In-vitro rat pineal cell calcification model and construction method thereof

Abstract

The invention relates to the technical field of biomedicine, and discloses an in-vitro rat pineal cell calcification model and a construction method thereof.The construction method comprises the following steps that S1, experimental materials are prepared, specifically, a modeling mouse, experimental reagents and experimental apparatuses are prepared; s2, extracting and culturing pineal body cells: carrying out coating treatment on the pore plate, and cleaning the pore plate after coating treatment; preparing a pine body cell culture medium, taking out the pine body of the modeling mouse, putting the pine body into the culture medium, taking out the pine body of the modeling mouse from the culture medium, removing peripheral connective tissues of the pine body of the modeling mouse, extracting primary pine body cells of the pine body of the modeling mouse, purifying the primary pine body cells, and paving the purified primary pine body cells into a coated pore plate; s3, calcification induction: preparing a phosphate solution, adding the phosphate solution into the cell culture medium after the cells are adhered to the wall, and inducing calcification of the pineal cells; and S4, carrying out calcification characterization. By adding high-concentration inorganic phosphate into a pine body cell culture medium, an in-vivo calcification environment and a calcification molecular mechanism can be simulated.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, and in particular to an in vitro rat pinealocyte calcification model and a construction method thereof. Background Art

[0002] Alzheimer's disease is a chronic neurodegenerative disease characterized by memory loss and cognitive decline. From a diagnostic perspective, the diagnostic method of Alzheimer's disease has evolved from syndromic diagnosis based on symptoms and signs to the construction of biomarkers based on the pathological characteristics of the disease. The diagnosis of early Alzheimer's disease mainly relies on the patient's clinical manifestations, such as memory loss, aphasia, apraxia, agnosia and other global dementia symptoms. However, these symptoms are not specific to Alzheimer's disease and may also occur in other types of dementia, so the accuracy of the diagnosis is limited. With the development of medical imaging and biomarker technology, amyloid-β deposition, pathological tau protein and neurodegeneration have become important biomarkers for the diagnosis of Alzheimer's disease.

[0003] Among the many research directions of Alzheimer's disease, the association between pineal calcification and Alzheimer's disease has gradually attracted attention in recent years. The pineal gland, as an important structure in the human brain, is mainly responsible for secreting melatonin, regulating the biological clock and sleep cycle. Many studies have shown that abnormal function of the pineal gland is closely related to the occurrence and development of a variety of neurological diseases. Especially in patients with Alzheimer's disease, imaging examination techniques such as computed tomography (CT) have found that the level of pineal calcification is significantly higher than that of healthy people. Although studies have found that the level of pineal calcification in patients with Alzheimer's disease is significantly higher than that in healthy controls, the lack of an in vitro cell model means that the causal relationship between pineal calcification and the onset of Alzheimer's disease remains unclear. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides an in vitro rat pineal cell calcification model and a construction method thereof, which solves the problem that the basic mechanism research of pineal calcification in the prior art is blank.

[0005] In order to achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is: a method for constructing an in vitro rat pineal cell calcification model, comprising the following steps:

[0006] S1, experimental material preparation: prepare model mice, experimental reagents and experimental equipment;

[0007] S2, extraction and culture of pineal cells: the well plate is coated and then washed; pineal cell culture medium is prepared, the pineal gland of the modeling mouse is taken out and placed in the culture medium, the pineal gland of the modeling mouse is taken out from the culture medium and the surrounding connective tissue is removed, primary pineal cells of the pineal gland of the modeling mouse are extracted, and the primary pineal cells are purified and then plated into the coated well plate;

[0008] S3, calcification induction: phosphate solution was prepared during the attachment of primary pineal cells to the well plate. After the cells attached to the well plate, phosphate solution was added to the cell culture medium. The phosphate solution was added to the culture medium every other day to induce calcification of primary pineal cells.

[0009] S4, calcification characterization: The calcified pinealocytes were stained with Alizarin red, and the extracellular calcium knot content, ALPL activity and osteogenesis-related protein were detected. The model was established.

[0010] Furthermore, the method for constructing the above-mentioned in vitro rat pineal cell calcification model, S2 is specifically as follows:

[0011] S21, coated well plate: coated the well plate with poly-L-lysine solution and placed it in a cell culture incubator overnight;

[0012] S22, cleaning the well plate: The next day, the coated well plate was washed twice with PBS and placed back into the cell culture incubator for later use;

[0013] S23, preparing culture medium: preparing pinealocyte culture medium, the culture medium includes 25 mmol / L HEPES, 1% penicillin-streptomycin double antibody and DMEM high glucose medium containing 10% FBS;

[0014] S24, extracting the pineal gland of the modeling mouse: the modeling mouse was killed by cervical dislocation, and the mouse was disinfected with 75% ethanol, and the pineal gland was taken out and placed in the culture medium in S23;

[0015] S25, processing the pineal gland: on a clean bench, transfer the pineal gland to a 6 cm dish, wash it twice with D-Hanks buffer, and use straight and curved ophthalmic forceps to remove the connective tissue around the pineal gland;

[0016] S26, cell extraction: Primary pineal cells were extracted from the treated pineal gland using the papain dissociation system kit, and the cells were resuspended and transferred to a 25 cm 2 The cells were cultured in a culture flask in a cell culture incubator for 16 hours;

[0017] S27, purification and plating: prepare a culture medium containing 1.5 μg / mL Ara-C to inhibit glial cell proliferation; after 16 hours, aspirate the culture medium and obtain pinealocytes after centrifugation; resuspend the cells in culture medium containing Ara-C and plate them into coated well plates, with 100,000 cells per well.

[0018] Furthermore, the method for constructing the above-mentioned in vitro rat pineal cell calcification model, S3 is specifically as follows:

[0019] S31, preparing a phosphate solution: during the process of primary pinealocytes adhering to the well plate, a phosphate solution is prepared under a sterile environment. The phosphate solution is specifically a buffer solution composed of NaH2PO4 and Na2HPO4 with a molar concentration of 1 mol / L;

[0020] S32, calcification induction treatment: after the cells adhere to the wall, add the phosphate solution in S31 to the culture medium to make the final concentration in the culture medium reach 3 mmol / L;

[0021] S33, continuous induction: 100 μL of phosphate solution was added to the culture medium every other day to continuously induce the calcification process. The total induction time was 8 days.

[0022] Furthermore, in the above-mentioned method for constructing the in vitro rat pineal cell calcification model, the well plate is a 24-well plate.

[0023] Furthermore, in the above-mentioned method for constructing the in vitro rat pineal cell calcification model, the modeling mice are 6-8 week old male SD rats.

[0024] Furthermore, in the above-mentioned method for constructing the in vitro rat pineal cell calcification model, the centrifugation in S27 is specifically to place the culture medium in a centrifuge and run it at a relative centrifugal force of 300×g for 5 minutes.

[0025] An in vitro rat pineal cell calcification model is constructed according to the above construction method.

[0026] The beneficial effects of the present invention are as follows: in the in vitro calcification experiment, the present invention can effectively simulate the calcification environment of the pineal gland in vivo by adding high concentrations of inorganic phosphate to the pineal cell culture medium. High concentrations of inorganic phosphate can induce osteogenic transdifferentiation of pineal cells, causing them to exhibit obvious osteogenic characteristics, including increased expression of osteogenic-related genes and proteins, such as osteopontin and osteogenic transcription factor Runx2. By studying the expression changes of these genes and proteins, researchers can gain a deeper understanding of the function of pineal cells in the calcification process and their regulatory mechanisms.

[0027] The increase in alkaline phosphatase activity further promotes the calcification process. Alkaline phosphatase is an important osteogenic marker, and its activity is closely related to calcium deposition. In a high concentration of inorganic phosphate environment, the increased activity of alkaline phosphatase means that the cells are in an active state during the mineralization process. This phenomenon provides an important biochemical indicator for studying the dynamic process of calcification and helps to evaluate the impact of different conditions on calcification.

[0028] Calcium deposition can be visually observed and quantified using the Alizarin Red staining technique. Alizarin Red is a specific dye that can bind to calcium to form a red complex, thus clearly showing the area and extent of calcium deposition under a microscope. By quantitatively analyzing the amount of calcium deposition, researchers can evaluate the calcification capacity of pineal cells under different experimental conditions.

[0029] The present invention can simulate the in vivo calcification environment and the molecular mechanism of calcification by adding high concentration of inorganic phosphate to the pineal cell culture medium, and provides an important experimental platform and theoretical basis for understanding the biological characteristics of pineal cells, the osteogenic transdifferentiation process and the study of calcification-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure is a flow chart of the model building method of the present invention. DETAILED DESCRIPTION

[0031] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0032] like Figure 1 As shown, this embodiment provides a method for constructing an in vitro rat pineal cell calcification model, comprising the following steps:

[0033] S1, experimental material preparation. Fifteen male SD rats aged 6-8 weeks were selected as modeling mice to ensure that the rats were in good health to reduce experimental errors. At the same time, the reagents and equipment required for the experiment were prepared, including poly-L-lysine solution, PBS buffer, DMEM high-glucose medium, FBS (fetal bovine serum), penicillin-streptomycin double antibody, papain dissociation system kit, Ara-C (cytosine arabinoside), phosphate solution (NaH2PO4 and Na2HPO4), Alizarin red staining solution, as well as cell culture incubators, centrifuges, microscopes and other experimental equipment.

[0034] S2, extraction and culture of pineal cells: the well plate is coated and then washed; pineal cell culture medium is prepared, the pineal gland of the modeling mouse is taken out and placed in the culture medium, the pineal gland of the modeling mouse is taken out of the culture medium and the surrounding connective tissue is removed, the primary pineal cells of the pineal gland of the modeling mouse are extracted, the primary pineal cells are purified and then plated into the coated well plate.

[0035] S2 is specifically:

[0036] S21, coating the well plate: coating the well plate with poly-L-lysine solution, and placing it in a cell culture incubator overnight to ensure uniform coverage of the well plate surface. In this embodiment, the well plate is a 24-well plate.

[0037] S22, washing the well plate: The next day, wash the coated well plate twice with PBS to remove unbound poly-L-lysine, and then put it back into the cell culture incubator for use.

[0038] S23, prepare culture medium: prepare pinealocyte culture medium, the culture medium includes HEPES at a concentration of 25 mmol / L, penicillin-streptomycin at a concentration of 1%, and DMEM high-glucose culture medium containing 10% FBS, and ensure that parameters such as pH value and osmotic pressure of the culture medium are suitable for cell growth.

[0039] S24, extracting the pineal gland of the modeling mouse: the modeling mouse was killed by cervical dislocation, and the mouse was disinfected with 75% ethanol, and the pineal gland was removed under sterile conditions and placed in the culture medium in S23 to maintain the activity of the cells.

[0040] S25, Processing of pineal gland: On a clean bench, transfer the pineal gland to a 6 cm dish and wash it twice with D-Hanks buffer to remove blood and impurities on the surface. Use straight and curved ophthalmic forceps to remove the connective tissue around the pineal gland to obtain pure pineal tissue.

[0041] S26, cell extraction: The treated pineal glands were enzymatically treated using a papain dissociation system kit to isolate primary pineal cells. The enzymatically treated cell suspension was centrifuged, and the supernatant was discarded to obtain a cell pellet. The cells were resuspended in culture medium and transferred to a 25 cm 2 The cells were cultured in a culture flask in a cell culture incubator for 16 hours.

[0042] S27, purification and plating: prepare a culture medium containing 1.5 μg / mL Ara-C to inhibit the proliferation of glial cells; after 16 hours, aspirate the culture medium, place the culture medium in a centrifuge and run it at a relative centrifugal force of 300×g for 5 minutes, discard the supernatant, and the precipitate is the pinealocytes; resuspend the cells in culture medium containing Ara-C, and plate them into the coated well plates, with 100,000 cells per well.

[0043] S3, calcification induction: phosphate solution was prepared during the attachment of primary pineal cells to the well plate. After the cells attached to the well plate, phosphate solution was added to the cell culture medium. The phosphate solution was added to the culture medium every other day to induce calcification of primary pineal cells.

[0044] S3 is specifically:

[0045] S31, preparing phosphate solution: during the process of primary pinealocyte attachment, preparing a phosphate solution with a molar concentration of 1 mol / L in a sterile environment. In this embodiment, NaH2PO4 and Na2HPO4 are selected as the source of phosphate to ensure the stability and biocompatibility of the solution.

[0046] S32, calcification induction treatment: after the cells adhere to the wall, add the phosphate solution in S31 to the culture medium to make the final concentration in the culture medium reach 3 mmol / L;

[0047] S33, continuous induction: In order to maintain a stable calcification environment and promote the calcification process, 100 μL of phosphate solution was added to the culture medium every other day. After 8 days of continuous induction, obvious cell calcification can be observed.

[0048] S4, Calcification characterization: The calcified pinealocytes were stained with Alizarin Red to visually observe the calcification. At the same time, experiments such as extracellular calcium knot content detection, ALPL (alkaline phosphatase) activity detection and osteoblast-related protein detection were performed to quantitatively evaluate the degree of cell calcification and the expression of calcification-related indicators. Through the comprehensive use of these characterization methods, the reliability and effectiveness of the constructed in vitro rat pinealocyte calcification model can be comprehensively and accurately evaluated, and the model is established.

[0049] By constructing an in vitro rat pineal cell calcification model, researchers can simulate the process of pineal cell calcification in vivo, thereby deeply exploring the molecular mechanism and influencing factors of calcification at the cellular level. By intervening in the expression of target genes in pineal cells, researchers can observe the effects of different genes on the calcification process, thereby determining which genes play a key role in pineal calcification, which not only helps to reveal the gene regulatory network of the calcification process, but also provides potential targets for the development of gene therapy for pineal calcification, which has important application value.

Claims

1. A method for constructing an in vitro rat pinealocyte calcification model, characterized in that: The following steps are involved: S1, experimental material preparation: prepare model mice, experimental reagents and experimental equipment; S2, pineal cell extraction and culture: the well plate is coated and then washed; preparing a pineal cell culture medium, taking out the pineal gland of the modeling mouse and placing it in the culture medium, taking out the pineal gland of the modeling mouse from the culture medium and removing the surrounding connective tissue, extracting primary pineal cells from the pineal gland of the modeling mouse, purifying the primary pineal cells and then staking them into the coated well plate; S3, calcification induction: phosphate solution was prepared during the attachment of primary pineal cells to the well plate. After the cells attached to the well plate, phosphate solution was added to the cell culture medium. The phosphate solution was added to the culture medium every other day to induce calcification of primary pineal cells. S4, calcification characterization: The calcified pinealocytes were stained with Alizarin red, and the extracellular calcium knot content, ALPL activity and osteogenesis-related protein were detected. The model was established.

2. The method for constructing an in vitro rat pinealocyte calcification model according to claim 1, wherein: The S2 is specifically: S21, coated well plate: coated the well plate with poly-L-lysine solution and placed it in a cell culture incubator overnight; S22, cleaning the well plate: The next day, the coated well plate was washed twice with PBS and placed back into the cell culture incubator for later use; S23, preparing a culture medium: preparing a pinealocyte culture medium, the culture medium comprising 25 mmol / L HEPES, 1% penicillin-streptomycin double antibody, and DMEM high-glucose culture medium containing 10% FBS; S24, extracting the pineal gland of the modeling mouse: the modeling mouse was killed by cervical dislocation, and the mouse was disinfected with 75% ethanol, and the pineal gland was taken out and placed in the culture medium in S23; S25, processing the pineal gland: on a clean bench, transfer the pineal gland to a 6 cm dish, wash it twice with D-Hanks buffer, and use straight and curved ophthalmic forceps to remove the connective tissue around the pineal gland; S26, cell extraction: Primary pineal cells were extracted from the treated pineal gland using the papain dissociation system kit, and the cells were resuspended and transferred to a 25 cm 2 The cells were cultured in a culture flask in a cell culture incubator for 16 hours; S27, purification and plating: prepare medium containing 1.5 μg / mL Ara-C to inhibit glial cell proliferation; after 16 hours, aspirate the medium and obtain pinealocytes after centrifugation; resuspend the cells in medium containing Ara-C and plate them into coated well plates, 100,000 cells per well.

3. The method for constructing an in vitro rat pinealocyte calcification model according to claim 1, characterized in that: The S3 is specifically: S31, preparing a phosphate solution: during the process of primary pinealocytes adhering to the well plate, a phosphate solution is prepared under a sterile environment. The phosphate solution is specifically a buffer solution composed of NaH2PO4 and Na2HPO4 with a molar concentration of 1 mol / L; S32, calcification induction treatment: after the cells adhere to the wall, add the phosphate solution in S31 to the culture medium to make the final concentration in the culture medium reach 3 mmol / L; S33, continuous induction: 100 μL of phosphate solution was added to the culture medium every other day to continuously induce the calcification process. The total induction time was 8 days.

4. The method for constructing an in vitro rat pinealocyte calcification model according to claim 1, wherein: The well plate is a 24-well plate.

5. The method for constructing an in vitro rat pinealocyte calcification model according to claim 1, characterized in that: The modeling mice were 6-8 week old male SD rats.

6. The method for constructing an in vitro rat pinealocyte calcification model according to claim 2, characterized in that: The centrifugation in S27 specifically includes placing the culture medium in a centrifuge and running it at a relative centrifugal force of 300×g for 5 minutes.

7. An in vitro rat pinealocyte calcification model, characterized in that: Constructed according to the construction method according to any one of claims 1 to 6.

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