Stem cell and preparation for repairing meniscus and preparation method of stem cell and preparation

Abstract

The invention relates to the technical field of bone joint repair, and provides a stem cell for repairing meniscus, a preparation and a preparation method thereof. It is found for the first time that hsacirc0000511 is used for promoting mesenchymal stem cells to be differentiated into osteoblasts and applied to meniscus injury repair, and the pharmaceutical composition prepared from hsacirc0000511 in cooperation with chondroitin sulfate and sodium hyaluronate can be remarkably used for meniscus repair, effectively relieves pain of patients and has wide application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of meniscus repair, and in particular to a stem cell for repairing meniscus, a preparation and a preparation method thereof. Background Art

[0002] The meniscus is a C-shaped buffer structure composed of elastic cartilage located between the two main bones that make up the knee joint. The meniscus can act as a buffer to prevent damage to the articular surface cartilage caused by impact. Meniscus injury can be caused by trauma or degeneration. Meniscus injury caused by severe trauma may be complicated by soft tissue damage in the knee, such as collateral ligament injury, cruciate ligament injury, joint capsule injury, cartilage surface injury, etc., which is often the cause of swelling after injury.

[0003] Therefore, there is an urgent need for a method that can effectively repair meniscus damage. With the development of regenerative medicine technology, stem cells have played an important role in the treatment of knee osteoarthritis due to their multidirectional differentiation, immunomodulation, anti-inflammatory and other properties, but there are few reports on stem cells in meniscus repair, and there is currently a lack of effective methods and drugs for meniscus repair. Summary of the invention

[0004] In view of the technical problems existing in the prior art, the present invention provides a stem cell, preparation and preparation method for meniscus repair. The present invention first discovered hsa_circ_0000511 to promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts, and applied it to meniscus injury repair. The pharmaceutical composition prepared with chondroitin sulfate and sodium hyaluronate can be significantly used for meniscus repair, effectively relieve the pain of patients, and has a broad application prospect.

[0005] Specifically, the present invention first provides an hsa_circ_0000511 that can promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts, and the nucleotide sequence of the hsa_circ_0000511 is shown in SEQ ID NO.1.

[0006] Furthermore, the present invention also provides a bone marrow mesenchymal stem cell overexpressing hsa_circ_0000511, in which hsa_circ_0000511 is overexpressed.

[0007] Furthermore, the present invention also provides the use of hsa_circ_0000511 or bone marrow mesenchymal stem cells overexpressing hsa_circ_0000511 in the preparation of a medicament for meniscus repair.

[0008] Furthermore, the present invention also provides a composition for meniscus repair, comprising bone marrow mesenchymal stem cells overexpressing hsa_circ_0000511, chondroitin sulfate and sodium hyaluronate.

[0009] Preferably, the composition comprises 1×10 4 -1×10 7 cells / g bone marrow mesenchymal stem cells overexpressing hsa_circ_0000511, 15-35mg / g chondroitin sulfate and 10-15mg / g sodium hyaluronate.

[0010] Preferably, the composition comprises 1×10 5 bone marrow mesenchymal stem cells overexpressing hsa_circ_0000511, 15 mg / g chondroitin sulfate, and 10 mg / g sodium hyaluronate.

[0011] Preferably, the composition comprises 1×10 6 bone marrow mesenchymal stem cells overexpressing hsa_circ_0000511, 25 mg / g chondroitin sulfate, and 12 mg / g sodium hyaluronate.

[0012] Preferably, the composition comprises 1×10 7 bone marrow mesenchymal stem cells overexpressing hsa_circ_0000511, 35 mg / g chondroitin sulfate, and 15 mg / g sodium hyaluronate.

[0013] Furthermore, the present invention also provides a method for preparing a composition for meniscus repair, the method comprising the following steps: 1) Isolate and obtain bone marrow mesenchymal stem cells from the bone marrow; 2) Subculturing the bone marrow mesenchymal stem cells prepared in step 1) for three consecutive subcultures before being used for later use; 3) Construction of overexpression vector of hsa_circ_0000511 gene; 4) The overexpression vector of the hsa_circ_0000511 gene constructed in step 3) is transferred into the bone marrow mesenchymal stem cells in step 2), and then freeze-dried for later use; 5) Weigh chondroitin sulfate and sodium hyaluronate in order by weight, mix them evenly and set aside; 6) Compounding the freeze-dried bone marrow mesenchymal stem cells in step 4) with the mixture obtained in step 5), mixing them evenly, and then drying and granulating them to obtain the pharmaceutical composition.

[0014] Furthermore, the present invention also provides the use of the composition in preparing a product for meniscus repair.

[0015] The advantages of the present invention are as follows: the present invention discovered for the first time hsa_circ_0000511 to promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts, and applied it to meniscus injury repair. The pharmaceutical composition prepared with chondroitin sulfate and sodium hyaluronate can be significantly used for meniscus repair, effectively relieve the pain of patients, and has a broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 .Analysis of differentiation of bone marrow mesenchymal stem cells into osteoblasts.

[0017] Figure 2 .Statistical analysis of meniscus repair scores. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0019] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0020] Example 1 The inventors previously analyzed the effect of transcriptome sequencing on cell differentiation by using 11. The specific method is as follows: hsa_circ_0000511 was artificially synthesized, and its nucleotide sequence was shown in SEQ ID NO. 1. An overexpression vector containing the hsa_circ_0000511 gene was constructed and transferred into bone marrow mesenchymal stem cells, and the cell differentiation of bone marrow mesenchymal stem cells was analyzed by RT-PCR experiment.

[0021] The results are as follows Figure 1 As shown: After overexpressing hsa_circ_0000511 in bone marrow mesenchymal stem cells, the expression of osteoblast markers RUNX2, COL-1 and ALP can be clearly detected to show an increasing trend. Compared with the blank control, overexpression of hsa_circ_0000511 in bone marrow mesenchymal stem cells significantly promotes their differentiation into osteoblasts. The above tests confirm that bone marrow mesenchymal stem cells overexpressing hsa_circ_0000511 can be used for meniscus repair treatment.

[0022] Example 2 A stem cell composition for repairing meniscus, comprising 1×10 5bone marrow mesenchymal stem cells overexpressing hsa_circ_0000511, 15 mg / g chondroitin sulfate, and 10 mg / g sodium hyaluronate.

[0023] A method for preparing a stem cell composition for repairing meniscus, the method comprising the following steps: 1) Isolate and obtain bone marrow mesenchymal stem cells from the bone marrow; 2) Subculturing the bone marrow mesenchymal stem cells prepared in step 1) for three consecutive subcultures before being used for later use; 3) Construction of overexpression vector of hsa_circ_0000511 gene; 4) The overexpression vector of the hsa_circ_0000511 gene constructed in step 3) is transferred into the bone marrow mesenchymal stem cells in step 2), and then freeze-dried for later use; 5) Weigh chondroitin sulfate and sodium hyaluronate in order by weight, mix them evenly and set aside; 6) Compounding the freeze-dried bone marrow mesenchymal stem cells in step 4) with the mixture obtained in step 5), mixing them evenly, and then drying and granulating them to obtain the pharmaceutical composition.

[0024] Example 3 A stem cell composition for repairing meniscus, comprising 1×10 6 bone marrow mesenchymal stem cells overexpressing hsa_circ_0000511, 25 mg / g chondroitin sulfate, and 12 mg / g sodium hyaluronate.

[0025] The steps for preparing the stem cell composition are the same as those in Example 2.

[0026] Example 4 A stem cell composition for repairing meniscus, comprising 1×10 7 bone marrow mesenchymal stem cells overexpressing hsa_circ_0000511, 35 mg / g chondroitin sulfate, and 15 mg / g sodium hyaluronate.

[0027] The steps for preparing the stem cell composition are the same as those in Example 2.

[0028] Verification Example Construction of meniscus injury model: 40 adult rats were selected, and their bilateral knee joints were shaved, iodine-prepared, and general anesthesia was performed intravenously. The rat's knee joint was moved to palpate the apex of the angle between the femur and the tibia behind the rat's knee joint. Based on this and the joint gap, the needle insertion position was marked about 3 mm forward along the joint gap at the posterior and lateral edge of the knee joint. Routine disinfection and laying of the towel were performed, and the marked point on the skin of the joint gap was vertically inserted to a depth of about 3 to 4 mm. During the insertion process, there was a relatively tough resistance at the beginning, and then the resistance decreased and disappeared. Cut the body of the lateral meniscus by sliding up and down in a direction perpendicular to the long axis of the meniscus. The blade was moved up and down to the bone of the tibia and femur, and the lateral meniscus was transected to complete the model construction.

[0029] The rats with successful modeling were divided into experimental and control groups, where experimental groups 1-3 were treated with the compositions in Examples 2-4 of the present invention, taking 15 mg / kg every morning and evening; the composition in the control group was compared with Example 2, except that the bone marrow mesenchymal stem cells were not transferred into the hsa_circ_0000511 overexpression vector, and the rest were the same. The experimental and control groups were continuously administered and monitored for one month. The meniscus repair scoring system was used to evaluate the results of meniscus defect repair.

[0030] The results are as follows Figure 2 As shown, compared with the control group, the meniscus repair in the experimental group was significantly improved, and the difference was extremely significant. Through the above comparison, it can be obtained that the composition obtained by the present invention can be used to repair meniscus damage.

[0031] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A hsa_circ_0000511 capable of promoting the differentiation of bone marrow mesenchymal stem cells into osteoblasts, characterized in that: The nucleotide sequence of hsa_circ_0000511 is shown in SEQ ID NO.

1.

2. A bone marrow mesenchymal stem cell overexpressing hsa_circ_0000511, characterized in that: hsa_circ_0000511 is overexpressed in the bone marrow mesenchymal stem cells.

3. Application of hsa_circ_0000511 or bone marrow mesenchymal stem cells overexpressing hsa_circ_0000511 in the preparation of drugs for meniscus repair.

4. A composition for meniscus repair, characterized in that: The composition includes bone marrow mesenchymal stem cells overexpressing hsa_circ_0000511, chondroitin sulfate and sodium hyaluronate.

5. The composition according to claim 4, characterized in that Includes 1×10 4 -1×10 7 cells / g bone marrow mesenchymal stem cells overexpressing hsa_circ_0000511, 15-35mg / g chondroitin sulfate and 10-15mg / g sodium hyaluronate.

6. The composition according to claim 5, characterized in that Includes 1×10 5 bone marrow mesenchymal stem cells overexpressing hsa_circ_0000511, 15 mg / g chondroitin sulfate, and 10 mg / g sodium hyaluronate.

7. The composition according to claim 5, characterized in that Includes 1×10 6 bone marrow mesenchymal stem cells overexpressing hsa_circ_0000511, 25 mg / g chondroitin sulfate, and 12 mg / g sodium hyaluronate.

8. The composition according to claim 5, characterized in that Includes 1×10 7 bone marrow mesenchymal stem cells overexpressing hsa_circ_0000511, 35 mg / g chondroitin sulfate, and 15 mg / g sodium hyaluronate.

9. A method for preparing a composition for meniscus repair, characterized in that: The method comprises the following steps: 1) Isolate and obtain bone marrow mesenchymal stem cells from the bone marrow; 2) Subculturing the bone marrow mesenchymal stem cells prepared in step 1) for three consecutive subcultures before being used for later use; 3) Construction of overexpression vector of hsa_circ_0000511 gene; 4) The overexpression vector of the hsa_circ_0000511 gene constructed in step 3) is transferred into the bone marrow mesenchymal stem cells in step 2), and then freeze-dried for later use; 5) Weigh chondroitin sulfate and sodium hyaluronate in order by weight, mix them evenly and set aside; 6) Compounding the freeze-dried bone marrow mesenchymal stem cells in step 4) with the mixture obtained in step 5), mixing them evenly, and then drying and granulating them to obtain the pharmaceutical composition.

10. Use of the composition according to any one of claims 4 to 8 in the preparation of a product for meniscus repair.

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