颌面部放疗致口干5例患者唇腺干细胞的分离培养与多向分化潜能鉴定

doi:10.6040/j.issn.1671-7554.0.2019.345

摘要/Abstract

摘要： 目的分离培养颌面部放疗患者唇腺间充质干细胞(MSCs),并对其增殖特征和多向分化潜能进行研究。方法收集颌面部放疗致口干患者5例,无菌条件下切取唇腺组织;用组织块贴壁法分离培养唇腺干细胞;CCK-8试剂盒检测细胞增殖曲线;流式细胞仪检测干细胞表面标志物;干细胞成骨向诱导分化后,以增殖培养基组为对照组,以成骨培养基组为成骨诱导组,用碱性磷酸酶(ALP)染色及茜素红染色检测成骨向分化潜能,real time-PCR检测成骨相关基因的mRNA表达;干细胞成脂向分化后以增殖培养基组为对照组,以成脂培养基组为成脂诱导组,用油红-O染色观察成脂向分化,real time-PCR检测成脂向相关基因的mRNA表达。结果分离获得了颌面部放疗致口干患者的原代唇腺干细胞,其增殖曲线符合干细胞“S型”增殖曲线的特征;干细胞表面标记物CD29、 CD44、 CD73、CD90 和CD105呈阳性表达,而CD34、CD45 和CD106均呈阴性表达,与对照组标记物表达无明显差异;成骨诱导7 d后,ALP染色法和茜素红染色法结果显示成骨诱导组唇腺干细胞中ALP和矿化结节均成强阳性表达,real time-PCR检测结果显示成骨诱导组与对照组成骨相关基因表达的差异有统计学意义;成脂诱导21 d后,油红-O染色结果显示成骨诱导组干细胞内可见大小不等的脂滴,real time-PCR检测成脂向相关基因表达显示成脂诱导组与对照组差异有统计学意义。结论颌面部放疗致口干患者唇腺中可分离获得MSCs,获得的MSCs具有增殖和多向分化潜能,可利用该唇腺干细胞作为唾液腺功能再建的种子细胞,为唾液腺组织工程提供新的思路。

关键词: 放疗, 唇腺, 干细胞, 多向分化潜能

Abstract: Objective To explore the proliferative characteristics and multi-directional differentiation potential of mesenchymal stem cells(MSCs)derived from labial glands in patients with dry mouth caused by maxillofacial radiotherapy. Methods Labial glands from the lower lip obtained during biopsy from 5 patients with dry mouth were excised under sterile conditions. MSCs were isolated and cultured using tissue adherent method. The cell proliferation curve was detected with CCK-8. Expressions of MSCs-related markers were examined in isolated cells with flow cytometry. After osteogenic differentiation, the proliferation medium group and osteogenic medium group were used as the control and osteogenic induction group, respectively. The osteogenic differentiation potential was assessed with alkaline phosphatase(ALP)staining and alizarin red staining. The mRNA expression of osteogenic-related genes was determined with real time-PCR. After adipogenic differentiation, the proliferation medium group and adipogenic medium group served as the control and adipogenic induction group, respectively. Then the adipogenic potential was observed with oil red-O staining, and the mRNA 山东大学学报 (医学版)57卷11期 -沈倍勇,等.颌面部放疗致口干5例患者唇腺干细胞的分离培养与多向分化潜能鉴定 \=-expression of adipogenic-related genes were detected with real time PCR. Results MSCs were successfully obtained. CCK8 assay showed the growth curve of MSCs presented an “S” shape, which was similar to other stem cells. The surface markers of CD29, CD44, CD73, CD90 and CD105 were positively expressed, while CD34, CD45 and CD106 were negatively expressed. After 7 days of osteogenic induction, ALP staining and alizarin red staining showed strong expression of ALP and mineralized nodules in the osteogenic stem cells compared with the control group. Real time PCR results showed the expression of osteogenic-related genes in the osteogenic induction group and control group were statistically significant. After 21 days of adipogenic induction, the results of oil red-O staining showed that lipid droplets of different sizes could be seen in stem cells of osteogenic induction group. There was significant difference in the expression of adipogenic-related genes between the adipogenic induction group and control group. Conclusion MSCs can be isolated from labial glands in patients with dry mouth caused by maxillofacial radiotherapy. The MSCs have the potential for proliferation and multi-directional differentiation. The labial stem cells can be used as seed cells for salivary gland function reconstruction.

Key words: Radiotherapy, Labial glands, Stem cells, Multipotential differentiation potential

中图分类号:

R783

沈倍勇,王晓飞,李军心,周琦. 颌面部放疗致口干5例患者唇腺干细胞的分离培养与多向分化潜能鉴定[J]. 山东大学学报 (医学版), 2019, 57(11): 71-77.

SHEN Beiyong, WANG Xiaofei, LI Junxin, ZHOU Qi. Isolation, culture and identification of labial stem cells from patients with dry mouth caused by maxillofacial radiotherapy[J]. Journal of Shandong University (Health Sciences), 2019, 57(11): 71-77.

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