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.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: http://journal11.magtechjournal.com/Jwk_jcyxylc/CN/10.16352/j.issn.1001-6325.2023.08.1193

http://journal11.magtechjournal.com/Jwk_jcyxylc/CN/Y2023/V43/I8/1193

参考文献

[1]Li Y, Teng D, Shi X, et al. Prevalence of diabetes recorded in mainland China using 2018 diagnostic criteria from the American Diabetes Association: national cross sectional study[J].BMJ, 2020, 369: m997. doi:10.1136/bmj.m997.
[2]Ablett AD, Boyle BR, Avenell A. Fractures in adults after weight loss from bariatric surgery and weight management programs for obesity: systematic review and Meta-analysis[J].Obes Surg, 2019, 29: 1327-1342. doi:10.1007/s11695-018-03685-4.
[3]Hu Z, Ma, C, Rong X, et al. Immunomodulatory ECM-like microspheres for accelerated bone regeneration in diabetes mellitus[J].ACS Appl Mater Interfaces, 2018, 10: 2377-2390. doi:10.1021/acsami.7b18458.
[4]Chen S, Shi J, Zhang M, et al. Mesenchymal stem cell-laden anti-inflammatory hydrogel enhances diabetic wound healing[J].Sci Rep, 2015, 5: 18104. doi:10.1038/srep18104.
[5]Hexter AT, Sanghani-Kerai A, Heidari N, et al. Mesenchymal stromal cells and platelet-rich plasma promote tendon allograft healing in ovine anterior cruciate ligament reconstruction[J].Knee Surg Sports Traumatol Arthrosc, 2021, 29: 3678-3688. doi:10.1007/s00167-020-06392-9.
[6]Fernandes G. Yang S. Application of platelet-rich plasma with stem cells in bone and periodontal tissue engineering[J].Bone Res, 2016, 4: 16036. doi:10.1038/boneres.2016.36.
[7]Niemeyer P, Fechner K, Milz S, et al. Comparison of mesenchymal stem cells from bone marrow and adipose tissue for bone regeneration in a critical size defect of the sheep tibia and the influence of platelet-rich plasma[J].Biomaterials, 2010, 31: 3572-3679. doi:10.1016/j.biomaterials.2010.01.085.
[8]Mautner K, Malanga GA, Smith J, et al. A call for a standard classification system for future biologic research: the rationale for new PRP nomenclature[J].PM R, 2015, 7: S53-S59. doi:10.1016/j.pmrj.2015.02.005.
[9]Marques LF, Stessuk T, Camargo IC, et al. Platelet-rich plasma (PRP): methodological aspects and clinical applications[J].Platelets, 2015, 26: 101-113.
[10]Georgakopoulos I, Tsantis S, Georgakopoulos P, et al. The impact of platelet rich plasma (PRP) in osseointegration of oral implants in dental panoramic radiography: texture based evaluation[J].Clin Cases Miner Bone Metab, 2014, 11: 59-66.
[11]孙慧颖, 朱崇元, 冯国栋,等. 含有白细胞的富血小板纤维蛋白的生物学特性及临床应用[J].基础医学与临床, 2020, 40: 1706-1710.
[12]Jiang G, Li S, Yu K, et al. A 3D-printed PRP-GelMA hydrogel promotes osteochondral regeneration through M2 macrophage polarization in a rabbit model[J].Acta Biomater, 2021, 128: 150-162.
[13]Wei B, Huang C, Zhao M, et al. Effect of mesenchymal stem cells and platelet-rich plasma on the bone healing of ovariectomized rats[J].Stem Cells Int, 2016, 2016: 9458396. doi:10.1155/2016/9458396.
[14]Wang Z, Wang Z, Zhang B, et al. Effect of activated platelet-rich plasma on chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells[J].Stem Cells Int, 2021, 2021: 9947187. doi:10.1155/2021/9947187.

[1]	王丽婷, 许岭翎, 李伟, 平凡, 张化冰, 马亚红, 李玉秀. 不同亚型1型糖尿病患者大血管病变临床特征及危险因素分析[J]. 基础医学与临床, 2023, 43(8): 1284-1288.
[2]	王鑫瑞, 秦燕. Ghrelin在代谢性疾病中作用的研究进展[J]. 基础医学与临床, 2023, 43(8): 1299-1303.
[3]	刘充, 刘迎迎, 郭兆安. 特异性促炎消退介质在糖尿病肾脏疾病中的研究进展[J]. 基础医学与临床, 2023, 43(7): 1152-1156.
[4]	陈纤纤, 冯烈, 周颖, 董向楠. 糖尿病肾病与其他类型肾病患者肾组织中Smad1蛋白表达的比较[J]. 基础医学与临床, 2023, 43(7): 1097-1103.
[5]	周悦, 林静娜. miR-199-3p通过靶向VegfA抑制大鼠糖尿病视网膜病变[J]. 基础医学与临床, 2023, 43(7): 1084-1089.
[6]	余卓颖, 杨静, 蒋烨, 李民. 痛性及非痛性糖尿病神经病变的临床研究进展[J]. 基础医学与临床, 2023, 43(6): 985-989.
[7]	尹乐, 王丽妍, 黄红东. 复方α-酮酸对行腹膜透析的糖尿病患者生活质量和自我感受负担的影响[J]. 基础医学与临床, 2023, 43(5): 790-793.
[8]	舒玲, 霍本念, 宋林. NT5C2在疾病及治疗中的研究进展[J]. 基础医学与临床, 2023, 43(5): 852-856.
[9]	张梦瑶, 牛姝, 蔡静, 赵志刚, 周艳. miR-140-5p靶向HMGB1/IκB-α轴对糖尿病大鼠视网膜病变的影响[J]. 基础医学与临床, 2023, 43(4): 615-620.
[10]	蒋烨, 余卓颖, 李民. 炎性反应在糖尿病神经病变发生中作用的研究进展[J]. 基础医学与临床, 2023, 43(4): 669-673.
[11]	张艺馨, 蔡善君, 李智立, 于伟泓, 张华. IgG N-糖基化修饰在糖尿病视网膜病变中作用的研究进展[J]. 基础医学与临床, 2023, 43(3): 509-513.
[12]	邹洁, 张德德, 陶毅, 赵静, 王琼, 徐乃佳. 葛根素减轻KKAy小鼠血管内皮细胞炎性反应[J]. 基础医学与临床, 2023, 43(3): 433-437.
[13]	王卉, 苏宪, 张赢赢, 褚战亚, 孙朝晖, 张金玲, 张倩倩. 异鼠李素减轻糖尿病视网膜病变大鼠视网膜细胞凋亡[J]. 基础医学与临床, 2023, 43(2): 265-270.
[14]	杨孟迪, 丁雨露, 王玉洁, 何风英, 孙琳楠, 甄东户. MicroRNA在糖尿病性骨质疏松症中作用的研究进展[J]. 基础医学与临床, 2023, 43(2): 311-316.
[15]	李瑜, 张雯, 袁昱, 李姝亭, 王哲, 贲亚琍, 戴飏. 小鼠内源性逆转录病毒Gag抗原在胰岛的表达及单克隆抗体的制备[J]. 基础医学与临床, 2023, 43(1): 59-67.

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

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价