摘要
目的探讨不同种类酰胺类局麻药对脂肪起源间充质干细胞成软骨能力影响。方法分离培养四月龄新西兰大白兔脂肪起源干细胞(ADMSCs)并向软骨方向诱导分化。取贴壁生长的第三代软骨方向分化ADMSCs,分别用磷酸缓冲液(对照组)、1%利多卡因(1%利多卡因实验组)、2%甲哌卡因(2%甲哌卡因实验组)、0.5%布匹卡因(0.5%布匹卡因实验组)和0.5%罗哌卡因(0.5%罗哌卡因实验组)浸泡30 min、60 min。5组在各时间点处理完成后,洗去局麻药,重新加入软骨方向诱导分化培养液,分别于培养第3天和第7天后采用流式细胞术检测细胞凋亡情况,并行番红-固绿染色评估软骨分化形态学变化,筛选出毒性最小的局麻药。将筛选出的毒性最小的一种局麻药稀释至25%,50%,75%,100%的浓度分别与软骨分化方向ADMSCs接触30 min和60 min,然后洗去局麻药重新加入软骨方向诱导分化培养液,在培养第3天和第7天后采用实时荧光定量(qPCR)和Western blot法检测软骨分化相关基因collagen Ⅰ、collagen Ⅲ、SOX9的表达,明确其对ADMSCs成软骨能力的影响。结果软骨方向分化的ADMSCs与对应的局麻药物接触后,1%利多卡因实验组、2%甲哌卡因实验组、0.5%布匹卡因实验组和0.5%罗哌卡因实验组软骨方向分化的ADMSCs凋亡率较对照组均显著增加,差异有统计学意义(P<0.05)。软骨分化方向ADMSCs与以上4个实验组接触60 min后的细胞凋亡率均高于组内接触30 min后,差异均有统计学意义(P<0.05);4个实验组培养第3天后的细胞凋亡率高于组内培养第7天后,差异均有统计学意义(P<0.05)。1%利多卡因处理组的细胞凋亡率低于2%甲哌卡因实验组、0.5%布匹卡因实验组和0.5%罗哌卡因实验组,差异均有统计学意义(P<0.05)。进一步研究显示,与不同浓度利多卡因接触后,collagen Ⅰ、collagen Ⅲ、SOX9的mRNA和蛋白表达均显著降低,差异均有统计学意义(P<0.05),且随着利多卡因�
Objective To identify the cytotoxicity of different local anesthetics (LAs) to the adipose-derived mesenchymal stem cells (ADMSCs) during early chondrogenic differentiation. Methods Adipose-derived stem cells (ADMSCs) were isolated from 4-month old New Zealand white rabbits, and cultured and induced to differentiate into cartilage. The third generation chondrogenic differentiating ADMSCs were isolated and incubated with 1% lidocaine, 2% methylpivacaine, 0.5% bupivacaine and 0.5% ropivacaine, respectively, for 30 min and 60 min, so as to screen out the LA of least toxicity. Control cells were incubated in phosphate buffer. The LAs were washed away and the chondrogenic differentiation medium was added to promote differentiation. Apoptosis and morphological changes during the chondrogenic differentiation were measured using flow cytometry and safranine-fast green double dyeing on the 3rd and 7th days of the culturing. The least toxic LA was diluted to 25%, 50%, 75% and 100% of the original concentration and incubated with ADMSCs for 30 and 60 minutes. On days 3 and 7 after the exposure, a quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) and western blotting were employed to analyze the expression of chondrogenesis-related genes including the collagen I, collagen III and SOX9. Results Apoptosis increased significantly after the lidocaine, ropivacaine, bupivacaine and mepivacaine treatments compared to the PBS control. LAs are cytotoxic to rADMSCs during early chondrogenesis in a type- and time-dependent manner. Lidocaine was the least toxic LA tested. The chondrogenesis-related markers all decreased significantly by days 3 and 7 after the exposure to lidocaine at different concentrations compared with the control group. Conclusions LAs are cytotoxic to ADMSCs during early chondrogenesis in a dose- and time-dependent manner. 1% lidocaine was the least toxic LA tested.
作者
吴涛
宋海新
李扬政
李建华
Wu Tao;Song Haixin;Li Yangzheng;Li Jianhua(Department of Rehabilitation Medicine,Sir Run Run Shaw Hospital,College of Medicine,Zhejiang University,Hangzhou 310016,China)
出处
《中华物理医学与康复杂志》
CAS
CSCD
北大核心
2019年第4期246-251,共6页
Chinese Journal of Physical Medicine and Rehabilitation
基金
浙江省自然科学基金2016年面上项目(LY16H170001).
关键词
干细胞
局麻药
细胞毒性
再生医学
Mesenchymal stem cells
Local anesthetics
Cytotoxicity
Regenerative medicine
