肥胖患者减重成功后其脂肪干细胞的生物学特性变化

Changes in biological characteristics of adipose-derived stem cells in obese patients post successful weight loss

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。

ObjectiveTo 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.MethodsThis 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.ResultsAt 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).ConclusionsCompared 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.