Composition containing PENK protein and application thereof

Abstract

The invention provides a composition containing PENK protein and application of the composition in promoting osteogenic differentiation by osteoblasts. Compared with the prior art, the proenkephalin PENK recombinant protein and the composition containing PENK have the advantages that after being applied to cells with osteogenic potential, the proenkephalin PENK recombinant protein and the composition containing PENK interact with CBFB protein to improve osteogenic performance, the effect of promoting osteogenic differentiation can be obviously achieved, and the proenkephalin PENK recombinant protein and the composition containing PENK have good application prospects. Compared with existing bone morphogenetic protein 2 (BMP) recombinant protein, the proenkephalin PENK recombinant protein is more excellent in osteogenesis promoting effect, and experiments prove that the osteogenic differentiation promoting effect of the proenkephalin PENK recombinant protein is dose-dependent. The application further verifies that the combined use of low doses of the PENK recombinant protein and the BMP recombinant protein has a more remarkable cell osteogenesis promoting effect compared with the independent use of high doses of the PENK recombinant protein and the BMP recombinant protein.

Description

Technical Field

[0001] This application relates to the technical field of protein compositions, and particularly relates to a composition containing PENK protein and its applications. Background Art

[0002] Proenkephalin (PENK), the gene encoding the precursor of enkephalin, was first discovered in the brain. This precursor protein can be hydrolyzed to produce a variety of protein products. These products include pentapeptide opioids: methionine (Met-enkephalin) and leucine (Leu-enkephalin), which are stored in synaptic vesicles and then released into the synapse, where they bind to opioid receptors to exert an analgesic effect. Other non-opioid cleavage products may have different biological activities. Studies have found that PENK receptors are present on osteocytes, and PENK is widely involved in bone development. It exists in undifferentiated cells in adult mouse bone tissue. After bone tissue injury, it can mimic its role during embryonic development and participate in tissue regeneration and pain control, but the mechanism is unclear. Moreover, the expression of PENK in osteoblasts is regulated by bone-targeting hormones and is downregulated during cell differentiation. In addition, it has been reported that PENK inhibits the migration of osteosarcoma cells by activating the PI3K / Akt signaling pathway. Furthermore, it has been reported that PENK acts on specific opioid receptors on various cells in the nervous, mucosal, skin, and immune systems, regulates the activities of keratinocytes and melanocytes, accelerates cell migration, and promotes wound healing. Although previous research results have not been entirely consistent, they all indicate that PENK is closely related to cell migration. In addition, it has been reported that PENK is involved in immune and various inflammatory processes, is associated with multiple cancers, and is a predictive biomarker for various diseases. The research on PENK is extensive, but the role of PENK in tooth development has not been reported. In addition, through literature retrieval, it was found that the PI3k and AKT signaling pathways downstream of PENK are closely related to the maintenance of periodontal ligament stem cells and periodontal homeostasis. Given the literature summary, PENK plays an important role in both gingival tissue and bone tissue. Therefore, it is speculated that PENK plays a key role in the development of periodontal soft and hard tissues. Summary of the Invention

[0003] In view of the above existing technical limitations, this application proposes a composition containing PENK protein and its applications; it overcomes the deficiencies and defects mentioned in the background art.

[0004] To achieve the above object, this application adopts the following technical solutions: The inventive point of this application is to provide proenkephalin PENK recombinant protein, and the amino acid sequence of the recombinant protein is as shown in SEQ ID No.1.

[0005] SEQ ID No.1: MARFLTLCTWLLLLGPGLLATVRAECSQDCATCSYRLVRPADINFLACVMECEGKLPSLKIWETCKELLQLSKPELPQDGTSTLRENSKPEESHLLAKRYGGFMKRYGGFMKKMDELYPMEPEEEANGSEILAKRYGGFMKKDAEEDDSLANSSDLLKELLETGDNRERSHHQDGSDNEEEVSKRYGGFMRGLKRSPQLEDEAKELQKRYGGFMRRVGRPEWWMDYQKRYGGFLKRFAEALPSDEEGESYSKEVPEMEKRYGGFMRF。

[0006] The second inventive point of the present application is to provide a gene encoding the above-mentioned preproenkephalin PENK recombinant protein, and the nucleotide sequence of the encoding gene is shown as SEQ ID No.2.

[0007] SEQ ID No.2: atggcgcgctttctgaccctgtgcacctggctgctgctgctgggcccgggcctgctggcgaccgtgcgcgcggaatgcagccaggattgcgcgacctgcagctatcgcctggtgcgcccggcggatattaactttctggcgtgcgtgatggaatgcgaaggcaaactgccgagcctgaaaatttgggaaacctgcaaagaactgctgcagctgagcaaaccggaactgccgcaggatggcaccagcaccctgcgcgaaaacagcaaaccggaagaaagccatctgctggcgaaacgctatggcggctttatgaaacgctatggcggctttatgaaaaaaatggatgaactgtatccgatggaaccggaagaagaagcgaacggcagcgaaattctggcgaaacgctatggcggctttatgaaaaaagatgcggaagaagatgatagcctggcgaacagcagcgatctgctgaaagaactgctggaaaccggcgataaccgcgaacgcagccatcatcaggatggcagcgataacgaagaagaagtgagcaaacgctatggcggctttatgcgcggcctgaaacgcagcccgcagctggaagatgaagcgaaagaactgcagaaacgctatggcggctttatgcgccgcgtgggccgcccggaatggtggatggattatcagaaacgctatggcggctttctgaaacgctttgcggaagcgctgccgagcgatgaagaaggcgaaagctatagcaaagaagtgccggaaatggaaaaacgctatggcggctttatgcgcttt。

[0008] The third inventive point of the present application is to provide a biological material, which contains the above-mentioned preproenkephalin PENK recombinant protein or the gene encoding the above-mentioned preproenkephalin PENK recombinant protein; the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector or engineered bacteria.

[0009] The fourth inventive point of the present application is to provide a composition containing PENK protein, which is prepared from the following proteins by weight: 0.8 - 1.2 parts of the above-mentioned PENK recombinant protein, 0.8 - 1.2 parts of BMP2 protein (preferably, the weight ratio of PENK recombinant protein to BMP2 protein is 1:1), and the amino acid sequence of the BMP2 protein is as shown in SEQ ID No.3.

[0010] SEQ ID No.3: MVAGTRCLLALLLPQVLLGGAAGLVPELGRRKFAAASSGRPSSQPSDEVLSEFELRLLSMFGLKQRPTPSRDAVVPPYMLDLYRRHSGQPGSPAPDHRLERAASRANTVRSFHHEESLEELPETSGKTTRRFFFNLSSIPTEEFITSAELQVFREQMQDALGNNSSFHHRINIYEIIKPATANSKFPVTRLLDTRLVNQNASRWESFDVTPAVMRWTAQGHANHGFVVEVAHLEEKQGVSKRHVRISRSLHQDEHSWSQIRPLLVTFGHDGKGHPLHKREKRQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR.

[0011] The fifth inventive point of the present application is to provide the application of the above-mentioned proenkephalin PENK recombinant protein, the above-mentioned coding gene, the above-mentioned biological material, and the above-mentioned composition in the preparation of products for promoting osteogenic differentiation of human dental mesenchymal cells.

[0012] The sixth inventive point of the present application is to provide the application of the above-mentioned proenkephalin PENK recombinant protein, the above-mentioned coding gene, the above-mentioned biological material, and the above-mentioned composition in the preparation of products for promoting osteogenic differentiation of human embryonic jawbone marrow stromal mesenchymal stem cells.

[0013] The seventh inventive point of the present application is to provide the application of the above-mentioned proenkephalin PENK recombinant protein, the above-mentioned coding gene, the above-mentioned biological material, and the above-mentioned composition in the preparation of products for promoting osteogenic differentiation of human embryonic long bone marrow mesenchymal cells.

[0014] Optionally, in the above application, the osteogenic administration concentration of the proenkephalin PENK recombinant protein is 10 - 200 ng / ml, and the preferred administration concentration is 100 ng / ml; the osteogenic administration concentration of bone morphogenetic protein 2 (BMP2) is 10 - 250 ng / ml, and the preferred administration concentration is 200 ng / ml.

[0015] The eighth inventive point of this application is to provide a marker for detecting the osteogenic activity of osteoblasts, where the osteoblasts are human dental-derived mesenchymal cells, human embryonic jawbone marrow stromal mesenchymal stem cells, or human embryonic long bone marrow mesenchymal cells administered with the above-mentioned proenkephalin PENK recombinant protein, the above-mentioned coding gene, the above-mentioned biomaterial, and the above-mentioned composition; the marker is selected from one or more of alkaline phosphatase ALP, transcription factor RUNX2, transcription factor SP7 / Osterix, type I collagen α1 (COL1α1), osteopontin OPN, osteocalcin OCN, and bone morphogenetic protein-2 (BMP2).

[0016] The ninth inventive point of this application is to provide the use of siRNA that inhibits PENK gene expression in the preparation of a product for inhibiting osteogenic differentiation, and the nucleotide sequence of the siRNA is as follows: siRNA - forward sequence: SEQ ID No.4; siRNA - reverse sequence: SEQ ID No.5.

[0017] SEQ ID No.4: GCAATCGAGATGGAACCAT; SEQ ID No.5: ATGGTTCCATCTCGATTGC.

[0018] The above siRNA can effectively interfere with the expression of PENK mRNA. After the reduction of PENK expression, the osteogenic activity decreases accordingly.

[0019] The sequence of PENK mRNA is as shown in SEQ ID No.6:

[0020] Compared with the prior art, the present application has the following advantages: The proenkephalin PENK recombinant protein and the composition containing PENK provided by the present application, after being applied to cells with osteogenic potential, achieve an improvement in osteogenic performance through interaction with the CBFB protein, can significantly achieve the effect of promoting osteogenic differentiation, and have a more excellent osteogenic effect compared with the existing bone morphogenetic protein 2 (BMP recombinant protein). Moreover, the osteogenic differentiation effect of the proenkephalin PENK recombinant protein on osteoblasts is verified by experiments and shows a dose dependence. The present application further verifies that the combined use of the PENK recombinant protein and the BMP recombinant protein at a low dose has a more significant cell osteogenic effect compared with the separate use of the two at a high dose. Brief Description of the Drawings

[0021] Figure 1 It is shown by human jawbone single-cell data that proenkephalin (PENK) has a certain co-expression relationship with the pre-osteoblast marker RUNX2. Functional enrichment shows that PENK + cells are closely related to ossification; Figure 1 A is a human jawbone single-cell umap map, Figure 1 B is a PENK expression feature map, Figure 1 C is a RUNX2 expression feature map, Figure 1 D is a PENK and RUNX2 co-expression feature map, Figure 1 E is a PENK + cell functional enrichment map.

[0022] Figure 2 It shows a summary of the expression of each marker gene at different times during the osteogenic induction of Human-DMSCs; the mRNA expression of osteogenic markers in different periods of the osteogenic differentiation of human bone marrow mesenchymal stem cells for 7 days was detected. The osteogenic markers ALPL, RUNX2, SP7, COL1A1, BMP2, and OCN mRNA increased to varying degrees, and the expression trend of PENK mRNA was similar to that of the osteogenic markers, showing an increase at 3 days.

[0023] Figure 3 It shows a summary of the expression of each marker gene at different times during the osteogenic induction of iliac bone mesenchymal stem cells in a public database; the applicant downloaded the osteogenic induction data of iliac bone mesenchymal stem cells from a public database and analyzed the expression of each osteogenic marker. The results showed that during the osteogenic induction process, the osteogenic markers ALPL, RUNX2, SP7, BMP2, and IBSP increased to varying degrees, and the expression trend of PENK was similar to that of the osteogenic markers, showing an increase at 3 days, a decline at 4 and 5 days, and a re-increase at 7 days.

[0024] Figure 2 and Figure 3 The results show that PENK has the potential to be an osteogenic marker.

[0025] Figure 4 It is shown that PENK protein promotes the early osteogenic differentiation of BMSCs, and the effect is better than that of BMP-2 protein; among them, Figure 4 A shows osteogenic induction with different concentrations of PENK protein, and bone morphogenetic protein-2 (BMP-2) is set as a positive control. At day 7, alkaline phosphatase (ALP) staining is performed. In the staining pictures, the upper row is the PENK protein applied, and the lower row is the BMP-2 protein applied, showing that the alkaline phosphatase staining effect in the PENK protein application group is significantly better than that in the BMP2 protein application group with the same concentration; specifically, as the concentration of PENK protein applied increases (25, 50, 100, 150, 200 ng / mL), the expression of ALPL increases, and the peak appears at 100 ng / ml; Figure 4 B shows alizarin red staining of the induced cells at day 14 of osteogenic induction, and it is found that the amount of calcium nodule formation in the PENK protein group is also significantly higher than that in the BMP-2 protein group. Specifically, it is found that after the application of PENK protein (25, 50, 100 ng / ml), it will promote earlier and more calcium nodule formation, showing a significant promoting effect on early osteogenesis, and the effect is better than that of BMP-2 protein with the same concentration; Figure 4 C shows the alkaline phosphatase quantitative experiment done at the same time, and it is found that there is a statistically significant difference between the PENK group and the BMP-2 group; Figure 4 D shows quantitative analysis after dissolving calcium nodules with cetylpyridinium chloride, and it is found that the content of calcium ions increases with the increase of PENK protein concentration, and the peak appears at 100 ng / ml. The calcium ion content detected after the application of PENK protein (25, 50, 100 ng / ml) is also higher than that in the group treated with BMP2 protein with the same concentration, and the difference is statistically significant.

[0026] Figure 5 It is shown that the osteogenic ability of cells decreases after the expression of PENK is interfered by siRNA. Among them, siRNA was used for transient transfection of BMSCs to interfere with the expression of its PENK mRNA, and then osteogenic induction was carried out. Alkaline phosphatase staining was detected at day 7, and the results showed that the staining area in the si-PENK interference group was significantly reduced ( Figure 5 A, Figure 5 C), and at the same time, the alkaline phosphatase activity was detected, and there was a statistically significant difference between the two groups ( Figure 5G); After 21 days, alizarin red staining was performed, and the staining area in the si-PENK interference group was significantly reduced ( Figure 5 B, Figure 5 D), After dissolving the calcium nodules with cetylpyridinium chloride for quantitative analysis, the difference was statistically significant ( Figure 5 H); Figure 5 E, Figure 5 F showed that the expression of PENK mRNA at 0 day and 7 days was significantly reduced, with statistical significance; Figure 5 I showed that on the 7th day of osteogenic induction, the osteogenic markers ALPL, RUNX2, SP7, OPN, COL1A1, BMP2, OCN, and IBSP were significantly reduced, and the differences were all statistically significant.

[0027] Figure 6 It was shown that the osteogenic ability of PENK-overexpressing immortalized cells was increased. Among them, A PENK-overexpressing immortalized cell line was constructed and verified by q-RT-PCR. The PENK mRNA in the cells was significantly increased ( Figure 6 C, Figure 6 D), indicating that the PENK-overexpressing immortalized cell line was successfully constructed; The cells were induced to differentiate into osteoblasts, and alkaline phosphatase staining was performed on the 7th day. The results showed that the alkaline phosphatase staining area in the OE-PENK group increased ( Figure 6 A); On the 14th day, alizarin red staining was performed, and the results showed that the staining area in the OE-PENK group increased ( Figure 6 B); Quantitative detection of alkaline phosphatase activity showed a significant increase in the OE-PENK group, and the difference was statistically significant ( Figure 6 E); On the 14th day, the calcium nodules were dissolved with cetylpyridinium chloride for quantitative analysis. The OE-PENK group increased significantly, and the difference was statistically significant ( Figure 6 F); Figure 6 G- Figure 6 O showed that the expression of osteogenic markers COL1A1, ALPL, RUNX2, OCN, SP7, BMP2, OPN, and DSPP mRNA in the OE-PENK group was higher than that in the NC (transfected with empty vector) group to varying degrees on the 3rd, 7th, and 14th days.

[0028] Figure 7 It showed the results of co-IP experiment, followed by proteomic analysis and identification verification after osteogenic induction of cells; Among them, Figure 7 A shows the protein bands after running gel and Coomassie brilliant blue staining after co-IP experiment in the OE-PENK induced for 7 days group, NC induced for 7 days group, and normal OE-PENK group. Figure 7Group B was the group induced by OE-PENK for 7 days and the group induced by NC for 7 days. After performing CO-IP experiments on the normal OE-PENK group and analyzing the protein profiles, a Venn diagram was drawn. It showed that there were 348 specific binding proteins in the group induced by OE-PENK for 7 days; Figure 7 Figure C shows the GO enrichment analysis of the 348 specific binding proteins in the group induced by OE-PENK for 7 days. The results showed that the specific binding proteins of PENK were closely related to osteoblast differentiation and the development of the skeletal system; Figure 7 Figure D shows that artificial intelligence predicted that there was a binding site between PENK and CBFB protein; Figure 7 Figure E shows that CO-IP experiments verified that after osteogenic induction, there was specific binding between PENK protein and CBFB protein.

[0029] Figure 8 The figure shows that si-CBFB significantly reduced the osteogenic activity of BMSCs, and the application of PENK recombinant protein failed to rescue the result. Among them, the experiment was divided into 4 groups: NC group (transfected with empty vector group), NC group applied with PENK protein, si-CBFB group knocking down CBFB mRNA, and si-CBFB group knocking down CBFB mRNA and applied with PENK protein; On the 7th day, alkaline phosphatase staining ( Figure 8 A) was performed. The staining area of the NC + PENK protein group was significantly higher than that of the NC group, the staining area of the si-CBFB group was significantly lower than that of the NC group, and the ALP staining area did not recover to the level of the NC group after applying PENK protein. Quantitative analysis of alkaline phosphatase activity found that there were statistically significant differences among the groups ( Figure 8 C); On the 14th day, alizarin red staining ( Figure 8 B) was performed. The staining area of the NC + PENK protein group was significantly higher than that of the NC group, the staining area of the si-CBFB group was significantly lower than that of the NC group, and the staining area did not recover to the level of the NC group after applying PENK protein. Quantitative analysis of calcium ion content found that there were statistically significant differences among the groups ( Figure 8 D); Figure 8 E- Figure 8 Figures E-H show the qRT-PCR verification of the expression of osteogenic markers RUNX2, ALPL, BMP2, and OCN mRNA. The expression of each osteogenic marker in the NC + PENK group was significantly higher than that in the NC group, the si-CBFB group was significantly lower than the NC group, and the mRNA expression of each osteogenic marker in the si-CBFB combined with PENK protein group increased slightly, but was significantly lower than that in the NC group, with significant statistical differences.

[0030] Figure 9It was shown that the combined use of PENK and BMP2 proteins significantly promoted the osteogenic activity of jaw BMSCs; among them, Figure 9 A. Osteogenic induction was performed on the Ctrl group, BMP2 protein group (50 ng / ml), PENK group (50 ng / ml), and combined BMP2 protein group (25 ng / ml) and PENK protein group (25 ng / ml). Alkaline phosphatase staining was performed on the 7th day. The results showed that the staining area of the combined BMP2 protein group (25 ng / ml) and PENK protein group (25 ng / ml) was significantly higher than that of the other three groups; on the 14th day, alizarin red staining was performed, and the results showed that the staining area of the combined BMP2 protein group (25 ng / ml) and PENK protein group (25 ng / ml) was also significantly higher than that of the other three groups; quantitative analysis of ALP activity found that the difference was statistically significant ( Figure 9 B); quantitative analysis of calcium ion content showed that the staining area of the combined BMP2 protein group (25 ng / ml) and PENK protein group (25 ng / ml) was also significantly higher than that of the other three groups, and the difference was statistically significant ( Figure 9 C); Figure 9 D- Figure 9 H. qRT-PCR was used to verify the expression of osteogenic markers RUNX2, ALPL, BMP2, and OCN. The expression of each osteogenic marker in the combined BMP2 protein group (25 ng / ml) and PENK protein group (25 ng / ml) was higher than that of the other three groups to varying degrees, and the difference was statistically significant. Detailed implementation mode

[0031] To make the purpose, technical solution, and advantages of this application clearer, the following further details this application. However, it should be understood that the description here is only used to explain this application and does not limit the scope of this application.

[0032] Unless otherwise defined, all technical terms and scientific terms used in this article have the same meaning as those commonly understood by those skilled in the technical field to which this application belongs. The terms used in the description of this application in this article are only for the purpose of describing specific embodiments and are not intended to limit this application. The reagents and instruments used in this article are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art and will not be elaborated here.

[0033] To further understand this application, the following further details this application with reference to the best embodiments.

[0034] Example 1 The proenkephalin PENK recombinant protein provided by this application has an amino acid sequence as shown in SEQ ID No.1.

[0035] The gene encoding the above-mentioned proenkephalin PENK recombinant protein has a nucleotide sequence as shown in SEQ ID No. 2.

[0036] The technical content protected by this application also relates to all biological materials containing the above-mentioned proenkephalin PENK recombinant protein or the gene encoding the above-mentioned proenkephalin PENK recombinant protein, including but not limited to recombinant DNA, expression cassette, transposon, plasmid vector, viral vector or engineered bacteria, etc.

[0037] The inventors also found that the PENK recombinant protein involved in this application can be co-administered with BMP2 protein (bone morphogenetic protein 2), and can further enhance its osteogenic effect. During the co-administration (i.e., used as a composition), the PENK recombinant protein and BMP2 protein are combined and administered at a weight ratio of 1:1. The amino acid sequence of BMP2 protein is as shown in SEQ ID No. 3.

[0038] The osteoblasts targeted by the provided proenkephalin PENK recombinant protein, encoding gene and biological materials containing both are human dental mesenchymal cells, human embryonic jaw bone marrow stromal mesenchymal stem cells and human embryonic long bone marrow mesenchymal cells; these three types of cells were found to have certain osteogenic differentiation potential in previous studies.

[0039] During the single administration process, the osteogenic administration concentration of the proenkephalin PENK recombinant protein is 10 - 200 ng / ml, and the preferred administration concentration is 100 ng / ml; the osteogenic administration concentration of bone morphogenetic protein 2 (BMP2) is 10 - 250 ng / ml, and the preferred administration concentration is 200 ng / ml.

[0040] During the co-administration process, the osteogenic administration concentration of the proenkephalin PENK recombinant protein is 10 - 200 ng / ml, and the preferred administration concentration is 25 ng / ml; the osteogenic administration concentration of bone morphogenetic protein 2 (BMP2) is 10 - 200 ng / ml, and the preferred administration concentration is 25 ng / ml.

[0041] During the above osteogenic experiment process, the applicant also found multiple markers for detecting the osteogenic activity of osteoblasts. The expression of these markers is all associated with the administration of PENK protein and BMP2 protein. The markers include alkaline phosphatase ALP, transcription factor RUNX2, transcription factor SP7 / Osterix, type I collagen α1 (COL1α1), osteopontin OPN, osteocalcin OCN, and bone morphogenetic protein-2 (BMP2).

[0042] To further verify the osteogenic promoting effect of PENK in osteoblasts, siRNAs specifically designed to inhibit the expression of the PENK gene were used. The nucleotide sequences of the siRNAs are as follows: siRNA-forward sequence: SEQ ID No.4; siRNA-reverse sequence: SEQ ID No.5. After interfering with the expression of PENK mRNA by siRNA, a significant decrease in osteogenic ability was observed.

[0043] The sequence of PENK mRNA is shown in SEQ ID No.6.

[0044] The applicant also constructed PENK-knockout dental mesenchymal stem cells (DMSCs), detected their phenotypes and various osteogenesis-related markers, and found that the expression of multiple markers decreased, indicating that PENK indeed has an osteogenic promoting effect.

[0045] Example 2 1. To verify that PENK can promote the osteogenic differentiation of human dental mesenchymal cells in a dose-dependent manner, an experiment on the osteogenic differentiation promoted by PENK recombinant protein was carried out. The specific experimental method is as follows: (1) Digest and count dental mesenchymal stem cells (DMSCs) and inoculate them into 12-well plates at a cell density of 1×10 4 cells / cm², and add 1 mL of normal complete medium to each well; (2) Place the 12-well plates in a CO2 incubator at 37°C, 5% CO2, and saturated humidity for culture; (3) On the second day, when the cell confluence reaches 70%, carefully aspirate the complete medium in the wells, and add 1 mL of OriCell human bone marrow mesenchymal stem cell osteogenic induction differentiation medium to the 12-well plates; (4) Replace with fresh OriCell human bone marrow mesenchymal stem cell osteogenic induction differentiation medium every 3 days; (5) After 14 days of induction, perform alizarin red staining.

[0046] On the 7th day of osteogenic induction, the alkaline phosphatase (ALP) activity of the cells was detected. The results showed that as the concentration of PENK protein increased (10, 25, 50 ng / mL), the expression of ALP increased. The quantitative experiment of alkaline phosphatase activity showed that the results were consistent with the staining, also showing a dose-dependent manner, and the differences between groups were statistically significant. On the 21st day of osteogenic induction, the efficiency of calcium nodule formation was detected by alizarin red staining. The results showed that as the concentration of PENK protein (10, 25, 50 ng / mL) increased, the area of alizarin red staining gradually increased. After dissolving the calcium nodules with cetylpyridinium chloride for quantitative analysis, it was found that the calcium ion content increased with the increase of PENK protein concentration, and the difference was statistically significant.

[0047] 2. Subsequently, as Figure 4 shown, bone morphogenetic protein-2 (BMP-2) was set as a positive control. After comparison, it was found that after the application of PENK protein, earlier and more calcium nodules were formed, showing a significant promotion of early osteogenic effect, and the effect was even better than that of BMP-2. On the 7th day of osteogenic induction, alkaline phosphatase staining was performed. The upper row was the applied PENK protein, and the lower row was the applied BMP-2 protein. It could be seen that the alkaline phosphatase staining effect of the PENK protein group was significantly better than that of the BMP2 protein group. At the same time, an alkaline phosphatase quantification experiment was conducted, and it was found that the difference between the PENK group and the BMP-2 group was statistically significant. On the 14th day of osteogenic induction, alizarin red staining was performed on the induced cells, and it was found that the amount of calcium nodules formed in the PENK protein group was also significantly higher than that in the BMP-2 protein group. However, at the late stage, on the 21st day of osteogenic induction, the difference between the two groups lost significance.

[0048] 3. To prove that PENK has a similar effect on different osteoblasts, it was verified in cells of different species at the same time. Human embryonic jawbone marrow stromal mesenchymal stem cells were used for the experiment, and the results were consistent with those in human embryonic mesenchymal cells. Verification was carried out in human embryonic long bone marrow mesenchymal cells, and the results of alizarin red at 14 days were obtained, indicating that the trend was consistent with the previous two types of cells; it was shown that PENK protein promoted the osteogenic differentiation of human jawbone marrow mesenchymal stem cells and was dose-dependent within a certain range (20 - 200 ng / ml, and the peak appeared at 100 ng / ml).

[0049] 4. siRNA was transiently transfected into BMSCs to interfere with the expression of its PENK mRNA, and then osteogenic induction was carried out. The results showed that the osteogenic ability of the cells decreased after siRNA interference with the expression of PENK ( Figure 5 ), indicating that siRNA effectively interfered with the expression of PENK mRNA. After the expression of PENK decreased, the osteogenic activity decreased accordingly.

[0050] 5. An immortalized cell line with overexpressed PENK was constructed and verified by q-RT-PCR. The PENK mRNA in the cells increased significantly, indicating that the immortalized cell line with overexpressed PENK was successfully constructed. After osteogenic induction of the cells, quantitative detection and analysis were carried out, and the results showed that the osteogenic ability of the immortalized cells with overexpressed PENK increased ( Figure 6 ), and the osteogenic activity of human dental-derived mesenchymal stem cells with overexpressed PENK increased significantly.

[0051] 6. Proteomic analysis showed that PENK protein could interact with CBFB protein to promote osteogenesis ( Figure 7); After siRNA interference of CBFB expression, the osteogenic activity of cells decreased, and exogenous application of PENK protein could not effectively rescue it, thus proving that PENK protein exerts its function through interaction with CBFB ( Figure 8 ).

[0052] 7. Alkaline phosphatase (ALP) is a marker of osteogenic activity of osteoblasts, and RUNX2 and SP7 / Osterix are important transcription factors during osteogenesis. qRT-PCR was used for detection to preliminarily explore the mechanism. It was found that PENK mRNA increased with the prolongation of induction time from 0 to 14 days and decreased on the 21st day. After application of PENK protein, the expression of PENK mRNA in the cells themselves also increased significantly, especially on the 7th day. The peak of PENK mRNA in the normal control group appeared on the 14th day, and after application of PENK protein, there was a very significant increase on the 7th day, and the expression was significantly advanced by a lot.

[0053] The expression of the osteoblast activity marker alkaline phosphatase ALP also increased significantly compared with the normal control group, and the peaks of both groups also appeared on the 7th day. At the same time, the transcription factors RUNX2 and SP7 / OSX were also detected, and it was found that their expressions increased significantly after application of PENK protein. The peak of RUNX2 also advanced to the 7th day, and it was approximately 10 times higher than that of the normal control group. The effect on SP7 / OSX was also very obvious. The expression of RUNX2 was earlier than that of SP7 / OSX. RUNX2 plays a role in the transformation of mesenchymal cells into pre-osteoblasts, and SP7 / OSX plays a role in the maturation stage of pre-osteoblasts into osteoblasts. The results of PCR preliminarily showed that PENK has an effect on both of these transcription factors, and it is speculated that it may play a role in both of these differentiation stages.

[0054] Single-cell data of human jawbones showed that proenkephalin (PENK) has a certain co-expression relationship with the pre-osteoblast marker RUNX2 ( Figure 1 ).

[0055] At the same time, other markers of osteoblast differentiation were also explored. They mainly include type I collagen a1, osteopontin OPN, and osteocalcin OCN. After application of PENK protein, the expressions of these markers all increased significantly.

[0056] The expression of each Marker gene during osteogenic induction of Human-DMSCs at different times is as Figure 2 shown.

[0057] The summary of the expression of each marker gene during osteogenic induction of iliac mesenchymal stem cells at different times in the public database is as Figure 3 shown.

[0058] 8. BMP2 combination application technical solution: PENK protein and BMP2 protein are formulated into a complex in a weight ratio of 1:1.

[0059] The specific application concentrations are: 50 ng / ml + 50 ng / ml and 25 ng / ml + 25 ng / ml.

[0060] The combined use of PENK protein and BMP2 protein significantly promotes the osteogenic activity of cells and achieves better osteogenic effects ( Figure 9 ).

[0061] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, or improvements made within the spirit and principles of the present application shall be included within the protection scope of the present application.

Claims

1. Proenkephalin PENK recombinant protein, characterized in that The amino acid sequence of the recombinant protein is shown in SEQ ID No.

1.

2. The gene encoding the proenkephalin PENK recombinant protein according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID No.

2.

3. A biomaterial, characterized in that: The biological material contains the proenkephalin PENK recombinant protein according to claim 1 or the gene encoding the proenkephalin PENK recombinant protein according to claim 2; the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector or engineered bacteria.

4. A composition containing PENK protein, characterized in that: The composition is prepared from the following proteins in parts by weight: 0.8-1.2 parts of the PENK recombinant protein according to claim 1, and 0.8-1.2 parts of the BMP2 protein, wherein the amino acid sequence of the BMP2 protein is shown in SEQ ID No.

3.

5. Use of the proenkephalin PENK recombinant protein according to claim 1, the encoding gene according to claim 2, the biomaterial according to claim 3, and the composition according to claim 4 in the preparation of a product for promoting osteogenic differentiation of human dental mesenchymal cells.

6. Use of the proenkephalin PENK recombinant protein according to claim 1, the encoding gene according to claim 2, the biomaterial according to claim 3, and the composition according to claim 4 in the preparation of a product for promoting osteogenic differentiation of human embryonic jaw bone marrow stromal mesenchymal stem cells.

7. Use of the proenkephalin PENK recombinant protein according to claim 1, the encoding gene according to claim 2, the biomaterial according to claim 3, and the composition according to claim 4 in the preparation of a product for promoting osteogenic differentiation of human embryonic long bone marrow mesenchymal cells.

8. The use according to any one of claims 5 to 7, characterized in that: The osteogenic administration concentration of the proenkephalin PENK recombinant protein is 10-200 ng / ml; the osteogenic administration concentration of the bone morphogenetic protein 2 is 10-250 ng / ml.

9. A marker for detecting osteoblast osteogenic activity, characterized in that: The osteoblasts are human dental mesenchymal cells, human embryonic jaw bone marrow stromal mesenchymal stem cells or human embryonic long bone bone marrow mesenchymal cells to which the proenkephalin PENK recombinant protein of claim 1, the encoding gene of claim 2, the biomaterial of claim 3 and the composition of claim 4 are administered; the markers are selected from alkaline phosphatase ALP, transcription factor RUNX2, transcription factor SP7 / Osterix, type I collagen α1, osteopontin OPN or osteocalcin OCN.

10. Use of siRNA that inhibits PENK gene expression in the preparation of a product that inhibits osteogenic differentiation, characterized in that: The nucleotide sequence of the siRNA is as follows: siRNA-forward sequence: SEQ ID No.4; siRNA-reverse sequence: SEQ ID No.5.

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