不同代次兔髓核细胞冻存后的生物学特性比较

Comparison of characteristics of different generations of rabbit nucleus pulposus cells after cryopreservation

中文关键词:  髓核细胞  冻存  不同代次  细胞活力  细胞培养  兔

英文关键词:Nucleus pulposus cells  Cryopreseryation  Different generation  Cell viability  Cell culture  Rabbit

中文摘要:

  【摘要】 目的：比较不同代次兔髓核细胞冻存后的生物学特性，找出适合作为种子细胞冻存的兔髓核细胞代次。方法：取3~4月龄新西兰大白兔10只，麻醉后在严格无菌条件下分离胸腰椎间盘（T1/2~L7/S1）髓核细胞，体外传代培养，分别取一、二、三、四、五代细胞冻存2~3个月后复苏，取一代未冻存细胞作为对照组（A组，n=10）；一、二、三、四、五代冻存后复苏的细胞作为实验组（分别为B、C、D、E、F组，各组n=10），在相同条件下培养2周，培养第2天时采用台盼蓝染色测定细胞成活率；培养第2d、5d、10d、14d时采用MTT法检测细胞生长活性，采用间苯三酚分光光度法测定蛋白多糖（PG）含量；培养第14d时采用阿利新兰染色法测定糖胺聚糖（GAG）合成总量，用RT-PCR法检测各组细胞Ⅱ型胶原基因的表达情况。检测细胞浓度均为1×105个/ml。采用SPSS 13.0软件进行统计学分析。结果：培养第2d时B、C组细胞成活率接近A组（P>0.05）；D、E、F三组细胞成活率明显低于A、B、C组（P<0.05）。不同时间点各组细胞均有不同程度的增殖，A、B、C、D四组生长趋势较接近，A、B、C三组相同时间点生长活性比较差异无统计学意义（P>0.05），D组与同一时间点A组比较有显著性差异（P<0.05），E组第10d和第14d以及F组各时间点间比较差异无显著性（P>0.05）；A、B、C三组各时间点的PG含量及GAG含量高于相应时间点D、E、F组（P<0.05）；A、B、C三组的Ⅱ型胶原基因表达量高于D、E组（P<0.05），明显高于F组（P<0.01）。结论：不同代次兔髓核细胞冻存后生物学特性不同，第一、二代细胞冻存后能较好保持髓核细胞特性，可作为种子细胞进行低温保存。

英文摘要:

  【Abstract】 Objective：To compare the characteristics of different generations of rabbit nucleus pulposus cells（NPC） after cryopreservate and search for the best generation suitable for cryopreseryation as seed cells.Method：10 New Zealand white rabbits（3-4 months old） were used，and the thoracic and lumbar（T1/2-L7/S1）NPC were separated immediately after general anesthesia under strictly aseptic conditions and cultured in vitro：control group（group A，n=10），the original generational cells；group B，C，D，E and F respectively representing the first，second，third，forth and fifth generation cells（each group n=10 and cell concentration 1×105/ml） were cryopreserved for 2-3 months and then continued for two weeks after recovery.The Trypan Blue and MTT were used for detecting cell survival rates and cell growth activity respectively；the proteoglycan（PG） synthesis and glycosaminoglycans（GAG） accumulation were tested；and the expression of type Ⅱ collagen gene were evaluated by RT-PCR.All the results were analyzed statistically using the SPSS 13．0 software package.Result：Cell survival rate of group B and C was close to that of group A on the 2nd day（P>0.05） and group D，E and F had obviously lower cell survival rate than that of group A，B and C（P<0.05）.Cells of each group proliferated in different degrees.The growth tendency of group A，B，C and D was closer，and group A，B and C showed no time-related difference（P>0.05），while group D had worse cell survival rate than group A（P<0.05）.The proliferation of group E on the 10th and 14th days as well as each time point of group F showed no significant difference（P>0.05）.PG synthesis and GAG accumulation of group A，B and C at each time point were higher than that of group D，E and F at the corresponding time point（P<0.05）.The collagen Ⅱgene expression of group A，B and C was higher than that of group D and E（P<0.05），and group F had lowest expression（P<0.01）.Conclusion：After cryopreservation，different generations of cells present varied characteristics.The first and second generations are suitable as seed cells after cryopreservation due to their better maintenance of NPC characteristics.