阅读说明：本技术 提高smurf1蛋白表达量的试剂在制备防治钙化性主动脉瓣疾病的药物中的应用 (Application of reagent for improving smurf1 protein expression quantity in preparing medicine for preventing and treating calcified aortic valve diseases ) 是由 王勇军 董念国 韩东 周廷文 于 2021-08-04 设计创作，主要内容包括：本发明涉及提高smurf1蛋白表达量的试剂在制备防治钙化性主动脉瓣疾病的药物中的应用。本发明经研究发现提高smurf1蛋白表达量可以有效下调瓣膜间质细胞中成骨分化标志基因RUNX2和Osterix的表达,抑制瓣膜间质细胞成骨分化,从而延缓或者抑制瓣膜钙化。进而提出了一种通过上调smurf1的表达水平以防治主动脉瓣钙化的方法,为钙化性主动脉瓣疾病的预防和治疗提供了新的非手术治疗方案。(The invention relates to application of a reagent for improving the expression level of smurf1 protein in preparing a medicament for preventing and treating calcified aortic valve diseases. According to the invention, researches show that the expression quantity of smurf1 protein is increased, so that the expression of osteogenic differentiation marker genes RUNX2 and Osterix in valve interstitial cells can be effectively reduced, and osteogenic differentiation of the valve interstitial cells is inhibited, thereby delaying or inhibiting valve calcification. Furthermore, a method for preventing calcification of the aortic valve by up-regulating the expression level of smurf1 is provided, which provides a new non-operative treatment scheme for preventing and treating calcified aortic valve diseases.)

1. Application of the reagent for improving the expression level of smurf1 protein in preparing medicines for preventing and treating calcified aortic valve diseases.

2. The use of the agent for increasing the expression level of smurf1 protein in the preparation of a medicament for preventing and treating calcified aortic valve diseases according to claim 1, wherein the agent for increasing the expression level of smurf1 protein comprises any one of the following:

smurf1 protein;

a recombinant vector containing a gene encoding smurf1 protein;

recombinant viruses containing genes encoding smurf1 protein;

smurf1 analogs.

3. The reagent for increasing the expression level of smurf1 protein according to claim 2, wherein the recombinant virus containing the gene encoding smurf1 protein is a recombinant lentivirus or a recombinant adenovirus containing the gene encoding the protein.

4. The agent for increasing the expression level of smurf1 protein according to claim 2, wherein the smurf1 analogue is recombinant smurf1 polypeptide.

5. The use of the reagent for increasing the expression level of smurf1 protein in the preparation of a medicament for preventing and treating calcified aortic valve diseases according to claim 1, wherein the reagent for increasing the expression level of smurf1 protein inhibits the osteogenic differentiation of valve interstitial cells by mediating the ubiquitination degradation of osteogenic differentiation related gene RUNX2, thereby delaying valve calcification.

6. The use of the agent for increasing the expression level of smurf1 protein in the preparation of the medicament for preventing and treating the calcified aortic valve diseases according to claim 1, wherein the medicament for preventing and treating the calcified aortic valve diseases is a pharmaceutical composition prepared from the agent for increasing the expression level of smurf1 protein and a conventional pharmaceutical carrier.

7. The medicine for preventing and treating calcified aortic valve diseases according to claim 6, wherein the pharmaceutical composition is capsule, granule, injection, sustained release tablet, buccal tablet or powder injection.

8. The use of the agent for increasing the expression level of smurf1 protein according to claim 1 in the preparation of a medicament for preventing and treating calcified aortic valve diseases, wherein the medicament for preventing and treating calcified aortic valve diseases is a medicament for increasing the expression level of smurf1 in aortic valve tissues.

9. A method for preventing aortic valve calcification, the method comprising up-regulating the expression level of smurf1 in a subject to prevent aortic valve calcification.

The technical field is as follows:

the invention relates to the field of biological medicine, in particular to application of a reagent for improving the expression level of smurf1 protein in preparation of a medicine for preventing and treating calcified aortic valve diseases.

Background art:

calcified Aortic Valve Disease (CAVD) is a progressive disease of high morbidity and mortality in the elderly, with major pathophysiological changes being fibroproliferative calcification of the aortic valve leaflets leading to valve stiffening and causing hemodynamic changes affecting cardiac function. From the past, CAVD is considered to be an age-related degenerative disease, which is a degenerative and hardening process of valve tissue with age. However, basic research in recent years indicates that CAVD is an active progress process involving complex pathological changes such as endothelial injury, inflammatory cell infiltration, extracellular matrix remodeling, and osteogenic differentiation of valve interstitial cells. Cells in valve tissue mainly include Valve Endothelial Cells (VECs), Valve Interstitial Cells (VICs), valve precursor cells, and the like, and research has proved that calcium salt increase caused by osteogenic differentiation of valve interstitial cells may be an important initiation factor for valve calcification. Currently, CAVD lacks an effective clinically available drug treatment regimen, the primary treatment of which is aortic valve replacement surgery. However, the patient who is subjected to the surgical operation inevitably bears a high medical operation risk and an economic burden. Therefore, exploring the specific pathogenesis of CAVD, the effective prevention and/or treatment of calcified aortic valve disorders by non-surgical methods is currently an urgent need for clinical treatment of CAVD.

SMAD specificity E3 ubiquitin protein ligase 1(smurf1) is a member of E3 ubiquitin protein ligase family mediating ubiquitination modification, and the research on the protein is mainly focused on the influence of the protein on osteoblast activity, bone homeostasis, mesenchymal stem cell osteogenic differentiation and the tumor field.

It was found that Smurf1 can be negatively correlated with osteoblast activity (Yamashita M, Ying SX, Zhang GM, Li C, Cheng SY, Deng CX, Zhang YE. ubiquitin lipid Smurf1 control osteoactive and bone hostats by targeting MEKK2 for degradation. cell 2005; 121(1): 101-13.). smurf1 Inhibits Osteoblast Differentiation by reducing Runx2 accumulation in osteoblasts (Shimazu J, Wei J, Karseny G.Smurf1 inhibitors Osteoblast Differentiation, Bone Formation, and Glucose homestasis through spring 148.Cell Rep 2016; 15(1): 27-35). These studies suggest that smurf1 may play an important role in the osteoblastic differentiation phenotype transformation of valve mesenchymal cells and aortic valve calcification, and the agent for improving the expression level of smurf1 protein has potential clinical value in the preparation of drugs for preventing or treating calcified aortic valve diseases.

The invention content is as follows:

technical problem to be solved

Aiming at the background, the research shows that the improvement of the expression level of smurf1 protein can effectively reduce the expression of osteogenic differentiation marker genes RUNX2 and Osterix in valve interstitial cells and inhibit the osteogenic differentiation of the valve interstitial cells, so that the valve calcification is delayed or inhibited, the application of the reagent for improving the expression level of the smurf1 protein in the preparation of the medicine for preventing and treating the calcified aortic valve diseases is provided, and a novel non-operative treatment scheme is further provided for the prevention and treatment of the calcified aortic valve diseases.

(II) technical scheme

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of a reagent for improving the expression level of smurf1 protein in preparing a medicament for preventing and treating calcified aortic valve diseases.

Further, the reagent for improving the expression level of smurf1 protein comprises any one of the following reagents:

smurf1 protein;

a recombinant vector containing a gene encoding smurf1 protein;

recombinant viruses containing genes encoding smurf1 protein;

smurf1 analogs.

Furthermore, the recombinant virus containing the smurf1 protein coding gene is a recombinant lentivirus or a recombinant adenovirus containing the protein coding gene.

Further, the smurf1 analogue is a recombinant smurf1 polypeptide.

The reagent for improving the expression level of smurf1 protein inhibits the osteogenic differentiation of valve interstitial cells by mediating the ubiquitination degradation of osteogenic differentiation related gene RUNX2, thereby delaying the calcification of valves.

Furthermore, the medicine for preventing and treating calcified aortic valve diseases is a medicine composition prepared from a reagent for increasing the expression level of smurf1 protein and a conventional medicinal carrier.

Further, the pharmaceutical composition is capsules, granules, injection, sustained release tablets, buccal tablets or powder injection.

Further, the medicament for preventing and treating the calcified aortic valve diseases is a medicament for improving the expression level of smurf1 in aortic valve tissues.

The invention also provides a method for preventing and treating aortic valve calcification, which comprises the step of up-regulating the expression level of smurf1 in a treatment subject so as to prevent and treat aortic valve calcification.

(III) advantageous effects

The invention has the following beneficial effects: the invention provides an application of a reagent for improving smurf1 protein expression quantity in preparing a medicament for preventing and treating calcified aortic valve diseases, and researches show that improving smurf1 protein expression quantity can effectively reduce the expression of an osteogenic differentiation marker gene RUNX2 in valve interstitial cells and inhibit osteogenic differentiation of the valve interstitial cells, thereby delaying or inhibiting valve calcification, further providing a method for preventing and treating aortic valve calcification by increasing the expression level of smurf1, and providing a new scheme for treating calcified aortic valve diseases.

Description of the drawings:

in order to more clearly illustrate the technical solution in the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly described below.

FIG. 1: quantitative analysis of smurf1 in calcified aortic valve versus normal aortic valve tissue;

wherein figure 1A is a timely fluorescent quantitative PCR showing that smurf1 is significantly reduced in expression in calcified aortic valve tissue compared to normal aortic valve tissue; FIG. 1B shows that SMurf1 expression was measured in normal and calcified aortic valve tissue using Western blot, showing that SMurf1 protein levels were significantly reduced in calcified aortic valve tissue;

FIG. 2: quantitative analysis of smurf1 in the process of inducing human aortic valve interstitial cell osteogenesis differentiation by using an osteogenesis induction culture medium;

wherein fig. 2A is a timely fluorescent quantitative PCR showing that smurf1 shows a time-dependent significant decrease in expression in human valve stromal cells induced in osteogenic induction medium for 0 day, 1 day, 3 days, 5 days, 7 days, and 14 days, as compared to the control group; FIG. 2B shows that the expression of smurf1 protein levels in human valve stromal cells induced by osteogenic induction medium for 0 day, 1 day, 3 days, 5 days, 7 days and 14 days showed significant time-dependent decrease compared to the control group in Western blot assay;

FIG. 3: overexpression of smurf1 improved the in vitro osteogenic differentiation phenotype mapping of human valve stromal cells.

Wherein figure 3A is alizarin red showing a significant reduction in calcium salt nodules in human aortic valve stromal cells of the smurf 1-overexpressing group compared to the control group; FIG. 3B is a graph showing that calcium deposition in human aortic valve stromal cells is significantly reduced after overexpression of smurf1 by calcium quantitation; FIG. 3C is a graph showing that alkaline phosphatase activity was significantly decreased in human aortic valve stromal cells in the smurf 1-overexpressed group compared to the control group;

FIG. 4: knocking down smurf1 promotes osteogenic differentiation of human aortic valve interstitial cells.

Wherein figure 4A is alizarin red showing a significant increase in calcium salt nodules in human aortic valve stromal cells in the knockdown smurf1 group compared to the control group; FIG. 4B is a graph showing that calcium deposition in human aortic valve interstitial cells is significantly increased after knocking down smurf1 for calcium quantitation; FIG. 4C is a graph showing that alkaline phosphatase activity assay shows that alkaline phosphatase activity of interstitial cells of human aortic valve is significantly increased in the knockdown smurf1 group compared to the control group;

FIG. 5: smurf1 plays a role in inhibiting osteogenic differentiation of human aortic valve interstitial cells by inhibiting expression of osteogenic differentiation marker protein Runx 2.

Wherein, FIG. 5A shows that Runx2 expression changes after the western blot detects the overexpression of smurf1, and the result shows that the Runx2 expression is obviously reduced after the overexpression of smurf 1; FIG. 5B shows that Runx2 expression changes after the detection of western blot knockdown of smurf1 show that Runx2 expression is obviously increased after the detection of the reduction of smurf 1;

the specific implementation mode is as follows:

the technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

Example 1, tissue level expression assay of smurf 1.

In the embodiment, the aortic valve tissues (a mild calcification group and a severe calcification group) of the aortic valve calcification patient and the aortic valve tissues (a control group) of the heart transplantation patient are collected, the real-time fluorescence quantitative PCR and western blot methods are used for respectively detecting the mRNA and protein expression level changes of smurf1 in the aortic valve tissues, and the result shows that the mRNA (shown in figure 1A) and protein (shown in figure 1B) expression levels of smurf1 are obviously reduced in the calcified aortic valve tissues. Wherein the smurf1 real-time fluorescent quantitative PCR primer sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

Example 2, construction of a calcification-inducing medium for inducing osteoblastic differentiated cell models of human valve stromal cells.

Taking human valve interstitial cells with the culture density of about 70 percent, starving the human valve interstitial cells overnight by using a DMEM high-sugar culture medium containing 2 percent fetal calf serum, and performing osteogenic differentiation induction on the human valve interstitial cells by newly configuring an osteogenic induction culture medium (50mg/mL vitamin C, 5mmol/L beta-glycerophosphate, 100nmol/L dexamethasone and a high-sugar DMEM culture medium containing 2 percent fetal calf serum) on the second day. The effect of different treatments on the osteogenic differentiation of valve stromal cells was examined after 14 days of induction.

Example 3, osteogenic induction medium induced inhibition of smurf1 expression in human valve stromal cells.

In the embodiment, by adopting the scheme in the embodiment 2, after osteogenic differentiation of human valve interstitial cells is induced at different time points of 0 day, 1 day, 3 days, 5 days, 7 days and 14 days, the change of the expression levels of mRNA and protein of smurf1 in the osteogenic differentiation induced cells at different time points is respectively detected by using a real-time fluorescence quantitative PCR and western blot method, and the result shows that the expression levels of mRNA (shown in figure 2A) and protein (shown in figure 2B) of smurf1 are both remarkably reduced in a time-dependent manner in an osteogenic induction culture group.

Example 4 overexpression of smurf1 inhibited osteoblastic differentiation of human valve stromal cells.

In the present example, valvular stromal cells were isolated and cultured, and smurf1 overexpression adenovirus was constructed. After transfusing valvular interstitial cells by the over-expression adenovirus for 72 hours, inducing the valvular interstitial cells by using an osteogenic differentiation induction culture medium to construct an in vitro osteogenic differentiation model. And (3) evaluating the calcification degree of the valve interstitial cells by using alizarin red staining, calcium quantitative analysis and alkaline phosphatase activity. Alizarin red staining and calcium quantitative analysis show that compared with a control group, overexpression of smurf1 can obviously inhibit the increase effect (P < 0.05) of calcium salt nodules (figure 3A) and calcium salt deposition (figure 3B) of valve interstitial cells induced by an osteogenic induction medium. Alkaline phosphatase activity detection found that overexpression of smurf1 significantly inhibited the valvular interstitial alkaline phosphatase activity-increasing effect induced by osteogenic induction medium compared to the control group (fig. 3C).

Example 5, knockdown of smurf1 promoted osteogenic differentiation of human valve stromal cells.

In the embodiment, the valvular interstitial cells are isolated and cultured, and after the small interfering RNA (si-smurf1) of smurf1 is used for transfecting the valvular interstitial cells and knocking down smurf1, the valvular interstitial cells are induced by an osteogenic differentiation induction culture medium for 14 days to construct an in vitro osteogenic differentiation model. And (3) evaluating the calcification degree of the valve interstitial cells by using alizarin red staining, calcium quantitative analysis and alkaline phosphatase activity. Alizarin red staining and calcium quantitative analysis show that compared with a control group, knocking-down smurf1 can obviously promote the increase effect (P < 0.05) of calcium salt nodules (figure 4A) and calcium salt deposition (figure 4B) of valve interstitial cells induced by an osteogenic induction medium. Alkaline phosphatase activity assay found that knocking down smurf1 significantly promoted increased alkaline phosphatase activity of valve stromal cells induced by osteoinduction medium compared to control (fig. 4C). Wherein the sequence of the smurf1 small interfering RNA (si-smurf1) is shown in SEQ ID NO: 3, and (b) is shown in the specification.

Example 6, smurf1 inhibited osteoblastic differentiation of human valve stromal cells by inhibiting Runx2 expression.

In the embodiment, the valvular interstitial cells are isolated and cultured, the smurf1 overexpression adenovirus is constructed, the valvular interstitial cells are transfected by the overexpression adenovirus for 72 hours, and then the in vitro osteogenic differentiation model is constructed by inducing the valvular interstitial cells by using the osteogenic differentiation induction culture medium for 14 days. Then, the expression change of an osteogenic differentiation marker protein Runx2 is detected, and the result of western blot shows that the expression of Runx2 is obviously reduced after smurf1 is over-expressed (FIG. 5A). Then, after SMurf1 small interfering RNA (si-SMurf1) is used for transfecting valvular interstitial cells to knock down SMurf1, an osteogenic differentiation induction culture medium is used for inducing the valvular interstitial cells for 14 days to construct an in vitro osteogenic differentiation model. Then, the expression change of an osteogenic differentiation marker protein Runx2 is detected, and the result of western blot shows that the expression of Runx2 is obviously increased after the smurf1 is knocked down (FIG. 5B).

The results prove that smurf1 can effectively inhibit osteogenic differentiation of human valve interstitial cells, and can play a role in inhibiting osteogenic differentiation of the valve interstitial cells by inhibiting expression of Runx 2.

Finally, it should be noted that the above examples are only used for illustrating the present invention and do not limit the protection scope of the present invention. In addition, after reading the technical content of the invention, the skilled person can make various changes, modifications or variations to the invention, and all the equivalents thereof also belong to the protection scope defined by the claims of the present application.