CN116115736A - Application of a kind of BMP9 in the preparation of preparation or medicine for inhibiting osteoblast senescence - Google Patents

Abstract

The invention relates to application of BMP9 in preparation of preparations or medicines for inhibiting bone cell aging, which proves that the BMP9 can reduce bone loss of old mice, has the functions of protecting bone microstructures of the old mice, improving fracture resistance and the like, and has application prospects in treating senile osteoporosis and the like as an anti-aging medicine.

Description

Translated fromChinese

一种BMP9在制备抑制成骨细胞衰老的制剂或药物中的应用Application of a kind of BMP9 in the preparation of preparation or medicine for inhibiting osteoblast senescence

技术领域technical field

本发明属于生物医药领域，特别涉及一种BMP9在制备抑制成骨细胞衰老的制剂或药物中的应用。The invention belongs to the field of biomedicine, and in particular relates to an application of BMP9 in preparing preparations or medicines for inhibiting osteoblast aging.

背景技术Background technique

随着人口老龄化加剧，年龄相关性骨质疏松症和骨折已成为日益严重的全球性公共卫生问题。最新的数据显示，在2000年，有900万例骨质疏松性骨折(160万例髋部骨折，170万例前臂骨折和140万例椎骨骨折)发生；到2050年，全球髋部骨折的发病率预期在女性和男性中将分别增加240％和310％。在女性中，1年内发生骨折的人数将超过乳腺癌、心肌梗死或中风的人数总和。Age-related osteoporosis and fractures have become a growing global public health problem as the population ages. The latest data show that in 2000, there were 9 million osteoporotic fractures (1.6 million hip fractures, 1.7 million forearm fractures and 1.4 million vertebral fractures); by 2050, the global incidence of hip fractures Rates are expected to increase by 240% in women and 310% in men. Among women, more fractures occur within a year than breast cancer, myocardial infarction or stroke combined.

发明内容Contents of the invention

本发明所要解决的技术问题是提供一种一种BMP9在制备抑制成骨细胞衰老的制剂或药物中的应用。The technical problem to be solved by the present invention is to provide an application of BMP9 in the preparation of preparations or medicines for inhibiting osteoblast aging.

本发明的一种BMP9在制备抑制成骨细胞衰老的制剂或药物中的应用。An application of the BMP9 of the present invention in the preparation of preparations or medicines for inhibiting osteoblast aging.

本发明的一种BMP9在制备抑制连续传代和/或过氧化氢刺激诱导的成骨细胞衰老的制剂或药物中的应用。Application of a BMP9 of the present invention in the preparation of preparations or medicines for inhibiting osteoblast senescence induced by continuous passage and/or hydrogen peroxide stimulation.

本发明的一种BMP9在制备老年骨质疏松症制剂或药物中的应用。An application of the BMP9 of the present invention in the preparation of preparations or medicines for senile osteoporosis.

本发明的一种BMP9在制备老年抗骨折制剂或药物中的应用。An application of the BMP9 of the present invention in the preparation of anti-fracture preparations or medicines for the elderly.

本发明的一种所述应用，所述制剂或药物包括单方制剂或复方制剂。In one application of the present invention, the preparation or medicine includes a single preparation or a compound preparation.

有益效果Beneficial effect

本发明中BMP9可减少老年小鼠的骨量丢失、具有保护老年小鼠骨骼微结构的作用、提高其抗骨折能力等。表明了BMP9在制备老年性骨质疏松症制剂或药物中的应用和制备老年性抗骨折制剂或药物中的应用。In the present invention, BMP9 can reduce the bone mass loss of aged mice, protect the bone microstructure of aged mice, improve its anti-fracture ability, and the like. The application of BMP9 in the preparation of senile osteoporosis preparations or medicines and the application of BMP9 in the preparation of senile anti-fracture preparations or medicines are shown.

附图说明Description of drawings

图1为小鼠体内BMP9过表达的验证；其中A.分别对6月龄和20月龄小鼠进行过表达BMP9的AAV2/9尾静脉注射，12周后进行验证；B.肝脏BMP9 mRNA表达水平；C.6月龄小鼠尾静脉注射后肝脏BMP9蛋白表达水平；D.20月龄小鼠尾静脉注射后肝脏BMP9蛋白表达水平；E.6月龄和20月龄小鼠尾静脉注射后血清BMP9水平。6M+AAV-CON组n＝9；6M+AAV-BMP9组n＝7；20M+AAV-CON组n＝9；20M+AAV-BMP9组n＝8。数据表示为平均值±标准差。*P<0.05；**P<0.01；***P<0.001；Figure 1 is the verification of BMP9 overexpression in mice; where A. 6-month-old and 20-month-old mice were injected into the tail vein of AAV2/9 overexpressing BMP9, and verified after 12 weeks; B. BMP9 mRNA expression in liver Level; C. BMP9 protein expression level in liver after tail vein injection of 6-month-old mice; D. BMP9 protein expression level in liver of 20-month-old mice after tail vein injection; E. Tail vein injection of 6-month-old and 20-month-old mice Post-serum BMP9 levels. 6M+AAV-CON group n=9; 6M+AAV-BMP9 group n=7; 20M+AAV-CON group n=9; 20M+AAV-BMP9 group n=8. Data are presented as mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001;

图2为BMP9减少老年小鼠骨量流失；其中A.成年和老年小鼠分别过表达BMP9后股骨2D图像、松质骨和皮质骨3D重建图像；B.成年和老年小鼠分别过表达BMP9，对照组尾静脉注射空载AAV；C.股骨松质骨体积骨密度；D.股骨皮质骨体积骨密度。6M+AAV-CON组n＝9；6M+AAV-BMP9组n＝7；20M+AAV-CON组n＝9；20M+AAV-BMP9组n＝8。数据表示为平均值±标准差。*P<0.05；**P<0.01；***P<0.001。Figure 2 shows that BMP9 reduces bone loss in aged mice; A. 2D images of femur, 3D reconstruction images of cancellous bone and cortical bone after overexpression of BMP9 in adult and aged mice; B. overexpression of BMP9 in adult and aged mice , The control group was injected with no-load AAV through the tail vein; C. Femoral cancellous bone volumetric bone density; D. Femoral cortical bone volumetric bone density. 6M+AAV-CON group n=9; 6M+AAV-BMP9 group n=7; 20M+AAV-CON group n=9; 20M+AAV-BMP9 group n=8. Data are presented as mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001.

图3为BMP9改善老年小鼠骨骼微结构；A.成年和老年小鼠分别过表达BMP9后股骨松质骨骨体积分数；B.股骨骨小梁厚度；C.骨小梁离散度；D.松质骨结构模式指数；E.股骨皮质骨骨体积分数；F.股骨皮质骨厚度；G.成年和老年小鼠分别过表达BMP9椎骨HE染色。数据表示为平均值±标准差。6M+AAV-CON组n＝9；6M+AAV-BMP9组n＝7；20M+AAV-CON组n＝9；20M+AAV-BMP9组n＝8。*P<0.05；**P<0.01；***P<0.001。Figure 3 shows that BMP9 improves bone microstructure in aged mice; A. Femoral cancellous bone volume fraction after overexpressing BMP9 in adult and aged mice; B. Femoral trabecular bone thickness; C. Dispersion of trabecular bone; D. Cancellous bone structure pattern index; E. femoral cortical bone volume fraction; F. femoral cortical bone thickness; G. HE staining of vertebrae overexpressing BMP9 in adult and aged mice, respectively. Data are presented as mean ± standard deviation. 6M+AAV-CON group n=9; 6M+AAV-BMP9 group n=7; 20M+AAV-CON group n=9; 20M+AAV-BMP9 group n=8. *P<0.05; **P<0.01; ***P<0.001.

图4为BMP9改善老年小鼠骨生物力学参数，A.弹性模量；B.弯曲强度；C.最大弯曲负荷；D.断裂能量。6M+AAV-CON组n＝9；6M+AAVBMP9组n＝7；20M+AAV-CON组n＝9；20M+AAV-BMP9组n＝8。数据表示为平均值±标准差。*P<0.05；**P<0.01；***P<0.001。Figure 4 shows that BMP9 improves bone biomechanical parameters in aged mice, A. elastic modulus; B. bending strength; C. maximum bending load; D. fracture energy. 6M+AAV-CON group n=9; 6M+AAVBMP9 group n=7; 20M+AAV-CON group n=9; 20M+AAV-BMP9 group n=8. Data are presented as mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001.

图5为BMP9提高改善老年小鼠骨转换状态；其中A.老年小鼠过表达BMP9后血清骨形成标志PINP水平(n＝7)；B.老年小鼠过表达BMP9后血清骨吸收标志CTX-1水平(n＝7)；C.老年小鼠过表达BMP9后骨组织成骨细胞特异基因表达(n＝6)；D.老年小鼠过表达BMP9后骨组织破骨细胞特异基因表达(n＝6)；E.骨组织TRAP染色。数据表示为平均值±标准差。*P<0.05；**P<0.01；***P<0.001。Figure 5 shows that the improvement of BMP9 improves the bone turnover status of aged mice; where A. the level of serum bone formation marker PINP after overexpressing BMP9 in aged mice (n=7); B. serum bone resorption marker CTX- after overexpressing BMP9 inaged mice 1 level (n=7); C. Osteoblast-specific gene expression in bone tissue after overexpressing BMP9 in aged mice (n=6); D. Osteoclast-specific gene expression in bone tissue after overexpressing BMP9 in aged mice (n =6); E. TRAP staining of bone tissue. Data are presented as mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001.

图6为BMP9改善成骨细胞复制性衰老，促成骨分化，A.连续传代后的MC3T3E1细胞给予小鼠重组BMP9蛋白处理，β-半乳糖苷酶染色检测衰老细胞；B.计数β-半乳糖苷酶染色阳性细胞比例；C.免疫荧光检测γ-H2AX阳性细胞D.qPCR检测P21、P53的mRNA表达；E.qPCR检测Ccl5、Nfkb1、Mmp的mRNA表达；F.qPCR检测Vcam1、Hmgb1、Cxcl5的mRNA表达；G.成骨分化诱导7天后ALP染色检测各组细胞分化程度；H.成骨分化关键转录因子Runx2、Osx的mRNA表达；I.成骨细胞特异基因Alp、Ocn、Col1a1的mRNA表达；J.成骨分化关键转录因子Runx2、Osx的蛋白表达。N＝3次生物学重复。数据表示为平均值±标准差。*P<0.05；**P<0.01；***P<0.001。Figure 6 shows that BMP9 improves the replicative senescence of osteoblasts and promotes osteogenic differentiation. A. MC3T3E1 cells after continuous passage were treated with mouse recombinant BMP9 protein, and senescent cells were detected by β-galactosidase staining; B. Counting β-galactosidase Proportion of positive cells stained with glycosidase; C. Immunofluorescence detection of γ-H2AX positive cells D. qPCR detection of mRNA expression of P21, P53; E. qPCR detection of mRNA expression of Ccl5, Nfkb1, Mmp; F. qPCR detection of Vcam1, Hmgb1, Cxcl5 G. ALP staining to detect the degree of cell differentiation in each group after 7 days of osteogenic differentiation induction; H. mRNA expression of key transcription factors Runx2 and Osx in osteogenic differentiation; I. mRNA expression of osteoblast-specific genes Alp, Ocn and Col1a1 Expression; J. Protein expression of key transcription factors Runx2 and Osx in osteogenic differentiation. N=3 biological replicates. Data are presented as mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001.

图7为BMP9改善H2O2诱导的成骨细胞衰老，促成骨分化；A.H2O2诱导衰老的MC3T3E1细胞给予小鼠重组BMP9蛋白处理，β-半乳糖苷酶染色检测衰老细胞；B.计数β-半乳糖苷酶染色阳性细胞比例；C.免疫荧光检测γ-H2AX阳性细胞；D.qPCR检测P21、P53的mRNA表达；E.qPCR检测Ccl5、Nfkb1、Mmp的mRNA表达；F.qPCR检测Vcam1、Hmgb1、Cxcl5的mRNA表达；G.成骨分化诱导7天后ALP染色检测各组细胞分化程度；H.成骨分化关键转录因子Runx2、Osx的mRNA表达；I.成骨细胞特异基因Alp、Ocn、Col1a1的mRNA表达；J.成骨分化关键转录因子Runx2、Osx的蛋白表达。N＝3次生物学重复。数据表示为平均值±标准差。*P<0.05；**P<0.01；***P<0.001。Figure 7 shows that BMP9 improves H2O2-induced osteoblast senescence and promotes osteogenic differentiation; A.H2O2-induced aging MC3T3E1 cells were treated with mouse recombinant BMP9 protein, and senescent cells were detected by β-galactosidase staining; B. Counting β-half Proportion of positive cells stained with lactosinase; C. Immunofluorescence detection of γ-H2AX positive cells; D. qPCR detection of mRNA expression of P21 and P53; E. qPCR detection of mRNA expression of Ccl5, Nfkb1, Mmp; F. qPCR detection of Vcam1 and Hmgb1 , Cxcl5 mRNA expression; G. ALP staining to detect the degree of cell differentiation in each group after 7 days of osteogenic differentiation induction; H. mRNA expression of key transcription factors Runx2 and Osx in osteogenic differentiation; I. Osteoblast-specific genes Alp, Ocn, Col1a1 J. Protein expression of key transcription factors Runx2 and Osx in osteogenic differentiation. N=3 biological replicates. Data are presented as mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001.

具体实施方式Detailed ways

下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

实施例1Example 1

分别对6月龄的成年小鼠和20月龄老年小鼠进行腺相关病毒尾静脉注射，其中实验组小鼠注射BMP9过表达的腺相关病毒，对照组小鼠注射空载病毒(图1A)。尾静脉注射12周后，分别取血和肝脏进行BMP9过表达的验证。qPCR和WB结果显示肝脏中BMP9的表达量明显上调(图1.B-D)，Elisa结果提示实验组成年和老年小鼠血清中BMP9含量均显著升高(图1.E)，证明BMP9在小鼠体内过表达成功。Adeno-associated virus was injected into the tail vein of 6-month-old adult mice and 20-month-old mice respectively. The mice in the experimental group were injected with BMP9-overexpressed adeno-associated virus, and the mice in the control group were injected with empty virus (Figure 1A). . After 12 weeks of tail vein injection, blood and liver were collected to verify the overexpression of BMP9. The results of qPCR and WB showed that the expression of BMP9 in the liver was significantly up-regulated (Fig. 1.B-D), and the results of ELISA indicated that the serum BMP9 levels in the aged and aged mice of the experimental group were significantly increased (Fig. 1.E), which proved that BMP9 in mice In vivo overexpression was successful.

对小鼠股骨进行micro-CT检测。2D和3D重组成像图提示，对照组老年小鼠与对照组成年小鼠相比，股骨远端松质骨结构明显稀疏、骨量减少，股骨中段皮质骨厚度明显变薄；过表达BMP9的老年小鼠松质骨结构和皮质骨厚度均显著增加，而过表达BMP9的成年小鼠骨量则无明显变化(图2A)。另外，分别对松质骨和皮质骨进行骨密度的检测，发现BMP9在老年小鼠的过表达可显著提高骨密度，在成年小鼠身上这一作用并不明显(图2.B-D)。提示BMP9可减少老年小鼠的骨量丢失。Micro-CT detection of mouse femurs. The 2D and 3D reconstruction images showed that compared with the adult mice in the control group, the cancellous bone structure of the distal femur was significantly sparser, the bone mass decreased, and the thickness of the cortical bone in the middle femur was significantly thinner in the aged mice in the control group; the aged mice overexpressing BMP9 Both cancellous bone structure and cortical bone thickness were significantly increased in mice, while bone mass was not significantly changed in adult mice overexpressing BMP9 (Fig. 2A). In addition, the bone density of cancellous bone and cortical bone was detected separately, and it was found that overexpression of BMP9 in aged mice could significantly increase bone density, but this effect was not obvious in adult mice (Fig. 2.B-D). It is suggested that BMP9 can reduce bone loss in aged mice.

一般老年小鼠的骨骼微结构遭到破坏，其中松质骨体积分数(BV/TV)和小梁骨厚度(Tb.Th)，而小梁骨离散度(Tb.Sp)和结构模式指数(SMI)在老年小鼠均升高；过表达BMP9后，以上参数均得到显著改善(图3.A-D)。皮质骨方面，发现老年小鼠的骨体积分数(BV/TV)和皮质骨厚度(Ct.Th)较成年小鼠显著下降；BMP9体内过表达可提高皮质骨体积分数和厚度，差异均具有统计学意义(图3.E,F)。同时通过对小鼠椎骨切片进行HE染色可直观地看到，老年鼠小梁骨厚度变薄、离散度增加，而BMP9过表达则可明显改善这一现象。成年小鼠过表达BMP9则未引起明显的骨微结构变化(图3.G)。这些结果提示BMP9具有保护老年小鼠骨骼微结构的作用。In general, the bone microstructure of aged mice was destroyed, among which cancellous bone volume fraction (BV/TV) and trabecular bone thickness (Tb.Th), while trabecular bone dispersion (Tb.Sp) and structural pattern index ( SMI) were all elevated in aged mice; after overexpression of BMP9, the above parameters were significantly improved (Fig. 3.A-D). In terms of cortical bone, it was found that the bone volume fraction (BV/TV) and cortical bone thickness (Ct.Th) of aged mice were significantly lower than those of adult mice; overexpression of BMP9 in vivo could increase the cortical bone volume fraction and thickness, and the differences were statistically significant. Scientific significance (Fig. 3.E,F). At the same time, through HE staining of mouse vertebral sections, it can be seen intuitively that the thickness of trabecular bone in aged mice becomes thinner and the dispersion increases, while the overexpression of BMP9 can significantly improve this phenomenon. Overexpression of BMP9 in adult mice did not cause obvious changes in bone microstructure (Fig. 3.G). These results suggest that BMP9 plays a role in protecting the bone microstructure of aged mice.

随年龄增长，小鼠骨量减少、抗骨折能力降低。与成年小鼠相比，老年鼠股骨生物力学参数值下降，其中骨弹性模量、弯曲强度、最大弯曲负荷和断裂能量等均显著降低。BMP9在老年鼠的过表达则可明显提升这些生物力学参数(图4)，证明BMP9改善老年鼠生物力学表现，提高其抗骨折能力。As the mice grow older, their bone mass decreases and their ability to resist fractures decreases. Compared with adult mice, the biomechanical parameters of the femur in aged mice decreased, among which bone elastic modulus, bending strength, maximum bending load and fracture energy were all significantly decreased. The overexpression of BMP9 in aged mice can significantly improve these biomechanical parameters (Figure 4), which proves that BMP9 improves the biomechanical performance of aged mice and improves their fracture resistance.

骨骼系统处于骨形成和骨转换的动态平衡状态，随年龄增长，骨转换水平下调。对老年小鼠进行BMP9过表达后进一步检测血清骨形成和骨吸收标志物PINP和CTX-1，BMP9显著提高PINP在血清的水平，差异具有统计学意义；CTX-1则呈下降趋势，但未达统计学差异(图5.A,B)。进一步测量了骨组织中成骨细胞和破骨细胞特异性的标志基因的表达，发现BMP9可显著提高老年小鼠骨OSX、ALP、OCN和Col1a1的表达；而破骨细胞标志基因中只有Nfatc1和Dc-stamP的表达成下降趋势(图5.C,D)。TRAP染色结果则显示BMP9作用下老年小鼠骨组织中破骨细胞数量相对减少，但差异并不十分显著(图5.E)。以上提示BMP9对老年小鼠骨转换的调控作用更多地以促进骨形成为主。同样，这一作用在成年小鼠身上并不明显。The skeletal system is in a state of dynamic balance between bone formation and bone turnover, and the level of bone turnover is down-regulated with age. After BMP9 was overexpressed in aged mice, the serum bone formation and bone resorption markers PINP and CTX-1 were further detected. BMP9 significantly increased the level of PINP in the serum, and the difference was statistically significant; CTX-1 showed a downward trend, but not Statistical difference was reached (Figure 5.A, B). Further measured the expression of osteoblast and osteoclast-specific marker genes in bone tissue, and found that BMP9 could significantly increase the expression of OSX, ALP, OCN and Col1a1 in aged mouse bone; while only Nfatc1 and The expression of Dc-stamP showed a downward trend (Figure 5.C,D). The results of TRAP staining showed that the number of osteoclasts in the bone tissue of aged mice was relatively reduced under the action of BMP9, but the difference was not very significant (Fig. 5.E). The above suggests that the regulation of BMP9 on bone turnover in aged mice is more about promoting bone formation. Again, this effect was not apparent in adult mice.

为了进一步探讨BMP9对衰老成骨细胞的保护作用，首先在体外利用反复传代的成骨细胞系MC3T3E1构建复制性衰老的细胞模型。在体外培养过程中发现，快速反复传代至P15以后的MC3T3E1细胞出现衰老的形态变化，具体表现为细胞胞体变大、胞内颗粒增多、增殖变慢等，因此利用P17代细胞作为衰老细胞模型，用P5-7代细胞作为正常对照进行后续实验。细胞分组设为：P7+Vehicle、P17+Vehicle、P17+BMP9组，其中P17+BMP9组给予100ng/ml的BMP9处理3天，P17+Vehicle组给予同等体积分数的PBS缓冲液处理同样时间。分别通过半乳糖苷酶染色、衰老基因表达测定、DNA损伤修复标志γ-H2AX检测和SASPs的表达验证反复传代后的成骨细胞衰老表型以及BMP9的作用；利用碱性磷酸酶(ALP)染色、成骨分化特异性基因的表达评价不同分组的成骨细胞分化能力的改变。In order to further explore the protective effect of BMP9 on aging osteoblasts, a replicative aging cell model was first constructed in vitro using the repeatedly passaged osteoblast cell line MC3T3E1. In the process of in vitro culture, it was found that MC3T3E1 cells that were rapidly and repeatedly passaged to P15 showed senescent morphological changes, specifically manifested as cell body enlargement, intracellular granules increased, and proliferation slowed down. Therefore, P17 generation cells were used as senescent cell models. Subsequent experiments were performed with P5-7 generation cells as normal controls. The cell grouping was set as: P7+Vehicle, P17+Vehicle, P17+BMP9 groups, wherein the P17+BMP9 group was treated with 100ng/ml BMP9 for 3 days, and the P17+Vehicle group was treated with the same volume fraction of PBS buffer for the same time. The aging phenotype of osteoblasts after repeated passages and the role of BMP9 were verified by galactosidase staining, aging gene expression measurement, DNA damage repair marker γ-H2AX detection and SASPs expression; using alkaline phosphatase (ALP) staining , Osteogenic differentiation-specific gene expression to evaluate the changes of osteoblast differentiation ability in different groups.

β-半乳糖苷酶检测提示反复传代后衰老的成骨细胞数量显著增多，对染色阳性的细胞进行计数，衰老细胞的比例可达48.69％，BMP9处理后这一比例则下降至19.45％，差异具有统计学意义(图6.A,B)。免疫荧光检测提示γ-H2AX阳性的细胞数量在反复传代后也明显增多，BMP9干预可减少这一类细胞的产生(图6C)。另外利用qPCR检测衰老特异性基因P16、P21和P53的表达，发现反复传代后的成骨细胞以P21的升高为主，P53的表达并无明显增多趋势，而P16基因在MC3T3E1细胞中的表达丰度较低，考虑成骨细胞的衰老可能主要由P21进行介导；BMP9的加入则显著降低P21的表达水平(图6.D)。衰老相关分泌性因子Ccl5、Mmp9、Nfkb1、Vcam1、Hmgb1和Cxcl5的表达水平也在P17细胞中明显升高，并可被BMP9下调(图6.E,F)。以上细胞表型说明BMP9对反复传代引起的成骨细胞衰老具有改善作用。The detection of β-galactosidase indicated that the number of senescent osteoblasts increased significantly after repeated passages, and the proportion of senescent cells was as high as 48.69% after counting the positive cells, but decreased to 19.45% after BMP9 treatment. Statistically significant (Fig. 6.A,B). Immunofluorescence detection indicated that the number of γ-H2AX-positive cells also increased significantly after repeated passages, and BMP9 intervention could reduce the generation of this type of cells (Fig. 6C). In addition, qPCR was used to detect the expression of aging-specific genes P16, P21, and P53, and it was found that the osteoblasts after repeated passages were mainly increased by P21, and the expression of P53 did not increase significantly, while the expression of P16 gene in MC3T3E1 cells The abundance is low, considering that the senescence of osteoblasts may be mainly mediated by P21; the addition of BMP9 significantly reduces the expression level of P21 (Fig. 6.D). The expression levels of senescence-associated secretory factors Ccl5, Mmp9, Nfkb1, Vcam1, Hmgb1 and Cxcl5 were also significantly increased in P17 cells and could be down-regulated by BMP9 (Fig. 6.E,F). The above cell phenotypes indicate that BMP9 can improve osteoblast senescence caused by repeated passage.

碱性磷酸酶染色提示反复传代后的MC3T3E1细胞成骨分化程度减低，BMP9可逆转这一变化(图6.G)。反复传代后，成骨细胞分化特异性的转录因子Runx2和Osx在RNA和蛋白水平的表达均降低，BMP9则能上调二者的表达；同时介导成骨分化的Alp、Ocn和Col1a1在BMP9的作用下也得到上调(图6.HJ)。以上结果说明BMP9在体外改善成骨细胞复制性衰老状态，提高成骨分化能力。Alkaline phosphatase staining indicated that the degree of osteogenic differentiation of MC3T3E1 cells after repeated passages was reduced, and BMP9 could reverse this change (Fig. 6.G). After repeated passages, the expression of osteoblast differentiation-specific transcription factors Runx2 and Osx decreased at the RNA and protein levels, while BMP9 could up-regulate the expression of both; at the same time, Alp, Ocn and Col1a1, which mediate osteogenic differentiation, were expressed in BMP9. It was also up-regulated under the effect of (Fig. 6.HJ). The above results indicate that BMP9 improves the replicative senescence state of osteoblasts in vitro and improves the osteogenic differentiation ability.

利用H2O2诱导MC3T3E1细胞衰老，验证BMP9的作用。H2O2 was used to induce senescence of MC3T3E1 cells to verify the role of BMP9.

细胞分组设为：正常对照组、H2O2组、H2O2+BMP9组，结合CCK8细胞活性测定结果筛选合适的H2O2浓度作用于MC3T3E1细胞2小时，随后用PBS缓冲液清洗细胞后，更换为正常完全培养液再培养3天，即可进行衰老表型的检测。H2O2+BMP9组的处理为在更换正常完全培养液的同时，加入终浓度100ng/ml的小鼠BMP9重组蛋白，共同培养3天。The cell groups were set as: normal control group, H2O2 group, H2O2+BMP9 group, combined with the results of CCK8 cell activity assay to select the appropriate concentration of H2O2 to act on MC3T3E1 cells for 2 hours, then washed the cells with PBS buffer, and replaced with normal complete culture medium After culturing for another 3 days, the aging phenotype can be detected. The treatment of the H2O2+BMP9 group was to add mouse BMP9 recombinant protein at a final concentration of 100ng/ml while replacing the normal complete culture medium, and co-culture for 3 days.

使用200nM浓度的H2O2处理后进行β半乳糖苷酶染色并计数衰老细胞个数，有高达64.16％的成骨细胞显示出衰老状态；BMP9处理可显著下调衰老细胞比例至36.65％，差异具有统计学意义(图7.A,B)。免疫荧光检测提示γ-H2AX阳性的细胞数量在H2O2处理后也显著增多，BMP9则明显抑制这一趋势(图7.C)。同时，衰老特异性基因P21的表达在H2O2作用下显著升高，BMP9能有效下调其在RNA和蛋白水平的表达。有意思的是，在这一衰老模型中同样观察到P53的表达并未随H2O2的诱导而显著上调，基于衰老表型在不同种类细胞中的特异性表现，考虑对于MC3T3E1细胞来说，P53并非导致衰老的关键调控基因(图7.D)。另外也对衰老相关分泌性因子Ccl5、Mmp9、Nfkb1、Vcam1、Hmgb1和Cxcl5的表达进行了检测，发现在H2O2作用下升高的这些SASPs均可被BMP9下调(图7.E,F)。以上说明BMP9对H2O2引起的成骨细胞衰老具有改善作用。After treatment with 200nM H2O2, stain with β-galactosidase and count the number of senescent cells. As high as 64.16% of osteoblasts showed senescence; BMP9 treatment can significantly down-regulate the proportion of senescent cells to 36.65%, and the difference is statistically significant Significance (Fig. 7.A,B). Immunofluorescence detection showed that the number of γ-H2AX positive cells also increased significantly after H2O2 treatment, and BMP9 significantly inhibited this trend (Fig. 7.C). At the same time, the expression of senescence-specific gene P21 was significantly increased under the action of H2O2, and BMP9 could effectively down-regulate its expression at the RNA and protein levels. Interestingly, in this aging model, it was also observed that the expression of P53 was not significantly up-regulated with the induction of H2O2. Based on the specific expression of aging phenotypes in different types of cells, it is considered that for MC3T3E1 cells, P53 does not cause Key regulatory genes of aging (Fig. 7.D). In addition, the expression of aging-related secretory factors Ccl5, Mmp9, Nfkb1, Vcam1, Hmgb1, and Cxcl5 was also detected, and it was found that these SASPs that were increased under the action of H2O2 could be down-regulated by BMP9 (Fig. 7.E, F). The above shows that BMP9 can improve the aging of osteoblasts induced by H2O2.

进一步分析了成骨细胞分化功能的改变。碱性磷酸酶染色提示H2O2作用下的MC3T3E1细胞成骨分化的能力减弱，BMP9可重新上调其分化水平(图7.G)。衰老组成骨细胞分化特异性的转录因子Runx2和Osx在RNA和蛋白水平的表达均降低，BMP9可上调二者的表达；同时介导成骨分化的Alp、Ocn和Col1a1在BMP9的作用下也得到上调(图7.H-J)。说明BMP9在对H2O2诱导的成骨细胞衰老的改善作用。证明了BMP9对不同诱导条件下的成骨细胞衰老状态均存在抑制作用。Alterations in osteoblast differentiation function were further analyzed. Alkaline phosphatase staining indicated that the osteogenic differentiation ability of MC3T3E1 cells under the action of H2O2 was weakened, and BMP9 could re-up-regulate its differentiation level (Fig. 7.G). The expression of Runx2 and Osx, the specific transcription factors of osteocyte differentiation in aging, decreased at the RNA and protein levels, and BMP9 could up-regulate the expression of both; at the same time, Alp, Ocn and Col1a1, which mediate osteogenic differentiation, were also reduced under the action of BMP9. Upregulation (Fig. 7.H-J). It shows that BMP9 can improve the osteoblast senescence induced by H2O2. It is proved that BMP9 has an inhibitory effect on the senescent state of osteoblasts under different induction conditions.

Claims (5)

1. Use of BMP9 in the preparation of a formulation or medicament for inhibiting bone cell aging.

2. Use of BMP9 in the preparation of a formulation or medicament for inhibiting serial passage and/or hydrogen peroxide stimulation induced osteoblast senescence.

3. Use of BMP9 in the preparation of senile osteoporosis preparations or medicaments.

4. Use of BMP9 in the preparation of an senile fracture resistant preparation or medicament is provided.

5. The use according to any one of claims 1 to 4, wherein the formulation or medicament comprises a single formulation or a compound formulation.

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