Application of WAC knock-down adenovirus in preparation of medicine for preventing and treating arthritis cartilage injury

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses application of WAC knock-down adenovirus in preparation of medicine for preventing and treating arthritis cartilage injury, WAC protein genes are inserted into PLVX lentiviral vectors to construct transfection plasmids, the transfection plasmids and virus component expression plasmids PxpaX2 and PMD2G are co-transfected in HEK 293T cells, virus supernate is collected after 48 hours, and the WAC knock-down adenovirus is obtained. The amino acid sequence of the WAC protein gene is as shown in SEQ ID NO. 1. Compared with the prior art, after the WAC is knocked down, the generation of cartilage cell matrix metalloproteinases MMP3 and MMP13 and chemotactic factors CCL2 and CCL3 is reduced, so that the cartilage injury caused by arthritis can be prevented and treated; compared with the existing clinical therapy, the technology starts from the pathogenesis of arthritis, and the purpose of treating the arthritis cartilage injury is achieved by a method of reducing cell WAC protein expression and reducing abnormal secretion of cartilage cells; finally, compared with the existing lentivirus / adenovirus therapy, the invention has stronger targeting to cartilage cells, and is safer and more effective.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the use of a WAC knockout adenovirus in the preparation of a drug for preventing and treating arthritis cartilage damage. Background Art

[0002] Cartilage is an important component of synovial joints. Chondrocytes are the only cells in cartilage and are embedded in a dense and highly organized extracellular matrix. The extracellular matrix (ECM) of cartilage is synthesized by chondrocytes and consists of a collagen network, mainly containing type II collagen, as well as glycosaminoglycans such as hyaluronic acid and various proteoglycans. Chondrocytes embedded in the ECM cause changes in the ECM through direct or indirect effects, destroying the integrity of the cartilage and even having a profound impact on adjacent cells and tissues, ultimately leading to joint dysfunction. Osteoarthritis (OA) and rheumatoid arthritis (RA) are the two most common diseases of articular cartilage damage, which have a serious impact on social development, medical expenses and the daily lives of many patients. Although they have different backgrounds and causes of onset, the production of inflammatory mediators such as TNF and IL1B and damage to articular cartilage are their common characteristics. In RA and OA, cartilage damage has a self-reinforcing and self-perpetuating effect. Under the stimulation of inflammatory mediators, chondrocytes will produce enzymes that can degrade cartilage matrix, such as matrix metalloproteinases (MMP3, MMP13, etc.) and chemokines (CCL2, CCL3, etc.). The degraded matrix and chemotactic inflammatory cells will further aggravate cartilage inflammation. However, there is still a lack of research on the common mechanism of cartilage damage in arthritis such as RA and OA. A more in-depth study of this mechanism may be of great significance for the prevention and treatment of cartilage damage in arthritis.

[0003] Abnormal secretion of matrix-degrading enzymes such as MMP family by chondrocytes degrades cartilage matrix and causes cartilage damage, which is also a common pathological manifestation of RA and OA. Although a large number of experiments have proved the core role of these enzymes in RA and OA, almost all clinical trials involving MMP inhibitors have failed, including MMP2, MMP3, MMP9, MMP13, etc., and the side effects of MMP inhibitors related to musculoskeletal toxicity have always been an unresolved problem.

[0004] Although the diagnosis and treatment of arthritis have made great progress in recent years, the exact cause of the disease has not yet been confirmed, and there is still no way to prevent and treat cartilage damage. Currently, the drugs for arthritis are mainly non-steroidal anti-inflammatory drugs. Although long-term use can inhibit the development of inflammation, it is difficult to stop or reverse the trend of cartilage damage, and patients eventually need surgery to solve the problem. Although a large number of experiments have proven the core role of matrix degrading enzymes such as the MMP family in RA and OA, almost all clinical trials involving MMP inhibitors have ended in failure. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides the use of WAC knock-down adenovirus in the preparation of drugs for preventing and treating arthritis cartilage damage.

[0006] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0007] One of the technical solutions of the present invention is to provide the use of WAC knock-down adenovirus in the preparation of a drug for preventing and treating arthritis cartilage damage. The WAC protein gene is inserted into a PLVX lentiviral vector to construct a transfection plasmid and co-transfected with viral component expression plasmids PxpaX2 and PMD2G into HEK 293T cells. After 48 hours, the viral supernatant is collected to obtain the WAC knock-down adenovirus. The amino acid sequence of the WAC protein gene is shown in SEQ ID NO.1.

[0008] The second technical solution of the present invention is to provide a drug for preventing and treating arthritis cartilage damage, comprising the WAC knock-down adenovirus.

[0009] Furthermore, the drug for preventing and treating arthritis cartilage damage also includes a pharmaceutically acceptable carrier and / or excipient.

[0010] Compared with the existing technology, the production of chondrocyte matrix metalloproteinases MMP3, MMP13 and chemokines CCL2, CCL3 is reduced after knocking down WAC, and therefore it can prevent and treat arthritis cartilage damage; compared with the current clinical therapies, this technology starts from the cause of arthritis and achieves the purpose of treating arthritis cartilage damage by reducing the expression of cellular WAC protein and reducing the abnormal secretion of chondrocytes; finally, compared with the existing lentivirus / adenovirus therapy, this invention has a stronger targeting of chondrocytes, and is safer and more effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : A is the protein quantitative omics detection process of the control group and TNF stimulation group of human knee joint-derived chondrocytes; B is the differential protein heat map analysis, showing that the protein expression changed significantly after stimulation; C is the GO-BP enrichment analysis result, showing that the changed genes are concentrated in the H2Bbu1 regulatory pathway; D is the expression of H2Bbu1 regulatory pathway molecules, showing that WAC changes are the most obvious.

[0012] Figure 2 RT-qPCR results of the control group and TNF-stimulated group of chondrocytes derived from human knee joints.

[0013] Figure 3 Western blot results of the control group and TNF-stimulated group of chondrocytes derived from human knee joint.

[0014] Figure 4 These are the results of safranin fast green staining of cartilage damage in arthritic mice in the rAAV2-control group and rAAV2-Sh-WAC group.

[0015] Figure 5 These are the results of tissue immunochemical staining of the secretory phenotype of cartilage damage in arthritic mice in the rAAV2-control group and rAAV2-Sh-WAC group. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0017] The WAC protein gene is an important intracellular connexin that plays an important role in regulating histone H2B monoubiquitination and cell autophagy. Its amino acid sequence is shown in SEQ ID NO. 1. Guangzhou Aiji Biotechnology Co., Ltd. was commissioned to synthesize the WAC protein gene fragment.

[0018] Guangzhou Aiji Biotechnology Co., Ltd. was commissioned to synthesize WAC knockdown adenovirus. The lentivirus preparation process is as follows:

[0019] 1. Construction of transfection plasmid:

[0020] Select vector: PLVX is selected as the vector. PLVX is an adenovirus vector commonly used for gene overexpression or knockdown. The vector sequence is shown in SEQ ID NO.2.

[0021] Cloning of target gene: Clone the target gene, WAC, into the PLVX vector by restriction endonuclease digestion and ligation. Synthesize the cDNA of WAC gene in advance and ligate it according to the multiple cloning sites provided by the vector. For gene overexpression construction, the target gene should have a promoter to drive its expression.

[0022] 2. Prepare viral vectors and viral component expression plasmids:

[0023] Virus packaging plasmid: The components required for virus packaging need to be transfected into HEK 293T cells. This step uses two plasmids, PxpaX2 and PMD2g (sequences see SEQ ID NO.3 and SEQ ID NO.4):

[0024] PxpaX2: Provides the envelope and core proteins of adenovirus, allowing efficient expression of adenovirus in HEK 293T cells.

[0025] PMD2g: ​​Provides adenovirus packaging proteins and enzymes required for the viral life cycle.

[0026] 3. Co-transfection of HEK 293T cells:

[0027] Cell culture: HEK 293T cells were cultured in an incubator maintained at 37°C and 5% CO. 2 until the cell density reaches 60-70%.

[0028] Transfection plasmids: Use a liposome transfection reagent (such as Lipofectamine 2000 or JetPRIME) to co-transfect three plasmids:

[0029] PLVX-Spartin

[0030] PxpaX2 (virus-encapsulated plasmid)

[0031] PMD2g (viral packaging plasmid)

[0032] During transfection, adjust the amount of DNA according to the instructions of the transfection reagent and ensure that the ratio of each plasmid is appropriate to obtain efficient virus packaging.

[0033] 4. Collection of viral supernatant:

[0034] Culture and collection of viral supernatant: 48 hours after transfection, wash HEK 293T cells with sterile PBS or culture medium, collect the viral supernatant in the culture medium, and obtain WAC knockdown adenovirus WAC ShRNA. At this point, the viral particles have been released outside the cells and can be used to infect target cells.

[0035] Virus Concentration: If necessary, the viral titer of WAC knockdown adenovirus WAC shRNA can be increased by concentrating the viral supernatant by centrifugation or ultracentrifugation.

[0036] 5. Virus infection target cells:

[0037] Infect target cells: The collected viral supernatant (WAC knockdown adenovirus WAC ShRNA) is transduced into target cells.

[0038] Transfection conditions: Viruses can be infected into target cells using a variety of methods such as centrifugation, chemical transfection, or direct inoculation.

[0039] Evaluation of infection effect: 48 hours after infection, check the phenotype or marker expression of cells, and use Western blot, RT-qPCR or immunofluorescence to verify whether the overexpression or knockdown of WAC is effective.

[0040] 6. Subsequent analysis:

[0041] Knockdown confirmation: The protein level of WAC was analyzed by Western blot, and RT-qPCR was used to confirm whether the transcription level of the WAC gene changed, and the corresponding function was detected.

[0042] 7. Storage and further use of viruses:

[0043] Virus storage: Virus supernatant can be stored at -80℃ for subsequent experimental use.

[0044] Virus titer determination: To quantify the infectivity of the virus, it may be necessary to determine the virus titer in cell culture (e.g., by qPCR or by infecting specific cells and measuring fluorescence or staining reactions).

[0045] Human knee joint chondrocytes were transfected with WAC knockdown adenovirus and control empty vector lentivirus for 48 hours, as follows. 5 The chondrocytes were plated in 12-well plates. Human knee joint chondrocytes were transfected with WAC shRNA and control lentivirus for 48 hours, and the culture medium containing 10 ng / mL tumor necrosis factor (TNF) was replaced to stimulate the human knee joint chondrocytes for 24 hours. The cells were sent to Lianchuan Biotechnology Co., Ltd. for proteomic detection, and the differential proteins between the two groups were compared. The results are as follows Figure 1 As shown by Figure 1 It can be seen that the protein is enriched in the histone H2B ubiquitination modification pathway. Further analysis of the changes in the expression levels of related molecules in this pathway showed that the expression difference of WAC was the most significant.

[0046] RT-qPCR was used to detect the expression of matrix metalloproteinases MMP3, MMP13 and chemokines CCL2 and CCL3 in chondrocytes. Figure 2 As shown; Western blot was used to detect the levels of chondrocyte matrix metalloproteinases MMP3, MMP13 and chemokines CCL2, CCL3 proteins, and histone H2B monoubiquitination. The results are shown Figure 3 shown by Figure 2 It can be seen that in human knee-derived chondrocytes, knockdown of Sh-WAC can reduce the production of matrix metalloproteinases MMP3, MMP13 and chemokines CCL2 and CCL3 after TNF stimulation; Figure 3 It can be seen that in human knee-derived chondrocytes, knockdown of Sh-WAC can reduce the production of matrix metalloproteinases MMP3, MMP13 and chemokines CCL2 and CCL3 after TNF stimulation.

[0047] As mentioned above, WAC knockdown can reduce the production of matrix metalloproteinases and chemokines in chondrocytes after being stimulated by inflammatory factors, and the production of matrix metalloproteinases and chemokines has been shown to be closely related to the occurrence and development of rheumatoid arthritis and osteoarthritis. Therefore, we reduce the expression of WAC in chondrocytes by injecting WAC knockdown adenovirus into the tail vein of arthritic mice to reduce the production of matrix metalloproteinases and chemokines, thereby alleviating the progression of rheumatoid arthritis and osteoarthritis. The specific plan is as follows:

[0048] 1) Construction of elderly osteoporosis mouse model and bone defect mouse model: 18-month-old male C57 mice were selected, and holes with a diameter of 2.5 mm and 1.0 mm were drilled in the skull and femur of the mice, respectively, and the wounds were sutured;

[0049] 2) Tail vein injection of WAC knockdown adenovirus: After the two arthritis models were constructed, they were divided into two groups, including the rAAV2-control group (rAAV2 vector was used to target transfect articular chondrocytes, and the rAAV2 vector was connected to the monomeric green or yellow fluorescent protein mNeonGreen, and the rAAV2 vector sequence is shown in SEQ ID NO.5) and the rAAV2-Sh-WAC group (rAAV2 vector was used to target transfect WAC knockdown adenovirus to articular chondrocytes, and the rAAV2 vector was connected to the monomeric green or yellow fluorescent protein mNeonGreen). The modeling time was taken as the 0th week, and the WAC knockdown adenovirus (4×10 11 GC; 2×10 13 GC / kg), injected once, and the knee and ankle joint specimens of mice were collected at 8 weeks.

[0050] 3) Detection of mouse cartilage damage and secretory phenotype: The mice were killed at the 8th week, and specimens of knee and ankle joints were collected to detect the expression of WAC by immunofluorescence, and the cartilage damage of mice was detected by safranin fast green staining. The production of matrix metalloproteinases and chemokines was detected by tissue immunochemical staining of secretory phenotype;

[0051] Tb.Th: trabecular bone thickness; Ct.Th: cortical bone thickness.

[0052] (1) Histone H2B monoubiquitination regulatory molecule WAC is upregulated in chondrocytes after stimulation by inflammatory factors. Knockdown of WAC reduces the production of chondrocyte matrix metalloproteinases MMP3, MMP13 and chemokines CCL2 and CCL3. The specific experimental results are as follows:

[0053] Safranin fast green staining results are as follows Figure 4 The results of tissue immunochemical staining of secretory phenotype are shown in Figure 5 As shown, Figure 4 , Figure 5In the experiment, safranin fast green staining showed that compared with the rAAV2-control group, the cartilage damage of arthritic mice in the rAAV2-Sh-WAC group was reduced, and immunohistochemical staining showed that the expression of WAC was reduced, proving the knockdown effect of rAAV2-Sh-WAC. Immunohistochemical staining showed that compared with the rAAV2-control group, the production of cartilage matrix metalloproteinases MMP3, MMP13 and chemokines CCL2, CCL3 in the arthritic mice in the rAAV2-Sh-WAC group was reduced, thereby reducing cartilage damage.

[0054] In summary, the WAC knockout adenovirus of the present invention can be used to prepare drugs for preventing and treating arthritis cartilage damage. In some embodiments, the drugs for preventing and treating arthritis cartilage damage may further include pharmaceutically acceptable carriers and / or excipients.

[0055] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Use of a WAC knockdown adenovirus in the preparation of a drug for preventing and treating arthritis cartilage damage, characterized in that: The WAC protein gene was inserted into the PLVX lentiviral vector to construct a transfection plasmid and co-transfected into HEK293T cells with the viral component expression plasmids PxpaX2 and PMD2G. After 48 hours, the viral supernatant was collected to obtain the WAC knockdown adenovirus. The amino acid sequence of the WAC protein gene is shown in SEQ ID NO.

1.

2. A drug for preventing and treating arthritis cartilage damage, characterized in that: Comprising the WAC knockout adenovirus as described in claim 1.

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

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