阅读说明：本技术 一种miRNA和人间充质干细胞在制备用于治疗关节炎的药物中的用途 (Application of miRNA and human mesenchymal stem cells in preparation of medicine for treating arthritis ) 是由 朱文敏 孙毅 陈伟 于 2021-08-11 设计创作，主要内容包括：本发明提供了一种miRNA在制备用于治疗关节炎的药物中的用途,所述miRNA的核苷酸序列如SEQ ID NO.1所示。还提供了一种表达上述miRNA的人间充质干细胞。还提供了上述的人间充质干细胞在制备用于治疗关节炎的药物中的用途。本发明已鉴定能够广泛靶向修复骨关节炎相关软骨基质及组织的miR-17-5p,并且在小鼠DMM模型中鉴定了miR-17-5p的骨关节炎治疗能力,并在临床样本中鉴定了骨关节炎严重程度与miR-17-5p表达水平高低的联系,同时还制备了能够自主、持续表达miR-17-5p的人间充质干细胞,建立以细胞为载体的miR-17-5p递送治疗系统,可作为慢性骨关节炎的治疗方法。(The invention provides an application of miRNA in preparing a medicament for treating arthritis, wherein the nucleotide sequence of the miRNA is shown in SEQ ID NO. 1. Also provides a human mesenchymal stem cell expressing the miRNA. Also provides the application of the human mesenchymal stem cells in preparing the medicament for treating arthritis. The invention identifies miR-17-5p capable of repairing cartilage matrix and tissue related to osteoarthritis in a wide-range targeted manner, identifies osteoarthritis treatment capacity of miR-17-5p in a mouse DMM model, identifies the relation between the osteoarthritis severity and miR-17-5p expression level in a clinical sample, simultaneously prepares human mesenchymal stem cells capable of autonomously and continuously expressing miR-17-5p, establishes a miR-17-5p delivery treatment system taking cells as a carrier, and can be used as a treatment method of chronic osteoarthritis.)

1. An application of miRNA in preparing a medicine for treating arthritis, wherein the nucleotide sequence of miRNA is shown in SEQ ID NO. 1.

2. A human mesenchymal stem cell expressing miRNA, wherein the nucleotide sequence of the miRNA is shown in SEQ ID NO. 1.

3. Use of the human mesenchymal stem cells of claim 2 in the manufacture of a medicament for the treatment of arthritis.

4. The application of miRNA in preparing biomarker for diagnosing osteoarthritis is disclosed, wherein the nucleotide sequence of miRNA is shown in SEQ ID NO. 1.

5. A drug delivery system is characterized in that mesenchymal stem cells are used as a vector, miRNA is expressed in the vector, and the nucleotide sequence of the miRNA is shown in SEQ ID NO. 1.

Technical Field

The invention belongs to the field of biomedicine, and relates to a biological small molecule and a cell, in particular to a microRNA (miRNA) for effectively delaying osteoarthritis deterioration and a human mesenchymal stem cell for autonomously and continuously expressing the miRNA.

Background

Osteoarthritis, also known as degenerative osteoarthropathy, is a chronic disease mainly involving damage of articular cartilage and affecting the whole joint tissues, and is clinically manifested as chronic disability caused by joint pain and related dysfunction. Because damaged articular cartilage has no self-repair capability, no obvious and effective method for preventing further deterioration of osteoarthritis exists in the current medicine, and most patients with advanced diseases can only select artificial joint replacement to improve the quality of life.

The structural integrity of articular cartilage is one of the important markers of cartilage health, primarily determined by the extracellular matrix of chondrocytes. Due to the stimulation of external biomechanics and biochemistry, a plurality of matrix degrading enzymes are locally generated, so that the cartilage matrix is gradually degraded, the structural integrity of the cartilage is damaged, an internal calcified layer is continuously abraded, and finally, the generation and progressive deterioration of osteoarthritis are caused.

Mesenchymal Stem Cells (MSCs) have wide sources, can self-renew and differentiate in multiple directions, have strong inflammation regulating capacity and homing capacity, migrate to tissue injury parts in a targeted manner, and secrete factors, exosomes and the like to regulate microenvironment; or the tissue cells are differentiated into specific types of tissue cells to carry out cell replacement, so that the repair of the damaged part is promoted, and the tissue cells have a wide application prospect in the treatment of various clinical diseases. However, MSCs show great heterogeneity due to differences in their donor sources, tissue sources, isolation methods and culture systems, and thus proper in vitro pretreatment-induced culture can modulate and improve the disease-specific therapeutic capabilities of MSCs. Among them, the MSCs derived from fat, skin and cord blood become ideal donors and tissue sources for MSCs culture due to easy acquisition, low tumor formation rate, high cost performance, little harm to patients and no ethical issues.

mirnas are small endogenous RNAs of about 20-24 nucleotides in length, and have a variety of important regulatory roles in vivo. The presently discovered mirnas can delay part of the pathological process of osteoarthritis through various mechanisms including regulation of target mRNA stability, endogenous inhibitory RNA pathway, etc., but are not satisfactory in terms of effective repair of chondrocytes and their matrix. Researches find that the skeletal dysfunction caused by miR-17-92 cluster gene dysfunction is accompanied with the shortening of bone units and the fusion of bone joints, and suggest that the miR-17-92 cluster gene can be a potential joint disease target. Meanwhile, the discovered miRNAs can only generate unidirectional targeting effect on specific single matrix degrading enzymes, and the discovery and identification of the miRNA which can widely target osteoarthritis-related matrix degrading enzymes are helpful for developing molecular therapeutic measures for effectively repairing osteoarthritis lesions.

At present, gradient treatment measures are clinically adopted for treating osteoarthritis, and combination treatment of staged physics, medicines and joint cavity repair surgery is adopted, and the measures can only slightly delay the development of osteoarthritis; when the disease worsens to an advanced stage, joint reconstructive surgery treatments, including expensive and complex joint replacement procedures, are required to relieve joint pain and restore joint mobility. With the development of joint replacement, more than 90% of cases have no advanced stage for more than 10 years after operation, but infection around the prosthesis at the early stage after operation and prosthesis loosening at the long stage after operation both need to be repaired again after operation, thus extremely reducing the life quality of patients. Therefore, a reversible therapeutic drug molecule or combined cell therapeutic measure with simple administration route, small wound and low operation difficulty is urgently needed to inhibit the progress of osteoarthritis.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides a microRNA (miRNA) and a human mesenchymal stem cell for effectively delaying osteoarthritis deterioration, and the miRNA and the human mesenchymal stem cell for effectively delaying osteoarthritis deterioration aim at solving the technical problem that the osteoarthritis treatment effect in the prior art is poor.

The invention provides application of miRNA (miR-17-5p) in preparation of a medicament for treating arthritis, wherein the nucleotide sequence of the miRNA is caaagugcuuacagugcagguag (shown as SEQ ID NO. 1).

The invention also provides a human mesenchymal stem cell for expressing miRNA, wherein the nucleotide sequence of the miRNA is caaagugcuuacagugcagguag (shown as SEQ ID NO. 1).

The invention also provides application of the human Mesenchymal Stem Cells (MSCs) in preparing a medicament for treating arthritis.

The invention also provides application of the miRNA in preparing a biomarker for diagnosing osteoarthritis, wherein the nucleotide sequence of the miRNA is caaagugcuuacagugcagguag (shown as SEQ ID NO. 1).

Specifically, the human mesenchymal stem cells are derived from human fat, skin or umbilical cord.

The invention also provides a drug delivery system, which takes the mesenchymal stem cells as a vector, wherein the vector autonomously and continuously expresses miRNA, and the nucleotide sequence of the miRNA is shown in SEQ ID NO. 1.

The invention provides a miRNA for effectively relieving osteoarthritis progression, and the connection between the level and osteoarthritis, the action and the treatment mechanism are demonstrated through an osteoarthritis (DMM) mouse model of medial meniscus injury; and has identified a link between the extent of cartilage tissue pathology in osteoarthritis patients and the levels of miRNA expression provided.

The miRNA provided by the invention and the human mesenchymal stem cell capable of effectively expressing and secreting the miRNA can be used for the following purposes, such as: can be used for treating osteoarthritis of different stages.

The principle that the miR-17-5p delays osteoarthritis deterioration and repairs the cartilage extracellular matrix of the bone joint comprises the following steps: the method has the advantages that four matrix degrading enzymes capable of destroying the articular cartilage extracellular matrix are subjected to wide targeted inhibition, namely matrix metallopeptidase MMP3 and MMP13, chondroprotein ADAMTS5 and nitric oxide synthase NOS2, and targeted inhibition is performed through recognition sites of 3' UTRs of the four matrix degrading enzymes (shown in figure 1); and maintaining the balance of anabolism and catabolism in the extracellular matrix of chondrocytes, and maintaining the structural integrity of articular cartilage.

The invention discloses a MSCs delivery system capable of autonomously and continuously expressing miR-17-5p, which is realized according to the following principle: intravenous or in situ infusion of MSCs can provide some disease relief; the Agomir is double-stranded small RNA which is modified by cholesterol at the 3' end, two thio frameworks at the 5' end and four full-strand methoxy groups at the 3' end according to a mature miRNA antisense chain, has higher affinity with a cell membrane, has expression time of up to one week after being transfected into cells, and has biosafety.

Compared with the prior art, the invention has remarkable technical progress. The invention identifies miR-17-5p capable of repairing cartilage matrix and tissue related to osteoarticular in a wide-target mode, identifies osteoarthritis treatment capacity of miR-17-5p in a mouse DMM model, identifies the relation between the osteoarthritis severity and miR-17-5p expression level in a clinical sample, simultaneously prepares human MSCs capable of autonomously and continuously expressing miR-17-5p, establishes a miR-17-5p delivery treatment system with cells as a carrier, can be used as a treatment method of chronic osteoarthritis, and has important clinical significance.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 shows that miR-17-5p targets the inhibition of four matrix degrading enzymes through the 3' UTR region.

FIG. 2 shows that the mouse DMM model is successfully constructed, and the expression level of miR-17-5p is closely related to the deterioration degree of cartilage degradation and osteoarthritis.

FIG. 3 shows that both DMM surgery and miR-17-92 cluster gene knockout can lead to increased osteoarthritis score, and the score is reduced after Agomir-17 injection, which indicates that miR-17-5p can inhibit osteoarthritis pathological process.

FIG. 4 shows that the expression level of miR-17-5p in normal human articular cartilage is far higher than that of osteoarthritis patients, about 2 times in early stage and 4 times in middle and late stages, and the similarity of the expression pattern and mechanism of miR-17-5p in human cartilage and mouse cartilage is suggested.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text. It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

Example 1

1. Test object

The C57BL/6 mouse is purchased from Shanghai Weitonglihua laboratory animal technology Limited company, miR-17-92^fl/flMouse and Col2CreER^TMice were purchased from Jackson laboratories and housed in the university of congruence laboratory animal center SPF grade environment. The internal operation training of the experimental animal center of college university has been performed in the experiment involving the operation, surgery and euthanization of the animals.

2. Experimental methods

The following cell experiment procedures were all completed in GMP laboratories in compliance with clinical standards; animal experiments were all done in an SPF environment.

2.1 miR-17-5p can be widely targeted to articular cartilage related matrix degrading enzymes

2.1.1 MiRanda database (http:// www.microrna.org /) provides the prediction information about miRNA target genes of human, Drosophila and zebrafish genomes and the expression profiles of miRNA in different tissues, and TargetScan (http:// www.targetscan.org /) predicts target genes by searching for mRNA sites matching each miRNA region; articular cartilage related matrix degrading enzymes of mRNA 3' UTR containing miR-17-5p recognition sites, including matrix metallopeptidase MMP3 and MMP13, chondroprotein ADAMTS5, nitric oxide synthase NOS2, are predicted by miRanda and TargetScan, and the binding sites are shown in figure 1A, and specific site mutation (mutation) is carried out according to the binding sites; the wt 3'UTR sequence and the mutant 3' UTR are synthesized by biological engineering Limited company and loaded into a fluorescent reporter gene vector psiCHECK-2, and the vector is purchased from Promega company and can monitor the change of the expression of a target gene fused with a reporter gene (Renilla luciferase);

caaagugcuuacagugcagguag (shown as SEQ ID NO. 1) provided by the invention can be prepared from miRBase (A), (B), (C) and C)http://www.mirbase.org/) And (6) database query acquisition.

2.1.2 luciferase reporter experiments were performed on HEK293T cells (purchased from ATCC, CRL-11268), HEK293T cells were seeded in 96-well plates and cultured, the transfection reagent Lipofectamine 2000 was purchased from Invitrogen, transfection procedures were performed according to the instructions, 10pmol of miR-17-5p and 10ng of 3' UTR-psiCHECK-2 were transfected per well, cells were collected after 48 hours of transfection, and luciferase activity was assayed according to the instructions of the Dual luciferase reporter assay kit (purchased from Promega). As shown in FIG. 1B, miR-17-5p can significantly reduce the luciferase activity of wt 3'UTR, but cannot reduce the luciferase activity of mutant 3' UTR, and similar level changes are observed in matrix metallopeptidase MMP3 and MMP13, chondroprotein ADAMTS5, and nitric oxide synthase NOS 2. Therefore, the miR-17-5p has wide matrix degrading enzyme targeting property, and simultaneously targets and regulates matrix metallopeptidase MMP3 and MMP13, chondroprotein ADAMTS5 and nitric oxide synthase NOS 2.

2.2 in the constructed mouse DMM model, the expression level of miR-17-5p is related to the development degree of osteoarthritis

2.2.1 mice of 10 weeks of age were anesthetized with ketamine and xylazine and subjected to a surgical procedure that transects the medial meniscal ligament of the left knee, destroying the cartilage of the left knee and inducing osteoarthritis associated with meniscal instability. The mice in the control group were only subjected to sham surgery treatment without transecting the medial meniscal ligament of the left knee joint;

2.2.2 two groups of mice are euthanized at 0, 4, 8 and 12 weeks after the operation, the left knee joint is obtained and stained with safranin O-fast green and Hematoxylin Eosin (HE), and the expression of microRNA-17 is detected by Fluorescence In Situ Hybridization (FISH);

the method comprises the following specific steps:

2.2.2.1 tissue fixation and Paraffin embedding

After collecting the left knee joint of the mouse, fixing the left knee joint for 48 hours by paraformaldehyde with the mass-volume ratio concentration (g/ml) of 4 percent, then decalcifying the left knee joint for 10 days by an Ethylene Diamine Tetraacetic Acid (EDTA) solution with the mass-volume ratio concentration (g/ml) of 10 percent, and placing the trimmed tissue and the corresponding label in a dehydration box;

putting the dehydration box into a dehydrator, and dehydrating with gradient alcohol (volume percentage concentration) for 90 minutes, 85 minutes, 90 minutes, 60 minutes and 60 minutes respectively by using 50 percent alcohol and 70 percent alcohol;

and (3) transparency: 5-10 minutes of alcohol benzene, 5-10 minutes of xylene I and 5-10 minutes of xylene II;

paraffin penetration: benzene wax 90 minutes, paraffin I60 minutes, paraffin II 60 minutes, and paraffin III 60 minutes;

embedding: embedding the left knee joint tissue permeated with the paraffin in an embedding machine, taking out the wax block from the embedding box after the paraffin is solidified, and finishing the wax block;

slicing: slicing the cartilage tissue on a paraffin slicer, slicing the cartilage tissue on a sagittal plane or a coronal plane with the thickness of 6 mu m, and storing the cartilage tissue at room temperature for later use after baking;

2.2.2.2 safranin O-fast Green staining

Basophilic cartilage tissue is combined with basic dye safranin O to be red, while acidophilic bone is combined with acid dye fast green to form green or blue, when cartilage is damaged, glycoprotein in the cartilage can be released, so that matrix components are unevenly distributed, and accordingly, the safranin O is slightly or not stained.

Dewaxing the paraffin section to distilled water conventionally, preparing Weigart dye liquor according to the conventional freshness, and dyeing for 3-5 minutes;

differentiation was carried out for 15 seconds by a hydrochloric acid-ethanol solution, followed by light washing with distilled water for 1 minute;

dyeing with a fast green dye for 5 minutes, and then washing with distilled water for 1 minute;

dip dyeing with safranin o (safranin o) dye for 2 minutes followed by light washing with distilled water for 1 minute;

the sections were washed gently with acetic acid solution for 1 minute to further remove residual dye, and then washed gently with distilled water for 1 minute;

dehydrating the mixture by ethanol with the volume percentage concentration of 95% and absolute ethanol in sequence, then performing xylene transparency, and sealing the mixture by using optical resin for subsequent observation and evaluation;

2.2.2.3 HE staining

Dewaxing the paraffin sections to distilled water conventionally, and preparing hematoxylin and eosin ethanol dyes according to the conventional method;

staining with hematoxylin for 10-30 min, and slightly washing with tap water to turn blue;

carrying out differentiation for about 15 seconds by using a hydrochloric acid-ethanol solution until the slices become light red, and then slightly washing by using tap water flow to recover the color to blue;

and (3) dehydration I: sequentially dehydrating the mixture by using 50 percent ethanol by volume, 70 percent ethanol by volume and 80 percent ethanol by volume for 3 to 5 minutes respectively;

re-dyeing for 1-3 minutes by using an eosin ethanol dye with the mass-volume ratio concentration (g/ml) of 0.5 percent;

and (II) dehydration: dehydrating the mixture by ethanol with the volume percentage concentration of 95% and absolute ethanol for 3-5 minutes respectively, carrying out dimethylbenzene transparentization twice after air drying, and sealing the mixture by using optical resin for subsequent observation and evaluation;

2.2.2.4 miR-17-5p-FISH detection

FISH of miR-17-5p is performed according to the international standard protocol. Digoxigenin (DIG) -labeled miR-17-5p oligonucleotide probes were purchased from Qiagen for labeling miR-17-5p molecules in tissue sections; DAPI staining was used to visualize the nuclei.

In brief, after collecting the left knee joint of the mouse, fixing the left knee joint by using 4% paraformaldehyde with mass-volume ratio concentration (g/ml), decalcifying the left knee joint by using 10% EDTA solution with mass-volume ratio concentration (g/ml), immersing the left knee joint in sucrose solution for gradient dehydration, embedding and freezing the left knee joint in a compound (OCT) with the optimal cutting temperature, and slicing the left knee joint in a thickness of 10 mu m; sections were permeabilized with proteinase K for 5 minutes at 37 ℃ and treated with 3% hydrogen peroxide water by volume concentration to block endogenous peroxidase activity; slides bearing frozen sections of cartilage tissue were then incubated overnight with the relevant probe (1 nM); finally, carrying out color development by using digoxin-peroxidase resistance (DIG-POD resistance) and Cy 3-tyramide (dissolved in hydrogen peroxide water with the volume percentage concentration of 0.001%), carrying out DAPI staining for cell nucleus color development, and further observing the statistical result;

2.2.3 introduction of the OARSI score of the International Association for osteoarthritis, scored by two observers to assess the extent of cartilage destruction, with higher scores evidencing greater severity of destruction;

2.2.4 As shown in FIG. 2A, with safranin O-fast green staining, the DMM mouse model is increased with time, and the distribution of cartilage tissues deeply stained on the surface of the bone joint is disordered and even faulted, which shows that the integrity of the cartilage tissues in the bone joint is progressively destroyed in the model; as shown in fig. 2A, loss of tissue integrity and continuity of the articular cavity surface was also observed by HE staining, indicating that the degree of articular cartilage degradation in mice progressively worsened after DMM surgery; FISH detection shows that the miR-17-5p expression quantity is remarkably reduced along with progressive worsening of the mouse articular cartilage degradation degree; as shown in fig. 2B, the OARSI score of mice in the operative group was about 2-fold that of the control group at week 4 after the operation, and stabilized at weeks 8 and 12, which was about 5-fold that of the control group; the number of miR-17-5p positive cells on the articular surface of mice in the operation group is about one half of that in the control group at the 4 th week after the operation, and the cells tend to be stable at the 8 th and 12 th weeks and are about one quarter of that in the control group. The legend and the experimental results prove that the mouse DMM model is successfully constructed, and the expression level of miR-17-5p is closely related to the deterioration degree of cartilage degradation and osteoarthritis.

2.3 miR-17-5p can inhibit osteoarthritis pathological process

2.3.1 miR-17-5p analogue can effectively repair bone joint soft tissue injury and dysfunction in mouse DMM model

2.3.1.1 mice of 10 weeks of age were anesthetized with ketamine and xylazine and subjected to surgery to transect the medial meniscal ligament of the left knee, destroying the cartilage of the left knee and inducing osteoarthritis associated with meniscal instability. The mice in the control group were only subjected to sham surgery treatment without transecting the medial meniscal ligament of the left knee joint;

2.3.1.2 Agomir-17 (1.5 nmol) was dissolved in 8. mu.l of phosphate buffer and injected into the mouse joint cavity 1 time a week for 4 times starting 4 weeks after DMM surgery; injecting Agomir-NC into mice in a control group;

2.3.1.3 at 8 weeks after DMM surgery, the left knee joints of three groups of mice (sham surgery group, surgery and Agomir-NC injection group, surgery and Agomir-17 injection group) were collected after euthanasia, fixed for 48 hours after 4% paraformaldehyde in mass-to-volume concentration (g/ml), then decalcified for 10 days with 10% Ethylene Diamine Tetraacetic Acid (EDTA) solution in mass-to-volume concentration (g/ml), the trimmed tissues and corresponding labels were placed in a dehydration box, embedded with paraffin after dehydration, further sliced and dewaxed to distilled water, and the structural integrity of articular cavity cartilage tissues was examined by safranin O-fast green staining, the operation was the same as 2.2.2.1-2 in example 1;

2.3.1.4 As shown in figure 3A, in the mouse DMM model, after 4 weeks of joint cavity injection treatment of miR-17-5p analogue Agomir-17, the articular cartilage layer structure is complete, while the cartilage tissue in the Agomir-NC treatment group is unevenly distributed and discontinuous, and the integrity is destroyed; accordingly, the OARSI score for the Agomir-17 treatment group was significantly lower than for the Agomir-NC treatment group, tending towards the sham group;

2.3.2 knocking out miR 17-92 cluster gene leads to pathological changes similar to osteoarthritis in joint cavity, and miR-17-5p analogue can effectively reverse corresponding pathological changes

2.3.2.1 miR-17～92^fl/flMouse and Col2CreER^TMouse hybridization to obtain Col2CreER^T；miR-17～92^fl/+Mice, and in turn Col2CreER^T；miR-17～92^fl/+Mice and miR-17-92^fl/flMouse is hybridized to obtain Col2CreER^T；miR-17～92^fl/flIn mice (miR-17-92 cKO mice), miR-17-92 cluster genes in chondrocytes and other cells which usually express endogenous type II collagen genes in miR-17-92 cKO mice can be inductively knocked out;

2.3.2.2 activator tamoxifen of estrogen receptor ER is purchased from Sigma company, 7-week-old miR-17-92 cKO mice receive tamoxifen with a dose of 100 mug/g through intraperitoneal injection, the dose is once a day for 6 days, and miR-17-5p is inductively knocked out;

2.3.2.3 Agomir-17 (1.5 nmol) was dissolved in 8. mu.l of phosphate buffer, and 1 Agomir-17 dilution was injected into the mouse joint cavity 4 times a week from 4 weeks after induction knockout; the control group mice are injected with Agomir-NC, and the blank control group is miR-17-92^fl/flA mouse;

2.3.2.4 at 8 weeks after induction knockout, collecting the left knee joints of three groups of mice (blank control group, induction and injection of Agomir-NC group, induction and injection of Agomir-17 group) after euthanasia, fixing for 48 hours after 4% paraformaldehyde with mass-volume ratio concentration (g/ml), decalcifying for 10 days with 10% Ethylene Diamine Tetraacetic Acid (EDTA) solution with mass-volume ratio concentration (g/ml), placing the trimmed tissues and corresponding labels in a dehydration box, embedding paraffin after dehydration, further slicing and dewaxing into distilled water, and checking the structural integrity of articular cavity cartilage tissues by safranin O-fast green staining, which is the same as 2.2.2.1-2 in example 1;

2.3.2.5 As shown in figure 3B, after miR-17-5p is knocked out, joint cavity injection treatment of miR-17-5p analogue Agomir-17 is carried out for 4 weeks, the structure of the articular cartilage layer is complete, and cartilage tissues in an Agomir-NC treatment group are unevenly and discontinuously distributed and the integrity is damaged; correspondingly, the OARSI score of the Agomir-17 treated group was significantly lower than that of the Agomir-NC treated group, tending toward the blank control group;

in conclusion, the expression level of miR-17-5p is highly related to the pathological deterioration degree of the bone joint in a mouse DMM model, and miR-17-5p can widely target 4 matrix degrading enzymes related to the bone joint cartilage matrix, including matrix metallopeptidase MMP3 and MMP13, cartilage proteoglycan ADAMTS5 and nitric oxide synthase NOS2, and maintain the structural integrity and the function of the joint cartilage tissue.

Example 2

1. Test object

Non-arthritic normal knee joint or hip joint cartilage tissue was surgically obtained from 6 osteosarcoma or trauma patients (1 male and 5 female, median age 53 years), and osteoarthritic cartilage specimens were obtained from 18 osteoarthritic patients (4 male and 14 female, median age 66 years) within 2 hours after joint reconstruction, and patients were graded and diagnosed according to the osteoarthritis diagnostic criteria of the american college of rheumatology. The collection of waste human tissue was approved by the research ethics committee of the affiliated hospital of xu medical university, and all patients participating in the study provided written informed consent.

2. Experimental methods

2.1 extraction of total RNA from human cartilage tissue and specific reverse transcription

The collected human cartilage Tissue was disrupted to homogenate by Tissue lyser-48L (purchased from kyoto new biomedical limited, shanghai), total RNA extraction was performed according to RNeasy fibers Tissue Mini Kit (purchased from Qiagen), then 0.5 μ g of total RNA was used for reverse transcription to synthesize cDNA library, and the reaction system was configured according to the reverse transcription Kit instructions of Takara in combination with the designed miR-17-5p specific stem-loop primers, substantially as follows:

components	Volume of addition
		5×PrimeScript buffer	4μl
PrimeScript RT Enzyme Mix I	1μl
		Stem loop primer	1μl
Total RNA	0.5μg
		RNase Free dH2O	Up to 20μl

Wherein the stem-loop Primer sequences are as follows (designed using software Primer Premier 5.0):

5'-gtcgtatccagtgcagggtccgaggtattcgcactggatacgacctacct-3', respectively; (as shown in SEQ ID NO. 2)

After fully mixing, centrifuging for a short time, placing on a PCR instrument, and running a program, wherein the reverse transcription program comprises the following steps:

15 minutes at 37 ℃; 5 seconds at 85 ℃; infinity at 4 ℃;

the reverse transcribed cDNA products were immediately used for Quantitative RT-PCR (qRT-PCR) detection or stored at-20 ℃;

2.2 detection of miR-17-5p relative expression level by human cartilage tissue qRT-PCR

A reaction system with a total volume of 20. mu.l was prepared according to the SYBR Green mix qRT-PCR kit from TaKaRa, all primers were diluted to 10. mu.M, and the cDNA library synthesized in example 2 at 2.1 was diluted at a ratio of 1:10, as follows:

components	Volume of addition
		cDNA	1μl
2×SYBR Green qPCR Super Mix	10μl
		Upstream primer (10. mu.M)	0.5μl
Upstream primer (10. mu.M)	0.5μl
		ROX	0.4μl
RNase Free dH2O	7.6μl

Wherein the upstream and downstream Primer sequences are respectively (designed by software Primer Premier 5.0):

an upstream primer: 5'-cgcaaagtgcttacagtgc-3', respectively; (as shown in SEQ ID NO. 3)

A downstream primer: 5'-agtgcagggtccgaggtatt-3' are provided. (as shown in SEQ ID NO. 4)

After loading, centrifugation was carried out at 2000rpm for 1 minute, and the program was run in an ABI 7500 Fast System (Applied Biosystems) apparatus:

10 minutes at 95 ℃ (15 seconds at 95 ℃, 30 seconds at 56 ℃, 30 seconds at 72 ℃) multiplied by 40 cycles, and a dissolution curve is added to analyze the specificity of the primer;

2.3 data analysis

After the amplification of the qRT-PCR instrument is finished, observing a dissolution curve and copying a CT value, and according to a fluorescent quantitative PCR relative quantitative formula 2^-△△CtSample fold expression calculation was performed, and the results were analyzed using SPSS software for Student's t-test, which is considered to be when P<0.05 has statistical significance.

2.4 As shown in figure 4, the expression level of miR-17-5p in normal human articular cartilage is far higher than that of osteoarthritis patients, about 2 times of the early stage and 4 times of the middle and late stages, which suggests that the expression patterns of miR-17-5p in human cartilage and mouse cartilage are similar. These findings indicate that miR-17-5p can be used for clinical osteoarthritis treatment.

Example 3

The following cellular experimental procedures were all performed in a GMP laboratory in compliance with clinical standards.

1. The safety of the single miR-17-5p micromolecule injection or miR-17-5p analogue Agomir-17 injection in the joint cavity is difficult to control, and the side effect is difficult to evaluate; clinical trials on the treatment of the MSCs are carried out in various diseases, and the safety, reliability and effectiveness are evaluated to a certain extent; agomir-17 is pretreated by MSCs to obtain miR-17-5p autonomous and continuous expression capacity, and miR-17-5p combined cell therapy is established by taking the MSCs as a carrier, so that the method is a safer and more effective potential strategy for clinically treating osteoarthritis.

2. Preparation of MSCs (messenger ribonucleic acids) and preparation of MSCs for autonomously and continuously expressing miR-17

2.1 preparation and quality control of MSCs

2.1.1 human umbilical cord tissue samples are placed in physiological saline buffer containing antibiotics, transported to GMP cells on ice, washed with physiological saline buffer containing antibiotics, transferred to a clean petri dish, stripped to remove vascular tissue, the Fahrenheit gel fraction is taken, washed and cut into about 1mm³Adding collagenase with the mass-volume ratio concentration (g/ml) of 0.2% into the tissue blocks, transferring the tissue blocks into a 15ml centrifuge tube, and putting the centrifuge tube into a water bath kettle at 37 ℃ for digestion for 1 hour, wherein similarly, the tissue sources such as human fat, skin and the like are also suitable;

2.1.2 preparing an alpha-MEM complete medium containing 5% by volume of KSR (human serum replacement), 0.032% by volume of sodium heparin, terminating the digestion, followed by filtering out the undigested tissue mass with a 70 μm cell sieve and centrifuging for 5 minutes at 1200rpm, the centrifuge setting parameter;

2.1.3 washing with complete Medium 2 times, after counting according to 5X 10⁶Cell density per ml plated at 37 ℃ in 5% CO₂Culturing in a humid incubator; removing non-adherent cells when the cells grow to reach the confluence degree of about 80%, and carrying out passage according to a ratio of 1:3 after digestion;

2.1.4 performing mycoplasma pollution detection, and determining that the cultured cells are not polluted by mycoplasma;

2.1.5 the cultured cells can grow adherent to the wall through visual observation; the cultured cells have the capacity of differentiating into fat cells, chondroblasts and osteoblasts through a three-line differentiation test; the cultured cells expressed positive cell surface markers CD44, CD73, CD90 and CD105 on the surface and did not express negative cell surface markers CD45, CD19, CD11b, CD34 and HLA-DR by flow identification of the cells. The three points prove that the cultured cells are MSCs;

2.2 preparation of MSCs containing miR-17-5p expression modules

2.2.1 synthetic Agomir-17 from RiboBio (RiboBio) science and technology, Inc., Guangzhou;

2.2.2 MSCs cultured in step 2.1 of example 3 were cultured to a density of 70% and the KSR halved medium was replaced 1 hour before transfection;

2.2.3 willHD (purchased from Promega corporation) reagent and Agomir-17 solution were equilibrated to room temperature;

2.2.41.5 nmol of Agomir-17 was dissolved in 50. mu.l of RNase-free ultrapure water and stored at a concentration of 30. mu.M, and 1. mu.l was diluted in 49. mu.l of Opti-MEM medium at the time of transfection to a final concentration of 600 nM; getHD 0.1μl/cm²Mixing with the Agomir-17 diluent, incubating at room temperature for 5-15min, and adding into cell culture system;

2.2.5, continuing to culture for 6-16h after transfection, and replacing the culture medium with a complete culture medium to obtain the successfully prepared MSCs containing the miR-17-5p expression components.

Sequence listing

<110> cinnarizine

<120> application of miRNA and human mesenchymal stem cells in preparation of medicine for treating arthritis

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

caaagugcuu acagugcagg uag 23

<210> 2

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacctacct 50

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgcaaagtgc ttacagtgc 19

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

agtgcagggt ccgaggtatt 20