CN110974940A - Application of Ihh in preparation of medicine for preventing or treating growth plate damage - Google Patents
Application of Ihh in preparation of medicine for preventing or treating growth plate damage Download PDFInfo
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Abstract
The invention relates to an application of Ihh in preparing a medicament for preventing and/or treating growth plate damage, and the invention discovers that the Ihh medicament is used for treating the growth plate damage, and the Ihh medicament can inhibit the formation of new bones in a damaged area of the growth plate at an early stage or inhibit the process of converting cartilage into bone, namely prevent the formation of a bone bridge after the growth plate is damaged, promote the proliferation of chondrocytes at an epiphyseal damaged part and promote the regeneration of the cartilage.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and relates to an application of Ihh in preparation of a medicament for preventing and/or treating growth plate damage.
Background
The Growth Plate (Growth Plate), also called epiphyseal (epiphyses) or epiphyseal cartilage (epiphyses cartilage), is a structure specific to the skeletal system of children, is located in the cartilage region growing actively between the epiphyseal and metaphyseal, is in the form of a wavy thin Plate, and can be divided into a Resting Zone (RZ), a Proliferative Zone (PZ), a Calcified Zone (CZ) and a HypertrophyZone (HZ) extending from the epiphyseal to the metaphyseal. With the continuous division and proliferation of chondrocytes in the growth plate, cartilage matrix is generated, meanwhile, the chondrocytes move to a mature area and a fat area, interstitial calcification is carried out, the chondrocytes are necrotic and decomposed and are gradually replaced by metaphyseal bone tissue, and then the longitudinal growth of the diaphysis is maintained. Growth Plate Injury (GPI) is a common disease of childhood osteoarticular diseases caused by trauma, infection, tumor, developmental disturbance, genetic deformity, etc., and has the characteristics of osteopontia and metaphyseal weak epiphyseal tissue, namely, after Injury, bone bridge formation and calcium salt deposition, so that the epiphyseal tissue is pathologically healed to different degrees, such as unequal limb length, tibia angulation and rotation deformity, and the limb morphology and function are seriously affected. The repair of the damage of the growth plate is an important problem in the process of basic and clinical research of children, how to prevent or avoid the formation of damaged bone bridges and the occurrence of sequela deformity becomes a research hotspot of clinical orthopedic physicians, and the repair of the damage of the growth plate is a great problem in the experimental research and clinical treatment of pediatric orthopedic surgery. At present, in basic application research, the method of dissociative vascularization plate cartilage transplantation, non-cartilage tissue engineering material, cartilage cell and tissue engineering cartilage transplantation and the like is used for repairing growth plate damage, but the dissociative growth plate cartilage transplantation is difficult to apply due to lack of blood supply; the blood circulation problem is solved by the transplantation of the vascularization growth plate, but the source of the autologous transplantation donor is limited, and the allogeneic transplantation has immunological rejection; the non-cartilage tissue material can only prevent the formation of a bone bridge in the defect of the small-area growth plate and has no regeneration and repair capability. In clinical treatment, epiphyseal injury and residual deformity are treated mainly by surgical excision of bone bridge (epiphyseal block), limb lengthening (orthopedic osteotomy) and other methods. These are open surgical procedures that have not yet been found to be ideal, despite the fact that people have been constantly exploring for over a century the repair of damage to growth plates. If the specific action mechanism of the bone bridge formation and cartilage repair process after the growth plate is damaged can be discussed and determined from the aspect of molecular biology, the key point for preventing and delaying the bone bridge formation after the growth plate is damaged so as to prevent the limb growth deformity is realized.
Disclosure of Invention
In view of the above, the present invention aims to provide an application of Ihh in the preparation of a medicament for preventing and/or treating growth plate damage.
In order to achieve the purpose, the invention provides the following technical scheme:
according to the invention, through constructing a Growth Plate Injury (GPI) animal model and injecting rhIhh, the over-expressed rhIhh is found to effectively inhibit the cartilage to osteogenesis, promote the growth of growth plate chondrocytes and promote cartilage repair to a certain extent, and can effectively become an important treatment means for repairing diseases related to growth plate injury.
Use of Ihh in the manufacture of a medicament for the prevention and/or treatment of growth plate damage.
Further, the use is for Ihh to reduce the generation of bone bridges in growth plate injuries.
Further, the application is that the Ihh promotes the proliferation of the chondrocytes.
Further, the application is that Ihh accelerates regeneration and healing of a cartilage layer of a growth plate.
Further, Ihh promotes cartilage regeneration.
Further, the Ihh is a preparation capable of over-expressing the Ihh.
Further, the preparation for over-expressing the Ihh is inactivated virus package, detoxified Ihh recombinant protein or over-expressed Ihh plasmid.
Further, the Ihh is recombinant human indian hedgehog protein (rhIhh).
The invention also provides application of the Ihh combined basement membrane matrix in preparing a medicament for preventing and/or treating growth plate injury.
According to the invention, 80ug/ml rhIhh-matrix prepared by using a basement membrane matrix as a solvent is found after injection of a Growth Plate Injury (GPI) animal model, the bone bridge formation of the damaged area of the growth plate of the 80ug/ml rhIhh-matrix group is less, and the difference compared with other contrast groups has statistical significance (P <0.05), so that the high-dose group (80ug/ml rhIhh-matrix group) can inhibit the bone formation of the area of the growth plate at an early stage or convert cartilage into bone, namely prevent the formation of the bone bridge at an early stage. Clustered red growth plate chondrocytes were observed in the high dose treatment group at 8 weeks post-dose, with greater red-stained chondrocyte area in the damaged area of the 80ug/ml rhIhh-Matrigel group compared to the Saline and Matrigel groups; in contrast, the area of trabecular (neo-bone bridge) tissue formation was smaller in the 80ug/ml rhIhh-matrix group lesion area, and the difference was statistically significant (". P <0.05,. P < 0.01"). Fully indicates that the rhIhh can inhibit the formation of the bone bridge after the growth plate is damaged, has the capability of repairing cartilage and can become the key for preventing and delaying the formation of the bone bridge after the growth plate is damaged so as to prevent the growth deformity of limbs.
Further, the Ihh is a preparation capable of over-expressing the Ihh.
The preparation for over-expressing the Ihh comprises but is not limited to recombinant protein, and can also be inactivated virus package, detoxified Ihh recombinant protein or over-expressed Ihh plasmid.
Further, the Ihh is recombinant human indian hedgehog protein (rhIhh).
The invention has the beneficial effects that: the invention discovers that the over-expression Ihh medicament, namely Ihh recombinant protein, is used for treating the damage of the growth plate, the over-expression Ihh medicament can inhibit the formation of new bones in the damaged area of the growth plate at an early stage or inhibit the process of converting cartilage into bone, namely prevent the formation of bone bridges at the damaged part of the growth plate at an early stage, has the functions of promoting the proliferation of chondrocytes at the damaged part of epiphyseal and promoting the regeneration of cartilage, provides a medicament for effectively preventing and/or treating the damage of the growth plate from the aspect of non-operative treatment, and provides a new treatment strategy for repairing the damage of the growth plate.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is an electron microscope scanning image of GPI after successful modeling;
FIG. 2 shows two-dimensional results (left) and three-dimensional reconstruction (right) after GPI modeling is successful;
FIG. 3 shows the formation of new bone in the damaged area of the growth plate of each treatment group 2 weeks after the Micro-CT quantitative analysis of each experimental group;
FIG. 4 shows a two-dimensional map (left) and a three-dimensional reconstructed map (right) of Micro-CT of each experimental group;
FIG. 5 is a gross photograph of tibial plateau of rats in 8 weeks after surgery in each experimental group;
FIG. 6 shows Safrain O/Fast Green staining of proximal tibial cartilage of knee joint on the left side of rats in each experimental group;
FIG. 7 is a bar graph comparing the areas of red-stained chondrocytes in the damaged area of each experimental group and the area of trabecular bone (new bone bridge) tissue formation in the damaged area of each group;
FIG. 8 is a graph showing HE staining and Masson staining of sections of each experimental group;
FIG. 9 is a bar graph comparing chondrocytes and osteoblasts under HE staining and Masson staining for each experimental group;
FIG. 10 is a graph of OCN immunohistochemical staining of Saline control and high dose groups 2 weeks post-surgery;
FIG. 11 is a graph of OCN immunohistochemical staining of Saline control and high dose groups at 8 weeks post-surgery;
FIG. 12 is a photograph of COL II immunohistochemical staining of Saline control and high dose groups at 8 weeks post-surgery;
FIG. 13 is a histogram comparing the results of immunohistochemical staining for post-operative 2w OCN, post-operative 8w OCN, and post-operative 8w COL II in Saline control group and 80. mu.g/ml rhIhh-Matrigel group.
FIG. 14 shows the results of RT-PCR of tissues in the damaged area of rat growth plate in each group.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturers.
Experimental part materials and instruments:
1) recombinant Protein Indian Hedgehog Homolog (rhIhh) Ihh Recombinant Protein: r & DSsystems, Specification: 25 ug/count, usa;
2) matrigel: basement Membrane matrix, Basement Membrane gel, also called BME (basal Membrane Extract). Cultrex Basement Membrane Extract (BME) U.S. Trevigen cat No.: 3431 001-02;
3) ioversol: jiangsu Hengrui medicine, Inc. (national Standard H20067896), Jiangsu, China;
4) hematoxylin-eosin (HE) staining solution, bi yun tian biotechnology limited, china;
5) masson staining, Safrain O/Fast Green, Sigma, USA;
6) rabbit primary antibodies against mouse Agg, COL II, COL X, OCN, OPG, RANKL, BMP-2, RUNX 2: bosch de bioengineering, ltd, wuhan, china;
7) prime script RT Master Mix kit: takara corporation, japan;
8) SYBR Premix Ex TaqTM II kit: takara corporation, japan;
9) β -actin, Agg, COL II, COL X, OCN, OPG, RANKL, BMP-2, RUNX2 primers Shanghai, China
10) Leica M720 OH5 surgical microscope: leica (Leica) microsystem Germany;
11) MicroCT/uCT: SCANCO Medical AG, Switzerland;
12) an X-ray imaging system: UltraFocus100X ray cabinet, Faxitron, USA;
13) scanning Electron Microscope (SEM): EVO MA 15/LS 15, Calzaisi, Germany.
Example 1 construction of a successful growth plate damage (GPI) animal model
100 healthy 5-week Sprague Dawley (SD) rats are selected and randomly and averagely divided into 5 groups, each group comprises 20 rats, the experimental group carries out central drilling on the inner platform of the left proximal tibia of the rat through a 2.0mm needle to establish a Growth Plate Injury (GPI) model, and the sham operation group adopts operation to incise knee joint capsules without drilling the proximal tibia.
The GPI model adopts a mode of drilling a hole by using a 2.0mm syringe needle after opening a left knee joint capsule to establish a successful growth plate damage model, and the specific operation process of the molding mode is as follows: a) taking the left knee joint, preparing skin, sterilizing by a conventional method, and paving; b) a knee joint is vertically cut, a patellar ligament is fully exposed, a joint cavity is opened along the side of the patellar ligament, and a medial meniscus is removed; c) rotating a needle head of a 2.0mm syringe to destroy a rat growth plate; d) the 2.0mm hole damage position can be seen; e) 20ul of medicine is injected; f) after the operation, the skin is sutured. After 1 day of operation, 1 rat in each group was sacrificed randomly, and the proximal tibia was cut horizontally along the medial and lateral tibial plateau parallel to the long axis vector position and observed under a scanning electron microscope. As shown in fig. 1, the result of the electron microscope is shown in fig. 1, where a is a graph showing that the drilled hole injury position successfully penetrates the growth plate (red arrow), the injury is in a regular circle (diameter ═ 2.0mm) and the injury area occupies about 30.00 ± 0.0070% of the medial tibial plateau and 15.59 ± 0.0017% of the total tibial plateau, and it is confirmed that the 2.0mm needle-punched growth plate injury is effective, and the injury area is more than 10% of the total plateau area. FIG. 1, panel B, shows the results of tibial proximal growth plate tissue (shown by the right arrow) and trabecular bone results (shown by the left arrow) for 5-week-old rats. In order to determine the successful injection of the medicine, the contrast agent ioversol is injected immediately after the drilling hole is damaged in the operation, the joint cavity and the surface skin are sutured immediately, the rat is placed on a Micro-CT immediately, the position condition of the contrast agent is observed, the ioversol contrast agent is filled in the drilling hole defect position, a high-density image is seen, as shown in figure 2, wherein the two-dimensional result (the left image in figure 2) and the three-dimensional reconstruction (the right image in figure 2) result of the CT show that the ioversol contrast agent is filled in the drilling hole defect position (the high-density image) at the proximal tibia of the rat, the ioversol contrast agent, the peripheral bone tissue and the growth plate tissue are clear in level, only a small amount of peripheral omission exists, the ioversol contrast.
Example 2
rhIhh-Matrigel: rhIhh was used as a solute, and mixed and diluted with Matrigel gel to prepare 40ug/ml and 80ug/ml rhIhh-Matrigel mixed solutions, respectively, for experimental injection treatment.
Matrigel acts here as a gel solvent and has no therapeutic effect.
The experiments are divided into five groups, including: (1) sham (Sham); (2) model group control group (injected with 20 μ l of physiological Saline, reference numeral Saline); (3) blank control group (injection of 20. mu.l Matrigel, reference numeral Matrigel) Matrigel; (4) low dose group (injected with 20. mu.l of 40ug/ml Recombinant Ihh Protein (rhIhh) -Matrigel, reference numeral 40ug/ml rhIhh-Matrigel), (5) high dose group (injected with 20. mu.l of 80ug/ml Recombinant Ihh Protein (rhIhh) -Matrigel, reference numeral 80ug/ml rhIhh-Matrigel). Here the high and low doses are used only to distinguish the groups 2 in the experiment.
The rats subjected to the surgical drilling injury in the embodiment 1 are immediately injected with the corresponding drugs of each group respectively, the injury part of the growth plate is gradually replaced by the bone bridge tissue at the 2 nd week after the surgery, 3 rats of each group at the 2 nd week after the surgery are taken, the rats are subjected to down Micro-CT examination, the bone bridge formation at the defect part of the growth plate is quantitatively analyzed, and the bone bridge formation conditions of each group are compared. Micro-CT quantitative analysis of new bone formation in the damaged area of growth plate of each treatment group 2 weeks after surgery, and table 1 shows the new bone mass data of each experimental group, as shown in fig. 3, compared with Saline group, 80ug/ml rhIhh-matrix group had less bridge formation and the difference was statistically significant (P < 0.05).
TABLE 1 Micro-CT quantification of post-operative 2 weeks new bone formation
FIG. 4 is a two-dimensional graph (left) and a three-dimensional reconstruction graph (right) of each group of Micro-CT, wherein different amounts of new bones (bone bridges) in a damaged area of the growth plate can be seen in comparison of each small graph, and the replacement area of bone tissues of each layer of the coronal part of the damaged area of the growth plate is quantitatively analyzed by using Micro-CT analysis software, and the results show that: the matrix, 40ug/ml rhIhh-matrix and 80ug/ml rhIhh-matrix groups had fewer bridges formed in the damaged area of the growth plate than the Saline group, with statistical differences (P < 0.05); compared with the rest groups, the 80ug/ml rhIhh-matrix group has less bone bridge formation in the damaged area of the growth plate, the difference has statistical significance (P <0.05), and the initial explanation shows that the high-dose group (80ug/ml rhIhh-matrix group) can inhibit the bone formation in the area of the growth plate or inhibit the process of converting cartilage into bone, namely prevent the formation of bone bridge in the early stage.
Example 3
Fixing with 4% paraformaldehyde for 1-2 days, and decalcifying with 15% EDTA-2Na at 20 deg.C for 20 days (taking soft bone tissue or needle penetrating as stop point). Paraffin embedding: after decalcification is finished, a slicing knife is used for cutting along the sagittal section of the inner and outer platforms, the inner platform is reserved, the section is attached to the embedding box, and the tissue placement direction is vertical or parallel to the edge of the embedding box. Making paraffin sections: removing redundant paraffin wax around a paraffin wax block by a slicing knife, freezing for 10-15min in an ice block, adjusting the thickness of the slice to 5um, cutting off, putting on a 50 ℃ slice spreading machine, after the paraffin wax slice is completely unfolded (observing the damaged part on the slice by naked eyes or a microscope after the slice is fished, and searching the damaged plane layer by layer one by layer), fishing the slice by using a glass slide, drying at room temperature, and baking at 70 ℃ for 1 h.
Safrain O/Fast Green staining was performed on the proximal cartilage of the tibia of the left knee joint of each group of rats, and the staining results are shown in FIG. 6, wherein in FIG. 6, A is a Sham group, B is a Saline group, C is a Matrigel group, D is a 40ug/ml rhIh-Matrigel group, and E is an 80ug/ml rhIh-Matrigel group, which shows that there is a significant difference between the combined treatment with high dose of 80ug/ml rhIh-Matrigel and the Saline-treated rats, and the damaged areas of the growth plates of the 80ug/ml rhIh-Matrigel-treated rats have more cartilage red staining areas than the damaged areas of the growth plates treated with Matrigel and 40ug/ml rhIh-Matrigel; compared with the other treatment groups, the rhIhh-Matrigel group with the concentration of 80ug/ml has more chondrocytes in the damaged area, clusters, distributes growth plate layers and shows cartilage red staining; saline group had no obvious red staining, no obvious chondrocyte filling was seen in the injured area, and a new bone cell cluster was seen locally. The pathological evaluation of Matrigel and 40ug/ml rhIhh-Matrigel was intermediate between the former two, but the chondrocyte clustering degree of the 40ug/ml rhIhh-Matrigel group was significantly better than that of the Matrigel group. FIG. 7 is a bar graph comparing the area of red-stained chondrocytes in each group of damaged areas and the area of trabecular bone (new bone bridge) tissue formation in each group of damaged areas, and it can be seen from FIG. 7 that the clustered red-stained growth plate chondrocytes are observed in the high dose treatment group at week 8 after surgery, and the area of red-stained chondrocytes in the damaged area of 80ug/ml rhIhh-Matrigel group is larger than that of the Saline group and Matrigel group; in contrast, the area of trabecular (neo-bone bridge) tissue formation was smaller in the 80ug/ml rhIhh-matrix group lesion area, and the difference was statistically significant (". P <0.05,. P < 0.01"). Fully indicates that the rhIhh can inhibit the formation of the bone bridge after the growth plate is damaged, has the capability of repairing cartilage and can become the key for preventing and delaying the formation of the bone bridge after the growth plate is damaged so as to prevent the growth deformity of limbs.
Example 4
Sections prepared in example 3 were HE stained and Masson stained and observed under light. The HE staining and Masson staining of the sections of each group were observed under a light microscope, and the staining results are shown in FIG. 8, in which the upper panel is the HE staining, the lower panel is the Masson staining, A is the Sham group, B is the Saline group, C is the Matrigel group, D is the rhIhh-Matrigel group of 40ug/ml, and E is the 80ug/ml rhIhh-Matrigel group. As shown in FIG. 8, the Sham group growth plate has a complete structure, the chondrocytes are regularly arranged and uniformly distributed along the growth plate in a transverse row, the layers are clear, the number of the chondrocytes is normal, and no obvious cluster is found. 80ug/ml rhIhh-Matrigel group can be seen in the clustering, clustered chondrocytes along the damaged area, and staggered with a small amount of osteoblasts and trabeculae, and the area of chondrocytes is larger, and the area of bone bridge cells is smaller; the Saline group is interrupted in the growth plate area, a large amount of osteoblasts in the damaged area are filled, obvious bone bridge formation is shown, most of the damaged area is filled, and no obvious chondrocyte clustering is shown; in the Matrigel group, damage causes the interruption of a growth plate area, a small amount of osteoblasts are filled in the area, a bone bridge is less formed, and obvious chondrocyte filling is not seen; the HE staining and Masson staining of the 40ug/ml rhIhh-matrix group was intermediate between the Matrigel and 80ug/ml rhIhh-matrix treated groups, but the cartilage number was higher and the osteoblast number was lower in the 80ug/ml rhIhh-matrix group, and the difference was statistically significant (P <0.05), see FIG. 9 for a histogram comparing chondrocytes and osteoblasts under HE staining and Masson staining for each group. The primary results of HE staining and Masson staining show that the over-expression of Ihh can effectively inhibit the transformation of cartilage to osteogenesis, promote cartilage repair to a certain extent, and can effectively become an important treatment means for repairing diseases related to growth plate injury (such as epiphyseal fracture, underdeveloped cartilage and the like).
Example 5
At 2 weeks after surgery, on immunohistochemical staining of OCN (Osteocalcin), 80. mu.g/ml rhIhh-Matrigel group had less local bone tissue staining, lighter staining, less new bone tissue (i.e., less bridge formation) in the lesion area, and OCN immunohistochemical staining was lower than that of Saline group in the lesion area, with statistical differences (P <0.05), as shown in FIG. 10. At 8 weeks after surgery, on immunohistochemical staining of OCN (osteogenic Marker), 80ug/ml rhIhh-Matrigel group had less local staining of bone tissue in the injured area, less staining, and a small amount of chondrocyte filling, and the difference was statistically significant (P <0.05) (as shown in FIG. 11), compared to Saline group. At 8 weeks after surgery, in COL II immunohistochemical staining, cartilage cells were stained more deeply with collagen and larger areas than those in Saline group at 80. mu.g/ml rhIhh-Matrigel, and more cartilage tissues were generated, and the difference was statistically significant (P <0.05), as shown in FIG. 12. FIG. 13 is a histogram comparing the post-operative 2w OCN, post-operative 8w OCN and post-operative 8w COL II immunohistochemical staining results for the Saline group and the 80. mu.g/ml rhIhh-Matrigel group, with P <0.05 indicating that the differences were statistically significant compared to the Saline group. Fully shows that the over-expression Ihh can inhibit the cartilage osteogenesis process at the damaged part of the growth plate and promote the growth of growth plate chondrocytes, for example, the growth plate can be used for treating epiphyseal injury diseases in the development stage, and can achieve better treatment effect and delay the occurrence of complications (such as bone angulation deformity or short deformity) after the epiphyseal injury.
Example 6
Real-time quantitative PCR (RT-PCR)
Every group randomly selects 3 rats after 8 weeks of operation, only keeps the proximal part of tibia, eliminates the tissue on the surface, cuts open the bone with an operation blade under an operation microscope, removes periosteum, a proximal cartilage layer and a bone layer, exposes the complete cartilage tissue of a growth plate, keeps the tissue of the cartilage (including a static region, a proliferation region, a hypertrophic region and a calcification region) of the full-layer growth plate with the operation microscope, and cuts out the damaged part (the area is slightly wider than the damaged part, and the color is different from that of the normal cartilage) with the operation blade. RT-qPCR method is adopted to detect mRNA expression conditions of Agg, COL II, COL X, OCN, OPG, RANKL, BMP-2 and RUNX2 of a Sham group, a salt group, a Matrigel group, a 40ug/ml rhIh-Matrigel group and an 80ug/ml rhIh-Matrigel group, each group of samples is subjected to 3 times of repeated experiments, and the data of the experiment results are expressed as 'average +/-standard error (X +/-s)'. SPSS 22.0 data software is used for processing, and single-factor analysis of variance is adopted for comparing group data with group data; p <0.05 indicates that the difference is statistically significant. The primer sequences used in the RT-qPCR are shown in Table 1.
TABLE 1 RT-qPCR primer sequences
The results of RT-PCR on the tissue of the damaged area of the rat growth plate in each group are shown in FIG. 14: on the mRNA expression of Agg and COL II, the mRNA expression of 80ug/ml rhIh-Matrigel group Agg and COL II is the highest, and the dosage dependence relationship is formed, namely the mRNA expression of 80ug/ml rhIh-Matrigel group is higher than that of 40ug/ml rhIh-Matrigel group, and the difference has statistical significance (P < 0.05); and the expression of the salt group Agg and COL II is the lowest, which indicates that the rhIhh-Matrigel has the function of promoting the regeneration of cartilage.
On COL X expression, the 80ug/ml rhIhh-Matrigel group had slightly higher mRNA expression than the Sham group, but both were not statistically significant (P < 0.05); the mRNA expression of the Saline group, the Matrigel group and the 40ug/ml rhIhh-Matrigel group has no obvious difference, but the mRNA expression of the COL X in the three groups is higher than that of the 80ug/ml rhIhh-Matrigel group, and the difference has statistical significance (P < 0.05); the expression of mRNA in OCN, OPG, RANKL, BMP-2, and RUNX2 was significantly higher in the Saline group than in the 80ug/ml rhIhh-Matrigel, 40ug/ml rhIhh-Matrigel, and Matrigel groups. On the mRNA expression of OCN, OPG, RANKL and BMP-2, the treatment groups are in a dose-dependent relationship, the mRNA expression of OCN, OPG, RANKL and BMP-2 in Matrigel, 40ug/ml rhIh-Matrigel and 80ug/ml rhIh-Matrigel groups is reduced in sequence, and the difference has statistical significance (P is less than 0.05); the Matrigel group was reduced in expression of RUNX2 compared to the 40ug/ml rhIhh-Matrigel group, with the differences being statistically significant (P < 0.05). The results show that: the rhIhh and Matrigel combined have obvious effects of inhibiting the expression of bone-related factors and promoting the expression of cartilage-related factors, and the high-dose rhIhh and Matrigel combined have better effects of inhibiting the bone-related factors and promoting the cartilage factors, and have better effects of inhibiting the formation of bone bridges and promoting the cartilage repair of growth plates.
The invention proves that the direct injection of the recombinant protein Ihh can inhibit the cartilage osteogenesis process at the damaged part of the growth plate, promote the growth of the chondrocytes of the growth plate, treat the epiphyseal injury disease in the development period, achieve better treatment effect and delay the occurrence of complications (such as angulation deformity or shortening deformity) after the epiphyseal injury.
The pharmaceutical form includes but is not limited to recombinant protein Ihh, and different methods can be used to construct over-expressed Ihh preparations, such as inactivated virus package, detoxified Ihh recombinant protein, or over-expressed Ihh plasmid package. The treatment method can adopt direct intra-articular injection and infiltration to chondrocytes along the fracture injury, or intravenous injection and infiltration to the injury part through blood vessels.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (9)
- Use of Ihh in the manufacture of a medicament for the prevention and/or treatment of growth plate damage.
- 2. The use according to claim 1, wherein said use is Ihh to reduce bone bridge formation at a damaged site following growth-plate damage.
- 3. Use according to claim 1, characterized in that said use is the promotion of chondrocyte proliferation by Ihh.
- 4. The use according to claim 1, wherein said use is Ihh accelerating healing of growth plate cartilage layer regeneration.
- 5. Use according to claim 1, characterized in that Ihh is an agent capable of overexpressing Ihh.
- 6. The use according to claim 1, wherein Ihh is recombinant human indian hedgehog protein.
- The application of the Ihh combined basement membrane matrix in preparing the medicines for preventing and/or treating the damage of the growth plate.
- 8. Use according to claim 6, characterized in that the Ihh is an agent capable of overexpressing Ihh.
- 9. The use according to claim 6, wherein the Ihh is recombinant human indian hedgehog protein.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114934052A (en) * | 2022-06-14 | 2022-08-23 | 贵州医科大学附属医院 | Application of long-chain non-coding RNA AABR07017227 |
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