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中华细胞与干细胞杂志(电子版)

中华细胞与干细胞杂志(电子版) ›› 2022, Vol. 12 ›› Issue (06) : 321 -328. doi: 10.3877/cma.j.issn.2095-1221.2022.06.001

论著

浓缩血小板提高冻存脂肪来源间充质干细胞活性的研究
吴顺1, 刘雪君2, 张一帆3, 孙家明3, 郭亮3,†()   
  1. 1. 430079　武汉，湖北肿瘤医院乳腺中心
    2. 430079　武汉，湖北肿瘤医院超声影像科
    3. 430022　武汉，华中科技大学同济医学院附属协和医院整形外科
  • 收稿日期:2022-07-16 出版日期:2022-12-01
  • 通信作者: 郭亮
  • 基金资助:
    国家自然科学基金(81401607)

Human platelet concentrate improved the bioactivity of mesenchymal stem cells derived from cryopreserved fat tissues

Shun Wu1, Xuejun Liu2, Yifan Zhang3, Jiaming Sun3, Liang Guo3,†()   

  1. 1. Department of Breast Surgery, Hubei Cancer Hospital, Wuhan 430079, China
    2. Department of Ultrasound and Radiology, Hubei Cancer Hospital, Wuhan 430079, China
    3. Department of Plastic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
  • Received:2022-07-16 Published:2022-12-01
  • Corresponding author: Liang Guo
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引用本文：

吴顺, 刘雪君, 张一帆, 孙家明, 郭亮. 浓缩血小板提高冻存脂肪来源间充质干细胞活性的研究[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(06): 321-328.

Shun Wu, Xuejun Liu, Yifan Zhang, Jiaming Sun, Liang Guo. Human platelet concentrate improved the bioactivity of mesenchymal stem cells derived from cryopreserved fat tissues[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(06): 321-328.

目的

探讨浓缩血小板（PC）对冻存脂肪组织（CF）来源的间充质干细胞（ADSCs）活性的促进作用。

方法

将CF来源的ADSCs分别使用含胎牛血清（FBS）、低、中、高浓度（9.75×1010/L、1.95×1011/L、3.90×1011/L）血小板培养基进行培养，记为CF+FBS、CF+LP、CF+MP、CF+HP。新鲜脂肪（FF）来源的ADSCs采用含FBS培养基进行培养，记为FF+FBS。检测PC对P3代冻存ADSCs增殖活性、克隆形成率及分化（成脂、成骨、成软骨）活性的影响。动物实验采用Coleman脂肪注射技术，根据注射脂肪的类型把雄性裸鼠分为新鲜脂肪组（FF）、冻存脂肪组（CF）、添加低浓度血小板的冻存脂肪组（LP+CF）。移植28 d后，测量注射移植除油脂后的各组移植脂肪团的湿重与移植前脂肪重量之比计算湿重保留率，并对除油脂后的各组移植脂肪团行HE和CD31免疫组化染色。两组间比较采用Dunnett-t检验，多组间比较采用单因素方差分析。

结果

与CF +LP比较，FF+ FBS、CF+ FBS体外克隆形成率[（5.29±0.64）%，（2.87±0.64）%比（98.72±0.59）%]、油红O染色阳性区域面积比[（2.46±0.93）%，（1.30±0.54）%比（15.96±5.59）%]降低，差异有统计学意义（P均< 0.05）；与CF+FBS相比，FF+FBS和CF +LP甲苯胺蓝染色阳性区域面积比[（20.49±3.16）%，（24.67±3.02）%比（7.65±4.76）%]、茜素红染色562 nm处的吸光度值（0.22±0.01，0.21±0.00比0.19±0.01）升高，差异有统计学意义（P均< 0.05）。脂肪移植后28 d，与FF相比，CF与LP+CF的湿重保留率[（17.79±2.872）%，（19.62±2.65）%比（34.22±4.22）%]均降低，差异有统计学意义（P均< 0.05）。与FF相比，CF及LP+CF的HE染色红染面积比[（25.75±7.11）%，（33.83±9.17）%比（14.14±3.97）%]升高，差异有统计学意义（P均< 0.05）；与FF比较，CF移植脂肪团中的血管数[（2.20±3.35）比（14.60±8.11）个]减少，差异有统计学意义（P均< 0.05），与LP+CF比较差异无统计学意义。

结论

PC能有效促进冻存ADSCs的增殖与分化活性，并可在一定程度上改善冻存脂肪的移植效果，但是与新鲜脂肪仍有一定差距。

关键词: 冻存脂肪, 脂肪来源间充质干细胞, 浓缩血小板, 富血小板血浆, 脂肪移植
Objective

To investigate whether platelet concentrate (PC) improved the viability of ADSCs from cryopreserved fat tissues (CF) .

Methods

The adipose-derived stem cells (ADSCs) obtained from CF were cultured with fetal bovine serum, low (390×109/ L) , middle (195×109/L) , and high (97.5×109/L) concentrations of PC and named CF+ FBS, CF+LP, CF+MP and CF+HP. The ADSCs derived from fresh fat tissues (FF) were cultured with FBS, named FF+FBS. The 3rd passage cells were evaluated by cell counting kit-8 (CCK-8) assay, cloning forming unit assay and adipogenic, osteogenic and chondrogenic differentiation. According to Coleman fat technique, the male nude mice were randomized and injected with FF, CF and CF with a low concentrations of PC (LP+CF) , respectively. Twenty-eight days after transplantation, the grafted samples were excised for graft retention rate, hematoxylin-eosin staining and immunohistochemistry. One-way analysis of variance (ANOVA) was used for comparison between multiple groups.

Results

Batter proliferation of ADSCs from cryopreserved fat (CF) tissues were found in the group, which was treated with PC, compared with the group without platelet supplement. Compared with FF+FBS, CF+FBS, The CF+LP was significantly increased in cloning forming unit assay [ (5.29±0.64) %, (2.87±0.64) %vs (98.72±0.59) %] and adipogenic differentiation[ (2.46±0.93) %, (1.30±0.54) %vs (15.96±5.59) %] (P < 0.05) . Compared with CF+FBS, FF+FBS and FF+FBS were significantly increased in osteogenic (0.22±0.01, 0.21±0.00 vs 0.19±0.01) and chondrogenic differentiations [ (20.49±3.16) %, (24.67±3.02) % vs (7.65±4.76) %] (P < 0.05) . The wet weight retention rate of the FF group was significantly higher than CF and CF+LP [ (17.79±2.872) %, (19.62±2.65) % vs (34.22±4.22) %] at 28 days after fat transplantation (P < 0.05) . The results of histological examinations in the FF group were inferior to CF+LP and CF groups[ (25.75±7.11) %, (33.83±9.17) %vs (14.14±3.97) %] (P < 0.05) . Compared with the FF, the number of vessels in CF (2.20±3.35 vs 14.60±8.11) was reduced (P < 0.05) .

Conclusions

PC can effectively improve the bioactivity of ADSCs isolated from cryopreserved adipose tissue. And the application of PC can improve the transplantation of cryopreserved fat to some extent.

Key words: Cryopreserved fat tissue, Adipose-derived stem cells, Platelet concentrate, Platelet-rich plasma (PRP), Fat grafts
图1 不同血小板浓度组P1、P2代冻存ADSCs细胞培养注：a ~ e图为不同血小板浓度组P1代冻存ADSCs细胞形态；f ~ j图为不同血小板浓度组的P2代冻存ADSCs细胞形态
图2 不同血小板浓度组的冻存脂肪来源P1、P2及P3代ADSCs细胞培养注：a图为不同血小板浓度组的冻存脂肪来源P3代ADSCs生长曲线；b图为第11天不同血小板浓度组的冻存脂肪来源P3代ADSCs光吸收值，与CF+LP比较，aP < 0.05，ns为差异无统计学意义
图3 皿底形成的细胞集落面积对比注：a~b图为FF+FBS及CF+FBS的细胞均在皿底形成明显可见的细胞克隆集落，c图为CF+LP形成的细胞克隆集落几乎爬满皿底，21 d后细胞瑞士-姬姆萨染色情况；与FF+FBS比较，aP < 0.01，与CF+FBS和FF+FBS比较，bP < 0.001
图4 ADSCs成脂诱导后油红O染色及其半定量检测注：a ~ c图分别为FF+FBS、CF+FBS和CF+LP成脂诱导21 d后行油红O染色，各组均呈阳性，细胞内有丰富脂滴形成；d图为油红O染色阳性区域面积对比，与CF+LP比较，aP < 0.001，ns为差异无统计学意义
图5 ADSCs成骨诱导后茜素红染色及其半定量检测注：a ~ c图分别为FF+FBS、CF+FBS和CF+LP成骨诱导后14 d进行茜素红染色，均可见大量红染的钙结节形成；d图为562 nm处吸收光密度（OD）值对比，与CF+FBS比较，aP < 0.01，ns为差异无统计学意义
图6 ADSCs成软骨诱导后甲苯胺蓝染色及半定量检测注：a ~ c图分别为FF+FBS、CF+FBS和CF+LP成软骨诱导21 d后甲苯胺蓝染色；b图为ADSCs形态紊乱而不规则，无明显细胞形态；a、c图均可见大量染色呈阳性的细胞，且细胞形态良较好；d图为各组染色阳性细胞的面积对比，与CF+FBS比较，aP < 0.01，ns为差异无统计学意义
图7 脂肪团在移植后第28天的湿重保留率检测注：a ~ c图分别为FF组、CF组和CF+LP组脂肪移植后第28天大体照片；d图为各组移植脂肪团测量；e图为各组移植脂肪团的湿重保留率对比，与FF组比较，aP < 0.001，ns为差异无统计学意义
图8 移植脂肪团的HE染色及组间纤维化区域的面积对比注：a ~ c图为移植脂肪团的HE染色（scale bar- 200 μm），FF组靠近中心区域的脂肪细胞排列杂乱，且形态不规则，外层近包膜区域的脂肪则形态规整，排列致密，局部纤维化程度较轻；b ~ c图为CF组及CF+LP组移植脂肪团中，具有正常脂肪细胞形态的脂肪细胞数量少于FF组，且排列疏松，脂肪细胞或空泡由大量红染的纤维组织所包绕；与FF组比较，aP < 0.05，ns为差异无统计学意义
图9 移植脂肪团的CD31免疫组化染色及血管计数注：a ~ c图为正置显微镜下观察CD31免疫组化染色切片；a图为移植脂肪团中心及外层区域均可见血管结构，主要分布于外层近包膜区域；b图为血管结构减少；c图为移植脂肪团中心及外层区域均可见明显血管结构，在部分纤维增生区域；图中红色三角所指为CD31+的血管结构；d图为组间的血管数对比，与FF组比较，aP < 0.05，ns为差异无统计学意义
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