Application of specific drug-activated receptor and / or expression reagent thereof in preparation of reagent for improving parathyroid hormone level

Abstract

The invention relates to application of a specific drug-activated receptor and / or an expression reagent thereof in preparation of a reagent for improving the level of parathyroid hormone secreted by parathyroid gland. The invention finds that a common Gi / Gq signal channel exists in regulation of nerve cell excitability and synthesis and secretion of PTH by parathyroid gland main cells through chemical genetics, the parathyroid gland is injected with lentivirus to transfect the parathyroid gland main cells to express a specific drug-activated receptor, and PTH secretion of the parathyroid gland main cells can be regulated through a specific drug. And the possibility of regulating the PTH secretion level of the in-situ parathyroid gland tissue by clinically applying a chemical genetics regulation and control technology is explored.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine technology and relates to the application of a specific drug-activated receptor and / or an expression reagent thereof in the preparation of a reagent for increasing the level of parathyroid hormone. Background Art

[0002] Parathyroid hormone (PTH) is secreted by parathyroid chief cells and is an important hormone that maintains calcium-phosphorus balance in the body. It regulates calcium-phosphorus balance and bone metabolism balance by directly acting on bones and kidneys and indirectly acting on the intestines. Hypoparathyroidism is a group of clinical symptoms caused by insufficient secretion of PTH. Its clinical manifestations are mainly increased neuromuscular excitability, mental anxiety, irritability, hallucinations, personality changes and epilepsy, nutritional damage to ectodermal organs, ectopic calcification, etc. In addition, female patients with this disease are more likely to have complications during pregnancy, which may affect the patient's quality of life or endanger the patient's life. The more common hypoparathyroidism is postoperative hypoparathyroidism and idiopathic hypoparathyroidism. Postoperative hypoparathyroidism is mainly caused by accidental injury to the parathyroid gland during thyroid surgery or parathyroidectomy during hyperparathyroidism surgery, and occasionally by other neck surgeries such as neck radiotherapy that damage the parathyroid gland. The cause of idiopathic hypoparathyroidism is still unclear, but it may be related to autoimmunity.

[0003] At present, the main treatments for hypoparathyroidism include internal medicine drug therapy, PTH-related peptide replacement therapy and surgical transplantation therapy. Internal medicine drug therapy: such as calcium and vitamin D and its derivatives supplementation, which is the main treatment method at present. Although taking drugs can correct hypocalcemia, it is difficult to obtain a stable calcium-phosphorus balance, and there is a risk of increased urine calcium causing nephrocalcinosis and even renal damage; PTH-related peptide replacement therapy: such as using PTH (1-34) pump therapy to treat hypoparathyroidism, its replacement effect is closer to the physiological state, but the half-life of PTH after entering the circulation is only 5 minutes, how to effectively regulate it for 24 hours still needs further research. In addition, peptide hormones can only be injected, not taken orally, and are expensive and difficult to promote; surgical transplantation treatment, mainly including autologous transplantation and allogeneic transplantation. The advantage of autologous transplantation is that there is no rejection reaction, but because most patients with postoperative hypoparathyroidism do not show symptoms until 24 to 48 hours after surgery, it is difficult to immediately detect and implement autologous transplantation in time during surgery. In theory, allogeneic transplantation can function more quickly and effectively solve the problem of insufficient graft sources, but the immune rejection reaction of the transplant cannot be solved and there are certain ethical issues.

[0004] In summary, how to make the PTH in the body more in line with physiological needs, be able to carry out collective regulation, and play a role more safely, stably and long-lastingly is one of the urgent problems to be solved in the treatment of hypoparathyroidism. Summary of the invention

[0005] In order to solve the problems of poor specificity of the effects of existing hypoparathyroidism drugs and uncontrollable parathyroid hormone (PTH) secretion after parathyroid transplantation treatment, the present invention provides the use of specific drug-activated receptors and / or their expression reagents in the preparation of reagents for increasing the level of parathyroid hormone, and specifically regulates the function of parathyroid chief cells in vivo based on precise chemical genetics regulation technology to promote the release of PTH by chief cells, regulates the blood calcium concentration by targeted regulation of the PTH secretion level of parathyroid cells, and improves the related symptoms caused by hypoparathyroidism.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides the use of a specific drug-activated receptor and / or an expression reagent thereof in the preparation of a reagent for increasing the level of parathyroid hormone secreted by the parathyroid gland.

[0008] In the present invention, it is found that there is a common Gi / Gq signaling pathway in the chemical genetics regulation of nerve cell excitability and the synthesis and secretion of PTH by parathyroid chief cells. Further, based on the chemical genetics regulation technology, the parathyroid chief cells are transfected with lentivirus injected into the parathyroid glands to express specific drug-activated receptors, and the PTH secretion of the parathyroid chief cells is regulated by specific drugs.

[0009] Preferably, the specific drug activated receptor comprises hM3Dq (human modified M3 muscarinic receptor).

[0010] Preferably, the specific drug includes clozapine-N-oxide (CNO).

[0011] It is understood that based on the present invention, any agent that can express a specific drug-activated receptor in the parathyroid gland is suitable for use in the present invention.

[0012] Preferably, the expression reagent includes a reagent for expressing the specific drug-activated receptor in parathyroid cells, specifically including an expression vector containing a gene encoding the specific drug-activated receptor.

[0013] It is understood that hM3Dq refers to a modified form of the human M3 muscarinic (hM3) receptor, which can be activated by azidopine-N-oxide (CNO). hM3Dq and its encoding gene with similar functions known in the art are applicable to the present invention without special limitation.

[0014] Preferably, the expression vector comprises a lentiviral vector.

[0015] Preferably, the agent for increasing the level of parathyroid hormone secreted by the parathyroid gland includes a drug for treating a disease associated with parathyroid hormone.

[0016] In the present invention, parathyroid chief cells are transfected with parathyroid disease chronic virus to express artificially designed receptors, and the PTH secretion of parathyroid chief cells is regulated by a specific drug CNO. Specifically, the parathyroid tissue of rats is chemically and genetically regulated at the in vitro and in vivo levels to confirm the promoting effect of the regulation on PTH release, thereby verifying that the technology can be applied to disease treatment.

[0017] Preferably, the parathyroid hormone-related disease includes at least one of hypoparathyroidism, hypocalcemia, hyperphosphatemia or osteoporosis.

[0018] In a second aspect, the present invention provides a kit for increasing the level of parathyroid hormone secreted by the parathyroid gland, the kit comprising an expression vector containing a gene encoding hM3Dq and azidopine-N-oxide.

[0019] In a third aspect, the present invention provides use of the specific drug-activated receptor and / or its expression reagent described in the first aspect in increasing the level of parathyroid hormone secreted by the parathyroid gland for non-disease treatment purposes.

[0020] In the present invention, it is found that chemical genetics regulation technology can be used to regulate the PTH secretion level of parathyroid tissue in situ, which can be applied to the treatment of related diseases and can also be used for non-disease treatment purposes, such as in basic research related to PTH, constructing an experimental model of PTH abnormality, etc.

[0021] Preferably, the specific drug-activated receptor comprises hM3Dq.

[0022] In a fourth aspect, the present invention provides a method for increasing the level of parathyroid hormone secreted by the parathyroid gland for the purpose of non-disease treatment, the method comprising:

[0023] An expression vector containing the gene encoding hM3Dq was transduced into parathyroid tissue, and the parathyroid tissue was treated with azidopine-N-oxide.

[0024] Preferably, the method specifically comprises:

[0025] The lentivirus carrying the hM3Dq coding gene is injected into the parathyroid gland of the target body, and after the virus infects the parathyroid cells, azidopine-N-oxide is injected intraperitoneally.

[0026] Compared with the prior art, the present invention has at least the following beneficial effects:

[0027] The present invention expresses the DREADDSs gene vector on parathyroid chief cells, and functionally regulates the secretion of PTH of parathyroid chief cells in in vitro tissues and in vivo animals through a specific drug CNO. The phenomenon that CNO promotes the secretion of parathyroid chief tissue can be observed at the in vitro level; at the in vivo level, the PTH secretion level of the bone defect animal model increases after drug regulation, achieving bone repair of osteoporosis, and verifying the feasibility of clinically applying chemical genetics regulation technology to regulate the PTH secretion level of parathyroid tissue in situ. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the fluorescence imaging of rat parathyroid tissue in vitro;

[0029] Figure 2 This is the result of lentiviral transfection rate of rat parathyroid tissue in vitro;

[0030] Figure 3 This is the result of the secretion of PTH by rat parathyroid tissue in vitro;

[0031] Figure 4 This is the imaging result of rat femur;

[0032] Figure 5 This is the result of rat femoral bone density. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0034] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0035] In the present invention, the core of chemical genetics technology is that specific drugs activate specific artificially designed receptors (Designer receptors exclusively activated by designer drugs, DREADDSs), and reversibly remotely control the activity of cell groups and neural circuits. G protein-coupled receptors (GPCRs) are the largest known class of cell surface receptors, which specifically respond to synthetic small molecule ligands rather than natural ligands. DREADDs have almost no affinity for the endogenous ligand acetylcholine, but can be highly selectively activated by the biologically inert ligand azidopine-N-oxide.

[0036] Example 1

[0037] This example performs an in vitro tissue test.

[0038] Purchase lentivirus LV-CMV-hM3D(Gq)-EYFP carrying the hM3Dq encoding gene.

[0039] The rat parathyroid tissue was infected with lentivirus, and the specific process included:

[0040] Rat parathyroid glands were isolated in vitro under a sterile environment, and the virus was diluted 200 times with serum-free DMEM medium, and then cultured in vitro with virus-containing medium (the medium liquid surface can completely cover the tissue block). After the virus was incubated for 5 hours, 5 times the volume of serum-containing DMEM medium was added to continue the culture. After 6 days, green fluorescence was observed under a fluorescence microscope, indicating that the in vitro tissue had been successfully transfected with the virus. An empty shell lentivirus carrying only the green fluorescent protein gene EYFP without the hM3Dq coding gene was used for infection under the same conditions as the control group (EYFP).

[0041] CNO (MedChemExpress, HY-17366) was used to stimulate parathyroid tissue in vitro. The specific process includes:

[0042] CNO was dissolved in DMSO (dimethyl sulfoxide) to a 0.1 mg / μL CNO stock solution and stored frozen at -20°C. When used, the stock solution was diluted to 25 μM with serum-containing DMEM medium, and the original medium was directly replaced with the DMEM medium containing CNO for in vitro tissue culture.

[0043] The cultured parathyroid tissue was stained. The specific process included (1. The parathyroid gland was removed from the culture medium and placed in a PFA solution for 12 hours of tissue fixation; 2. After fixation, the tissue was removed and placed in a 30% sucrose solution for 18 hours of desugaring; 3. After the tissue was precipitated with sugar, it was removed and cryo-embedded in OCT; 4. After embedding, the tissue was made into 25 μm thick tissue sections; 5. The tissue sections were immunofluorescently stained with parathyroid hormone (PTH). The virus expression was monitored by observing the fluorescence expression, and the virus transfection efficiency was calculated by cell counting. The results are shown in Figure 2. Figure 1 and Figure 2 As shown, the green fluorescence labeling indicates that the cell has been successfully transfected with the lentivirus, and the red fluorescence indicates that the cell is a parathyroid chief cell that secretes PTH. It can be seen that the transfection rate of the two groups of lentiviruses on the parathyroid chief cells is about 75%. The culture medium was collected at various time points to detect the PTH concentration (PRE indicates before CNO stimulation, and the timing starts from 0h when CNO is added). The PTH concentration was detected using an Elisa kit (LSBio, LS-F5548). The test results are shown in Figure 3 As shown in the figure, CNO drug stimulation can effectively promote PTH release in a short period of time, and it returns to normal after 24 hours.

[0044] The above results show that the present invention successfully transfected and expressed hM3Dq in in vitro cultured parathyroid tissue using chronic diseases, and that CNO stimulation can promote the secretion of PTH by the parathyroid main tissue, indicating that the controllable characteristics of specific drug CNO injection can be used to accurately regulate the PTH synthesis and secretion of parathyroid cells.

[0045] Example 2

[0046] This example conducts a study on the tissue structure of the carrier

[0047] Eight osteoporotic rats (12 weeks, female, Guangdong Weitong Lihua) were randomly divided into two groups. Lentivirus carrying the hM3Dq coding gene was directly injected into the parathyroid gland of the rats as the experimental group, and empty lentivirus carrying the green fluorescent protein gene EYFP without the hM3Dq coding gene was used as the control group. 1 μL of virus was injected into each parathyroid gland. Three weeks after the virus injection, CNO (1 mg / kg) was injected intraperitoneally every two days for in vivo stimulation. After continuous injection for one and a half months, the femurs of the rats were collected for micro-CT analysis. The femur imaging results are shown in Figure 4 The bone mineral density (BMD) results are shown in Figure 5 As shown, compared with the control group, the osteoporosis symptoms of rats stimulated by CNO were improved after transfection with lentivirus carrying the hM3Dq encoding gene, indicating that chemical genetics regulation technology can be used to regulate the PTH secretion level of parathyroid tissue in situ and treat osteoporosis and other related diseases.

[0048] Example 3

[0049] This embodiment provides an agent for increasing the level of parathyroid hormone secreted by the parathyroid gland.

[0050] Based on the verification results of Example 1 and Example 2, reagents for increasing the level of parathyroid hormone secretion by the parathyroid gland can be further designed, such as using a lentivirus carrying the hM3Dq encoding gene and CNO and corresponding auxiliary reagents or consumables to prepare a kit, wherein the auxiliary reagents or consumables can be preservatives, buffers, packaging materials, and other substances that are conducive to the storage and transportation of lentiviruses and CNO.

[0051] In summary, the present invention expresses DREADDSs gene vectors on parathyroid chief cells, and functionally regulates the secretion of PTH of parathyroid chief cells in vitro and in vivo through a specific drug CNO. The phenomenon that CNO promotes the secretion of parathyroid chief tissue can be observed at the in vitro level; at the in vivo level, the PTH secretion level of the bone defect animal model increases after drug regulation, achieving bone repair of osteoporosis, exploring the possibility of clinically applying chemical genetics regulation technology to regulate the PTH secretion level of parathyroid tissue in situ, and providing new ideas for the treatment of PTH-related diseases.

[0052] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. Use of a specific drug-activated receptor and / or an agent for its expression in the preparation of an agent for increasing the level of parathyroid hormone secreted by the parathyroid gland.

2. The use according to claim 1, characterized in that: The specific drug activated receptors include hM3Dq.

3. The use according to claim 1 or 2, characterized in that: The expression reagent includes a reagent for expressing the specific drug-activated receptor in parathyroid cells, and specifically includes an expression vector containing a gene encoding the specific drug-activated receptor.

4. The use according to claim 3, characterized in that: The expression vector includes a lentiviral vector.

5. The use according to claim 1, characterized in that: The agents that increase the level of parathyroid hormone secreted by the parathyroid glands include drugs for treating diseases related to parathyroid hormone.

6. The use according to claim 5, characterized in that: The diseases related to parathyroid hormone include at least one of hypoparathyroidism, hypocalcemia, hyperphosphatemia or osteoporosis.

7. A kit for increasing the level of parathyroid hormone secretion by the parathyroid gland, characterized in that: The kit comprises an expression vector containing a gene encoding hM3Dq and azido-N-oxide.

8. Use of the specific drug-activated receptor and / or its expression reagent as claimed in claim 1 in increasing the level of parathyroid hormone secreted by the parathyroid gland for non-disease treatment purposes.

9. A method for increasing the level of parathyroid hormone secreted by the parathyroid gland for the purpose of non-disease treatment, characterized in that: The method comprises: An expression vector containing the gene encoding hM3Dq was transduced into parathyroid tissue, and the parathyroid tissue was treated with azidopine-N-oxide.

10. The method for increasing the level of parathyroid hormone secreted by the parathyroid gland according to claim 9, characterized in that: The method specifically comprises: The lentivirus carrying the hM3Dq coding gene is injected into the parathyroid gland of the target body, and after the virus infects the parathyroid cells, azidopine-N-oxide is injected intraperitoneally.

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