徐学振,于占革,杨 威,魏 伟,李念龙,张 东.不同代次兔髓核细胞冻存后的生物学特性比较[J].中国脊柱脊髓杂志,2011,(8):680-684. |
不同代次兔髓核细胞冻存后的生物学特性比较 |
中文关键词: 髓核细胞 冻存 不同代次 细胞活力 细胞培养 兔 |
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。 |
Comparison of characteristics of different generations of rabbit nucleus pulposus cells after cryopreservation |
英文关键词:Nucleus pulposus cells Cryopreseryation Different generation Cell viability Cell culture Rabbit |
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. |
投稿时间:2011-04-11 修订日期:2011-05-30 |
DOI:10.3969/j.issn.1004-406X.2011.8.680.4 |
基金项目:基金项目:黑龙江省攻关课题项目(编号:GC06C414) |
|
摘要点击次数: 3211 |
全文下载次数: 2743 |
查看全文 查看/发表评论 下载PDF阅读器 |
关闭 |