内毒素／脂多糖对人脐带间充质干细胞增殖及凋亡的影响

Effects of endotoxin/lipopolysaccharide on proliferation and apoptosis of human umbilical cord mes- enchymal stem cells

目的体外观察不同浓度LPS对人脐带间充质干细胞（hUCMSC）增殖以及凋亡的影响，并探讨可能机制。方法取足月剖宫产健康胎儿的脐带组织，采用组织块法体外分离培养hUCMSC，实验选用第3代细胞。按随机数字表法将细胞分为对照组及0．1、1．0、10．0、100．0μg／mL LPS干预组，各LPS干预组细胞培养液中添加对应浓度的LPS，对照组细胞培养液中不添加LPS。对LPS干预组细胞，刺激12、24、48h采用噻唑蓝法检测细胞增殖活性（每组每时相点样本数为5），数据以吸光度值表示；刺激24h采用吖啶橙．溴化乙啶（AO-EB）染色观察细胞凋亡情况（每组样本数为4），并采朋流式细胞仪检测细胞凋亡率（每组样本数为5）。对照组细胞于相同时相点行相应检测。对数据行单因素方差分析、LSD-t检验。结果（1）刺激12h，各组细胞增殖活性无明显差异（t值为-1．67～1．33，P值均大于0．05）。与对照组比较，0．1、1．0、10．0μg／mL LPS干预组刺激24、48h细胞增殖活性均明显增高（t值为-l3．42～17．34，P〈0．05或P〈0．01），其中以1．0μg／mL LPS干预组最明显；100．0μg／mL LPS干预组刺激24、48h细胞增殖活性明显降低（t值分别为8．64、17．34，P值均小于0．01）。（2）AO-EB染色显示，对照组和0．1、1．0、10．0μg／mL LPS干预组未见明显细胞凋产，100．0μg／mLLPS干预组可见明昆细胞凋亡。（3）流式细胞仪显示，对照组及0．1、1．0、10．0、100．0μg／mL LPS干预组细胞凋亡率分别为（3．1±O．6）％、（2．6±0．7）％、（2．9±0．8）％、（3．1±0．4）％、（25．1±2．7）％，组问比较差异有统计学意义（F=272．19，P〈0．01）。0．1、1．0、10．0μg／mL LPS干预组细胞凋亡率与对照组相近（t值分别为1．22、0．57、-0．14，P值均大于0．05），100．0μg／mL LPS干预组细胞凋亡率明显高于对照组（t=-17．63，P〈0．01）。结论低浓度水平LPS促进hUCMSC增殖，但随着LPS浓度逐渐增大，hUCMSC增殖活性下降甚至凋亡，这可能与不同浓度LPS激活的主要分子信号通路各异有关。

Objective To investigate the effects of different concentrations of lipopolysaccharide （LPS） on proliferation and apoptosis of human umbilical cord mesenchymal stem cells （bUCMSCs） in vitro, and to explore their possible mechanism. Methods hUCMSCs from umbilical cord tissue of full-term healthy fetus delivered by caesarean section were isolated and cultured in vitro using tissue attachment meth- od. The 3rd passage hUCMSCs were used in the study. Cells were divided into groups A, B, C, D, and E, which were treated with DMEM/FI2 medium containing0, 0.1, 1.0, 10. 0, and 100.0 μg/mL of LPS re- spectively. In groups B, C, D, and E, methyl-thiazole-tetrazolium assay was used to detect proliferative ac- tivity of hUCMSCs at post treatment hour （PTH） 12, 24, and 48 （denoted as absorption value）, with 5 samples in each group at each time point ; apoptosis of hUCMSCs at PBH 24 was identified with acridine or- ange-ethidium bromide （AO-EB） staining, with 4 samples in each group; apoptotic rate of hUCMSCs was determined by flow cytometer, with 5 samples in each group. Above-mentioned indexes were determined in group A at the same time points. Data were processed with analysis of variance and LSD- t test. Results（ 1 ） There was no statistically significant difference in proliferative activity of hUCMSCs at PTI-I 12 among groups A, B, C, D, and E （ with t values from - 1.67 to 1.33, P values above 0.05）. Compared with that of group A, proliferative activity of hUCMSCs was increased in groups B, C, and D at PTH 24 and 48 （with t values from -13.42 to 17.34, P 〈0. 05 or P 〈 0.01 ） , especially so in group C. Proliferative activity of hUCMSCs was lower in group E at PTH 24 and 48 than in group A （with t values respectively 8.64 and 17.34, P values below 0. O1 ）. （2） Obvious apoptosis of hUCMSCs was observed in group E but not in the other 4 groups with AO-EB staining. （3） Apoptosis rates of hUCMSCs in groups A, B, C, D, and E were respec- tively （3.1 ±0.6）%, （2.6 ±0.7）%, （2.9 ±0.8）%, （3.1 ±0.4）%, （25.1 ±2.7）% （ F =272.19, P 〈 0.01 ）. Apoptotic rate of hUCMSCs in group B, C, or D was respectively close to that in group A （with t values respectively 1.22, 0.57, - 0.14, P values above 0.05） , but it was higher in group E than in group A （ t = -17.63, P 〈0.01）. Conclusions hUCMSCs proliferation may be promoted by low concentration of LPS. hUCMSCs proliferation is inhibited or induced to apoptosis along with the increase in concentration of LPS, and it may be related to activation of different major molecular signaling pathways by different con- centrations of LPS.