深低温保存温度及时间对椎间盘生物活性的影响

Effects of storage temperatures and times on cell viability of cryopreserved intervertebral disc

中文关键词:  椎间盘  深低温保存  细胞活性  细胞存活率

英文关键词:Intervertebral disc  Cryopreserve  Cytoactive  Survival ratio of cell

中文摘要:

  【摘要】 目的：探讨深低温保存温度及保存时间对椎间盘生物活性的影响。方法：取1岁龄Beagle犬新鲜椎间盘72个，分为对照组（A组，n=8）、-80℃保存组（B组，n=32）和-196℃保存组（C组，n=32）。B和C组在冷冻保存液中分别保存于-80℃低温冰箱及-196℃液氮中，在保存2w、2m、6m及12m时每组每个时间点取8个椎间盘，通过溴化吡啶/二乙酸荧光素（EB/FDA）荧光染色法检测椎间盘组织中不同部位的细胞存活率，以二甲基亚甲蓝（DMMB）染色法检测髓核组织蛋白多糖（PG）含量，以羟脯氨酸检测法测定纤维环组织中的总胶原含量；对照组取标本后直接进行以上检测。结果：B和C组椎间盘在保存至2w时各部位细胞存活率较A组明显下降，2m~12m保存期间，B、C两组椎间盘各部位的细胞存活率均维持在相对恒定的水平，但均较2w时下降；B组及C组外层纤维环细胞存活率在保存2w和2m时无明显差别（P>0.05），在保存6m及12m时C组明显高于B组（P<0.05）；C组内层纤维环及髓核组织细胞存活率在各检测时间点均明显高B组（P<0.05）。B、C两组在各检测时间点PG含量均较A组明显降低，在保存2m~12m期间，C组PG含量均高于B组（P<0.05）。B、C两组总胶原蛋白含量在保存期间随时间逐渐降低，但两组间未见明显差异（P>0.05）。结论：利用-196℃保存椎间盘组织，无论在细胞总的存活率及PG含量上均优于-80℃保存；-196℃保存12个月时椎间盘细胞存活率及PG含量均维持在相对稳定的水平，能满足同种异体椎间盘移植的要求。

英文摘要:

  【Abstract】 Objective：To investigate effects of different storage temperatures and times on cell viability of cryopreserved intervertebral disc and to obtain the optimal storage condition.Method：72 fresh canine intervertebral discs were divided into control group（group A，n=8），-80℃ group（group B，n=32） and -196℃ group（group C，n=32）.The intervertebral discs in group B and C were immerged into the freezing preservation medium and stored in -80℃ low temperature refrigerator and liquid nitrogen respectively.At each time-point of the 2md week，2nd month，6th month and 12th month，the survival ratio of different position at disc was detected by EB/FDA，the proteoglycans（PG） content of nucleus pulposus （NP） tissue was detected by DMMB，and the collagen content was detected by hydroxyproline.Result：The survival ratio of cell in group A was higher than that in group B and C at any time-points.Moreover，the survival ratios of cell in group B and C remained in a lower level.At 2th week and 2th month，the survival ratio in outer layer annulus fibrosus did not differ significantly in group B and C（P>0.05）.At the 6th month and 12th month，the survival ratio in outer layer annulus fibrosus in group B was higher than that in group C（P<0.05）.The survival ratio in inner layer annulus fibrosus and nucleus pulposus in group B was higher than that in group C at any time-point（P<0.05）.The PG content reduced gradually in 2-month storage.While from the 2nd to 12th storage month，the PG content in group C was higher than that in group B（P<0.05）.The collagen content in both groups showed no difference（P>0.05）.Conclusion：Both the ratio of cell survival and PG content of intervertebral disc stored in liquid nitrogen are higher than those in -80℃.During 1 year storage in liquid nitrogen，the ratio of cell survival and PG content maintain stably，which is suitable for allogenic transplantation.