at present, especially to the research of clinical test patient screening and predictive biomarkers of target drugs. However, xenograft model tumors, in addition to growing in situ, occasionally metastasize distally, fail to study spontaneous development of distant or local (intrahepatic) metastases, and are also more difficult to mimic the recurrence of tumor-matrix hardness environment interactions, while also being greatly limited by immune response dysfunction or loss.
3) Transgenic or genetically engineered liver cancer models (GEMs):
the transgenic liver cancer model refers to that the animal receives exogenous gene by using transgenic technology, and the gene is stably integrated in the genome of the chromosome and can be inherited to offspring. The conventional hepatitis B virus transgenic liver cancer model, monkey virus 40 (sv 40) transgenic animal liver cancer model, TGF transgenic mouse model and the like are mainly used for researching the functions of special genes and the interactions (such as oncogenes and cancer suppressor genes) of different genes in the development process of hepatocellular carcinoma, and can also be used for researching the relationship between the special genes and liver-specific oncogenes. However, transgene expression is in all liver cells, including tumor microenvironment cells, and such gene mutations are already present at the time of embryogenesis, potentially resulting in activation of some compensatory molecular pathways. In addition, the transgenic animal liver cancer model has high technical requirements and high price, limits the application of the model, and the model also cannot simulate the liver cirrhosis process and is difficult to be used for researching the interaction between tumor and matrix hardness.
The gene engineering liver cancer model comprises a c-Myc and Mtdh liver cell specific model and the like, in the GEMs of double overexpression of Mtdh and Myc, invasive HCC and lung metastasis occur in mice, the GEM model induces specific mutation to reproduce the expression of HCC, and the liver cancer model is mostly used for researching the occurrence of liver cancer, but the model also can not simulate the liver cancer occurrence process on the basis of liver cirrhosis.
The CRISPR-Cas model also belongs to a genetically engineered liver cancer model, and the model introduces a CRISPR-Cas system into a mouse fertilized egg by means of RNA injection to generate site-directed mutation in an embryo, namely, a CRISPR-Cas technology is used for targeting tumor suppressor genes (such as PTEN, p53 or Ctnnb1 oncogene). The technology can directly mutate tumor suppressor or oncogene so as to facilitate the study of the tumor suppressor or oncogene in the functional genomics of HCC, in addition, the method has no limitation on the genetic strain of mice, can delete large fragment genome DNA, and can achieve the effect of generating multiple gene mutations in the same mouse or rat by injecting RNA sequences for different genes simultaneously. The germ line transfer capability of the genetically modified animal constructed by the method is obviously higher than that of the germ line transfer capability of the conventional method, and the genetically modified animal constructed by the method is a new method for reliably, efficiently and quickly constructing a knockout animal model. However, the process of hepatitis, liver cirrhosis and liver cancer is avoided in the molding process, so that research on the interaction between tumor and matrix background environment cannot be performed, and the cost of the model is high.
4) Rabbit liver cancer animal model: belongs to a liver cancer model of liver allograft, adopts VX2 tumor cell strain as tumor source, and inoculates the liver of rabbit to prepare an in-situ liver cancer model. The model is the current largest animal vaccinatable liver tumor model; is a blood-rich tumor, and the blood supply artery is mainly a hepatic artery; pathological tumors are giant block solid tumors, grow in an infiltrative manner, have abundant blood supply and are similar to human giant block liver cancer; the model is simple to manufacture, the price is relatively low, the inoculation success rate is high, and the experimental period is short. Is suitable for local treatment such as intubation, liver tumor imaging diagnosis and experimental study of interventional radiotherapy. However, the rabbit is transplanted liver cancer, the liver cirrhosis background is lacking, the interaction between tumor and matrix background environment cannot be studied, and VX2 tumor is easy to transfer and spread.
In summary, although liver cancer research has been successful in establishing different types of mouse/rat liver cancer models, ideal liver cancer models with different liver hardness backgrounds are still lacking at present. In the prior art, although the in-situ liver cancer model of the different liver cirrhosis backgrounds Buffalo rats provides a reliable animal model for researching the malignant characteristics of the matrix hardness-regulated liver cancer, the in-situ liver cancer model is limited by the high cost, difficult acquisition, long molding cycle, relatively complex characteristics and the like of the Buffalo rats. Therefore, further development of more practical and efficient novel animal models has been aimed at solving the key problems that the hardness-related research is urgently needed to be explored and solved. In view of the poor tolerance of mice, the liver cirrhosis mice often die due to ascites, liver failure and the like or cannot tolerate the subsequent liver in-situ tumor transplantation, and the molding success rate is low. The method selects a common easily-obtained SD rat, adopts a combined modeling mode, firstly induces the SD rat with high liver hardness through carbon tetrachloride, then injects Matrigel mixed Buffalo rat liver cancer cells McA-RH7777 under the liver capsule of the SD rat with high liver hardness, and combines with immunosuppressant for short-term use to successfully obtain the SD rat liver cancer model with high liver hardness background. Compared with other liver cancer models of mice, SD rats and Buffalo rat liver cancer cells McA-RH7777 used by the model are easy to obtain and low in cost, and the model has the advantages of high tumorigenesis rate, controllable tumor number and position, short tumorigenesis time, high metastasis and the like, can better simulate malignant pathological features of liver cancer with high liver hardness background through detection and display, can analyze mechanism analysis such as gene function, radiotherapy resistance, immunosuppression and the like under the hardness microenvironment, and provides a practical and efficient liver cancer animal model for the research of regulating and controlling invasion and metastasis of liver cancer by matrix hardness mechanical signals.
Disclosure of Invention
The first aim of the invention is to provide a construction method of an in-situ liver cancer model of a SD rat with high liver hardness background, which better simulates and reproduces clinical pathology and biochemical characteristics of a liver cirrhosis patient, solves the problems of high cost, difficult acquisition, long modeling period, relatively complex characteristics and the like of a Buffalo rat, and forms a more practical and efficient novel substitute animal model. Therefore, the method selects the commonly available SD rat, adopts a combined modeling mode, firstly induces the SD rat with high liver hardness through carbon tetrachloride, then injects Matrigel mixed Buffalo rat hepatoma cells McA-RH7777 under the liver capsule of the SD rat with high liver hardness, and combines with small dose of glucocorticoid for short-term use to obtain the SD rat in-situ liver cancer model with high liver hardness background.
The second purpose of the invention is to provide the application of the SD rat in-situ liver cancer model with high liver hardness background, and the pathological characteristics of a liver cancer patient simulating clinical liver cirrhosis background in the rat body are used for researching the mechanism related to the invasion and metastasis of the matrix hardness-regulated liver cancer.
In order to achieve the first object, the present invention adopts the following technical scheme:
the invention provides a construction method of an in-situ liver cancer model of a high liver hardness background SD rat, which comprises the following steps:
(1) Injecting carbon tetrachloride subcutaneously in the abdomen of SD rat to induce the formation of SD rat with high liver hardness;
(2) Injecting low growth factor Matrigel mixed rat liver cancer cells under the liver capsule of the SD rat with high liver hardness in the step (1), and simultaneously combining with injection of small dose of glucocorticoid to form massive liver cancer after 12-14 days, so as to obtain an SD rat in-situ liver cancer model with high liver hardness background.
In a preferred embodiment, in the step (1), the SD rat is a 4-6 week old male SD rat.
In the step (1), carbon tetrachloride is injected subcutaneously into the abdomen of the SD rat for the first time according to the dosage of 3ml/kg, and then carbon tetrachloride-olive oil solution with the volume ratio of 1:1 is injected according to the dosage of 2ml/kg for 2 times per week for 12 weeks, so that the SD rat with high liver hardness is induced.
As a preferable technical scheme, in the step (2), the rat liver cancer cell is Buffalo rat liver cancer cell McA-RH7777, 1.6X10 is adopted 6 The volume ratio of the rat hepatoma cells/100 ul PBS suspension to low growth factor Matrigel is 1:1 are injected after being gently mixed.
In the preferred technical scheme, in the step (2), the glucocorticoid is dexamethasone, and the injection is carried out intramuscularly according to a dosage of 5mg/kg, and the injection of the liver cancer cells of the rat from the SD rat with high liver hardness is started two days before the injection of the liver cancer cells of the rat until the 2 nd day (d-2-d 2) after the operation, which is 5 days in total.
As an optimal technical scheme, the step of establishing the SD rat in-situ liver cancer model with high liver hardness background in the step (2) comprises the following steps:
(2-1) intramuscular injection of dexamethasone at a dose of 5mg/kg, starting from two days before the injection of liver cancer cells of the rat into the SD rat with high liver hardness until the 2 nd day (d-2-d 2) after operation, for 5 days;
(2-2) the high liver hardness SD rats were anesthetized with 2% sodium pentobarbital at a dose of 3ml/kg on the day of surgery, and the abdominal operation area was shaved;
(2-3) taking the above-mentioned high liver hardness SD rat abdomen median incision under aseptic super clean bench to open abdomen, and fully exposing liver, 1.6X10 6 The volume ratio of the rat hepatoma cells/100 ul PBS suspension to low growth factor Matrigel is 1:1 gently mixing to obtain a suspension, sucking the suspension by an insulin needle, slowly injecting the suspension into the lower part of a liver capsule, inclining the needle inserting direction from the lower part of a liver lobe to the upper direction, inserting the needle along the liver surface capsule, wherein the needle inserting depth is 0.5-1 cm, and after the injection is finished, a transparent nearly circular bulge appears under the liver capsule, then withdrawing the needle, pressing the injection by a dry cotton ball for a few seconds, preventing cells from flowing out from a puncture point, and observing the abdomen closing layer by layer after no bleeding of the liver;
(2-4) intramuscular injection of penicillin according to 8 ten thousand U/dose after operation, and forming massive liver cancer after 12-14 days from the beginning of the operation day to the next day (d 0-2) after operation.
According to the method, early pre-experiments are carried out, SD rats which are easy to obtain and good in tolerance are determined from a plurality of candidate animals (including SD rats, wistar rats and the like), meanwhile, early modeling experience (Chinese patent application No. ZL201410025039.9 and ZL 201310127532.7) is referred to, the liver cirrhosis induction of the SD rats is combined with the in-situ transplantation of liver tumors of the SD rats by adopting a combined modeling mode, meanwhile, small-dose glucocorticoids are used for temporarily forming transplantation tumors in situ on the SD rats by Buffalo liver cancer cells McA-RH7777, the intrahepatic tumor formation is accelerated by adopting a mode of mixing cells and Matrigel with liver capsule under-injection, meanwhile, intrahepatic diffusion of liver cancer cells caused by operation factors is avoided, an in-situ liver cancer model of the SD rats with high liver hardness background is successfully constructed, clinical pathology and biochemical characteristics of liver cirrhosis patients can be well simulated and reproduced, and the hepatoma formation and lung transfer capacity of the SD rat model with high liver hardness and mechanical induction molecules (Interin beta 1 or Piezo 1) are found to be obviously lower than that of the SD rat model with high liver hardness. In addition, compared with a normal liver hardness SD rat liver cancer model, the high liver hardness background SD rat in-situ liver cancer model has obvious resistance to radiotherapy, and the application proves that the increase of the matrix hardness can obviously enhance the malignant characteristics of liver cancer cells, thereby providing a novel animal model for discussing the related pathological mechanism of the matrix hardness for regulating and controlling the occurrence and development of liver cancer.
The invention also provides application of the SD rat in-situ liver cancer model with high liver hardness background in mechanism analysis research for simulating malignant pathological characteristics of a patient with liver cancer with high liver hardness background and gene functions, radiotherapy resistance and immunosuppression under a hardness mechanics microenvironment.
Compared with the prior art, the invention has the beneficial effects that:
(1) Compared with the existing rat liver cancer model, the SD rat and Buffalo rat liver cancer cells McA-RH7777 adopted by the SD rat in-situ liver cancer model with high liver hardness background are easy to obtain and low in cost, have the advantages of high tumor formation rate, controllable tumor number and position, short tumor formation time, high metastasis and the like, can better simulate malignant pathological characteristics of a liver cancer patient with high liver hardness background by detection and analysis of mechanisms such as gene function, radiotherapy resistance, immunosuppression and the like under a hardness mechanics microenvironment, greatly solve the technical difficulty and cost puzzling the establishment of the liver cancer animal model with hardness background, and provide a practical and efficient novel liver cancer animal model for the research of regulating and controlling invasion and metastasis of liver cancer by a matrix hardness mechanics signal.
(2) The tumor source adopted by the invention is Buffalo rat hepatoma cell McA-RH7777, the allotype transplantation tumor is formed in situ in SD rat by short-time small dose of glucocorticoid, and intrahepatic and pulmonary metastasis can occur, and the invention can be better used for researches on relevant mechanisms of local or remote metastasis of liver cancer, drug treatment response and the like.
(3) The SD rat in-situ liver cancer model with high liver hardness background constructed by the invention has the advantages of high tumor formation rate, controllable tumor quantity and position, short tumor formation time, high metastasis, lower cost, easy operation and the like, can be widely applied to related researches on liver cancer metastasis regulated by matrix hardness, and has better economic benefit and application value.
Drawings
FIG. 1 is a schematic flow chart of constructing an SD rat in situ liver cancer model with a high liver hardness background in the examples.
FIG. 2 is an in situ liver cancer model of a high liver hardness background SD rat constructed in the examples; wherein: (a) liver cirrhosis SD rat liver is generally observed; (B) Transparent bulge formed after liver cancer cells are injected under liver capsule of SD rat with high liver hardness; (C) The liver orthotopic tumor of the SD rat orthotopic liver cancer model with high liver hardness background is general.
FIG. 3 is an in situ liver cancer model validation of a high liver hardness background SD rat constructed in the examples; wherein: (A) HE staining of liver cancer tissue of SD rat with high liver hardness; (B) Liver function conditions of an in-situ liver cancer model of a SD rat with high liver hardness background; (C-D) immunohistochemical detection of liver LOX and COL1 expression in normal liver hardness SD rats and high liver hardness SD rats; (E-F) liver cirrhosis background SD rat orthotopic liver cancer model lung metastasis (< p <0.05, < p <0.01, < p <0.005, < p < 0.001).
FIG. 4 is a graph showing the effect of hardness mechanics induction molecules Integlin beta 1 and Piezo1 on liver cancer invasion and metastasis of SD rat in situ liver cancer model with high liver hardness background in the examples; wherein: (A) Rat liver cancer cell line with intelgrinβ1 knockdown (right) and Piezo1 knockdown (left); (B) Influence of Integlin beta 1 or Piezo1 expression on tumor proliferation of SD rats with high liver hardness background, and the tumors are in general shape; (C) tumor volume differences for each group of high liver hardness rats; (D) Influence of Integlin beta 1 or Piezo1 expression on tumor lung metastasis of SD rat with high liver hardness background; (E) Influence of Integlin beta 1 or Piezo1 expression on tumor proliferation of SD rats with normal liver hardness background, and the tumors are in general shape; (F) differences in tumor volume in normal liver hardness rats of each group; (G) differences in tumor weight in normal liver hardness rats of each group; (H) Effect of intel β1 or Piezo1 expression on tumor lung metastasis in SD rats with normal liver cirrhosis background (< p <0.05, < p <0.01, < p <0.005, < p < 0.001).
FIG. 5 is a graph showing that matrix hardness promotes liver cancer proliferation and radiotherapy resistance in hepatocellular carcinoma in the examples; wherein: (A) The effects of stromal hardness and radiation therapy on tumor proliferation, tumors are generally rough; (B) differences in tumor weight for each group of rats; (C) tumor volume differences for each group of rat tumors; (D) Different liver hardness background SD rats have tumor weight change conditions under radiotherapy stimulation; (E) Different liver hardness background SD rats showed change in tumor volume under radiotherapy stimulation (< 0.05, < p <0.01, < p <0.005, < p < 0.001).
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention fall within the protection scope of the present invention.
Example 1
The method for constructing the SD rat in-situ liver cancer model with high liver hardness background according to the method shown in figure 1 comprises the following steps:
first step, inducing SD rats with high liver hardness:
selecting 4-6 week old male SD rats, injecting carbon tetrachloride subcutaneously into the abdomen of the SD rats, and the dosage is as follows: pure carbon tetrachloride (3 ml/kg) was injected first, followed by carbon tetrachloride-olive oil solution (1:1), 2ml/kg, 2 times a week for 12 weeks, and rat liver conditions after induction are shown in FIG. 2A.
Secondly, establishing an in-situ liver cancer model of a SD rat with high liver hardness background:
and (3) cells: amplifying the rat liver cancer cells McA-RH 7777;
glucocorticoid pretreatment: intramuscular injection of dexamethasone, the dosage is 5mg/kg, 5 days is total, and rats begin 2 days before in-situ liver cancer implantation until postoperative day 2 (d-2-d 2);
preoperative preparation: the day of surgery was anesthetized with 2% sodium pentobarbital, dose: 3ml/kg. Shaving off the hair in the abdominal operation area;
subcapsular injection: the operation is performed under a sterile super clean bench, and the abdomen is cut off by taking the median incision of the abdomen, and the liver is fully exposed, 1.6X10 6 The cells (0.1 ml PBS suspension) were mixed with low growth factor Matrigel (1:1, gently mixed), the suspension was sucked with an insulin needle and slowly injected under the liver capsule, taking care that the needle insertion direction was inclined upward from the lower part of the liver lobe, inserting the needle along the liver surface capsule, the needle insertion depth was about 0.5-1 cm, and a transparent nearly circular protrusion was seen under the liver capsule after the injection (FIG. 2B). Along with itThe needle is withdrawn, the dry cotton ball is used for pressing and injecting for a few seconds, so that cells are prevented from flowing out from the puncture point, and the abdomen is closed layer by layer after the liver is observed to be free from bleeding; penicillin is intramuscular injected after operation, the dosage is 8 ten thousand U/patient, nodular liver cancer can be formed after 12-14 days from the beginning of the operation day to the next day (d 0-2) after the operation (figure 2C).
Example 2
The in-situ liver cancer model of the SD rat with high liver hardness background in the example 1 is verified, and the steps are as follows: the liver hardness of the pre-formed high liver hardness SD rats is evaluated, and 6 high liver hardness SD rats are randomly selected for liver elasticity ultrasonic detection of the liver hardness, so that the average liver hardness value of the high liver hardness SD rats is 17.03KPa (table 1), and the liver hardness value of the human liver cirrhosis can be better simulated.
Table 1: liver elasticity ultrasonic hardness value of SD rat in-situ liver cancer model with high liver hardness background
| |
1 | 2 | 3 | 4 | 5 | 6 | Mean |
| Stiffness value(KPa) | 15.45 | 19.68 | 15.02 | 17.67 | 17.85 | 16.54 | 17.03 |
Serum liver function indexes of an in-situ liver cancer model of a SD rat with high liver hardness background are detected, and liver enzymes (ALT and AST) of the SD rat with high liver hardness group are found to be increased, the albumin level is reduced, the albumin/globulin ratio is inverted (figure 3B), and the serum phenotype of the SD rat with high liver hardness background is similar to that of a clinical liver cirrhosis patient, so that the liver fibrosis/liver cirrhosis expression is indicated.
Immunohistochemical staining of liver tissues of SD rats with high liver hardness and SD rats with normal liver hardness, selecting two indexes of LOX and COL1 as judging standards, observing liver matrix hardness conditions of two groups of rats (figures 3C-D), finding that the staining intensity of the liver tissues of SD rats with high liver hardness is obviously stronger than that of liver tissues of normal control groups, indicating that the expression of the liver tissues of SD rats with high liver hardness is obviously increased, and prompting the increase of matrix hardness.
In situ liver cancer tissues of the SD rat with high liver hardness are subjected to HE staining, and the tumor morphology is observed, which indicates that the liver cancer constructed by the model is hepatocellular carcinoma (figure 3A).
The lung tissue of the high liver hardness SD rat in situ liver cancer model was taken for continuous section and HE staining, and the lung metastasis condition was observed, and the lung metastasis can be found in the rat model (FIG. 3E-F).
Example 3
In this embodiment, the in-vivo and in-vitro research experiments related to the influence of the matrix microenvironment on liver cancer are performed by the SD rat in-situ liver cancer model with high liver hardness background, and the steps are as follows:
the method is used for constructing a rat liver cirrhosis model, preparing liver cirrhosis rats, and dividing the liver cirrhosis rats into four groups: (1) liver cirrhosis rats, WT; (2) liver cirrhosis rats, NC; (3) liver cirrhosis rats, short 1; (4) liver cirrhosis rat, shPiezo1.
Culturing rat liver cancer cells McA-RH777 in culture flask, collecting cells in logarithmic proliferation phase, washing with PBS, counting with counting plate to obtain 1.6X10 6 100ul of Marigel is added into 100ul of the liver cancer cell/100 ul of cell suspension to be blown and evenly mixed, so that bubbles are avoided.
The cell implantation surgery under the liver capsule was performed on each group of rats according to the above method, and after 2 weeks, the rats were sacrificed, and the tumor size, weight, lung metastasis and the like were observed, measured and recorded. The influence of hardness mechanics induction molecules Intigrin beta 1 and Piezo1 on invasion and metastasis of liver cancer is evaluated, and the tumor formation and lung metastasis capacities of the high liver hardness SD rat liver cancer model which is down-regulated by the Intigrin beta 1 or Piezo1 are found to be obviously lower than those of the high liver hardness SD rat liver cancer model of a control group (figures 4A-D).
Rats of normal liver hardness (without carbon tetrachloride-induced cirrhosis) were prepared simultaneously and divided into four groups: (1) normal liver hardness rats, WT; (2) normal liver hardness rats, NC; (3) normal liver hardness rats, short 1; (4) normal liver hardness rat, shPiezo1. Repeating the steps according to the method, constructing a rat liver cancer model with different expression levels of normal liver hardness SDINTEGRIN beta 1 and Piezo1, observing, measuring and recording the tumor size, weight, lung metastasis and other conditions. The influence of Integlin beta 1 and Piezo1 on liver cancer invasion and metastasis in normal liver hardness SD rats is evaluated, and the tumor formation and lung metastasis capacities of the liver cancer models of the normal liver hardness SD rats which are downregulated by the Integlin beta 1 or Piezo1 are found to be obviously lower than those of the liver cancer models of the normal liver hardness SD rats of the control group. In addition, no lung metastasis occurred in the normal liver hardness SD rat liver cancer model, suggesting that the normal liver hardness SD rat liver cancer model had lower lung metastasis capacity than the high liver hardness SD rat liver cancer model (fig. 4E-H).
Example 4
Relevant in-vitro and in-vivo research experiments on the influence of liver matrix hardness on the radiotherapy resistance of liver cell liver cancer are carried out by using a SD rat in-situ liver cancer model with high liver hardness background in the embodiment 1, and the steps are as follows:
an SD rat liver cirrhosis model was constructed by the method of example 1, and a liver cirrhosis rat and a control normal rat were prepared. The four groups are: (1) normal rats, no irradiation (N); (2) normal rats, irradiated (n+ir); (3) a rat suffering from liver cirrhosis, which has been treated with a drug,non-irradiation (H); (4) liver cirrhosis rats were irradiated with (h+ir). McA-RH777 was cultured in flasks and cells were scored into two groups during log proliferation: control and irradiation groups. Sealing the culture flask, and horizontally transferring the cells to an accelerator machine room; the irradiation method comprises the following steps: 6MV-X, dose rate 300MV/min, frame angle 180 deg., 1.5cm compensation film under the lower part, once irradiating with 4Gy, collecting supernatant, PBS cleaning, adding pancreatin, stopping reaction with the collected culture solution, collecting cells, centrifuging at 1000rpm for 3min, adding PBS, re-suspending, counting with counting plate, and making into 1.6X10 6 100ul of Marigel is added into 100ul of cell suspension to be blown and mixed uniformly, so that bubbles are avoided.
The method is used for carrying out cell implantation operation under liver capsule on each group of rats, killing the rats after 2 weeks, observing, measuring and recording the tumor size, weight, lung metastasis and other conditions, evaluating the influence degree of liver matrix hardness on liver cell liver cancer radiotherapy resistance (figure 5), and finding that compared with a normal liver hardness SD rat liver cancer model, a high liver hardness background SD rat in-situ liver cancer model has obvious resistance to radiotherapy.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (1)
1. The application of the high liver hardness background SD rat in-situ liver cancer model in the mechanism analysis research of simulating the malignant pathological characteristics of a high liver hardness background liver cancer patient and gene function, radiotherapy resistance and immunosuppression under the hardness mechanics microenvironment is characterized in that the construction method of the high liver hardness background SD rat in-situ liver cancer model comprises the following steps:
(1) Injecting carbon tetrachloride subcutaneously in the abdomen of SD rat to induce the formation of SD rat with high liver hardness; wherein:
the SD rats are 4-6-week-old male SD rats, pure carbon tetrachloride is injected according to the dose of 3mL/kg for the first time, and then the injection volume ratio is 1 according to the dose of 2 mL/kg: 1, 2 times per week for 12 weeks, inducing the formation of high liver hardness SD rats;
(2) Injecting low growth factor Matrigel mixed rat liver cancer cells under the liver capsule of the SD rat with high liver hardness in the step (1), and simultaneously combining short intramuscular injection of small dose of glucocorticoid to form massive liver cancer after 12-14 days, so as to obtain an SD rat in-situ liver cancer model with high liver hardness background; wherein:
the rat liver cancer cell is Buffalo rat liver cancer cell McA-RH7777, 1.6X10 6 The volume ratio of the rat hepatoma cell/100 uLPBS suspension to the low growth factor Matrigel is 1:1, injecting after lightly mixing;
the glucocorticoid is dexamethasone, and is injected into muscle according to a dosage of 5mg/kg, and the liver cancer cells of the rats are injected from the SD rats with high liver hardness from two days before the injection to the 2 nd day after the operation, and the total time is 5 days;
the specific process is as follows: the SD rat with high liver hardness is anesthetized with 2% sodium pentobarbital according to the dosage of 3mL/kg on the same day, hair in the abdominal operation area is shaved, the abdomen incision of the SD rat with high liver hardness is opened under a sterile super clean bench, and the liver is fully exposed, 1.6X10 6
The volume ratio of the rat hepatoma cells/100 uL PBS suspension to the low growth factor Matrigel is 1:1 gently mixing to obtain a suspension, sucking the suspension by an insulin needle, slowly injecting the suspension into the lower part of a liver capsule, inclining the needle inserting direction from the lower part of a liver lobe to the upper direction, inserting the needle along the liver surface capsule, wherein the needle inserting depth is 0.5-1 cm, and after the injection is finished, observing that a transparent nearly circular bulge appears under the liver capsule, then withdrawing the needle, pressing the injection by a dry cotton ball for a few seconds, preventing cells from flowing out from an injection point, and observing that the liver is closed layer by layer after no bleeding; penicillin is injected into muscle after the operation according to the dose of 8 ten thousand U/dose, and massive liver cancer is formed after 12-14 days from the beginning of the operation day to the next day after the operation.
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