A composition containing a penk protein and its use

Abstract

This application provides a composition containing PENK protein and its application in promoting osteoblast osteogenic differentiation. Compared with the prior art, this application has the following advantages: The pro-PENK recombinant protein and the PENK-containing composition provided in this application, after being applied to cells with osteogenic potential, enhance osteogenic performance through interaction with CBFB protein, and can significantly promote osteogenic differentiation. Compared with the existing bone morphogenetic protein 2 (BMP recombinant protein), the osteogenic effect is superior. Furthermore, experiments have verified that the osteogenic differentiation effect of pro-PENK recombinant protein on osteoblasts is dose-dependent. This application further verifies that the combined use of low doses of PENK recombinant protein and BMP recombinant protein has a more significant cellular osteogenic effect than the use of either at high doses alone.

Description

Technical Field

[0001] This application relates to the field of protein composition technology, specifically to a composition containing PENK protein and its application. Background Technology

[0002] Proenkephlin (PENK), the gene encoding a precursor to enkephalin, was first discovered in the brain. This precursor protein, upon hydrolysis, yields various protein products. These products include pentapeptide opioids: methionine and leucine, which are stored in synaptic vesicles and then released into the synapse, where they bind to opioid receptors to exert analgesic effects. The cleavage products of other non-opioid drugs may have different biological activities. Studies have found PENK receptors on osteoblasts. PENK is widely involved in bone development and is present in undifferentiated cells in adult mouse bone tissue. After bone injury, it can mimic its embryonic developmental role, participating in tissue regeneration and pain control, although the mechanism remains unclear. Furthermore, PENK expression in osteoblasts is regulated by bone-targeting hormones and is downregulated during cell differentiation. Furthermore, reports indicate that PENK inhibits osteosarcoma cell migration by activating the PI3K / Akt signaling pathway. Additionally, reports suggest that PENK acts on specific opioid receptors on various cells in the nervous, mucosal, skin, and immune systems, regulating the activity of keratinocytes and melanocytes, accelerating cell migration, and promoting wound healing. Although previous research results are not entirely consistent, they all indicate that PENK is closely related to cell migration. There are also reports that PENK participates in immune and various inflammatory processes, is associated with multiple cancers, and is a predictive biomarker for various diseases. Research on PENK is extensive, but its role in tooth development has not been reported. Furthermore, a literature search revealed that the downstream PI3K and AKT signaling pathways of PENK are closely related to periodontal ligament stem cells and the maintenance of periodontal homeostasis. Based on the literature summary, PENK plays an important role in both gingival and bone tissues; therefore, it is hypothesized that PENK plays a crucial role in the development of periodontal soft and hard tissues. Summary of the Invention

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

[0004] To achieve the above objectives, this application adopts the following technical solution:

[0005] The inventive point of this application is to provide a recombinant enkephalinogen PENK protein, the amino acid sequence of which is shown in SEQ ID No. 1.

[0006] SEQ ID No. 1:

[0007] MARFLTLCTWLLLLGPGLLATVRAECSQDCATCSYRLVRPADINFLACVMECEGKLPSLKIWETCKELLQLSKPELPQDGTSTLRENSKPEESHLLAKRYGGFMKRYGGFMKKMDELYPMEPEEEANGSEILAK RYGGFMKKDAEEDDSLANSSDLLKELLETGDNRERSHHQDGSDNEEEVSKRYGGFMRGLKRSPQLEDEAKELQKRYGGFMRRVGRPEWWMDYQKRYGGFLKRFAEALPSDEEGESYSKEVPEMEKRYGGFMRF.

[0008] The second inventive point of this application is to provide a gene encoding the above-mentioned proenkephalin PENK recombinant protein, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0009] SEQ ID No. 2:

[0010] .

[0011] The third inventive point of this application is to provide a biological material containing the above-mentioned proenkephalin PENK recombinant protein or the above-mentioned gene encoding proenkephalin PENK recombinant protein; the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector or engineered bacteria.

[0012] The fourth inventive point of this application is to provide a composition containing PENK protein, said composition being prepared from the following proteins in parts by weight: 0.8-1.2 parts of the above-mentioned recombinant PENK protein, and 0.8-1.2 parts of BMP2 protein (preferably the weight ratio of recombinant PENK protein to BMP2 protein is 1:1), wherein the amino acid sequence of said BMP2 protein is shown in SEQ ID No. 3.

[0013] SEQ ID No. 3:

[0014] MVAGTRCLLALLLPQVLLGGAAGLVPELGRRKFAAASSGRPSSQPSDEVLSEFELRLLSMFGLKQRPTPSRDAVVPPYMLDLYRRHSGQPGSPAPDHRLERAASRANTVRSFHHEESLEELPETSGKTTRRFFFNLSSIPTEEFITSAELQVFREQMQDALGNNSSFHHRINIYEIIKPATANSKFPVTRLLDTRLVN QNASRWESFDVTPAVMRWTAQGHANHGFVVEVAHLEEKQGVSKRHVRISRSLHQDEHSWSQIRPLLVTFGHDGKGHPLHKREKRQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR.

[0015] The fifth inventive point of this application is to provide the use of the above-mentioned proenkephalin PENK recombinant protein, the above-mentioned encoding gene, the above-mentioned biomaterial, and the above-mentioned composition in the preparation of a product for promoting osteogenic differentiation of human dental mesenchymal cells.

[0016] The sixth inventive point of this application is to provide the use of the above-mentioned proenkephalin PENK recombinant protein, the above-mentioned encoding gene, the above-mentioned biomaterial, and the above-mentioned composition in the preparation of a product for promoting osteogenic differentiation of human embryonic jawbone bone marrow stromal mesenchymal stem cells.

[0017] The seventh inventive point of this application is to provide the use of the above-mentioned proenkephalin PENK recombinant protein, the above-mentioned encoding gene, the above-mentioned biomaterial, and the above-mentioned composition in the preparation of a product for promoting osteogenic differentiation of human embryonic long bone bone marrow mesenchymal cells.

[0018] Optionally, in the above applications, the bone-promoting concentration of the pro-PENK recombinant protein is 10-200 ng / ml, preferably 100 ng / ml; and the bone-promoting concentration of bone morphogenetic protein 2 (BMP2) is 10-250 ng / ml, preferably 200 ng / ml.

[0019] The eighth inventive point of this application is to provide a biomarker for detecting osteoblast osteogenic activity, wherein the osteoblasts are human dental mesenchymal cells, human embryonic jawbone bone marrow stromal mesenchymal stem cells, or human embryonic long bone bone marrow mesenchymal cells that have been administered the above-mentioned proenkephalin PENK recombinant protein, the above-mentioned encoding gene, the above-mentioned biological material, and the above-mentioned composition; the biomarker is selected from one or more of alkaline phosphatase ALP, transcription factor RUNX2, transcription factor SP7 / Osterix, type I collagen α1 (COL1α1), osteopontin OPN and osteocalcin OCN, and bone morphogenetic protein-2 (BMP2).

[0020] The ninth inventive point of this application is to provide the application of siRNA that inhibits PENK gene expression in the preparation of products that inhibit osteogenic differentiation, wherein the nucleotide sequence of the siRNA is as follows: siRNA-forward sequence: SEQ ID No. 4; siRNA-reverse sequence: SEQ ID No. 5.

[0021] SEQ ID No.4:

[0022] GCAATCGAGATGGAACCAT;

[0023] SEQ ID No. 5:

[0024] ATGGTTCCATCTCGATTGC.

[0025] The aforementioned siRNA can effectively interfere with the expression of PENK mRNA. As PENK expression decreases, osteogenic activity also decreases.

[0026] The sequence of PENK mRNA is shown in SEQ ID No. 6:

[0027]

[0028] Compared with the prior art, this application has the following advantages:

[0029] The pro-PENK recombinant protein and PENK-containing compositions provided in this application, when applied to cells with osteogenic potential, enhance osteogenic performance through interaction with CBFB protein, significantly promoting osteogenic differentiation. Compared to existing bone morphogenetic protein 2 (BMP recombinant protein), the osteogenic effect is superior. Experiments have verified that the osteogenic differentiation-promoting effect of pro-PENK recombinant protein is dose-dependent. This application further verifies that the combined use of low doses of PENK recombinant protein and BMP recombinant protein exhibits a more significant cellular osteogenic effect compared to the use of either at high doses alone. Attached Figure Description

[0030] Figure 1 The data obtained from single-cell analysis of human jawbone showed that proenkephalin (PENK) and the pre-osteoblast marker RUNX2 were co-expressed. Functional enrichment analysis revealed that PENK... + Cells are closely related to ossification; Figure 1 A is a single-cell UMP image of the human jawbone. Figure 1 B is the PENK expression feature map. Figure 1 C represents the RUNX2 representation feature map. Figure 1 D represents the co-expression feature map of PENK and RUNX2. Figure 1 E is PENK + Cellular function enrichment diagram.

[0031] Figure 2 The data summarizes the expression of various marker genes at different time points during osteogenic induction of Human-DMSCs. The expression of osteogenic marker mRNA was detected at different time points during 7 days of osteogenic differentiation of human bone marrow mesenchymal stem cells. Osteogenic markers ALPL, RUNX2, SP7, COL1A1, BMP2, and OCN mRNA showed varying degrees of increase. The expression of PENK mRNA showed a similar trend to that of osteogenic markers, increasing at 3 days.

[0032] Figure 3The data presents a summary of the expression of various marker genes at different time points during osteogenic induction of iliac bone mesenchymal stem cells from a public database. The applicant downloaded osteogenic induction data of iliac bone mesenchymal stem cells from the public database and analyzed the expression of various osteogenic markers. The results showed that during osteogenic induction, the osteogenic markers ALPL, RUNX2, SP7, BMP2, and IBSP increased to varying degrees. The expression trend of PENK was similar to that of the osteogenic markers, increasing for 4 days after 3 days, decreasing for 5 days, and then increasing again on 7 days.

[0033] Figure 2 and Figure 3 The results suggest that PENK has the potential to serve as an osteogenic marker.

[0034] Figure 4 The results showed that PENK protein promoted early osteogenic differentiation of BMSCs, with a better effect than BMP-2 protein; among them...

[0035] Figure 4 A. Osteogenesis induction was induced by applying different concentrations of PENK protein, with bone morphogenetic protein-2 (BMP-2) as a positive control. On day 7, alkaline phosphatase (ALP) staining was performed. In the staining image, the top row shows the applied PENK protein, and the bottom row shows the applied BMP-2 protein. It shows that the ALP staining effect of the PENK protein group was significantly better than that of the BMP-2 protein group at the same concentration. Specifically, as the concentration of applied PENK protein increased (25, 50, 100, 150, 200 ng / mL), the expression of ALPL increased, with the peak appearing at 100 ng / mL.

[0036] Figure 4 B. When osteogenic induction was performed for 14 days, the induced cells were stained with Alizarin Red. It was found that the amount of calcium nodule formation in the PENK protein group was significantly higher than that in the BMP-2 protein group. Specifically, the application of PENK protein (25, 50, 100 ng / ml) promoted the formation of calcium nodules earlier and more, showing a significant effect of promoting early osteogenic formation, which was better than that of BMP-2 protein at the same concentration.

[0037] Figure 4 C represents the quantitative alkaline phosphatase experiment conducted during the same period, which revealed a statistically significant difference between the PENK group and the BMP-2 group.

[0038] Figure 4 D represents the quantitative analysis performed after dissolving calcium nodules with hexadecylpyridine chloride. The results showed that the calcium ion content increased with the increase of PENK protein concentration, with the peak occurring at 100 ng / ml. The calcium ion content detected after PENK protein application (25, 50, 100 ng / ml) was also higher than that of the BMP2 protein treatment group at the same concentration, and the difference was statistically significant.

[0039] Figure 5 The results showed that siRNA interference with PENK expression led to a decrease in osteogenic capacity of cells.

[0040] BMSCs were transiently transfected with siRNA to interfere with the expression of their PENK mRNA, followed by osteogenic induction. Alkaline phosphatase staining was performed on 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 alkaline phosphatase activity was also measured, and the difference between the two groups was statistically significant. Figure 5 G); Alizarin red staining was performed 21 days later, and the staining area in the si-PENK interference group was significantly reduced ( Figure 5 B. Figure 5 D), quantitative analysis was performed after dissolving calcium nodules with hexadecylpyridine chloride, and the differences were statistically significant. Figure 5 H); Figure 5 E, Figure 5 F showed that PENK mRNA expression was significantly reduced on days 0 and 7, which was statistically significant; Figure 5 I-values ​​showed that on day 7 of osteogenic induction, the levels of osteogenic markers ALPL, RUNX2, SP7, OPN, COL1A1, BMP2, OCN, and IBSP were significantly reduced, with statistically significant differences.

[0041] Figure 6 The results showed increased osteogenic capacity in immortalized cells with PENK overexpression.

[0042] A PENK-overexpressing immortalized cell line was constructed, and q-RT-PCR verification showed a significant increase in PENK mRNA levels in the cells. Figure 6 C Figure 6 D), indicating that the PENK overexpression immortalized cell line was successfully constructed; the cells were induced to osteogenic origin, and alkaline phosphatase staining was performed on day 7. The results showed that the alkaline phosphatase staining area was increased in the OE-PENK group ( Figure 6 A); On day 14, alizarin red staining was performed, and the results showed that the staining area increased in the OE-PENK group ( Figure 6 B); Quantitative detection of alkaline phosphatase activity was performed, and the OE-PENK group showed a significant increase, with statistically significant differences. Figure 6 E); On day 14, calcium nodules were dissolved using hexadecylpyridine chloride for quantitative analysis. The OE-PENK group showed a significantly increased number of nodules, with a statistically significant difference. Figure 6 F); Figure 6 G- Figure 6O results showed that the expression of osteogenic markers COL1A1, ALPL, RUNX2, OCN, SP7, BMP2, OPN, and DSPP mRNA was significantly higher in the OE-PENK group than in the NC (transfected empty vector) group on days 3, 7, and 14.

[0043] Figure 7 The results are shown as follows: after osteogenic induction, CO-IP experiment was performed on cells, followed by proteomic analysis and identification verification.

[0044] Figure 7 A represents the OE-PENK induction group for 7 days and the NC induction group for 7 days. After CO-IP experiment, the normal OE-PENK group was run on gel and Coomassie brilliant blue staining showed protein bands.

[0045] Figure 7 B represents the OE-PENK-induced 7-day group, the NC-induced 7-day group, and the normal OE-PENK group. After CO-IP experiments, protein proteomic analysis was performed, and Venn diagrams were plotted. The results show that the OE-PENK-induced 7-day group contains 348 specific binding proteins.

[0046] Figure 7 C represents the GO enrichment analysis of 348 proteins specifically bound by OE-PENK induction for 7 days. The results show that PENK-specific binding proteins are closely related to osteoblast differentiation and skeletal system development.

[0047] Figure 7 D indicates that artificial intelligence predicts that PENK and CBFB proteins have binding sites.

[0048] Figure 7 E is a CO-IP experiment that verified that PENK protein and CBFB protein specifically bind after osteogenic induction.

[0049] Figure 8 The figure shows that si-CBFB significantly reduces osteogenic activity of BMSCs, and the application of PENK recombinant protein failed to salvage the results. The experiment was divided into 4 groups: NC group (transfected with empty vector), NC group with PENK protein, si-CBFB knockdown of CBFB mRNA group, and si-CBFB knockdown of CBFB mRNA with PENK protein group.

[0050] On day 7, alkaline phosphatase staining was performed. Figure 8 A) The staining area in the NC+PENK protein group was significantly higher than that in the NC group, while the staining area in the si-CBFB group was significantly lower than that in the NC group. After applying PENK protein, the ALP staining area failed to recover to the level of the NC group. Quantitative analysis of alkaline phosphatase activity revealed statistically significant differences among the groups. Figure 8 C);

[0051] On day 14, alizarin red staining was performed. Figure 8 B), the staining area in the NC+PENK protein group was significantly higher than that in the NC group, while the staining area in the si-CBFB group was significantly lower than that in the NC group. After applying PENK protein, the staining area failed to recover to the level of the NC group. Quantitative analysis of calcium ion content revealed statistically significant differences among the groups. Figure 8 D);

[0052] Figure 8 E- Figure 8 H was used to verify the expression of osteogenic markers RUNX2, ALPL, BMP2, and OCN mRNA by qRT-PCR. The expression of each osteogenic marker in the NC+PENK group was significantly higher than that in the NC group, while that in the si-CBFB group was significantly lower than that in the NC group. The mRNA expression of each osteogenic marker in the si-CBFB combined with PENK protein group was slightly increased, but significantly lower than that in the NC group, and the differences were statistically significant.

[0053] Figure 9 The combined use of PENK and BMP2 proteins significantly promoted osteogenic activity of jawbone BMSCs; among which...

[0054] Figure 9 Osteogenic induction was performed in groups A: Ctrl, BMP2 protein group (50 ng / ml), PENK group (50 ng / ml), and a combination of BMP2 protein group (25 ng / ml) and PENK protein group (25 ng / ml). On day 7, alkaline phosphate staining was performed, and the results showed that the staining area of ​​the combination of BMP2 protein group (25 ng / ml) and PENK protein group (25 ng / ml) was significantly higher than the other three groups. On day 14, alizarin red staining was performed, and the staining area of ​​the combination of BMP2 protein group (25 ng / ml) and PENK protein group (25 ng / ml) was again significantly higher than the other three groups. ALP activity quantification revealed statistically significant differences. Figure 9 B); Quantitative analysis of calcium ion content showed that the staining area of ​​the BMP2 protein group (25 ng / ml) combined with the PENK protein group (25 ng / ml) was significantly higher than that of the other three groups, and the difference was statistically significant. Figure 9 C);

[0055] Figure 9 D- Figure 9 H was used to verify the expression of osteogenic markers RUNX2, ALPL, BMP2, and OCN by qRT-PCR. The expression of each osteogenic marker in the BMP2 protein group (25 ng / ml) and the PENK protein group (25 ng / ml) was significantly higher than that in the other three groups. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0058] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0059] Example 1

[0060] The recombinant enkephalinogen PENK protein provided in this application has the amino acid sequence shown in SEQ ID No. 1.

[0061] The nucleotide sequence of the gene encoding the aforementioned proenkephalin PENK recombinant protein is shown in SEQ ID No. 2.

[0062] The technical content protected by this application also relates to all biological materials containing the above-mentioned proenkephalin PENK recombinant protein or the above-mentioned proenkephalin PENK recombinant protein encoding gene, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors or engineered bacteria, etc.

[0063] The inventors also discovered that the PENK recombinant protein involved in this application can be combined with BMP2 protein (bone morphogenetic protein 2) and can further enhance its bone-promoting effect. In the process of combined application (i.e. use of the composition), the PENK recombinant protein and BMP2 protein are combined and applied in a 1:1 weight ratio. The amino acid sequence of BMP2 protein is shown in SEQ ID No. 3.

[0064] The osteoblasts targeted by the provided pro-PENK recombinant protein, encoding gene, and biomaterials containing both are human dental mesenchymal cells, human embryonic jawbone bone marrow stromal mesenchymal stem cells, and human embryonic long bone bone marrow mesenchymal cells; these three cell types have been found to have certain osteogenic differentiation potential in previous studies.

[0065] When administered alone, the osteogenic concentration of proenkephalin PENK recombinant protein is 10-200 ng / ml, preferably 100 ng / ml; the osteogenic concentration of bone morphogenetic protein 2 (BMP2) is 10-250 ng / ml, preferably 200 ng / ml.

[0066] During combined administration, the osteogenic concentration of proenkephalin PENK recombinant protein is 10-200 ng / ml, preferably 25 ng / ml; the osteogenic concentration of bone morphogenetic protein 2 (BMP2) is 10-200 ng / ml, preferably 25 ng / ml.

[0067] During the aforementioned osteogenic experiments, the applicant also discovered several biomarkers for detecting osteoblast osteogenic activity. The expression of these biomarkers was associated with the administration of PENK and BMP2 proteins. These biomarkers included alkaline phosphatase (ALP), transcription factor RUNX2, transcription factor SP7 / Osterix, type I collagen α1 (COL1α1), osteopontin OPN and osteocalcin OCN, and bone morphogenetic protein-2 (BMP2).

[0068] To further verify the osteogenic role of PENK in osteoblasts, a corresponding siRNA was designed to inhibit the expression of the PENK gene. The nucleotide sequences of the siRNA are shown below: 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 capacity was observed.

[0069] The sequence of PENK mRNA is shown in SEQ ID No. 6.

[0070] The applicant also constructed PENK knockout dental mesenchymal stem cells (DMSCs), examined their phenotype and various osteogenic markers, and found that the expression of multiple markers decreased, indicating that PENK does indeed have an osteogenic promoting effect.

[0071] Example 2

[0072] 1. To verify that PENK can promote osteogenic differentiation of human dental mesenchymal cells in a dose-dependent manner, an experiment was conducted to promote osteogenic differentiation using recombinant PENK protein. The specific experimental method was as follows:

[0073] (1) Digest and count dental mesenchymal stem cells (DMSCs) at a rate of 1×10⁻⁶. 4 Cells were seeded at a density of cells / cm² into 12-well plates, with 1 mL of standard complete culture medium added to each well.

[0074] (2) Place the 12-well plate in a CO2 incubator at 37°C, 5% CO2, and saturated humidity for incubation;

[0075] (3) On the second day, when the cell fusion rate reached 70%, carefully remove the complete culture medium from the well and add 1 ml of LOriCell human bone marrow mesenchymal stem cell osteogenic induction differentiation medium to the 12-well plate.

[0076] (4) Replace the medium with fresh OriCell human bone marrow mesenchymal stem cell osteogenic induction differentiation medium every 3 days;

[0077] (5) After 14 days of induction, stained with alizarin red.

[0078] Seven days after osteogenic induction, cellular alkaline phosphatase (ALP) activity was measured. The results showed that ALP expression increased with increasing PENK protein concentration (10, 25, 50 ng / mL). Quantitative alkaline phosphatase activity assays confirmed the results, showing a dose-dependent relationship, and the differences between groups were statistically significant. Twenty-one days after osteogenic induction, alizarin red staining was used to assess calcium nodule formation efficiency. The results showed that the area stained with alizarin red gradually increased with increasing PENK protein concentration (10, 25, 50 ng / mL). Quantitative analysis after dissolving calcium nodules with hexadecylpyridine chloride revealed that calcium ion content increased with increasing PENK protein concentration, and the difference was statistically significant.

[0079] 2. Subsequently, as Figure 4 As shown, bone morphogenetic protein-2 (BMP-2) was used as a positive control. Comparison revealed that PENK protein application promoted earlier and more abundant calcium nodule formation, exhibiting a significant effect in promoting early osteogenic development, even surpassing the effect of BMP-2. At 7 days of osteogenic induction, alkaline phosphatase staining was performed; the top row shows the applied PENK protein, and the bottom row shows the applied BMP-2 protein. It can be seen that the alkaline phosphatase staining effect in the PENK protein group was significantly better than that in the BMP-2 protein group. Simultaneously, a quantitative alkaline phosphatase experiment was performed, revealing a statistically significant difference between the PENK and BMP-2 groups. At 14 days of osteogenic induction, Alizarin Red staining of the induced cells showed that the amount of calcium nodule formation in the PENK protein group was also significantly higher than that in the BMP-2 protein group. However, in the late stage, at 21 days of osteogenic induction, the difference between the two groups lost its statistical significance.

[0080] 3. To demonstrate that PENK has similar effects on different osteoblasts, it was validated in different cell lines. Human embryonic jawbone bone marrow mesenchymal stem cells were used in the experiment, and the results were consistent with those in human embryonic mesenchymal cells. Validation in human embryonic long bone bone marrow mesenchymal cells yielded alizarin red results after 14 days, indicating a trend consistent with the previous two cell types. This suggests that PENK protein promotes osteogenic differentiation of human jawbone bone marrow mesenchymal stem cells in a dose-dependent manner within a certain range (20-200 ng / ml, peak at 100 ng / ml).

[0081] 4. Transient transfection of BMSCs with siRNA interfered with the expression of PENK mRNA, followed by osteogenic induction. The results showed that the osteogenic capacity of cells decreased after siRNA interference with PENK expression. Figure 5 This indicates that siRNA effectively interferes with the expression of PENK mRNA, and osteogenic activity decreases as PENK expression decreases.

[0082] 5. A PENK-overexpressing immortalized cell line was constructed and verified by q-RT-PCR. The PENK mRNA level in the cells was significantly increased, indicating the successful construction of the PENK-overexpressing immortalized cell line. Quantitative analysis after osteogenic induction showed that the osteogenic capacity of the PENK-overexpressing immortalized cells was increased. Figure 6 Overexpression of PENK human dental mesenchymal stem cells significantly increased osteogenic activity.

[0083] 6. Protein proteomic analysis showed that PENK protein can interact with CBFB protein to promote osteogenic formation. Figure 7 After siRNA interfered with CBFB expression, osteogenic activity 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 ).

[0084] 7. Alkaline phosphatase (ALP) is a marker of osteoblast activity, and RUNX2 and SP7 / Osterix are important transcription factors in osteogenic processes. qRT-PCR was used to investigate the mechanism. It was found that PENK mRNA expression increased with increasing induction time from day 0 to 14, and then decreased at day 21. After application of PENK protein, the expression of PENK mRNA in cells also increased significantly, especially on day 7. In the normal control group, the peak PENK mRNA level occurred on day 14, while after PENK protein application, there was a very significant increase on day 7, indicating a much earlier expression rate.

[0085] The expression of alkaline phosphatase (ALP), a marker of osteoblast activity, was significantly increased compared to the normal control group, with peak values ​​appearing on day 7 in both groups. The transcription factors RUNX2 and SP7 / OSX were also measured, and their expression was significantly increased after PENK protein administration. The peak expression of RUNX2 also occurred earlier, on day 7, and was approximately 10 times higher than in the normal control group. The effect on SP7 / OSX was also very significant, with RUNX2 expression occurring earlier than SP7 / OSX. RUNX2 plays a role in the transformation of mesenchymal cells into proosteoblasts, while SP7 / OSX plays a role in the maturation of proosteoblasts into osteoblasts. Preliminary PCR results indicate that PENK has an effect on both transcription factors, suggesting that it may play a role in both differentiation stages.

[0086] Single-cell data from human jawbone showed that proenkephalin (PENK) and the pre-osteoblast marker RUNX2 were co-expressed. Figure 1 ).

[0087] Other markers of osteoblast differentiation were also explored, including type I collagen α1, osteopontin OPN, and osteocalcin OCN. All of these markers showed a significant increase after the application of PENK protein.

[0088] The expression of various marker genes at different time points during osteogenic induction of Human-DMSCs is as follows: Figure 2 As shown.

[0089] A summary of the expression of various marker genes at different time points during osteogenic induction of iliac bone mesenchymal stem cells from public databases is as follows: Figure 3 As shown.

[0090] 8. BMP2 combined application technology solution:

[0091] PENK protein and BMP2 protein were formulated into a complex in a 1:1 weight ratio.

[0092] The specific application concentrations are: 50 ng / ml + 50 ng / ml and 25 ng / ml + 25 ng / ml.

[0093] The combined use of PENK and BMP2 proteins significantly promotes osteogenic activity in cells, achieving better osteogenic effects. Figure 9 ).

[0094] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A composition containing PENK protein, characterized in that, The composition is prepared from the following proteins in parts by weight: 1 part of PENK recombinant protein and 1 part of BMP2 protein; the amino acid sequence of the PENK recombinant protein is shown in SEQ ID No. 1 and the amino acid sequence of the BMP2 protein is shown in SEQ ID No.

3.

2. The composition according to claim 1, characterized in that, The nucleotide sequence encoding the PENK recombinant protein is shown in SEQ ID No.

2.

3. The use of the composition of claim 1 in the preparation of a product for promoting osteogenic differentiation of human dental mesenchymal cells.

4. The use of the composition of claim 1 in the preparation of a product for promoting osteogenic differentiation of human embryonic jawbone bone marrow stromal mesenchymal stem cells.

5. The use of the composition of claim 1 in the preparation of a product for promoting osteogenic differentiation of human embryonic long bone bone marrow mesenchymal cells.

6. The application according to any one of claims 3-5, characterized in that, The osteogenic concentration of the PENK recombinant protein is 10-200 ng / ml; the osteogenic concentration of the BMP2 protein is 10-250 ng / ml.

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