PI3K/AKT通路在促红细胞生成素调控钙磷陶瓷表面成骨中的作用

doi:10.3969/j.issn.1006-5725.2026.13.017

摘要/Abstract

摘要：

目的探讨EPO/pDA/BCP复合支架的促成骨效能及分子机制，重点验证其是否通过激活PI3K/AKT通路并上调MTSS1与CYTL1，从而促进骨髓间充质干细胞（BMSCs）的成骨分化及体内临界骨缺损修复。方法采用双相磷酸钙支架并接枝多巴胺和促红细胞生成素构建EPO/pDA/BCP复合材料；体外实验分4组：空白对照组、BCP组、EPO/pDA/BCP组及加入PI3K/AKT通路抑制剂的EPO/pDA/BCP + LY294002组，通过CCK-8法、碱性磷酸酶活性测定、RT-qPCR和免疫印迹法评估细胞增殖及成骨分化能力。通过RT-qPCR和免疫印迹检测PI3K/AKT通路及下游基因MTSS1、CYTL1的表达水平；体内实验通过建立SD大鼠股骨髁临界骨缺损模型，随机分为空白组、BCP组和EPO/pDA/BCP组进行干预。术后4周和12周进行Micro-CT、苏木精-伊红染色、马松染色及免疫组化分析骨再生情况。结果 CCK-8实验证实EPO/pDA/BCP复合材料具有良好的生物相容性。碱性磷酸酶活性测定、RT-qPCR及Western blot结果显示，与BCP组和对照组相比，EPO/pDA/BCP组能够显著促进BMSCs的成骨分化（P 0.05）。而加入抑制剂LY294002后，该促成骨效应明显减弱，且PI3K/AKT信号通路相关分子（p-PI3K、p-AKT、CYTL1和MTSS1）表达下调（P 0.05）。Micro-CT扫描显示，EPO/pDA/BCP组骨缺损区域的新生骨体积分数和骨密度等均显著高于空白组与BCP组（P 0.05）；组织学染色结果提示，相较于其他两组，EPO/pDA/BCP组骨组织再生更为完整和成熟；免疫组化染色进一步显示，EPO/pDA/BCP组骨缺损区内成骨标志物骨形态发生蛋白-2与骨钙素的阳性染色强度均显著高于BCP组和空白对照组。结论 EPO/pDA/BCP生物陶瓷在体内外均具有良好的促成骨性能，该作用可能与激活PI3K/AKT信号通路蛋白表达有关。

关键词: 双相钙磷陶瓷, 促红细胞生成素, 骨髓间充质干细胞, 成骨分化, PI3K/AKT信号通路

Abstract:

Objective To explore the osteogenic efficacy and molecular mechanism of the EPO/pDA/BCP composite scaffold， this study focuses on verifying whether it promotes the osteogenic differentiation of bone marrow mesenchymal stem cells （BMSCs） and the repair of critical bone defects in vivo by activating the PI3K/AKT pathway and up-regulating MTSS1 and CYTL1. Methods The EPO/pDA/BCP composite material was fabricated by grafting dopamine and erythropoietin onto a biphasic calcium phosphate scaffold. In in-vitro experiments， four groups were established： the control group， the BCP group， the EPO/pDA/BCP group， and the EPO/pDA/BCP + LY294002 group with the addition of a PI3K/AKT pathway inhibitor. The cell proliferation and osteogenic differentiation capacity were assessed using the CCK-8 method， alkaline phosphatase activity assay， RT-qPCR， and Western blotting. The expression levels of the PI3K/AKT pathway and its downstream genes MTSS1 and CYTL1 were determined by RT-qPCR and Western blotting. In in-vivo experiments， a critical bone defect model of the femoral condyle in SD rats was created and randomly assigned to the blank group， the BCP group， and the EPO/pDA/BCP group for intervention. At 4 and 12 weeks after surgery， Micro-CT， hematoxylin-eosin staining， Masson staining， and immunohistochemical analysis were carried out to evaluate bone regeneration. Results The CCK-8 experiment verified that the EPO/pDA/BCP composite material exhibited excellent biocompatibility. The outcomes of the alkaline phosphatase activity assay， RT-qPCR， and Western blot demonstrated that， in comparison with the BCP group and the control group， the EPO/pDA/BCP group could notably promote the osteogenic differentiation of BMSCs （P 0.05）. Following the addition of the inhibitor LY294002， the osteogenic effect was markedly weakened， and the expression of molecules associated with the PI3K/AKT signaling pathway （p-PI3K， p-AKT， CYTL1， and MTSS1） was down-regulated （P 0.05）. Micro-CT scanning revealed that the new bone volume fraction and bone density in the bone defect area of the EPO/pDA/BCP group were significantly higher than those of the blank group and the BCP group （P 0.05）. Histological staining results suggested that the bone tissue regeneration in the EPO/pDA/BCP group was more comprehensive and mature than that in the other two groups. Immunohistochemical staining further indicated that the positive staining intensities of the osteogenic markers bone morphogenetic protein-2 and osteocalcin in the bone defect area of the EPO/pDA/BCP group were significantly higher than those of the BCP group and the blank control group. Conclusion The EPO/pDA/BCP bioceramic exhibits excellent osteogenic properties both in vitro and in vivo， and this effect might be associated with the activation of the expression of PI3K/AKT signaling pathway proteins.

Key words: biphasic calcium phosphate, erythropoietin, bone marrow mesenchymal stem cells, osteogenic differentiation, PI3K/AKT signaling pathway

中图分类号:

R318

向雪飞,高鹤友,陈芷芸,胡清川,何流,王宇. PI3K/AKT通路在促红细胞生成素调控钙磷陶瓷表面成骨中的作用[J]. 实用医学杂志, 2026, 42(13): 2404-2418.

Xuefei XIANG,Heyou GAO,Zhiyun CHEN,Qingchuang HU,Liu HE,Yu WANG. Study on the role of the PI3K/AKT pathway in the regulation of osteogenesis on the surface of calcium phosphate ceramics by erythropoietin[J]. The Journal of Practical Medicine, 2026, 42(13): 2404-2418.

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基因	引物序列（5′-3′）
Runx2	正义链：AAG GAG CAG ACC GTC AAA GG 反义链：CCC AGA GTC CAC CTC TCT GA
Osterix	正义链：GCA TCT GAA AGC CCA CTT GC 反义链：AGT GGT CGC TTC GGG TAA AG
OPN	正义链：GAT GAC GAC GAC GAT GAC GA 反义链：GCT GGC AGT GAA GGA CTC AT
CYTL1	正义链：CAC CGT CCT CCA GAG TGA AC 反义链：CGG ACC CAG TTT GGA TGG AA
MTSS1	正义链；GCG GTT TTC TCT GAC CCA AG 反义链：CCC CAA GTC CCA ATC CGA AA
GAPDH	正义链：GAC ATG CCG CCT GGA GAA AC 反义链：AGC CCA GGA TGC CCT TTA GT

组别	第1天	第3天	第7天
空白组	0.28 ± 0.03	0.47 ± 0.06	1.02 ± 0.07
BCP组	0.32 ± 0.01	0.47 ± 0.06	0.99 ± 0.06
EPO/pDA/BCP组	0.27 ± 0.01	0.41 ± 0.01	1.04 ± 0.04

组别	第3天	第7天
空白组	0.02 ± 0.00	0.05 ± 0.00
BCP组	0.04 ± 0.01^*	0.07 ± 0.00^*
EPO/pDA/BCP组	0.06 ± 0.01^*#	0.08 ± 0.00^*#
EPO/pDA/BCP+LY组	0.04 ± 0.00^△	0.05 ± 0.01^△

组别	p-PI3K/PI3K	p-AKT/AKT	CYTL1	MTSS1
空白组	0.29 ± 0.13	0.24 ± 0.07	0.46 ± 0.03	0.41 ± 0.08
BCP组	0.47 ± 0.07	0.48 ± 0.06	0.48 ± 0.04	0.51 ± 0.10
EPO/pDA/BCP组	0.85 ± 0.09^*#	0.80 ± 0.03^*#	0.61 ± 0.05^*#	0.71 ± 0.05^*#
EPO/pDA/BCP+LY组	0.49 ± 0.13^△	0.39 ± 0.12^△	0.39 ± 0.05^△	0.40 ± 0.06^△

组别	CYTL1	MTSS1
空白组	1.01 ± 0.17	1.00 ± 0.15
BCP组	2.50 ± 0.55	2.56 ± 0.41
EPO/pDA/BCP组	4.10 ± 0.75^*#	3.34 ± 0.12^*#
EPO/pDA/BCP+LY组	2.11 ± 0.12^△	2.21 ± 0.11^△