Changes in biological characteristics of adipose-derived stem cells in obese patients post successful weight loss
-
摘要:
目的 探索肥胖患者减重成功后其脂肪干细胞（ASC）的生物学特性变化,为该类ASC在难愈性创面修复中的临床应用提供参考。 方法 该研究为实验研究。将2021年4月—2023年4月郑州大学第一附属医院收治的12例肥胖减重成功后行腹壁皮肤松弛矫正术的患者纳入减重组[女8例、男4例,年龄为（50±9）岁],将该单位同期收治的行腹部抽脂后面部填充术的12名健康志愿者纳入健康组[女10例、男2例,年龄为（50±9）岁]。收集减重组患者与健康组志愿者的脂肪组织并提取ASC,取第4、5代ASC进行实验。于培养0（即刻）、24、48、72 h,采用噻唑蓝法检测细胞增殖水平。行细胞划痕试验,计算划痕后12、24 h细胞迁移率。行细胞Transwell试验,培养24 h后计数迁移细胞。行成脂诱导、成骨诱导实验,分别于诱导18、21 d后观测细胞成脂、成骨分化水平。采用实时荧光定量反转录PCR法检测细胞中脂蛋白脂肪酶（LPL）、过氧化物酶体增殖物激活受体γ（PPARγ）、Runt相关转录因子2（Runx2）、骨桥蛋白、碱性磷酸酶（ALP）、基质金属蛋白酶9（MMP-9）、转化生长因子β（TGF-β）的mRNA表达。各实验样本数均为12。 结果 培养0 h,减重组患者与健康组志愿者细胞增殖水平分别为1.022±0.056、1.000±0.144,组间比较,差异无统计学意义（P>0.05）。培养24、48、72 h,减重组患者细胞增殖水平分别为1.366±0.030、1.353±0.012、1.390±0.016,明显低于健康组志愿者的1.755±0.077、1.737±0.014、1.700±0.023（t值分别为16.27、71.35、38.56,P值均<0.05）。细胞划痕试验中,划痕后12、24 h,减重组患者细胞迁移率均低于健康组志愿者,但差异均无统计学意义（P>0.05）。细胞Transwell试验中,培养24 h后,健康组志愿者和减重组患者细胞迁移数比较,差异无统计学意义（P>0.05）。成脂诱导18 d后,减重组患者细胞成脂分化水平明显低于健康组志愿者（t=27.81,P<0.05）；成骨诱导21 d后,减重组患者细胞成骨分化水平明显低于健康组志愿者（t=14.85,P<0.05）。与健康组志愿者比较,减重组患者细胞中LPL、PPARγ、TGF-β和Runx2的mRNA表达均明显降低（t值分别为59.48、146.10、46.10、3.13,P<0.05）,而骨桥蛋白、ALP和MMP-9的mRNA表达均无明显变化（P>0.05）。 结论 相对于健康志愿者,肥胖患者减重成功后其ASC的增殖功能明显减弱,分化功能相对较弱,与成脂、成骨分化对应的部分基因表达水平下降,这可能会影响用这些ASC治疗由烧伤、糖尿病和放射性损伤等导致的难愈性创面的效果,故在临床应用时,需要考虑ASC的供体差异。 Abstract:Objective To explore the changes in biological characteristics of adipose-derived stem cells (ASCs) in obese patients post successful weight loss, so as to provide a reference for the clinical application of these ASCs in refractory wound repair. Methods This study was an experimental study. Twelve obese patients (8 females and 4 males, aged (50±9) years) who underwent abdominal skin tightening surgery after successful weight loss and were admitted to the First Affiliated Hospital of Zhengzhou University from April 2021 to April 2023 were included in weight loss group, and 12 healthy volunteers (10 females and 2 males, aged (50±9) years) who underwent abdominal liposuction and facial fat grafting surgery during the same period in the same institution were included in healthy group. Adipose tissue was collected from patients in weight loss group and volunteers in healthy group, and ASCs were extracted. Experiments were conducted using ASCs at passages 4 and 5. Cell proliferation levels were assessed using the methyl thiazolyl tetrazolium assay at 0 (immediately), 24, 48, and 72 hours of culture. The cell scratch test was performed and the cell migration rates at 12 and 24 hours after scratching were calculated. The cell Transwell assay was performed and the number of migration cells at 24 hours after culture was counted. Adipogenic and osteogenic induction assays were carried out, and the adipogenic and osteogenic differentiation levels of cells were detected after 18 and 21 days of induction, respectively. Real-time fluorescence quantitative reverse transcription polymerase chain reaction was employed to measure the mRNA expressions of lipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma (PPARγ), Runt-related transcription factor 2 (Runx2), osteopontin, alkaline phosphatase (ALP), matrix metalloproteinase 9 (MMP-9), and transforming growth factor beta (TGF-β). The sample number of each experiment was 12. Results At 0 hour of culture, the cell proliferation levels of patients in weight loss group and volunteers in healthy group were 1.022±0.056 and 1.000±0.144, respectively, with no statistically significant difference between the groups (P>0.05). At 24, 48, and 72 hours of culture, the cell proliferation levels of patients in weight loss group were 1.366±0.030, 1.353±0.012, and 1.390±0.016, respectively, which were significantly lower than 1.755±0.077, 1.737±0.014, and 1.700±0.023 of volunteers in healthy group (with t values of 16.27, 71.35, and 38.56, respectively, P values all <0.05). In the cell scratch test, at 12 and 24 hours after scratching, the cell migration rates of patients in weight loss group were lower than those of volunteers in healthy group, but the differences were not statistically significant (P>0.05). In the cell Transwell assay, after 24 hours of culture, there was no statistically significant difference in the number of migrated cells between patients in weight loss group and volunteers in healthy group (P>0.05). After 18 days of adipogenic induction, the cell adipogenic differentiation level of patients in weight loss group was significantly lower than that of volunteers in healthy group (t=27.81, P<0.05). After 21 days of osteogenic induction, the cell osteogenic differentiation level of patients in weight loss group was significantly lower than that of volunteers in healthy group (t=14.85, P<0.05). Compared with those of volunteers in healthy group, the mRNA expressions of LPL, PPARγ, TGF-β, and Runx2 of patients in weight loss group were significantly reduced (with t values of 59.48, 146.10, 46.10, and 3.13, respectively, P<0.05), while there were no statistically significant changes in the mRNA expressions of osteopontin, ALP, or MMP-9 (P>0.05). Conclusions Compared with healthy volunteers, the proliferative capacity of ASCs in obese patients after successful weight loss is significantly diminished, the differentiation potential is relatively weak, and the expression levels of some genes corresponding to adipogenic and osteogenic differentiation are decreased, which may affect the therapeutic efficacy of these ASCs in treating refractory wounds caused by burns, diabetes, or radiation injuries. Therefore, the donor differences of ASCs need to be considered in clinical application. -
参考文献
(35) [1] 邢楠,霍然,王海涛,等.脂肪干细胞基质胶促进创面愈合的研究进展[J].中华烧伤与创面修复杂志,2023,39(1):81-84.DOI: 10.3760/cma.j.cn501120-20211204-00404. [2] WangJM,GuY,PanCJ,et al.Isolation, culture and identification of human adipose-derived stem cells[J].Exp Ther Med,2017,13(3):1039-1043.DOI: 10.3892/etm.2017.4069. [3] Lopez-YusM,García-SobrevielaMP,Del Moral-BergosR,et al.Gene therapy based on mesenchymal stem cells derived from adipose tissue for the treatment of obesity and its metabolic complications[J].Int J Mol Sci,2023,24(8):7468.DOI: 10.3390/ijms24087468. [4] 李畅,冷清阳,冷华卿,等.基于生物信息学筛选通过调控脂肪细胞功能改善肥胖的中药单体[J].解放军医学杂志,2022,47(8):771-780.DOI: 10.11855/j.issn.0577-7402.2022.08.0771. [5] ZhuXY,MaS,EirinA,et al.Functional plasticity of adipose-derived stromal cells during development of obesity[J].Stem Cells Transl Med,2016,5(7):893-900.DOI: 10.5966/sctm.2015-0240. [6] BrazelCB,SimonJC,TuckermannJP,et al.Inhibition of 11β-HSD1 expression by insulin in skin: impact for diabetic wound healing[J].J Clin Med,2020,9(12):3878.DOI: 10.3390/jcm9123878. [7] YangHJ,KimKJ,KimMK,et al.The stem cell potential and multipotency of human adipose tissue-derived stem cells vary by cell donor and are different from those of other types of stem cells[J].Cells Tissues Organs,2014,199(5/6):373-383.DOI: 10.1159/000369969. [8] ChenS,HeZ,XuJ.Application of adipose-derived stem cells in photoaging: basic science and literature review[J].Stem Cell Res Ther,2020,11(1):491.DOI: 10.1186/s13287-020-01994-z. [9] MirzaeiA,DeyhimfarR,Azodian GhajarH,et al.Quercetin can be a more reliable treatment for metastatic prostate cancer than the localized disease: an in vitro study[J].J Cell Mol Med,2023,27(12):1725-1734.DOI: 10.1111/jcmm.17783. [10] ZhangC,JiangT,JiangG,et al.White adipose tissue-derived small extracellular vesicles: a new potential therapeutic reagent for accelerating diabetic wound healing[J].FASEB J,2023,37(12):e23314.DOI: 10.1096/fj.202301549R. [11] 周晓洁,于湄,田卫东.前成脂细胞及其临床应用潜能研究进展[J].解放军医学杂志,2022,47(2):178-185.DOI: 10.11855/j.issn.0577-7402.2022.02.0178. [12] 包郁露,孔维诗,沈纵,等.脂肪间充质干细胞的抗衰老作用[J].中华整形外科杂志,2023,39(10):1147-1152.DOI: 10.3760/cma.j.cn114453-20220218-00043. [13] 杨洪秋,邓映,杜晓霜,等.脂肪干细胞在面部抗衰老中的研究进展[J].中国医疗美容,2023,13(4):57-61.DOI: 10.19593/j.issn.2095-0721.2023.04.015. [14] 韩李念,王君.放射性皮肤损伤治疗的研究进展[J/CD].中华损伤与修复杂志(电子版),2023,18(6):533-537.DOI: 10.3877/cma.j.issn.1673-9450.2023.06.015. [15] 孙皓,蒲胤瑄,刘佳林,等.过表达miR-378a修饰骨髓间充质干细胞复合胶原蛋白海绵支架对大鼠股骨缺损的修复作用[J].解放军医学杂志,2023,48(2):175-182.DOI: 10.11855/j.issn.0577-7402.2023.02.0175. [16] PestelJ,BlangeroF,EljaafariA.Pathogenic role of adipose tissue-derived mesenchymal stem cells in obesity and obesity-related inflammatory diseases[J].Cells,2023,12(3):348.DOI: 10.3390/cells12030348. [17] 刘裴裴,丁世杰,宋文娟,等.NAC通过调控活性氧影响脂肪间充质干细胞增殖和分化[J].中国农业科学,2023,56(21):4330-4343.DOI: 10.3864/j.issn.0578-1752.2023.21.015. [18] WangT,LiY,ZhuY,et al.Anti-aging mechanism of different age donor-matched adipose-derived stem cells[J].Stem Cell Res Ther,2023,14(1):192.DOI: 10.1186/s13287-023-03415-3. [19] EirinA,MengY,ZhuXY,et al. The micro-RNA cargo of extracellular vesicles released by human adipose tissue-derived mesenchymal stem cells is modified by obesity[J].Front Cell Dev Biol,2021,9:660851.DOI: 10.3389/fcell.2021.660851. [20] WuX,MuY,YaoJ,et al.Adipose-derived stem cells from patients with ulcerative colitis exhibit impaired immunosuppressive function[J].Front Cell Dev Biol,2022,10:822772.DOI: 10.3389/fcell.2022.822772. [21] SohrabiK,AhmadiH,AminiA,et al.Promising improvement in infected wound healing in type two diabetic rats by combined effects of conditioned medium of human adipose-derived stem cells plus photobiomodulation[J].Lab Anim Res,2023,39(1):29.DOI: 10.1186/s42826-023-00178-z. [22] 岳强,刘立强.人体不同部位和个体差异对脂肪组织及其间充质干细胞的影响[J].中华整形外科杂志,2024,40(8):917-923.DOI: 10.3760/cma.j.cn114453-20221105-00349. [23] 高俊丽,徐静,余春丽,等.黄芪甲苷对高糖环境下脂肪干细胞衰老的延缓作用[J].中国临床医学,2023,30(3):460-467.DOI: 10.12025/j.issn.1008-6358.2023.20230192. [24] 陶宁,王华.间充质干细胞衰老的研究进展[J].中国医药生物技术,2023,18(1):56-59.DOI: 10.3969/j.issn.1673-713X.2023.01.011. [25] Lopes AlvesDV,Claudio-da-SilvaC,SouzaMCA,et al.Adipose tissue-derived mesenchymal stromal cells from ex-morbidly obese individuals instruct macrophages towards a M2-like profile in vitro[J].Int J Stem Cells,2023,16(4):425-437.DOI: 10.15283/ijsc22172. [26] 车丽娜,李凤娇,赵崇杰,等.长白猪骨髓和脂肪间充质干细胞复制性衰老过程中生物学特性和抗氧化损伤能力的比较[J].天津科技大学学报,2023,38(2):1-10.DOI: 10.13364/j.issn.1672-6510.20220177. [27] 杨超富,谭国庆,徐展望.骨质疏松中衰老相关分泌表型调控机制的研究进展[J].中国全科医学,2024,27(29):3685-3695.DOI: 10.12114/j.issn.1007-9572.2023.0721. [28] MikłoszA,ŁukaszukB,SupruniukE,et al.RabGAP AS160/TBC1D4 deficiency increases long-chain fatty acid transport but has little additional effect on obesity and metabolic syndrome in ADMSCs-derived adipocytes of morbidly obese women[J].Front Mol Biosci,2023,10:1232159.DOI: 10.3389/fmolb.2023.1232159. [29] CortezM,CarmoLS,RogeroMM,et al.A high-fat diet increases IL-1, IL-6, and TNF-α production by increasing NF-κB and attenuating PPAR-γ expression in bone marrow mesenchymal stem cells[J].Inflammation,2013,36(2):379-386.DOI: 10.1007/s10753-012-9557-z. [30] GriescheN,LuttmannW,LuttmannA,et al.A simple modification of the separation method reduces heterogeneity of adipose-derived stem cells[J].Cells Tissues Organs,2010,192(2):106-115.DOI: 10.1159/000289586. [31] FrazierTP,GimbleJM,DevayJW,et al.Body mass index affects proliferation and osteogenic differentiation of human subcutaneous adipose tissue-derived stem cells[J].BMC Cell Biol,2013,14:34.DOI: 10.1186/1471-2121-14-34. [32] Oregel-CortezMI,Frayde-GómezH,Quintana-GonzálezG,et al.Resistin induces migration and invasion in PC3 prostate cancer cells: role of extracellular vesicles[J].Life (Basel),2023,13(12):2321.DOI: 10.3390/life13122321. [33] 黎鑫,艾克拜尔·艾力,阿力木江·麦斯依提,等.外周血WBC和NLR对肥胖症合并胃食管反流病的临床诊断价值[J].解放军医学杂志,2023,48(10):1180-1185.DOI: 10.11855/j.issn.0577-7402.2153.2023.0410. [34] 王跃东,叶再元,莫大超,等.腹腔镜胃肠部分切除术治疗重度肥胖症[J].中华普通外科杂志,2009,24(4):307-309.DOI: 10.3760/cma.j.issn.1007-631X.2009.04.014. [35] AdemiH,Michalak-MickaK,MoehrlenU,et al.Effects of an adipose mesenchymal stem cell-derived conditioned medium and TGF-β1 on human keratinocytes in vitro[J].Int J Mol Sci,2023,24(19):14726.DOI: 10.3390/ijms241914726. -
图 1 划痕试验检测的减重组患者与健康组志愿者脂肪干细胞划痕后各时间点水平迁移情况 光学显微镜×50。1A、1B、1C、1D.分别为减重组划痕后0（即刻）、6、12、24 h的划痕面积,依次逐渐缩小；1E、1F、1G、1H.分别为健康组划痕后0、6、12、24 h的划痕面积,图1A与图1E、图1B与图1F、图1C与图1G、图1D与图1H划痕面积差别不明显
注：减重组患者为肥胖减重成功后行腹壁皮肤松弛矫正术的患者,健康组志愿者为行腹部抽脂后面部填充术的健康志愿者
图 2 Transwell试验检测的减重组患者与健康组志愿者脂肪干细胞培养24 h后垂直迁移情况 苏木精×200。2A.减重组迁移至Transwell下室的细胞（蓝色）数量较多；2B.健康组迁移至Transwell下室的细胞数量与图2A相近
注：减重组患者为肥胖减重成功后行腹壁皮肤松弛矫正术的患者,健康组志愿者为行腹部抽脂后面部填充术的健康志愿者
图 3 减重组患者与健康组志愿者脂肪干细胞成脂诱导18 d与成骨诱导21 d后成脂与成骨分化情况。3A.健康组细胞成脂分化水平较高 油红O×200；3B.减重组细胞成脂分化水平低于图3A 油红O×200；3C.健康组细胞成骨分化水平较高 茜素红×100；3D.减重组细胞成骨分化水平低于图3C 茜素红×100
注：减重组患者为肥胖减重成功行腹壁皮肤松弛矫正术的患者,健康组志愿者为行腹部抽脂后面部填充术的健康志愿者
Table 1. 减重组患者与健康组志愿者一般资料比较
组别 例数/人数 性别（例/名） 年龄（岁, 体重指数[kg/m2,M（Q1,Q3）] 男 女 减重组 12 4 8 50±9 22.95（22.00,23.58） 健康组 12 2 10 50±9 23.10（22.38,23.58） 统计量值 — t=0.02 Z=-0.29 P值 0.640 0.954 0.772 注：减重组患者为肥胖减重成功后行腹壁皮肤松弛矫正术的患者,健康组志愿者为行腹部抽脂后面部填充术的健康志愿者；“—”表示无此项 
下载: 导出CSV
基因名称 引物序列（5'→3'） 产物大小（bp） Runx2 上游：CACAAGTGCGGTGCAAACTT 154 下游：TGCTTGCAGCCTTAAATGACT ALP 上游：AGTGCTCTGCGCAGGATTG 219 下游：CGCCAGTACTTGGGGTCTTT 骨桥蛋白 上游：ATCTCCTAGCCCCACAGCAAT 279 下游：CATCAGACTGGTGAGAATCATC LPL 上游：CAGGATGTGGCCCGGTTTAT 159 下游：AGCTGGATCGAGGCCAGTAA PPARγ 上游：GGGCGCTTGGGTCGG 117 下游：GCTTCTTTCAAATCTGCGGC TGF-β 上游：CGTCTGCTGAGGCTCAAGTT 152 下游：CGGTAGTGAACCCGTTGATG MMP-9 上游：TTCTGCCCGGACCAAGGATA 158 下游：ATGCCATTCACGTCGTCCTT 18S核糖体 上游：GAAACTGCGAATGGCTCATTAAA 315 下游：CACAGTTATCCAAGTAGGAGAGG 注：Runx2为Runt相关转录因子2,ALP为碱性磷酸酶,LPL为脂蛋白脂肪酶,PPARγ为过氧化物酶体增殖物激活受体γ,TGF-β为转化生长因子β,MMP-9为基质金属蛋白酶9
下载: 导出CSV
Table 3. 减重组患者与健康组志愿者脂肪干细胞培养各时间点增殖水平比较（
组别 样本数 0 h（即刻） 24 h 48 h 72 h 减重组 12 1.022±0.056 1.366±0.030 1.353±0.012 1.390±0.016 健康组 12 1.000±0.144 1.755±0.077 1.737±0.014 1.700±0.023 t值 0.49 16.27 71.35 38.56 P值 0.628 <0.001 <0.001 <0.001 注：减重组患者为肥胖减重成功后行腹壁皮肤松弛矫正术的患者,健康组志愿者为行腹部抽脂后面部填充术的健康志愿者；分组因素主效应,F=351.83,P<0.001；时间因素主效应,F=378.99,P<0.001；两者交互作用,F=36.69,P<0.001
下载: 导出CSV
Table 4. 减重组患者与健康组志愿者脂肪干细胞划痕后各时间点迁移率比较（%,
组别 样本数 12 h 24 h 减重组 12 13±5 39±10 健康组 12 18±7 44±14 t值 1.94 0.90 P值 0.334 0.372 注：减重组患者为肥胖减重成功后行腹壁皮肤松弛矫正术的患者,健康组志愿者为行腹部抽脂后面部填充术的健康志愿者；分组因素主效应,F=0.02,P=0.894；时间因素主效应,F=78.36,P<0.001；两者交互作用,F=2.94,P=0.093
下载: 导出CSV
Table 5. 减重组患者与健康组志愿者脂肪干细胞中多种细胞因子mRNA表达比较（
组别 样本数 PPARγ LPL 骨桥蛋白 TGF-β Runx2 ALP MMP-9 减重组 12 0.284±0.012 0.271±0.022 0.99±0.04 0.31±0.03 0.96±0.04 1.23±0.05 0.96±0.04 健康组 12 0.955±0.010 1.000±0.036 1.01±0.04 0.97±0.04 1.06±0.11 1.24±0.03 0.96±0.03 t值 146.10 59.48 1.10 46.10 3.13 0.37 0.13 P值 <0.001 <0.001 0.283 <0.001 0.005 0.713 0.897 注：减重组患者为肥胖减重成功后行腹壁皮肤松弛矫正术的患者,健康组志愿者为行腹部抽脂后面部填充术的健康志愿者；PPARγ为过氧化物酶体增殖物激活受体γ,LPL为脂蛋白脂肪酶,TGF-β为转化生子因子β,Runx2为Runt相关转录因子2,ALP为碱性磷酸酶,MMP-9为基质金属蛋白酶9
下载: 导出CSV
-
魏志茹视频.mp4
-
图(4) / 表(5)
计量
- 文章访问数: 32
- HTML全文浏览量: 7
- PDF下载量: 1
- 被引次数: 0
