CN115725717A - Diagnosis and treatment target for osteoporosis and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and discloses a diagnosis and treatment target for osteoporosis and application thereof. The diagnosis and treatment target ALKBH5 for osteoporosis has a negative regulation and control effect on osteoclast differentiation of human osteoclast precursor cells, and the deficiency of ALKBH5 is an important reason for reducing the bone mass of osteoporosis patients. ALKBH5 levels are positively correlated with bone mass, with ALKBH5 positively regulating bone mass. The new medicine for treating osteoporosis is developed based on the new target spot, can relieve the condition of bone mass loss of osteoporosis patients, and has good effects of controlling progressive reduction of bone mass and preventing pathological fracture.

Description

Diagnosis and treatment target for osteoporosis and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a diagnosis and treatment target for osteoporosis and application thereof.

Background

Osteoporosis (OP) is the most common metabolic bone disease, is caused by imbalance of bone formation and bone resorption, and has the main pathological characteristics that proteins and other soluble bone calcium and minerals in bones are massively and chronically metabolized or lost, so that the bone mass is reduced, the bone brittleness is increased, and huge public health burden is brought to the society.

Osteoporosis includes three categories, primary, secondary and idiopathic. Caused by an imbalance between bone formation and resorption caused by various risk factors, such as aging, endocrine, metabolic and nutritional disorders, and by some drugs. At present, the drug therapy is the main means for treating osteoporosis, and the drugs clinically used for treating osteoporosis mainly comprise four types, wherein one type is a bone absorption inhibiting drug, such as bisphosphonates, sex estrogens, selective estrogen receptor modulators, nuclear factor-kappa B receptor activator ligand inhibitors, calcitonin and the like; the second is a bone formation promoting drug, such as parathyroid hormone analogue; the third is other mechanism medicine, such as active vitamin D and its analogue, anti-sclerostin monoclonal antibody, strontium salt, etc.; and the fourth is traditional Chinese medicine, such as drynaria total flavone, icariin, etc. However, the exact cause of osteoporosis still cannot be confirmed in the prior art, so that it is difficult to find a radical cure method. Although there are many clinically existing drugs for treating osteoporosis, these drugs have the disadvantages of poor curative effect and many side effects.

Therefore, there is a need to research and develop a novel osteoporosis therapeutic drug with better therapeutic effect, stronger specificity, fewer complications and higher safety.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a diagnosis and treatment target spot for osteoporosis and application thereof, so as to clinically control progressive reduction of bone mass and prevent pathological fracture.

In order to realize the purpose, the technical scheme adopted by the invention is as follows:

in a first aspect, the invention provides a diagnosis and treatment target for osteoporosis, wherein the target is ALKBH5, and the amino acid sequence of the target is shown as SEQ ID No. 1.

The ALKBH5 can be used as a new diagnosis target for osteoporosis. ALKBH5 has a negative regulation effect on osteoclast differentiation of human osteoclast precursor cells, the level of ALKBH5 is positively correlated with bone mass, ALKBH5 positively regulates bone mass, and ALKBH5 deficiency mediates enhancement of monocyte osteoclast differentiation to reduce bone mass of osteoporosis patients.

In a second aspect, the invention provides a small molecule drug for treating osteoporosis, which comprises ALKBH5 protein or ALKBH5 up-regulator.

The small molecular drug can relieve the condition of bone mass loss of osteoporosis patients, and has good effects of controlling progressive reduction of bone mass and preventing pathological fracture.

The preferred embodiment of the small molecule drug of the invention further comprises a pharmaceutically acceptable carrier.

Further, the carrier is at least one of diluent, adhesive, adsorbent, filler, disintegrant and additive.

In a third aspect, the invention provides a product for assessing osteoporosis, comprising a detection reagent for detecting the level of ALKBH5 expression in a biological sample.

The invention successfully separates and induces the osteoclast differentiation of osteoclast precursor cells in vitro cell research, and finds that ALKBH5 has negative regulation and control effect on the osteoclast differentiation of human osteoclast precursor cells by interfering the level of ALKBH 5. The occurrence and the development of osteoporosis can be evaluated by detecting the ALKBH5 expression level in a biological sample.

In a fourth aspect, the invention provides an application of the diagnosis and treatment target in preparing a kit for diagnosing osteoporosis.

As a preferred embodiment of the application, the kit is a kit for diagnosing osteoporosis by detecting ALKBH5 gene expression through RT-PCR, real-time quantitative PCR, immunodetection, in-situ hybridization, a chip or a high-throughput sequencing platform.

As a preferable embodiment of the application, the kit is a kit for diagnosing osteoporosis by detecting the expression of ALKBH5 protein through an immunoblotting method, an enzyme-linked immunosorbent assay, a chromatography method, a mass spectrometry method or a gel electrophoresis method.

In a fourth aspect, the diagnosis and treatment target and the small molecule drug are applied to preparation of a medicament for treating osteoporosis.

The new medicine for treating osteoporosis is developed based on the new target spot, can relieve the condition of bone mass loss of osteoporosis patients, and has good effects of controlling progressive reduction of bone mass and preventing pathological fracture.

Compared with the prior art, the invention has the beneficial effects that:

the invention successfully separates and induces the osteoclast differentiation of osteoclast precursor cells in vitro cell research, and finds that ALKBH5 has negative regulation and control effect on the osteoclast differentiation of human osteoclast precursor cells by interfering the level of ALKBH 5. Further constructing an ALKBH5 specific knockout mouse, and detecting the bone mass to find that the bone mass of the ALKBH5 specific knockout mouse is obviously lower than that of a wild type mouse, thereby indicating that the deficiency of ALKBH5 is an important reason for the bone mass reduction of the osteoporosis patient. And after ALKBH5 overexpression adeno-associated virus is injected into the tail vein of the ALKBH5 knockout mouse, the bone mass loss condition is obviously relieved, the bone mass of the ALKBH5 knockout mouse is close to that of a wild mouse, the ALKBH5 level and the bone mass are in positive correlation, and ALKBH5 positively regulates the bone mass. The occurrence of osteoporosis is related to the enhancement of osteoclast differentiation activity caused by the specific deletion of mouse osteoclast ALKBH5, the reduction of the bone mass of an osteoporosis patient is caused by the deletion of ALKBH5 mediated monocyte osteoclast differentiation enhancement, and ALKBH5 is an important target point for treating osteoporosis.

The new medicine for treating osteoporosis is developed based on the new target spot, can relieve the condition of bone mass loss of osteoporosis patients, and has good effects of controlling progressive reduction of bone mass and preventing pathological fracture.

Drawings

FIG. 1 is TRAP staining to detect osteoclastic differentiation in human monocytes;

FIG. 2 is TRAP staining to detect osteoclastic differentiation of mouse monocytes;

in both FIG. 1 and FIG. 2, the si group shows siRNA knockdown.

FIG. 3 shows the protein level of the human monocyte osteoclastic differentiation marker measured by Western blot.

Figure 4 is a schematic flow chart of an animal experiment scheme.

Fig. 5 is a mouse femur trabecula and cortex lycii Micro-CT scanning reconstruction diagram.

FIG. 6 is a statistical analysis chart of trabecular and cortical bone of mouse femur.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following examples. It should be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.

Example 1: cell experiments

Separating and inducing osteoclast differentiation of osteoclast precursor cells, and discovering that ALKBH5 has negative regulation effect on osteoclast differentiation of human osteoclast precursor cells by interfering the level of ALKBH 5.

The amino acid sequence of ALKBH5 is as follows:

MAAASGYTDLREKLKSMTSRDNYKAGSREAAAAAAAAVAAAAAAAA AAEPYPVSGAKRKYQEDSDPERSDYEEQQLQKEEEARKVKSGIRQMRLFSQ DECAKIEARIDEVVSRAEKGLYNEHTVDRAPLRNKYFFGEGYTYGAQLQKR GPGQERLYPPGDVDEIPEWVHQLVIQKLVEHRVIPEGFVNSAVINDYQPGGCIV SHVDPIHIFERPIVSVSFFSDSALCFGCKFQFKPIRVSEPVLSLPVRRGSVTVLSG YAADEITHCIRPQDIKERRAVIILRKTRLDAPRLETKSLSSSVLPPSYASDRLSG NNRDPALKPKRSHRKADPDAAHRPRILEMDKEENRRSVLLPTHRRRGSFSSE NYWRKSYESSEDCSEAAGSPARKVKMRRH。

the specific experiment is as follows:

(1) Isolation of monocytes

1) The mouse monocyte cell line RAW264.7 was purchased from the chinese institute cell bank.

2) Primary human-derived monocytes were extracted from healthy volunteers, and peripheral blood was collected from the forearms of healthy volunteers and about 50mL of peripheral blood was drawn with a syringe (previously wetted with a heparin sodium solution) after routine sterilization.

The operation is carried out 30 minutes after the ultraviolet disinfection biological safety cabinet is adopted in advance. After peripheral blood 1 was diluted with physiological saline, 20 15mL centrifuge tubes were taken, 5mL of Ficoll separation solution was added to each tube, 5mL of diluted blood was gently added to each 15mL centrifuge tube, and the mixture was centrifuged at 1500rpm for 20 min. The middle buffy coat layer was then aspirated using a pasteur pipette, supplemented with sterile PBS buffer to 10mL, placed in a centrifuge and centrifuged at 1800rpm for 8min. After centrifugation, the supernatant was decanted, 5mL of sterile PBS buffer was added to one centrifuge tube, the centrifuge tubes were washed one by one with 5 tubes to 1 tube, twice, and after washing, the tube was centrifuged at 1800rpm for 8min in a centrifuge. After centrifugation, the supernatant was decanted and resuspended in 1mL sterile PBS buffer. Then, CD14 monocytes were separated from Peripheral Blood Mononuclear Cells (PBMC) according to the CD14 magnetic bead sorting instruction, inoculated in a well plate with a-MEM medium at an appropriate density, and transferred with 5% CO ₂ Culturing in a constant temperature incubator at 37 ℃.

(2) Monocyte ALKBH5 level intervention

Human and murine monocytes were seeded in well plates, the original culture medium was discarded the next day after plating, the monocytes were transfected with ALKBH5 knockdown siRNA, and the induction medium was replaced 6h after transfection of SiRNA.

The human ALKBH5-1 siRNA sequence is as follows:

sense 5′-GACUGUGCUCAGUGGAUAUTT-3′，

antisense 5'-AUAUCCACUGAGCACAGUCTT-3′；

the human ALKBH5-2 siRNA sequence is as follows:

sense 5′-GCUAUGCUUCAGAUCGCCUTT-3′，

antisense 5'-AGGCGAUCUGAAGCAUAGCTT-3′；

the sequence of the mouse ALKBH5-1 siRNA is as follows:

sense 5′-GCUGCAAGUUCCAGUUCAATT-3′，

antisense 5'-UUGAACUGGAACUUGCAGCTT-3′；

the sequence of the mouse ALKBH5-2 siRNA is as follows:

sense 5′-GCGCGGUCAUCAACGACUATT-3′，

antisense 5'-UAGUCGUUGAUGACCGCGCTT-3′；

the control siRNA sequences were:

sense 5′-UUCUCCGAACGUGUCACGUTT-3′，

antisense 5′-ACGUGACACGUUCGGAGAATT-3′。

(3) Osteoclast differentiation induction by monocytes

The corresponding induction of differentiation was performed after knockdown of the cells. Adding osteoclast differentiation induction culture medium, and periodically replacing the culture medium for 1 time/3 days. The medium was replaced by discarding the old medium, washing with sterile PBS buffer 3 times, adding the new osteoclast induction medium, and adjusting to 5% CO ₂ Culturing in a constant temperature incubator at 37 ℃.

Wherein, the formula of the osteoclast differentiation induction culture medium is as follows: 10% FBS α -MEM, 25nM M-CSF, 50nM RNAKL, 100IU/mL penicillin-streptomycin.

(3) Monocyte osteoclast differentiation level assay

Osteoclast levels were measured at day 8 of osteoclast differentiation induction. Cells were examined for osteoclastic differentiation using TRAP staining. Protein levels of osteoclast differentiation related markers CTSK, TRAP and NFATc1 in human monocytes were detected by Western blot.

Tartrate-resistant acid phosphatase (TRAP) is a specific marker enzyme of osteoclast, TRAP staining detects the osteoclast differentiation of human monocytes as shown in fig. 1, and the TRAP staining intensity for characterizing the osteoclast differentiation degree after the ALKBH5 is knocked down in the human monocytes is enhanced, which indicates that ALKBH5 deficiency can effectively promote the osteoclast differentiation. The TRAP staining detection of mouse monocyte osteoclast differentiation is shown in FIG. 2, and in mouse monocytes, the TRAP staining intensity for representing osteoclast differentiation degree after ALKBH5 is knocked down is enhanced, which indicates that ALKBH5 deficiency can effectively promote osteoclast differentiation.

The result of detecting the protein level of the osteoclast differentiation related marker in the human monocyte by using Western blot is shown in figure 3, CTSK, TRAP and NFATc1 can enable cells to differentiate into osteoclasts and promote bone resorption, and ALKBH5 knock-down enables the protein level of CTSK, TRAP and NFATc1 in the human monocyte to be increased on average, and particularly the protein levels of CTSK and TRAP show extremely remarkable increase.

In human and murine monocytes, the protein level of the osteoclast differentiation marker was increased after the knockdown of ALKBH5, and the TRAP staining intensity was enhanced, indicating that ALKBH5 deletion can effectively promote osteoclast differentiation. Suggesting that ALKBH5 level is negatively related to an osteoclast differentiation marker, and ALKBH5 can negatively regulate osteoclast differentiation.

Example 2: animal experiments

The experimental protocol is shown in fig. 4, and specifically as follows:

(1) Construction of osteoporosis mouse animal model

Alkbh5 with C57BL/6 background ^fl/+ Mice were constructed by Cyagen (suzhou, china) using CRISPR/Cas-mediated genome engineering. The Alkbh5 gene (NCBI reference sequence: NM-172943, ensembl: ENSMUSG 00000042650) is located on mouse chromosome 11. Exon 1 was selected as the conditional knock-out region. To design targeting vectors, homology arms and conditional knock-out CKO regions were generated by PCR using BAC clone RP23-329 M3 as template. Cas9, guide RNA, and targeting vector were co-injected into fertilized eggs for CKO mouse production. Pups were genotyped by PCR and then sequenced.

CTSK-Cre transgenic mice were purchased from Jackson laboratories. CTSK-Cre mice and Alkbh5 ^fl/+ Mouse hybridization to obtain CTSK-Cre, alkbh5 ^fl/+ Mice were designated as heterozygous condition Alkbh5 knockout mice. By mating CTSK-Cre, alkbh5 ^fl/+ Male mouse and CTSK-Cre, alkbh5 ^fl/fl The female mice obtain CTSK-Cre and Alkbh5 ^fl/fl Mice were treated as homozygous conditional Alkbh5 knockout mice.

Genomic DNA extracted from mouse rat tail was subjected to PCR analysis to identify the genotype of transgenic mice.

Primers for the Alkbh5 knockout allele typing of floxed were as follows:

forward primer 5 'CAGGTTTGAAGTGGCCCATAGTAGTAGC-3',

reverse primer 5 'and GAGGCCAAGACAGGAGAATCAGAC 3'.

Primers for Cre transgenic genotyping were as follows:

forward primer 5 'GCTCTGATGTTGGCAAGGGGGGT-3',

reverse primer 5 'AACATCTTCTCAGGTTCTGCGGG-3'.

(2) Construction of ALKBH5 overexpression adeno-associated virus

The ALKBH5 overexpression adeno-associated virus is designed and constructed by Shanghai and Yuan Biotechnology Limited.

(3) Group administration

The wild type mouse and the ALKBH5 specific knockout mouse are divided into three groups respectively, including a wild type mouse group, an ALKBH5 knockout mouse group and an ALKBH5 knockout mouse + ALKBH5 overexpression group. After mouse tail identification to confirm mouse genotype, three groups were administered with the same volume of physiological saline and 5X 10 ¹¹ Empty vector and APPL1 overexpressing adenovirus tail vein injections, once every 2 weeks.

(4) Mouse femoral bone mass detection

Mice in three groups were sacrificed using cervical dislocation starting at 9 weeks after dosing. After sacrifice, the femur of the mouse is taken and subjected to Micro-CT scanning, and relevant indexes of the femur trabecula bone and the cortical bone, including bone volume fraction, cortical bone thickness, bone surface area/bone volume, trabecula bone thickness, trabecula bone number and trabecula bone separation degree, are analyzed.

No infection or abnormal death occurred in any of the three groups of mice. Comparison of reconstruction of Micro-CT scan of trabecular femur and cortical bone of mice in each group is shown in fig. 5, and statistical analysis of trabecular femur and cortical bone of mice is shown in fig. 6. Compared with wild mice, the bone number of the ALKBH5 specific knockout mouse is remarkably reduced, the bone surface area/the bone volume is remarkably increased, the trabecular thickness and the trabecular number are remarkably reduced, the trabecular separation degree is remarkably increased, the cortical thickness is reduced, the bone mass of the ALKBH5 specific knockout mouse is remarkably reduced, and the fact that an osteoporosis mouse model is successfully modeled is proved, and the ALKBH5 deficiency is an important cause of osteoporosis occurrence.

Compared with the ALKBH5 specific knockout mouse, after the overexpression adeno-associated virus is injected into the ALKBH5 knockout mouse through tail veins, the bone body integral number of the ALKBH5 overexpression specific knockout mouse is remarkably increased, the bone surface area/the bone body volume is remarkably reduced, the thickness of bone trabeculae and the number of the bone trabeculae are remarkably increased, the separation degree of the bone trabeculae is remarkably reduced, the thickness of cortical bone is increased, the bone mass reduction condition of the ALKBH5 specific knockout mouse is remarkably improved and is equivalent to the bone mass of a normal wild type mouse, namely the bone mass loss condition is remarkably relieved, the level of AKLBH5 is positively correlated with the bone mass, and the ALKBH5 positively regulates the bone mass.

The invention successfully separates and induces the osteoclast differentiation of osteoclast precursor cells in vitro cell research, and finds that ALKBH5 has negative regulation and control effect on the osteoclast differentiation of human osteoclast precursor cells by interfering the level of ALKBH 5. Further constructing an ALKBH5 specific knockout mouse, and detecting the bone mass to find that the bone mass of the ALKBH5 specific knockout mouse is obviously lower than that of a wild type mouse, thereby indicating that the deficiency of ALKBH5 is an important reason for the bone mass reduction of the osteoporosis patient. And after ALKBH5 overexpression adeno-associated virus is injected into the tail vein of the ALKBH5 knockout mouse, the bone mass loss condition is obviously relieved, the bone mass of the ALKBH5 knockout mouse is close to that of a wild mouse, the ALKBH5 level and the bone mass are in positive correlation, and ALKBH5 positively regulates the bone mass. The occurrence of osteoporosis is related to the enhancement of osteoclast differentiation activity caused by the specific deletion of mouse osteoclast ALKBH5, the reduction of the bone mass of an osteoporosis patient is caused by the deletion of ALKBH5 mediated monocyte osteoclast differentiation enhancement, and ALKBH5 is an important target point for treating osteoporosis.

The new medicine for treating osteoporosis is developed based on the new target spot, can relieve the condition of bone mass loss of osteoporosis patients, and has good effects of controlling progressive reduction of bone mass and preventing pathological fracture.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (9)

1. The diagnosis and treatment target for osteoporosis is characterized in that the target is ALKBH5, and the amino acid sequence of the target is shown as SEQ ID No. 1.

2. A micromolecular medicine for treating osteoporosis is characterized by comprising ALKBH5 protein or ALKBH5 up-regulating agent.

3. The small molecule drug of claim 2, further comprising a pharmaceutically acceptable carrier.

4. The small molecule drug according to claim 3, wherein the carrier is at least one of a diluent, a binder, an adsorbent, a filler, a disintegrant, and an additive.

5. A product for evaluating osteoporosis, comprising a detection reagent for detecting an expression level of akbh 5 in a biological sample.

6. The use of the diagnostic target of claim 1 for the preparation of a kit for the diagnosis of osteoporosis.

7. The use of claim 6, wherein the kit is a kit for diagnosing osteoporosis by detecting ALKBH5 gene expression through RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization, a chip or a high-throughput sequencing platform.

8. The use of claim 6, wherein the kit is a kit for diagnosing osteoporosis by detecting ALKBH5 protein expression by immunoblotting, ELISA, chromatography, mass spectrometry or gel electrophoresis.

9. The diagnostic target of claim 1 and the small molecule drug of any one of claims 2-4, for use in the preparation of a medicament for treating osteoporosis.

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