Application of lipophagy receptor protein SPARTIN overexpressed adenovirus in preparation of medicine for preventing and treating postmenopausal osteoporosis

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of a lipophage receptor protein SPARTIN overexpressed adenovirus in preparation of a medicine for preventing and treating postmenopausal osteoporosis, after a lipophage receptor protein SPARTIN gene is inserted into a PLVX lentiviral vector to construct a transfection plasmid, the transfection plasmid and virus component expression plasmids PxpaX2 and PMD2G are co-transfected in an HEK 293T cell, after 48 hours, a virus supernatant is collected, and the recombinant adenovirus is obtained. The amino acid sequence of the SPARTIN protein gene is as shown in SEQ ID NO. 1. The invention also discloses a preparation method of the SPARTIN overexpressed adenovirus. Compared with the prior art, overexpression of the lipophage receptor protein SPARTIN can supplement expression of cell lipophage receptor protein, enhance marrow lipolysis and promote bone formation, so that the purpose of treating postmenopausal osteoporosis is achieved. And finally, compared with the existing lentivirus / adenovirus therapy, the BMAs targeting property is higher, and the BMAs targeting agent is safer and more effective.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, in particular to the use of adenovirus overexpressing lipophage receptor protein SPARTIN in the preparation of a drug for preventing and treating postmenopausal osteoporosis. Background Art

[0002] Osteoporosis is a common degenerative disease characterized by decreased bone mass and increased bone fragility, accompanied by a large accumulation of bone marrow adipocytes. As the disease progresses, the probability of patients suffering from brittle fractures increases significantly, seriously affecting the survival prognosis and quality of life of patients. Bone marrow adipocytes (BMAs) are a group of terminally differentiated cells originating from bone marrow mesenchymal stem cells (MSCs) and are widely distributed in the bone marrow. BMAs themselves have the ability to store and decompose triglycerides and release free fatty acids. Bone formation in the human body requires osteoblasts to consume a lot of energy for osteogenic differentiation and secretion of bone matrix. BMAs, as the main energy source of the microenvironment in the bone marrow, mediate the bone formation process. In the physiological process, bone formation and bone resorption are balanced to maintain bone mass at a certain level, but as women age, estrogen secretion decreases, bone marrow fat decomposition decreases, bone formation weakens, and osteoporosis follows.

[0003] Although good progress has been made in the diagnosis and treatment of osteoporosis in recent years, the exact cause of the disease has not yet been confirmed, and no radical cure has been found. As the disease progresses, the probability of brittle fractures in osteoporosis patients increases greatly, and they are more likely to fall, leading to brittle fractures, disability, and even death. All currently approved osteoporosis drugs have a large number of complications associated with their use, although they are rare, which limits patients' acceptance of them as osteoporosis therapies. At the same time, there are fewer drugs that promote bone formation, and there are many restrictions on their use. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides the use of adenovirus overexpressing lipophage receptor protein SPARTIN in the preparation of a drug for preventing and treating postmenopausal osteoporosis.

[0005] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0006] One of the technical schemes of the present invention is to provide an application of adenovirus overexpressing lipophage receptor protein SPARTIN in the preparation of a drug for preventing and treating postmenopausal osteoporosis. After the lipophage receptor protein SPARTIN gene is inserted into a PLVX lentiviral vector to construct a transfection plasmid, it is co-transfected with viral component expression plasmids PxpaX2 and PMD2G into HEK 293T cells. After 48 hours, the viral supernatant is collected to obtain an adenovirus overexpressing lipophage receptor protein SPARTIN. The amino acid sequence of the SPARTIN protein gene is shown in SEQID NO.1.

[0007] The second technical solution of the present invention is to provide a drug for preventing and treating postmenopausal osteoporosis, comprising an adenovirus overexpressing the lipophage receptor protein SPARTIN.

[0008] Furthermore, the drug for preventing and treating postmenopausal osteoporosis also includes a pharmaceutically acceptable carrier and / or excipient.

[0009] Compared with the existing technology, the overexpression of lipophagic receptor protein SPARTIN can supplement the expression of cellular lipophagic receptor protein, enhance bone marrow fat decomposition, and promote bone formation to achieve the purpose of treating postmenopausal osteoporosis. Finally, compared with the existing lentiviral / adenoviral therapy, this invention has a stronger targeting of BMAs, and is safer and more effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The TPM values ​​of the SPARTIN gene in the transcriptome sequencing of bone marrow adipocytes in the OVX group and the SHAM group established by C57BL / 6 mice: the OVX group was ovariectomized mice, and the SHAM group was a sham operation group.

[0011] Figure 2 The mRNA levels of SPARTIN after knockdown and overexpression of SPARTIN in adipogenically differentiated mesenchymal cells using adenovirus: Sh-NC group was the knockdown control group, Sh-SPARTIN was the SPARIN knockdown group, OE-NC group was the overexpression control group, and OE-SPARTIN was the SPARTIN overexpression group.

[0012] Figure 3 Western Blot protein level detection of SPARTIN expression in Sh-NC group, Sh-SPARTIN group, OE-NC group, and OE-SPARTIN group, as well as the corresponding quantitative analysis.

[0013] Figure 4 Oil Red O staining and Bodipy493 / 503 staining of Sh-NC group, Sh-SPARTIN group, OE-NC group, OE-SPARTIN group and the corresponding quantitative analysis results.

[0014] Figure 5 To detect and quantify the free fatty acids in the cell culture supernatant of the Sh-NC group, Sh-SPARTIN group, OE-NC group, and OE-SPARTIN group.

[0015] Figure 6 Transmission electron microscopy images of cells in the Sh-NC group, Sh-SPARTIN group, OE-NC group, and OE-SPARTIN group.

[0016] Figure 7 These are the fluorescence images of cells in the Sh-NC group, Sh-SPARTIN group, OE-NC group, and OE-SPARTIN group after lentivirus-overexpression of the PLIN1-mCherry-GFP lipophagy reporter molecule.

[0017] Figure 8 These are the immunofluorescence images of femoral bone marrow adipocytes in the SHAM group, OVX-rAAV8 group, and OVX-rAAV8-SPARTIN group of mice.

[0018] Fig. 9 These are the micro-CT longitudinal images and trabecular reconstruction images of the femurs of mice in the SHAM group, OVX-rAAV8-Control group, and OVX-rAAV8-SPARTIN group. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0020] The lipophagic receptor protein SPARTIN gene is a core element that mediates the binding of lipid droplets to autophagosomes in cells, and its amino acid sequence is shown in SEQ ID NO. 1. Guangzhou Aiji Biotechnology Co., Ltd. was commissioned to synthesize the lipophagic receptor protein SPARTIN gene fragment.

[0021] Guangzhou Aiji Biotechnology Co., Ltd. was commissioned to synthesize adenovirus for knocking down lipophagic receptor protein SPARTIN (SPARTINshRNA), and Shanghai Heyuan Biotechnology Co., Ltd. was commissioned to construct adenovirus for overexpressing lipophagic receptor protein SPARTIN. The adenovirus preparation process is as follows:

[0022] 1. Construction of transfection plasmid:

[0023] Select vector: PLVX is selected as the vector. PLVX is an adenovirus vector commonly used for gene overexpression or knockdown. The vector sequence is shown in SEQ ID NO.2.

[0024] Cloning of target gene: The target gene, lipophage receptor protein SPARTIN, is cloned into the PLVX vector by restriction endonuclease digestion and ligation. The cDNA of the SPARTIN gene is pre-synthesized and ligated according to the multiple cloning sites provided by the vector. For gene overexpression construction, the target gene should have a promoter to drive its expression.

[0025] 2. Prepare viral vectors and viral component expression plasmids:

[0026] Virus packaging plasmid: The components required for virus packaging need to be transfected into HEK 293T cells. This step uses two plasmids, PxpaX2 and PMD2g (sequences see SEQ ID NO.3 and SEQ ID NO.4):

[0027] PxpaX2: Provides the envelope and core proteins of adenovirus, allowing efficient expression of adenovirus in HEK 293T cells.

[0028] PMD2g: ​​Provides adenovirus packaging proteins and enzymes required for the viral life cycle.

[0029] 3. Co-transfection of HEK 293T cells:

[0030] Cell culture: HEK 293T cells were cultured in an incubator at 37°C and 5% CO2 until the cell density reached 60-70%.

[0031] Transfection plasmids: Use a liposome transfection reagent (such as Lipofectamine 2000 or JetPRIME) to co-transfect three plasmids:

[0032] PLVX-SPARTIN

[0033] PxpaX2 (virus-encapsulated plasmid)

[0034] PMD2g (viral packaging plasmid)

[0035] During transfection, adjust the amount of DNA according to the instructions of the transfection reagent and ensure that the ratio of each plasmid is appropriate to obtain efficient virus packaging.

[0036] 4. Collection of viral supernatant:

[0037] Cultivate and collect viral supernatant: 48 hours after transfection, wash HEK293T cells with sterile PBS or culture medium, collect the viral supernatant in the culture medium, and obtain adenovirus overexpressing lipophagy receptor protein SPARTIN. At this point, the viral particles have been released outside the cells and can be used to infect target cells.

[0038] Virus concentration: If necessary, the virus supernatant can be concentrated by centrifugation or ultracentrifugation to increase the virus titer.

[0039] 5. Virus infection target cells:

[0040] Infecting target cells: The collected viral supernatant (lipophagic receptor protein SPARTIN overexpressing adenovirus) is transduced into target cells.

[0041] Transfection conditions: Viruses can be infected into target cells using a variety of methods such as centrifugation, chemical transfection, or direct inoculation.

[0042] Evaluation of infection effect: 48 hours after infection, check the cell phenotype or marker expression, and use Western blot, RT-qPCR or immunofluorescence to verify whether the overexpression or knockdown of the lipophage receptor protein SPARTIN is effective.

[0043] 6. Subsequent analysis:

[0044] Confirmation of overexpression or knockdown: Analyze the protein level of SPARTIN by Western blot, confirm whether the transcription level of SPARTIN gene changes by RT-qPCR, and detect the corresponding functions, such as lipophagy.

[0045] 7. Storage and further use of viruses:

[0046] Virus storage: Virus supernatant can be stored at -80℃ for subsequent experimental use.

[0047] Virus titer determination: To quantify the infectivity of the virus, it may be necessary to determine the virus titer in cell culture (e.g., by qPCR or by infecting specific cells and measuring fluorescence or staining reactions).

[0048] Adipogenic differentiation of bone marrow mesenchymal stem cells was induced in vitro for 6 days using adipogenic induction medium, and adipocytes were transfected with lipophagic receptor protein SPARTIN knockdown adenovirus and lipophagic receptor protein SPARTIN overexpression adenovirus for 48 hours, respectively, as follows. 5 MSCs were plated in 12-well plates and induced into adipogenesis for 6 days using adipogenic induction medium. The cells were placed in a 1.5 ml centrifuge tube and centrifuged at 10,000 rpm for 10 seconds. The floating layer of the bone marrow centrifugation supernatant was carefully aspirated and sent to BGI Biotech Co., Ltd. for transcriptome detection. The results are as follows: Figure 1 As shown, Figure 1 The TPM values ​​of the SPARTIN gene in the transcriptome sequencing of bone marrow adipocytes in the OVX group and the SHAM group established by C57BL / 6 mice: the OVX group was ovariectomized mice, and the SHAM group was a sham operation group.

[0049] Western blot was used to detect the expression of SPARTIN protein in adipocytes. Figure 2 As shown, Figure 2 The mRNA level of SPARTIN was measured after knockdown and overexpression of SPARTIN in adipogenic mesenchymal cells using adenovirus: Sh-NC group was the knockdown control group, Sh-SPARTIN was the SPARIN knockdown group, OE-NC group was the overexpression control group, and OE-SPARTIN was the SPARTIN overexpression group; RT-qPCR was used to detect the expression of SPARTIN mRNA in adipocytes. Figure 3 As shown, Figure 3 Western Blot protein level detection of SPARTIN expression in Sh-NC group, Sh-SPARTIN group, OE-NC group, and OE-SPARTIN group, as well as corresponding quantitative analysis; Oil Red O and Bodipy staining kits were used to detect adipocyte lipid droplet levels. Figure 4 As shown, Figure 4 The results of Oil Red O staining and Bodipy493 / 503 staining and the corresponding quantitative analysis results of the Sh-NC group, Sh-SPARTIN group, OE-NC group, and OE-SPARTIN group were shown in the figure. The free fatty acid level in the cell supernatant was detected using a free fatty acid detection kit. Figure 5 As shown, Figure 5 To detect and quantify free fatty acids in the cell culture supernatants of the Sh-NC group, Sh-SPARTIN group, OE-NC group, and OE-SPARTIN group;

[0050] The cells were digested with trypsin and centrifuged. After being fixed with glutaraldehyde for 5 min, transmission electron microscopy was performed at Hubei Bios Biotechnology Co., Ltd. to observe the fusion level of intracellular lipid droplets and autophagosomes. Figure 6 As shown, Figure 6 Transmission electron microscopy images of cells in the Sh-NC group, Sh-SPARTIN group, OE-NC group, and OE-SPARTIN group.

[0051] 1x 10 5 MSCs were seeded in a 15 mm confocal dish. After 6 days of adipogenic induction using adipogenic induction medium, plin1-GFP-mCherry overexpression lentivirus was transfected for 48 hours. After 3 days, the lipophage reporter level was observed under a fluorescence microscope. Figure 7 shown.

[0052] Depend on Figure 1 It can be seen that transcriptome sequencing suggested that Spartin expression in bone marrow adipocytes of OVX mice was significantly downregulated compared with the TPM value of the SHAM group.

[0053] Depend on Figure 2 It can be seen that the Spartin mRNA level in the SPARTIN knockdown group was significantly downregulated compared with the Sh-NC group. The Spartin mRNA level in the SPARTIN overexpression group was significantly upregulated compared with the overexpression control group. This indicates that the SPARTIN mRNA knockdown and overexpression were successful.

[0054] Depend on Figure 3 It can be seen that the SPARTIN protein level in the SPARTIN knockdown group was significantly downregulated compared with the Sh-NC group. The SPARTIN protein level in the SPARTIN overexpression group was significantly upregulated compared with the overexpression control group. This indicates that SPARTIN knockdown and overexpression were successful at the protein level.

[0055] Depend on Figure 4 It can be seen that the Oil Red O staining and Bodipy493 / 503 staining of the SPARTIN knockdown group showed significant accumulation of intracellular lipid droplets compared with the Sh-NC group. The lipid droplets in the SPARTIN overexpression group were significantly reduced compared with the overexpression control group. This shows that the knockdown of SPARTIN leads to the accumulation of lipid droplets, while the overexpression leads to the elimination of lipid droplets.

[0056] Depend on Figure 5 It can be seen that the detection of free fatty acids in the cell culture supernatant of the SPARTIN knockdown group compared with the Sh-NC group indicated that the free fatty acids in the cell supernatant decreased. The free fatty acids in the cell supernatant of the SPARTIN overexpression group increased compared with the overexpression control group. This shows that the knockdown of SPARTIN leads to a decrease in the secretion of cellular free fatty acids, while the overexpression leads to an increase in the secretion of cellular free fatty acids.

[0057] Figure 6 The transmission electron microscope images of cells in the above Sh-NC group, Sh-SPARTIN group, OE-NC group, and OE-SPARTIN group. The 5um scale image reflects the overall fat content of the cell, and the 500nm scale image reflects the interaction between lipid droplets and autophagosomes in the subcellular structure of the cell. Compared with the Sh-NC group, the intracellular lipid droplets in the Sh-SPARTIN group increased significantly, and the contact between lipid droplets and autophagosomes decreased; compared with the OE-NC group, the intracellular lipid droplets in the OE-SPARTIN group decreased significantly, and the contact between lipid droplets and autophagosomes increased. This shows that the knockdown of SPARTIN leads to the weakening of lipophagic function, and the overexpression of SPARTIN leads to the enhancement of lipophagic function.

[0058] Figure 7The fluorescence images of the Sh-NC group, Sh-SPARTIN group, OE-NC group, and OE-SPARTIN group cells after lentiviral overexpression of PLIN1-mCherry-GFP lipophagy reporter molecules. The red light is mCherry, and the green light is GFP. Their co-localization level reflects the lipophagy level. Compared with the Sh-NC group, the yellow light after the fusion of red and green light in the cells of the Sh-SPARTIN group increased significantly, and the fusion protein did not enter the lysosome through the lipophagy pathway; compared with the OE-NC group, the green light in the cells of the OE-SPARTIN group decreased significantly, and the red light increased, indicating that the GFP protein underwent fluorescence quenching after entering the lysosome. This shows that the knockdown of SPARTIN leads to the weakening of lipophagy function, and the overexpression of SPARTIN leads to the enhancement of lipophagy function.

[0059] The above experiments prove that SPARTIN can promote lipophage in bone marrow adipocytes and promote the recovery of osteoporosis in postmenopausal osteoporotic mice.

[0060] As mentioned above, knocking down SPARTIN can lead to decreased lipophage in bone marrow adipocytes, reduced free fatty acid secretion, and decreased bone formation, which has been shown to be closely related to the occurrence and development of postmenopausal osteoporosis. Therefore, we supplemented BMAs SPARTIN expression by injecting SPARTIN-overexpressing adeno-associated virus into the tail vein of ovariectomized osteoporotic mice to enhance the lipophage of BMAs, thereby alleviating the progression of postmenopausal osteoporosis. The specific plan is as follows:

[0061] 1) Ovariectomized osteoporotic mice and construction of bone defect model: 2-month-old female C57BL / J mice were selected and underwent ovariectomy. The control group underwent sham surgery and the skin was incised and sutured.

[0062] 2) Grouping tail vein injection of adeno-associated virus: Ovariectomized osteoporotic mice were divided into two groups after construction, including the OVX-rAAV8-Control group (rAAV8 vector was used to target bone marrow adipocytes, and the rAAV8 vector was connected to the monomeric green or yellow fluorescent protein mNeonGreen, and the rAAV8 vector sequence is shown in SEQ ID NO.5) and the OVX-rAAV8-SPARTIN group (rAAV8 vector was used to target the lipophage receptor protein SPARTIN overexpression adenovirus to the bone marrow adipocytes, and the rAAV8 vector was connected to the monomeric green or yellow fluorescent protein mNeonGreen). Taking the modeling as the 0th week, the lipophage receptor protein SPARTIN overexpression adenovirus (4×10 11 GC; 2×10 13 GC / kg), injected once, and collected femur specimens of mice at 8 weeks; immunofluorescence results are shown in Figure 8 shown.

[0063] 3) Detection of the number of mouse trabeculae: At the 8th week, the mice were killed and the femurs were sampled. SPARTIN overexpression was detected by immunofluorescence, and the number of femoral trabeculae was detected by Micro-CT. BV / TV: relative bone volume; Tb.N: number of trabeculae; Tb.Sp: trabecular separation; Tb.Th: trabecular thickness; Ct.Th: cortical thickness. The results of Micro-CT trabeculae detection are as follows Fig. 9 shown.

[0064] Figure 8 These are immunofluorescence images of femoral bone marrow adipocytes in the SHAM, OVX-rAAV8-Control, and OVX-rAAV8-SPARTIN groups. Green fluorescence represents SPARTIN, red fluorescence represents PLIN1, and blue fluorescence represents DAPI cell nuclei. Green fluorescence is highly expressed in bone marrow adipocytes, indicating that adenovirus targets bone marrow adipocytes. Overexpression of SPARTIN in the OVX-rAAV8-SPARTIN group leads to a decrease in the number and volume of bone marrow adipocytes.

[0065] Fig. 9 The micro-CT longitudinal images and trabecular reconstruction images of the femur of mice in the SHAM group, OVX-rAAV8-Control group, and OVX-rAAV8-SPARTIN group are shown. The bar graph shows the BV / TV, BS / BV, Tb.T, Tb.N, and Tb.Sp trabecular analysis values. Compared with the SHAM group, the BV / TV, Tb.Th, and Tb.N of the OVX-rAAV8-Control group were significantly reduced, and the BS / BV and Tb.S were significantly increased, indicating that the OVX model was successfully established; compared with the OVX-rAAV8-Control group, the BV / TV and Tb.N of the OVX-rAAV8-SPARTIN group were significantly increased, and Tb.Sp was significantly decreased, but there was no significant difference in BS / BV and Tb.Th. This shows that the overexpression of SPARTIN in bone marrow adipocytes improved the bone loss of OVX to a certain extent and promoted bone formation.

[0066] The above data indicate that in the rAAV8-SPARTIN group, the expression of SPARTIN increased, the volume and number of adipocytes decreased, and the bone mass increased in the ovariectomized osteoporotic mice.

[0067] In summary, the adenovirus overexpressing the lipophage receptor protein SPARTIN of the present invention can be used to prepare drugs for preventing and treating postmenopausal osteoporosis. In some embodiments, the drugs for preventing and treating postmenopausal osteoporosis can also include pharmaceutically acceptable carriers and / or excipients.

[0068] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Use of adenovirus overexpressing lipophage receptor protein SPARTIN in the preparation of a drug for preventing and treating postmenopausal osteoporosis, characterized in that: The lipophage receptor protein SPARTIN gene was inserted into the PLVX adenovirus vector to construct a transfection plasmid, and then co-transfected with the virus component expression plasmids PxpaX2 and PMD2G into HEK 293T cells. After 48 hours, the viral supernatant was collected to obtain a lipophage receptor protein SPARTIN overexpression adenovirus. The amino acid sequence of the SPARTIN protein gene is shown in SEQ ID NO.

1.

2. A drug for preventing and treating postmenopausal osteoporosis, characterized in that: The invention comprises the adenovirus overexpressing the lipophage receptor protein SPARTIN as claimed in claim 1.

3. The drug for preventing and treating postmenopausal osteoporosis according to claim 2, characterized in that: Also included are pharmaceutically acceptable carriers and / or excipients.

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