富血小板血浆加速糖尿病大鼠骨损伤愈合

doi:10.16352/j.issn.1001-6325.2023.08.1193

基础医学与临床 ›› 2023, Vol. 43 ›› Issue (8): 1193-1200.doi: 10.16352/j.issn.1001-6325.2023.08.1193

富血小板血浆加速糖尿病大鼠骨损伤愈合

吴智钢^1,2*, 权冬², 常欣², 陈雪雪³, 张子茹¹

1.空军军医大学第二附属医院骨科, 陕西西安 710038;
2.中国人民解放军63750部队医院外科, 陕西西安 710005;
3.西安体育学院研究生院, 陕西西安 710068

收稿日期:2022-07-11 修回日期:2023-06-02 出版日期:2023-08-05 发布日期:2023-07-26
通讯作者: ^*wuzhigang77@163.com
基金资助:
中国博士后基金(2020M673663)

Platelet-rich plasma promotes bone injury repairment in rats with diabetes mellitus

WU Zhigang^1,2*, QUAN Dong², CHANG Xin², CHEN Xuexue³, ZHANG Ziru¹

1. Department of Orthopedics, the Second Affiliated Hospital of Air Force Medical University, Xi'an 710038;
2. Department of Surgery, Chinese People's Liberation Army Hospital 63750, Xi'an 710005;
3. Graduate School, Xi'an Physical Education University, Xi'an 710068, China

Received:2022-07-11 Revised:2023-06-02 Online:2023-08-05 Published:2023-07-26
Contact: ^*wuzhigang77@163.com

摘要/Abstract

摘要： 目的探讨富血小板血浆(PRP)能否通过促进糖尿病(DM)大鼠骨髓巨噬细胞(BMDMs)向M2型极化,从而诱导糖尿病大鼠脂肪干细胞(ADSCs)向成骨细胞分化,促进骨损伤愈合。方法分离培养DM大鼠BMDMs和ADSCs,采用流式细胞仪对其进行鉴定;BMDMs极化分组为:对照组(PRP 0%),PRP组(1%,5%,10%);ADSCs成骨分化分组为:对照组(control,ADSCs, 2×10⁵/mL,+同PRP组相同容积不含PRP血浆),ADSCs+BMDMs组(简称BMDMs组,2×10⁵/mL,+不含PRP血浆),ADSCs+10% PRP组(简称PRP组,加入PRP使培养液中含有10% PRP),ADSCs+BMDMs+10% PRP组(简称BMDMs+PRP组)。Transwell小室法鉴定极化的BMDMs促进ADSCs侵袭;RT-qPCR和Western blot鉴定BMDMs极化和ADSCs的成骨分化以及相关基因和蛋白的表达;免疫荧光检测成骨蛋白Osterix的表达;DM大鼠股骨干骺端构建3 mm×5 mm的骨缺损为骨损伤模型,将大鼠分为:对照组、ADSCs+巨噬细胞清除剂CLP组(简称ADSCs组)、ADSCs+BMDMs组(简称BMDMs组,无CLP注射),ADSCs+CLP+PRP组(简称PRP组),ADSCs+PRP组(简称BMDMs+PRP组,无CLP注射)。构建模型前48 h尾静脉注射CLP(5 mL/kg),术后每周注射2次,连续8周;在骨缺损处局部注射ADSCs(1×10⁷/mL)与藻酸盐凝胶混合物,骨缺损处PRP连续治疗4次,每次间隔2周,每次注射500 μL;采用micro-CT重建并定量分析DM大鼠骨缺损修复。结果 ADSCs阳性标志物Sca-1和CD29纯度达85.4%和99.9%,BMDMs阳性标志物CD11b和F4/80纯度达94%和90%;与对照组相比,随着PRP浓度的提高,BMDMs向M2型极化的蛋白和基因表达增高(P＜0.05);与PRP组相比,BMDMs+PRP组ADSCs迁移的数量显著增多(P＜0.05);与其他组相比,BMDMs+PRP组ADSCs的成骨分化活性更强,成骨蛋白和基因的表达显著提高(P＜0.05);与其他组相比,BMDMs+PRP组的骨体积/组织体积(BV/TV)、骨小梁数量(Tb.N)和厚度(Tb.Th)显著提高(P＜0.001)。结论体外实验表明,PRP通过促进BMDMs向M2极化从而促进ADSCs迁移和成骨分化;体内实验表明,BMDMs+PRP能够显著促进ADSCs向成骨细胞分化,加速骨形成,进而改善糖尿病骨缺损愈合不良的问题。

关键词: 富血小板血浆, 脂肪干细胞, 骨缺损, 糖尿病, 骨形成

Abstract: Objective To investigate whether platelet-rich plasma (PRP) can promote osteoblasts differentiation in diabetic mellitus(DM) adipose-derived stem cells(ADSCs) by M2 polarization of DM bone marrow derived macrophages(BMDMs). Methods PRP was extracted from the heart of rats according to the instructions of the kit. BMDMs and ADSCs of DM rats were isolated and cultured, then identified by flow cytometry. BMDMs were divided into control group (PRP 0%) and PRP group (1%, 5%, 10%); ADSCs were divided into: control group (control, ADSCs, 2×10⁵/mL, + same volume as PRP group without PRP plasma), ADSCs+BMDMs group (BMDMs group, 2×10⁵/mL, + plasma without PRP), ADSCs+10% PRP group (PRP group, medium with 10% PRP), and ADSCs+BMDMs+10% PRP group (BMDMs+PRP). Transwell was used to count ADSCs by polarized BMDMs in invasion experiment. The polarization of BMDMs and osteogenic differentiation of ADSCs were determined by RT-qPCR and Western blot. The expression of osteogenic protein Osterix was detected by immunofluorescence. The bone defect model of DM rats was constructed with 3 mm×5 mm at femoral epiphysis, and the rats were divided into: control group (DM rats), ADSCs+ clodronate liposomes (CLP)group (ADSCs group), ADSCs+BMDMs group (BMDMs group without CLP injection), ADSCs+CLP+PRP group (PRP group), ADSCs+PRP group (BMDMs+PRP group without CLP injection). CLP(5 mL/kg) was injected into tail vein of rat 48 hours before operation followed by injection twice a week after surgery for 8 weeks. ADSCs (1×10⁷/mL) and alginate gel mixture were injected into the bone defect, and PRP was treated once every 2 weeks with 500 μL PRP for 4 times. Micro-CT was used to reconstruct and quantitatively analyze the bone defect repair in DM rats. Results The purity of ADSCs positive markers Sca-1 and CD29 were 85.4% and 99.9% respectively and and the purity of BMDMs positive markers CD11b and F4/80 were 94% and 90%. Compared with the control group, BMDMs polarized into M2-type dependent on PRP concentration (P＜0.05). Compared with PRP group, the cell counting of ADSCs migrated in BMDMs+PRP group was significantly increased(P＜0.05). Compared with other groups, the osteogenic differentiation activity and the expressions of osteogenic proteins and genes were significantly increased in ADSCs with BMDMs+PRP treatment(P＜0.05). Bone volume/ tissue volume(BV/TV),trabecular number(Tb.N)and trabecular thickness(Tb.Th)were significantly increased in the BMDMs+PRP group compared with the other groups (P＜0.001). Conclusions PRP promotes ADSCs migration and osteogenic differentiation by stimulating the polarization of BMDMs towards M2 in vitro. BMDMs+PRP can significantly promote the differentiation of ADSCs into osteoblasts and accelerate bone formation in vivo, thus improving the poor healing of diabetic bone defects of rats.

Key words: platelet-rich plasma, adipose-derived stem cells, bone defect, diabetes mellitus, bone formation

中图分类号:

Q291

吴智钢, 权冬, 常欣, 陈雪雪, 张子茹. 富血小板血浆加速糖尿病大鼠骨损伤愈合[J]. 基础医学与临床, 2023, 43(8): 1193-1200.

WU Zhigang, QUAN Dong, CHANG Xin, CHEN Xuexue, ZHANG Ziru. Platelet-rich plasma promotes bone injury repairment in rats with diabetes mellitus[J]. Basic & Clinical Medicine, 2023, 43(8): 1193-1200.

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富血小板血浆加速糖尿病大鼠骨损伤愈合

Platelet-rich plasma promotes bone injury repairment in rats with diabetes mellitus

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