CN116726202A

CN116726202A - Application of reagent for increasing BMP7 expression quantity in preparation of medicine for treating stress cognitive impairment

Info

Publication number: CN116726202A
Application number: CN202310647541.2A
Authority: CN
Inventors: 钱令嘉; 谢方; 赵云; 王雪; 孙兆炜; 李峰
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2023-06-02
Filing date: 2023-06-02
Publication date: 2023-09-12

Abstract

The invention discloses an application of a reagent for increasing the expression level of BMP7 in preparing a medicament for treating stress cognitive impairment, and relates to the technical field of biological medicines. The invention prepares adeno-associated virus containing bone morphogenetic protein 7 (BMP 7) expression sequence, the virus is specifically expressed in neuron cells only, and can induce the neuron cells in an infected area to efficiently produce BMP7 protein after the mammal is infected. The invention discovers that the hippocampal region of a rat with cognitive impairment caused by injection stress of the adeno-associated virus constructed by the invention can obviously inhibit apoptosis of hippocampal structure cells of the stressed rat, lighten the level of oxidative stress, and strengthen synaptic plasticity, thereby improving the recognition capacity of new objects and enhancing learning and memory capacity, thereby proving that the reagent for improving the expression quantity of BMP7 can be used for preparing medicines for treating stress cognitive impairment.

Description

Application of reagent for increasing BMP7 expression quantity in preparation of medicine for treating stress cognitive impairment

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of a reagent for improving BMP7 expression quantity in preparation of a medicine for treating stress cognitive impairment.

Background

With the acceleration of the life rhythm of modern society and the aggravation of social competition, most people are under different degrees of stress load. Epidemiological surveys show that the prevalence of mild cognitive impairment and dementia is more than twice that of the same age non-stressed population in the stressed state for a long period of time. High levels of stress hormones can lead to structural and functional changes in multiple brain regions, including reduced hippocampal volume, reduced dendritic spine numbers in vertebral cells, synaptic plasticity abnormalities, blocked dentate gyrus, remodelling of the prefrontal cortex nerve loops, reduced spontaneous activity, etc., but drugs targeted to stress hormones have not been able to effectively prevent and treat stress-related cognitive dysfunction.

The body's response to the pressure source depends on the nature, intensity and duration of the pressure source, creating an inverted U-shaped continuous quantitative effect curve. Acute stress causes compensatory reactions in the body, such as increased alertness and sensitivity; as the duration of stress increases, the body system coordinates actions to maintain homeostasis, thereby actively adapting to the source of stress. The cumulative effects of further stress can lead to a disturbance of the homeostasis, leading to pathophysiological damage. This indicates that the body's function changes from adapting to injury during stress.

Bone Morphogenic Protein (BMP) is a multifunctional cytokine involved in the development and differentiation of various tissues, including mesodermal induction, neurogenesis and apoptosis. BMP is a member of the tgfβ superfamily of ligands, all of which transmit signals through a receptor complex consisting of type I and type II serine/threonine protein kinases. Bone morphogenic protein 7 (BMP 7) has a main function of participating in physiological processes such as regulating proliferation, differentiation, migration and the like of various germ cells in embryonic development, participating in skeletal development after birth, and inducing ectopic bone formation.

Gene therapy refers to the transfer of specific genetic material into specific target cells of a patient to ultimately achieve a therapeutic approach to prevent or alter a particular disease state. In recent years, gene therapy has made a major breakthrough in the field of biological therapy, and adeno-associated virus (AAV) has been attracting attention as a powerful tool for delivering gene therapy, and has the characteristics of immunogenicity, high safety, strong diffusivity, and the like. Medicaments using adeno-associated virus as a vector can be applied to the treatment of various diseases including malignant tumors. Up to now, 164 intervention clinical trials have been conducted on clinical Trial.

At present, there is no report on the treatment of stress cognitive impairment by increasing the BMP7 expression level.

Disclosure of Invention

The invention aims to provide application of an agent for improving the expression quantity of BMP7 in preparing a medicament for treating stress cognitive impairment, so as to solve the problems in the prior art, and the invention discovers that improving the expression of BMP7 can enhance the synaptic plasticity of neurons, maintain the survival and activity of neurons, relieve the oxidative stress level, promote the growth and development of dendrites and relieve the chronic stress-induced cognitive impairment, thereby playing a role in resisting and treating the stress cognitive impairment.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides an application of an agent for improving BMP7 expression quantity in preparing a drug for treating stress cognitive impairment.

The invention also provides application of the reagent for improving the BMP7 expression quantity in preparing a medicine for improving stress-induced intra-hippocampal apoptosis.

The invention also provides application of the reagent for improving the BMP7 expression level in preparing a medicament for reducing the oxidative stress level in the hippocampus.

The invention also provides the application of the reagent for improving the BMP7 expression quantity in preparing the medicine for improving the stress-induced hippocampal synaptic plastic damage

Further, the agent comprises a biomaterial that overexpresses BMP 7.

Further, the biological material comprises the substance described in any one of the following (1) to (3):

(1) BMP7 gene with nucleotide sequence shown in SEQ ID NO. 1;

(2) Recombinant plasmids comprising the BMP7 gene;

(3) Adeno-associated virus, including the recombinant plasmid.

The invention also provides a medicine for treating stress cognitive impairment, which comprises an agent for improving the expression level of BMP 7.

Further, the agent comprises a biomaterial that overexpresses BMP 7.

(1) BMP7 gene with nucleotide sequence shown in SEQ ID NO. 1;

(2) Recombinant plasmids comprising the BMP7 gene;

(3) Adeno-associated virus, including the recombinant plasmid.

Further, the medicament also comprises pharmaceutically acceptable auxiliary materials.

The invention discloses the following technical effects:

the invention prepares adeno-associated virus containing bone morphogenetic protein 7 (BMP 7) expression sequence, the virus is specifically expressed in neuron cells only, and can induce the neuron cells in an infected area to efficiently produce BMP7 protein after the mammal is infected. The virus has higher efficiency of expressing BMP7 protein, can realize the obvious neurotrophic effect on animal nerve tissues at the body level, and can be used for the treatment and research of various diseases.

The invention can obviously inhibit the apoptosis of the hippocampal structure cells of the stressed rats, lighten the oxidative stress level and strengthen the synaptic plasticity, thereby improving the recognition capability of new objects and enhancing the learning and memory capability. The adenovirus related virus provided by the invention has wide host range, the introduced BMP7 gene has high homology in mammals, the application object is not only limited to rats and mice, but also suitable for other mammals including human beings, and the adenovirus related virus over-expressed by the BMP7 has potential application value in treating cognitive function decline caused by various stress pressures.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the result of electrophoresis identification of BMP7 target gene obtained by PCR method; wherein 1 is a PCR product, and 2 is a Marker;

FIG. 2 is a diagram of the structure of a plasmid used for constructing a recombinant expression vector of a target gene;

FIG. 3 shows the results of vector cleavage; wherein 1 is 1kb DNAladder Marker,2 and 3 is a carrier enzyme-cut fragment;

FIG. 4 shows colony PCR identification results; wherein, 1: DL2,000 DNAMaroker; 2 to 7 are 6 selected transformants;

FIG. 5 is a graph showing changes in apoptosis levels of hippocampus cells in the brain under stress conditions; wherein A is a hippocampal clear-Caspase 3 protein band diagram; b is a clear-Caspase 3 protein band gray level statistical graph; c is a hippocampal Bax gene expression statistical graph;

FIG. 6 is a graph showing changes in stress levels of the sea Ma Yanghua in the brain under stress conditions; wherein A-C are superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and Malondialdehyde (MDA), respectively;

FIG. 7 shows changes in the expression of PSD95, GAP43 and Syp, which are related to hippocampal synapse proteins in the brain under stress conditions; wherein A is a hippocampal synapse-related protein PSD95, GAP43 and Syp protein band diagram; b is a protein band gray level statistical graph;

FIG. 8 is a graph showing changes in cognitive function in rats under stress conditions; A-C are the results of open field experiments, new object identification experiments and water maze experiments respectively;

FIG. 9 shows the results of verification of the results of the injection of AAV-oeBMP7 in the brain of rats with respect to the control group; wherein A is a BMP7 protein band diagram; b is a BMP7 protein band gray level statistical graph; c is a fluorescence image of the hippocampus infected with the virus;

FIG. 10 is a flow chart of an experiment for injecting virus;

FIG. 11 is the effect of over-expression of BMP7 on the level of hippocampal apoptosis in rats; wherein A is a hippocampal clear-Caspase 3 protein band diagram; b is a protein band gray level statistical result graph; c is a hippocampal Bax gene expression statistical graph;

FIG. 12 is a graph showing the effect of over-expression of BMP7 on the level of oxidative stress in the hippocampus of rats; wherein A-C are SOD, GSH-Px and MDA respectively;

FIG. 13 is the effect of BMP7 over-expression on rat hippocampal neurite plasticity; wherein A is dendritic spine density fluorescence; b is the density statistics result of the dendritic spines; c is a synapse-related protein PSD95, GAP43 and Syp protein band diagram; d is a PSD95 protein band gray level statistical graph; e is GAP43 protein band gray level statistical graph; f is a Syp protein band gray level statistical graph;

FIG. 14 is the effect of over-expression of BMP7 on chronic stress induced cognitive impairment; wherein A is an open field experimental track diagram; b is open field score; c is an object identification index; d is a water maze experiment track diagram; e is a water maze experiment table searching experiment; f is the number of times of water maze experiment.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

Example 1

1. Construction and identification of Bmp 7-containing expression vector

The primer is designed to obtain the coding gene of BMP7 through PCR, and then the coding gene is cloned into the gland related expression vector.

Cloning and purification of the target Gene

(1) CDS region sequence (NM-001191856.3, SEQ ID NO. 1) of BMP7 mRNA was obtained from NCBI database.

SEQ ID NO.1：

ATGCACGTGCGCTCGCTGCGCGCTGCGGCGCCACACAGCTTCGTGGCGCTCTGGGCGCCTCTGTTCTTGCTGCGCTCTGCCTTGGCCGACTTCAGCCTGGACAACGAGGTGCACTCCAGTTTCATCCACCGGCGCCTCCGCAGTCAGGAGCGGCGGGAGATGCAGCGGGAAATCCTGTCCATCTTGGGCTTGCCCCATCGTCCGCGCCCGCACCTCCAGGGAAAACATAATTCGGCGCCCATGTTCATGTTGGACCTGTACAACGCCATGGCGGTGGAGGAGAGTGGGCCGGACGGACAGGGCTTCTCCTACCCCTACAAGGCCGTCTTCAGTACCCAGGGTCCCCCTTTGGCCAGCCTGCAGGACAGCCACTTCCTCACCGACGCCGACATGGTCATGAGCTTCGTCAACCTAGTGGAGCACGACAAGGAATTCTTCCACCCTCGATACCACCATCGAGAGTTCCGGTTTGATCTTTCCAAGATCCCCGAGGGAGAGGCGGTGACCGCAGCCGAGTTCAGGATCTATAAGGACTACATCCGGGAGCGGTTTGACAACGAGACCTTCCAGATCACAGTCTATCAGGTGCTCCAGGAGCACTCAGGCAGGGAGTCCGACCTCTTCTTGCTGGACAGCCGTACCATCTGGGCTTCTGAGGAGGGCTGGTTGGTATTTGACATCACAGCCACCAGCAACCACTGGGTGGTCAACCCTCGGCACAACCTGGGCTTACAGCTCTCCGTGGAGACCCTGGATGGGCAGAGCATCAACCCCAAGTTGGCAGGCCTGATTGGACGGCATGGACCCCAGAACAAGCAACCCTTCATGGTGGCCTTCTTCAAGGCCACGGAGGTTCATCTCCGTAGCATCCGGTCCACGGGGGGCAAACAACGCAGCCAGAACCGCTCCAAGACTCCAAAGAACCAAGAGGCACTGAGGATGGCCAGTGTGGCAGAAAACAGCAGCAGTGACCAGAGGCAGGCCTGCAAGAAACACGAGCTGTATGTTAGCTTCCGAGACCTTGGCTGGCAGGACTGGATCATCGCACCTGAAGGCTATGCTGCCTACTACTGTGAGGGAGAGTGTGCCTTCCCTCTGAACTCCTACATGAACGCCACCAACCATGCTATCGTCCAGACACTGGTTCACTTCATCAACCCAGACACCGTACCCAAGCCCTGCTGTGCCCCCACCCAGCTCAACGCGATATCTGTCCTCTACTTCGACGACAGCTCCAACGTCATCCTGAAGAAGTACAGAAACATGGTGGTCCGGGCCTGTGGCTGCCACGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT。

(2) Design and Synthesis of cloning primers

The primer is designed to obtain the coding gene of BMP7 through PCR, and then the coding gene is cloned into the gland related expression vector. The primer sequences used for cloning were as follows:

BMP7-Forwardprimer(SEQ ID NO.2)：

GTCCGGCTAGCGCCACCATGCACGTGCGCTCGCTGCGCGCTGCGGCGCCACACAGCTT；

BMP7-Reverse primer(SEQ ID NO.3)：

CTTGCTCACCATGGATCCAGGTCCAGGGTTCTCCTCCACGTCTCCAGCCTGCTTCAGC。

the underlined sequence is the cleavage site (NheI/BamHI). Product length: 1391bp; the annealing temperature was 60 ℃. The peptide fragment produced by translation of the product is the full length of BMP7 protein. The amino acid sequence (SEQ ID NO. 4) is specifically as follows:

SEQ ID NO.4：

MHVRSLRAAAPHSFVALWAPLFLLRSALADFSLDNEVHSSFIhRLRSQERREMQREILSILGLPhPRPHLQGKHNSAPMFMLDLYNAMAVEESGPDGQGFSYPYKAVFSTQGPPLASLQDSHFLTDADMVMSFVNLVEHDKEFFHPRYHhEFRFDLSKIPEGEAVTAAEFRIYKDYIRERFDNETFQITVYQVLQEHSGRESDLFLLDSRTIWASEEGWLVFDITATSNHWVVNPRHNLGLQLSVETLDGQSINPKLAGLIGRHGPQNKQPFMVAFFKATEVHLRSIRSTGGKQRSQNRSKTPKNQEALRMASVAENSSSDQRQACKKHELYVSFRDLGWQDWIIAPEGYAAYYCEGECAFPLNSYMNATNHAIVQTLVHFINPDTVPKPCCAPTQLNAISVLYFDDSSNVILKKYRNMVVRACGCH。

(3) Amplification of the Bmp7 Gene using PCR: PCR was performed using high fidelity enzyme (Takara Co.) to obtain BMP7 fragment. The PCR reaction solution formulations are shown in table 1:

TABLE 1

Name of the name	Volume of
		5×PS Buffer	10μL
dNTPs(2.5mM each)	4μL
		PrimeSTAR HS DNA polymerase	0.5μL
Forward primer(10μM)	1μL
		Reverse primer(10μM)	1μL
Stencil (10 ng/. Mu.L)	1μL
		ddH ₂ O	32.5μL
Total volume of	50μL

The PCR reaction was performed in a PCR apparatus as follows: pre-denaturation at 98 ℃ for 5min; denaturation at 98℃for 10sec, annealing at 60℃for 10sec, extension at 72℃for 2min for 30sec, for a total of 35 cycles; finally, the reaction is carried out for 8min at 72 ℃.

(4) To the PCR product, 10. Mu.L of a 6×loading buffer was added, and after mixing, agarose gel electrophoresis was performed to confirm the size of the product (FIG. 1). The target strip was cut under an ultraviolet lamp and recovered and purified according to the protocol of the gel recovery kit (Tiangen biosystems).

Cleavage of fragments and vectors

(1) The adeno-associated expression vector used was AOV062 (fig. 2); the element sequence is pAAV-hSyn-EGFP-3xFLAG-WPRE, the vector carries a neuron-specific promoter hSyn, and because BMP7 is mainly expressed around neurons, the invention selects an expression vector with the neuron-specific promoter element to ensure that BMP7 is expressed only in neurons. Cloning site: nheI/BamHI.

(2) The recovered fragment and vector were cut with restriction enzyme NheI/BamHI (NEB Co.). The reaction solution was prepared as follows, and after mixing, the mixture was digested overnight in a 37℃water bath.

The system for fragment cleavage is shown in Table 2, and the system for vector cleavage is shown in Table 3.

TABLE 2

Name of the name	Volume of
		10×CutSmart Buffer	5μL
Fragments	20μL
		BamHI(10U/μL)	1μL
ddH ₂ O	23μL
		Total volume of	50μL

TABLE 3 Table 3

Name of the name	Volume of
		10×CutSmart Buffer	5μL
vector(1μg/μL)	2μL
		BamHI(10U/μL)	1μL
ddH ₂ O	42μL
		Total volume of	50μL

(3) To the cut products of the fragments and the vector, 10. Mu.L of 6×loading buffer was added, respectively, and agarose gel electrophoresis was performed, and the target band was cut under an ultraviolet lamp and recovered according to the protocol of the gel recovery kit (Tiangen) (FIG. 3).

Ligation of the fragment (III) to the vector

(1) 1. Mu.L of each of the recovered fragments and the carrier was subjected to agarose gel electrophoresis, gray scale quantification was performed, molar mass was calculated, and ligation was performed at a molar ratio of 3:1 of the fragments to the carrier.

(2) The reaction solution was prepared according to the system shown in Table 4, and the mixture was left at room temperature for 2 hours after being mixed uniformly.

TABLE 4 Table 4

Name of the name	Volume of
		2×T4 Buffer	2μL
T4 Strong ligase (Invitrogen Co.)	1μL
		Fragments	5μL
Carrier body	2μL
		Total volume of	10μL

(IV) transformation and screening of recombinant vectors

(1) One E.coli DH5 competent cell (Takara Co.) was taken and placed on ice for natural thawing. mu.L of ligation product was added to competent cells and left on ice for 20min. Heat shock is carried out for 90sec in a water bath at 42 ℃, and the mixture is immediately inserted into ice and is placed for 2min. 700. Mu.L of LB medium is added and the mixture is shaken by a shaker at 37℃for 45min.

(2) The bacterial liquid was spread evenly on ampicillin-containing agar plates. Firstly, placing the mixture in a baking oven at 37 ℃ for 30min, and then culturing the mixture for 16h in an inverted mode.

(3) A plurality of monoclonals are selected and respectively put into a shaking tube, 3mL of LB culture medium containing ampicillin is added, and shaking table shaking is carried out at 37 ℃ for 16 hours.

(V) identification of Positive recombinants

(1) The PCR primers were designed and synthesized and identified as follows:

P1：GTGACCGCAGCCGAGTTCA(SEQ ID NO.5)；P2：CATGGTGGCCTTCTTCAAGG(SEQ ID NO.6)。

the product length was 605bp and the annealing temperature was 60 ℃.

(2) PCR was performed using the bacterial liquid as a template, and the reaction system was as shown in Table 5:

TABLE 5

Name of the name	Volume of
		2 XPCR mix (Tiangen Co., ltd.)	10μL
P1 primer(10μM)	1μL
		P2 primer(10μM)	1μL
Bacterial liquid	1μL
		ddH ₂ O	7μL
Total volume of	20μL

The PCR reaction was performed in a PCR apparatus as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30sec, annealing at 60℃for 30sec, elongation at 72℃for 1min, 35 cycles in total; finally, the reaction is carried out for 10min at 72 ℃.

(3) The 6. Mu.LPCR product was subjected to agarose gel electrophoresis to confirm the size of the product (FIG. 4).

(4) The positive flora initially identified by PCR was sequenced (Shanghai Ji Kai biosystems, inc.) and compared with the target gene sequence to confirm that the recombinant plasmid sequence was correct and that no gene mutation was present.

(5) The bacterial liquid with correct sequencing is added into 10mL LB culture medium containing ampicillin, and the bacterial liquid is shaken by a shaker at 37 ℃ for 16h. The plasmid miniextract medium-quantity kit (Tiangen biology Co., ltd.) is used for extracting recombinant plasmid according to the instruction, thus obtaining pAAV-oeBMP7, and the plasmid is a neuron specific promoter, so that the expression in 293T cells can not be carried out, and the virus effect can be verified by carrying out infection in animals in sea horses.

2. Packaging adeno-associated virus

Recombinant expression plasmids pAAV-oeBMP7, pAAV-RC (helper plasmid carrying AAV replication and capsid genes, purchased from metazoan) and pHelper (helper plasmid carrying adenovirus-derived genes, purchased from metazoan) were co-transfected into AAV-293 cells, and after 3 days the cell supernatants and cells were collected and lysed to obtain AAV particles. The virus was concentrated and purified by CsCl density gradient centrifugation and ultrafiltration. Finally, the titer of the virus was detected by real-time PCR.

Cultivation of AAV-293 cells

a. DMEM medium containing 10wt% fbs (referred to as complete medium) was prepared for culturing AAV-293 cells.

b. 3mL of complete medium was added to a 10mL glass centrifuge tube.

c. Taking out the cells from the liquid nitrogen tank or the refrigerator at the temperature of minus 80 ℃, rapidly putting the cells into a water bath kettle at the temperature of 37 ℃, and slightly shaking the cells for 2min to completely melt the cells.

d. And taking the frozen storage tube into an ultra-clean bench, and wiping the surface with an alcohol cotton ball for disinfection. The cell suspension was added to a centrifuge tube prepared in advance.

e. Centrifuging for 800g×3min, removing supernatant, adding 2mL of fresh complete medium, gently blowing with a dropper to suspend cells, inoculating into 10cm culture dish containing 8mL of fresh complete medium, and standing at 37deg.C in 5% CO ₂ Culturing in incubator.

AAV-293 cell passaging

a. The growth state and density of gentleman cells were observed daily, and when the cell density reached 50%, passage was performed.

b. The original medium was aspirated, and the cells were washed twice with 10mL of physiological saline, added with 1mL of 0.5% trypsin solution, and put into a 37℃incubator for digestion for 1min until the cells were just detached from the dish.

c. Digestion was terminated by adding 3mL of complete medium and the cell suspension was transferred to a 10mL glass centrifuge tube.

d. Centrifuging for 800g×3min, and discarding supernatant. Adding 5mL of fresh complete medium, gently beating with a dropper to suspend cells, inoculating 1mL into 10cm culture dish containing 8mL of fresh complete medium, co-inoculating 5 bottles, placing at 37deg.C and 5% CO ₂ Culturing in incubator.

(3) Cryopreservation of AAV-293 cells

a. AAV-293 cells in logarithmic growth phase were taken, the original medium was aspirated, the cells were washed twice with 10mL of physiological saline, 1mL of 0.5% trypsin solution was added, and the cells were put into a 37℃incubator for digestion for 3min until the cells were just detached from the culture dish.

b. Digestion was terminated by adding 3mL of complete medium and the cell suspension was transferred to a 10mL glass centrifuge tube.

c. Centrifuging for 800g×3min, and discarding supernatant. Adding 3mL of cell cryopreservation solution (Suzhou New Saimei Co., ltd.) to resuspend cells, packaging into cryopreservation tube, 1 mL/tube, placing into a refrigerator at-80deg.C, and storing in liquid nitrogen tank for the next day for long term.

(II) transfection of cells

(1) Taking AAV-293 cells in logarithmic growth phase, inoculating into 10cm culture dish, placing at 37deg.C and 5% CO ₂ Culturing in incubator, and transfecting when the cell density reaches 70-80%.

(2) 1 of a 1.5mL EP tube was taken and 500. Mu.L CaCl was added ₂ The solution (0.3M) was then added with 10. Mu.g of each of pAAV-oeBMP7, pAAV-RC and pHelper, and gently mixed.

(3) 1 fresh 1.5mL EP tube was taken, 500. Mu.L of 2 XHBS solution was added, and DNA/CaCl was added dropwise ₂ The mixture is inverted and mixed evenly.

(4) Mixing the mixed DNA/CaCl ₂ The HBS solution was added dropwise to the cell culture dish while gently shaking the dish to uniformly distribute the solution in the medium, and placed in 5% CO ₂ Culturing in incubator at 37 ℃.

(5) After 6h, the medium was changed, 10mL of fresh complete medium was added, and the culture was continued for 72h.

(III) collecting adeno-associated virus

(1) Observing the cell morphology and the color change of the culture medium, and indicating successful virus packaging when part of cells are rounded and fall off and the color of the culture medium changes from red to orange or yellow. Viruses are typically collected three days after transfection.

(2) The cells were gently blown down with a dropper and transferred to a 15mL centrifuge tube along with the medium.

(3) The supernatant was transferred to a new 15mL centrifuge tube after centrifugation for 800 g.times.3 min, and 1mL of LPBS was added to the cell pellet for resuspension.

(4) The cell suspension was repeatedly placed in liquid nitrogen and 37 ℃ water bath, and freeze-thawed 4 times.

(5) The supernatant was transferred to a new EP tube by centrifugation at 10000 g.times.3 min.

(IV) Virus concentration

(1) PEG8000 was added to the supernatant of step (3) to a final concentration of 8%, and the mixture was left on ice for 2 hours, with the mixture being inverted and mixed every 15 minutes.

(2) Centrifugation was performed for 2500 g.times.30 min, the supernatant was discarded, PBS was added for resuspension, and the supernatant collected in step (5) of step (III) was combined.

(3) The supernatant was transferred to a new EP tube by centrifugation at 3000 g.times.30 min. The residual plasmid DNA was digested by adding Benzonase nuclease (final concentration: 50U/mL) (Merck Co.), mixed upside down and incubated at 37℃for 30min.

(4) The impurities in the solution were filtered off using a 0.45 μm filter.

(V) Virus purification

(1) To the virus concentrate was added CsCl at a concentration of about 6.5g/10mL and 1.41g/mL, and the mixture was dissolved by shaking.

(2) The sample was added to the ultracentrifuge tube and the remaining space of the tube was filled with a pre-configured CsCl solution (1.41 g/mL).

(3) The mixture was centrifuged for 175000 g.times.24 hours to form a density gradient, and solutions of different densities were collected in order, and the titer was measured to collect a composition containing AAV particles.

(4) The above steps are repeated once.

(VI) Ultrafiltration desalination

(1) 4mL of deionized water was added to an Amicon-15 ultrafiltration device to wet the membrane.

(2) The purified virus solution was added to an ultrafiltration device, the total volume was made up to 4mL with PBS, and the lid was closed.

(3) The remaining solution volumes were observed every 5min by centrifugation at 1500g until the final volume was 200. Mu.L.

(4) PBS was added to the remaining solution to make up the volume to 4mL.

(5) The above steps were repeated 3 times.

(6) The tube was centrifuged to give a final volume of virus solution of 0.5mL.

Glycerol (final concentration 5% by volume) was added to obtain adeno-associated virus AAV-oeBMP7, which was stored in sub-package at-80 ℃.

(seventh) Virus titre determination

(1) Using the Zfcas9 plasmid (2.45E+13Copies/mL), with ddH ₂ O gradient dilution to obtain standard substances with concentration of 2.45X10 respectively ¹⁰ 、2.45×10 ⁹ 、2.45×10 ⁸ 、2.45×10 ⁷ 、2.45×10 ⁶ Copies/mL。

(2) The gradient diluted virus solution was diluted as shown in table 6:

TABLE 6

Name of the name	Virus solution	ddH ₂ O
			Sample-2	1 mu L stock solution	99μL
Sample-3	1μLSample-2	9μL
			Sample-4	1μLSample-3	9μL
Sample-5	1μLSample-4	9μL

(3) The real-time PCR reaction solution was prepared, and the reaction system per well was as shown in Table 7:

TABLE 7

Name of the name	Volume of
		2×SYRB Green Master(ROX)(Takara)	10μL
Forward Primer(5μM)	0.5μL
		Reverse Primer(5μM)	0.5μL
ddH ₂ O	4μL
		Total volume of	15μL

(4) To a 96-well plate, 15. Mu.L of each well was added, and 5. Mu.L of standard or sample was added, and a multiplex well was set.

(5) After sealing the 96-well plate, putting the sealed membrane into a real-time PCR instrument, and reacting according to the following procedures: pre-denaturation at 95℃for 10min; denaturation at 95℃for 15sec, annealing at 60℃for 30sec and extension at 72℃for 30min, followed by 40 cycles in total; a dissolution profile was obtained by reacting at 95℃for 15sec,60 sec, and 95℃for 15 sec.

(6) Drawing a standard curve according to the logarithmic value and Ct average value of the standard substance concentration: y= -0.2981x+13.459, r ² ＝0.9998。

(7) The concentration was calculated from the Ct value of each Sample. Since the standard is double stranded DNA and the AAV viral particles are single stranded DNA, the viral stock concentration is equal to each Sample concentration divided by the dilution and multiplied by 2.

(8) The virus stock concentrations calculated for each Sample were averaged to give an AAV virus titer of 1.09E+13Copies/mL (Table 8).

TABLE 8

3. Application of overexpression BMP7 in stress cognitive injury treatment

The influence of chronic unpredictability Wen Heying stress (CUMS) on the cognitive ability of rats was tested by animal behavioral experiments using a rat stress agonist model established to test the effects of apoptosis levels, oxidative stress levels and dendritic plasticity in the hippocampus of rats. AAV-oeBMP7 and control viruses are injected into the inner hippocampus of the rats in the stress group at fixed points, the influence of apoptosis level, oxidative stress level and dendritic plasticity in the hippocampus is detected, the change of learning and memory related behaviours of animals is detected, and the application value of the over-expressed BMP7 in the treatment of stress cognitive impairment is clarified.

In this example, a Chronic Unpredictable Mild Stimulation (CUMS) stress model is taken as an example, and the application of overexpressing BMP7 in the treatment of stress cognitive impairment is described, but the application of overexpressing BMP7 is not limited to this stress model, but includes the treatment of cognitive impairment caused by other stress models.

Influence of stress on apoptosis in hippocampus, oxidative stress, dendritic plasticity and cognitive competence in rats

(1) An stress object model is established according to the following program: (1) night illumination, (2) light shielding for 12h, (3) damp padding for 12h, (4) padding removal for 12h, (5) fasted for 12h, (6) water forbidden for 12h, (7) ice swimming for 3min (4 ℃), (8) restraint for 4h, (9) cage shaking (70 r/min) for 2h, and plantar electric shock for 5min (0.2 mA). The stress pattern was randomly generated, two different stress source combinations were selected daily and the same stress pattern was not reused for 3 days. And (3) taking hippocampal tissues, extracting RNA and total protein, carrying out RT-qPCR and WB experiments, and detecting the influence of stress on Bax genes and clear-Caspase 3 protein in the hippocampus. The results showed that the level of apoptosis in the hippocampus of rats increased with the prolongation of the stress time (fig. 5).

(2) The elevated levels of oxidative stress in the hippocampus of stressed rats were confirmed by the detection of the levels of oxidative stress in the hippocampus of rats by superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and Malondialdehyde (MDA) detection kit (fig. 6).

(3) Changes in the expression of the synapse-associated proteins postsynaptic compact protein 95 (PSD 95), growth-related protein 43 (GAP 43) and synaptorin protein (Syp) were further clarified by WB experiments, confirming that the rat hippocampal synaptic plasticity was impaired with prolonged stress time (fig. 7).

(4) The influence of stress on the cognitive ability of rats is detected through open field experiments, new object identification experiments and water maze experiments.

(1) Open field experiments: the test was carried out in a black wooden box of 100X 45 cm. The wooden box floor was divided into 25 equal squares (20 x 20 cm). At the beginning of the experiment, rats were placed individually in the center of the open field bottom and observed for the number of squares and head-up frequency passed by the rats in 5min using an ANY-size behavioral analysis system. To ensure that the test results are not affected by any previous residues, alcohol is used to clean the interior of the open field prior to each test. The open field score is the sum of the number of squares passed by the rat and the number of heads lifted.

(2) New object identification experiment: the experimental device is a black wooden box (60×60×45 cm), and the whole test process comprises the following three steps: adaptation, training and testing. Adaptation segment: the rats were left free to move in a wooden box for 10min (no object) 1 day before the experiment. Training phase: on the day of the experiment, two identical objects are placed at the left and right ends of the side wall of the box body, and the rat is placed at the center of the field opposite to the two objects, so that the rat can freely explore for 10min in the box body. Testing: before testing, one of the two objects is replaced by another object with different color and shape, and the rat is put back into the wooden box for free exploration for 10min by the same method. The ANY-size behavior analysis system records the time the rat explores new and old objects, denoted Tnew and Told, respectively. The cognitive index CI is expressed as: CI= (Tnew-Told)/(Tnew+told).

(3) Water maze experiment: the experimental device is a black round plastic barrel (diameter 160cm, depth 75 cm), the cloth curtains around the device are stuck with stickers with different shapes and colors, and the water temperature is 22+/-2 ℃ as a spatial reference clue. During the training phase, rats were trained for 5 and a half days, at a frequency of once per day for 3min. In the training process, the rats are gently placed in water, the quadrants of each placement are different, and the rats are trained to find an underwater platform which is about 3cm away from the water surface. If the platform cannot be found within 3min, the rat is guided onto the platform and left on the platform for 10s. During the testing phase, the platform was removed and the rats were placed in water in the quadrant opposite the platform. Each rat was allowed to swim in water for 3min, and the time for the rat to find the original Platform position for the first time and the number of Platform passes within 3min were recorded as the Platform seeking latency (Escape latency) and Platform passing times (Platform passes), respectively.

The results showed that the open field score and new object recognition cognitive index of the stressed 6 week group rats were reduced and the water maze time to seek was increased compared to the control group, indicating that long term stress could lead to reduced cognitive ability of the rats (fig. 8).

(II) Effect of intra-hippocampal injection of AAV-oeBMP7 on intra-hippocampal apoptosis, oxidative stress, dendritic plasticity and cognitive ability in rats

As shown in fig. 10, AAV-oeBMP7 and AAV-NC viruses were injected into rat hippocampus by brain stereotactic injection technique, respectively, CUMS intervention and normal feeding were performed, respectively, and each group was designated as Ctrl-NC, ctrl-oeBMP7, CUMS-NC, and CUMS-oeBMP7 (see fig. 9 for virus results verification), followed by the following detection:

(1) And (3) taking hippocampal tissues, extracting RNA and total protein, carrying out RT-qPCR and WB experiments, and detecting the influence of overexpression of BMP7 on Bax genes and clear-Caspase 3 proteins in the hippocampus. The results showed that BMP7 can reduce the level of apoptosis in hippocampus (fig. 11).

(2) The level of oxidative stress in the hippocampus of the rat after the overexpression of BMP7 is detected by the SOD, GSH-Px and MDA detection kit, and the condition that the level of oxidative stress in the hippocampus of the stressed rat can be reduced is proved by the overexpression of BMP 7. (FIG. 12).

(3) Further, by WB experiments, changes in expression of the post-synaptic related proteins PSD95, GAP43 and Syp that overexpress BMP7 were confirmed to improve stress-induced plastic damage of the hippocampal synapses in rats (fig. 13).

(4) And detecting the influence of the overexpression of BMP7 on the cognitive ability of the stressed rats through an open field experiment, an object recognition experiment and a water maze experiment. The results show that the open field score and the object recognition cognitive index of BMP7 overexpressed rats are increased and the water maze homing time is reduced compared to the injection of control virus, suggesting that BMP7 overexpression can promote recovery of cognitive ability in stressed rats, suggesting that BMP7 may be a potential target for stress cognitive impairment treatment (fig. 14).

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. The application of an agent for improving the expression level of BMP7 in preparing a medicament for treating stress cognitive impairment.

2. The use of an agent that increases BMP7 expression levels in the manufacture of a medicament for ameliorating stress-induced apoptosis in hippocampus.

3. Use of an agent that increases BMP7 expression levels in the manufacture of a medicament for reducing oxidative stress levels in the hippocampus.

4. Use of an agent that increases BMP7 expression levels in the manufacture of a medicament for ameliorating stress-induced plastic damage to hippocampal synapses.

5. The use according to any one of claims 1 to 4, wherein the agent comprises a biological material over-expressing BMP 7.

6. The use according to claim 5, wherein the biological material comprises any one of the following (1) - (3):

(1) BMP7 gene with nucleotide sequence shown in SEQ ID NO. 1;

(2) Recombinant plasmids comprising the BMP7 gene;

(3) Adeno-associated virus, including the recombinant plasmid.

7. A medicament for treating stress cognitive impairment, comprising an agent that increases BMP7 expression level.

8. The medicament of claim 7, wherein the agent comprises a biological material that overexpresses BMP 7.

9. The drug according to claim 8, wherein the biological material comprises any one of the following (1) to (3):

(1) BMP7 gene with nucleotide sequence shown in SEQ ID NO. 1;

(2) Recombinant plasmids comprising the BMP7 gene;

(3) Adeno-associated virus, including the recombinant plasmid.

10. The medicament of claim 7, further comprising a pharmaceutically acceptable adjuvant.