胱抑素A对股骨头坏死的预测价值研究

doi:10.12114/j.issn.1007-9572.2024.0299

中国全科医学 ›› 2025, Vol. 28 ›› Issue (35): 4464-4472.DOI: 10.12114/j.issn.1007-9572.2024.0299

胱抑素A对股骨头坏死的预测价值研究

田佳庆¹^,², 彭鹏¹^,², 韦雨柔¹^,², 肖方骏¹^,², 何宪顺¹^,², 林锟¹^,², 陆舜¹^,², 何敏聪², 刘良燕³, 魏秋实²^,³^,⁴^,^*()

1．510006　广东省广州市，广州中医药大学
2．510006　广东省广州市，广东省中医骨伤研究院关节中心
3．515400　广东省揭阳市揭西县中医医院博士工作站骨科
4．510006　广东省广州市，中医证候全国重点实验室/骨科　广州中医药大学第三附属医院关节中心

收稿日期:2024-07-15 修回日期:2024-08-03 出版日期:2025-12-15 发布日期:2025-10-15
通讯作者: 魏秋实
作者贡献：
田佳庆提出主要研究目标，负责研究的构思与设计，研究的实施，撰写论文；彭鹏、韦雨柔、肖方骏、何宪顺进行数据的收集与整理，统计学处理，图、表的绘制与展示；林锟、陆舜负责临床标本的收集与整理；何敏聪、刘良燕、魏秋实进行论文的修订；魏秋实负责文章的质量控制与审查，对文章整体负责，监督管理。
基金资助:
国家自然科学基金资助项目(82274544，82004392); 广东省基础与应用基础基金资助项目(2023A1515010551); 广东省中医骨伤研究院开放课题重点项目(GYH202101-01，GYH202101-04)

Study on the Predictive Value of Cystatin A for Osteonecrosis of the Femoral Head

TIAN Jiaqing¹^,², PENG Peng¹^,², WEI Yurou¹^,², XIAO Fangjun¹^,², HE Xianshun¹^,², LIN Kun¹^,², LU Shun¹^,², HE Mincong², LIU Liangyan³, WEI Qiushi²^,³^,⁴^,^*()

1. Guangzhou University of Chinese Medicine, Guangzhou 510006, China
2. Joint Center, Guangdong Provincial Institute of Traditional Chinese Medicine Orthopedics and Traumatology, Guangzhou 510006, China
3. Orthopedics Department, Doctor Workstation, Jiexi County Hospital of Traditional Chinese Medicine, Jieyang 515400, China
4. Orthopedics Department, State Key Laboratory of Traditional Chinese Medicine Syndrome/Joint Center, Third Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510006, China

Received:2024-07-15 Revised:2024-08-03 Published:2025-12-15 Online:2025-10-15
Contact: WEI Qiushi

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摘要/Abstract

摘要： 背景股骨头坏死（ONFH）是一种临床难治性骨科疾病，发病率较高，预后差。目前磁共振成像（MRI）是诊断ONFH的"金指标"，但其检查流程烦琐，费用较高，无法作为常规筛查的工具。因此，亟待研发新型分子生物标志物用于ONFH的诊断。目的研究ONFH患者血清胱抑素A（CSTA）水平，评估其作为ONFH生物标志物的诊断价值。方法选取2023年5—11月于广州中医药大学第三附属医院就诊的21例ONFH患者为试验组，同期招募33例健康志愿者为对照组。收集受试者血清和股骨头组织样本，其中股骨头组织样本包括试验组17例ONFH患者和同期因股骨颈骨折手术的6例患者（FNF组），采用酶联免疫吸附法（ELISA）检查血清CSTA水平；采用苏木素-伊红（HE）染色观察骨组织形态，并使用CT扫描检测骨微结构；采用Trap染色评估不同骨组织中破骨细胞分化能力；通过免疫组织化学、免疫荧光染色和Western Blotting评估骨组织CSTA的表达水平；采用Spearman秩相关分析探究CSTA水平与国际骨循环研究协会（ARCO）分期之间的相关性；绘制受试者工作特征（ROC）曲线评估CSTA对ONFH的预测价值。结果血清CSTA检测结果显示，试验组ARCO Ⅱ ~ Ⅳ期患者血清CSTA水平高于对照组，ARCO Ⅱ期低于ARCO Ⅲ期，ARCO Ⅲ期低于ARCO Ⅳ期（P<0.05）；HE染色结果显示，ARCO Ⅱ、Ⅲ、Ⅳ期空骨陷窝率高于FNF组，ARCO Ⅲ、Ⅳ期高于ARCO Ⅱ期（P<0.05）；CT定量分析结果显示，ARCOⅡ、Ⅲ期骨体积分数（BV/TV）、骨小梁厚度（Tb.Th）、骨梁数量（Tb.N）低于FNF组，骨小梁分离度（Tb.Sp）高于FNF组（P<0.05）；Trap染色结果显示：ARCOⅡ、Ⅲ期Trap阳性细胞区域面积高于FNF组，且ARCOⅡ期高于ARCO Ⅳ期（P<0.05）；骨组织CSTA检测结果显示，ARCO Ⅱ、Ⅲ和Ⅳ期CSTA表达高于FNF组，且ARCO Ⅳ期高于ARCO Ⅱ期（P<0.05）；血清CSTA水平预测ONFH的ROC曲线下面积（AUC）为0.939 （P<0.001），灵敏度为78.79%，特异度为95.24%。结论 CSTA蛋白可通过调控破骨细胞的形成来影响股骨头内坏死骨的修复，在股骨头坏死患者中，CSTA表达升高与疾病的严重程度呈正相关。

关键词: 股骨头坏死, CSTA, 破骨细胞, 生物标志物, ARCO分期

Abstract:

Background

Osteonecrosis of the femoral head (ONFH) is a clinically refractory orthopedic disease with a relatively high incidence and poor prognosis. Currently, magnetic resonance imaging (MRI) is the "gold standard" for diagnosing ONFH, but its examination process is cumbersome and the cost is high, so it cannot be used as a tool for routine screening. Therefore, it is urgent to develop new molecular biomarkers for the diagnosis of ONFH.

Objective

To study the serum cystatin A (CSTA) level in ONFH patients and evaluate its diagnostic value as a biomarker for ONFH.

Methods

Twenty-one ONFH patients who visited the Third Affiliated Hospital of Guangzhou University of Chinese Medicine from May to November 2023 were selected as the experimental group, and 33 healthy volunteers were recruited as the control group at the same time. Serum and femoral head tissue samples were collected from the subjects. Among them, the femoral head tissue samples included 17 patients with ONFH from the experimental group and 6 patients who underwent surgery for femoral neck fractures during the same period, who were designated as the femoral neck fracture (FNF) group. The serum CSTA level was detected by ELISA; The bone tissue morphology was observed by HE staining, and the bone microstructure was detected by CT; Trap staining was used to evaluate the osteoclast differentiation ability in different bone tissues; The expression level of CSTA in bone tissues was evaluated by immunohistochemistry, immunofluorescence staining, and Western Blotting; Spearman rank correlation analysis was used to analyze the correlation between CSTA level and ARCO stage; The receiver operating characteristic (ROC) curve was drawn to evaluate the diagnostic value of CSTA.

Results

The results of serum CSTA detection showed that the serum CSTA levels of patients with ARCO stageⅡ-Ⅳ in the experimental group were higher than those in the control group (P<0.05). ARCO stage Ⅱ was lower than ARCO stage Ⅲ, and ARCO stage Ⅲ was lower than ARCO stage Ⅳ (P<0.05). The results of HE staining showed that there were empty bone depressions in ARCO stages Ⅱ, Ⅲ and Ⅳ. The litter rate was higher than that of the FNF group, and that of ARCO stage Ⅲ andⅣ was higher than that of ARCO stage Ⅱ (P<0.05) ; CT quantitative analysis results showed that the bone volume fraction (BV/TV), trabecular bone thickness (Tb.Th), and trabecular bone number (Tb.N) in ARCO Ⅱ and Ⅲ stages were lower than those in the FNF group, and the trabecular bone separation (Tb.Sp) was higher than that in the FNF group (P<0.05) ; Trap staining results showed that the area of Trap-positive cell regions in ARCO Ⅱ and Ⅲ groups was higher than that in the FNF group, and ARCO Ⅱ was higher than ARCO Ⅳ (P<0.05) ; The results of CSTA detection in bone tissues showed that the CSTA expression in ARCO Ⅱ, Ⅲ, and Ⅳ stages was higher than that in the FNF group, and ARCO Ⅳ stage was higher than ARCO Ⅱstage (P<0.05) ; The area under the ROC curve (AUC) of serum CSTA level in diagnosing ONFH was 0.939, with a sensitivity of 78.79% and a specificity of 95.24%.

Conclusion

The CSTA protein can affect the repair of necrotic bone in the femoral head by regulating the formation of osteoclasts. In patients with osteonecrosis of the femoral head, the increased expression of CSTA is positively correlated with the severity of the disease.

Key words: Osteonecrosis of the femoral head, CSTA, Osteoclast, Biomarker, ARCO staging

中图分类号:

R 68

田佳庆,彭鹏,韦雨柔,等. 胱抑素A对股骨头坏死的预测价值研究[J]. 中国全科医学, 2025, 28(35): 4464-4472. DOI: 10.12114/j.issn.1007-9572.2024.0299.
TIAN Jiaqing,PENG Peng,WEI Yurou, et al. Study on the Predictive Value of Cystatin A for Osteonecrosis of the Femoral Head[J]. Chinese General Practice, 2025, 28(35): 4464-4472. DOI: 10.12114/j.issn.1007-9572.2024.0299.

图/表 10

表1 对照组和试验组ARCO Ⅱ~Ⅳ期患者血清CSTA水平比较（±s，ng/L）

Table 1 Comparison of CSTA levels in serum of control group and experimental group with ARCO stages of Ⅱ-Ⅳ

组别	例数	CSTA水平
对照组	21	12.47±5.38
ARCO Ⅱ期	10	19.96±6.43^a
ARCO Ⅲ期	13	27.26±4.21^ab
ARCO Ⅳ期	10	33.76±5.55^abc
F值		233.374
P值		<0.001

表2 对照组和试验组ARCO Ⅱ~Ⅳ期患者空骨陷窝率和骨小梁参数定量分析比较（±s）

Table 2 The quantitative analysis comparison of empty lacunae rate and trabecular bone parameters between control group and experimental group with ARCO stages of Ⅱ-Ⅳ

组别	例数	空骨陷窝率（%）	骨小梁参数
组别	例数	空骨陷窝率（%）	BV/TV	Tb.N	Tb.Sp	Tb.Th
FNF组	6	5.36±1.77	71.21±2.34	1.71±0.03	0.48±0.05	0.43±0.04
ARCO Ⅱ期	6	25.38±2.78^a	41.53±2.42^a	1.04±0.14^a	0.71±0.03^a	0.38±0.02^a
ARCO Ⅲ期	5	76.26±3.35^ab	25.05±2.54^ab	0.87±0.03^a	1.11±0.07^ab	0.24±0.03^ab
ARCO Ⅳ期	6	85.40±4.45^ab	43.54±2.53^a	1.34±0.13	0.60±0.03^ac	0.38±0.04
F值		705.318	302.842	68.964	178.943	36.294
P值		<0.001	<0.001	<0.001	<0.001	<0.001

图1 骨组织HE染色和影像学比较注：ARCO=国际骨循环研究协会，FNF=股骨颈骨折；A~D为X线检查结果，E~H为大体标本外观图，I~L为苏木素-伊红（HE）染色图；黑色箭头所指为空骨陷窝。

Figure 1 Comparison of HE staining and imaging in bone tissue

图2 CT扫描图像分析注：A~D为股骨头大体标本直观图，E~H为CT扫描图，I~L为CT重建图。

Figure 2 CT scan image analysis

表3 Trap染色和骨组织CSTA表达定量分析（±s）

Table 3 The results of quantitative analysis of trap staining and CSTA expression in osseous tissue

组别	例数	Trap染色结果	CSTA
组别	例数	Trap染色结果	免疫组织化学染色	免疫荧光染色	Western Blotting
FNF组	6	0.18±0.04	0.04±0.03	0.04±0.01	0.36±0.02
ARCO Ⅱ期	6	1.00±0.10^a	0.40±0.15	0.20±0.06	0.37±0.01
ARCO Ⅲ期	5	0.60±0.10^a	0.67±0.15^ab	0.65±0.17^ab	0.67±0.05^ab
ARCO Ⅳ期	6	0.32±0.05	1.00±0.36^abc	1.00±0.24^abc	1.00±0.03^abc
F值		52.394	33.348	50.027	324.013
P值		<0.001	<0.001	<0.001	<0.001

图3 骨组织Trap染色结果（4×）注：A为FNF组，B为ARCOⅡ期，C为ARCO Ⅲ期，D为ARCO Ⅳ期；红色箭头所指为Trap染色阳性区域。

Figure 3 Trap staining results of osteonecrosis

图4 免疫组织化学检测骨组织CSTA表达注：A为股骨颈骨折组，B为ARCOⅡ期，C为ARCO Ⅲ期，D为ARCO Ⅳ期；红色箭头所指为阳性区域。

Figure 4 Immunohistochemical of CSTA expression in femoral head tissue

图5 免疫荧光检测骨组织CSTA的表达注：DAPI=4'，6-二脒基-2-苯基吲哚，FITC=异硫氰酸荧光素，Merge=FITC和DAPI合并结果；A为FNF组，B为ARCO Ⅱ期，C为ARCO Ⅲ期，D为ARCO Ⅳ期；蓝色荧光代表DAPI染色，绿色荧光代表CSTA蛋白FITC染色。

Figure 5 Immunofluorescence of CSTA expression in osseous tissue

图6 Western Blotting检测骨组织CSTA表达注：β-actin=β肌动蛋白。

Figure 6 Western Blotting of CSTA expression in osseous tissue

图7 血清CSTA水平诊断ONFH的ROC曲线

Figure 7 The ROC curve of CSTA levels in serum for the diagnosis of ONFH patients

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胱抑素A对股骨头坏死的预测价值研究

Study on the Predictive Value of Cystatin A for Osteonecrosis of the Femoral Head

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