【摘要】 目的:胚胎细胞脊髓内移植是判定冻存细胞最直接的证据。通过计算机图像处理系统判定细胞核质比例,以观察经深低温保存后大鼠胚胎脊髓细胞形态和存活率的变化。方法:实验于2004-12/2005-03在承德医学院临床技能实验室完成。剖腹取胎龄14d Wistar大鼠的胎鼠,分离胚胎脊髓,剪成1mm3的小块。将DMEM细胞培养液70mL、大鼠灭活血清20mL、DMSO10mL混合制成冻存液。取冻存管,加入冻存液10mL,并将剪好的胚胎脊髓组织10～20块放入其中,封口。放于冰箱中逐步降温保存,最后置于液氮中保存。胚胎脊髓在液氮中保存15d后,取出冻存管,放入37℃恒温水浴槽中快速复温,待完全融化后,以3000r/min离心3min,弃去上清液,再加入等量DMEM细胞培养液,静置3min,冲洗,并植入正常大鼠脊髓内。以台盼蓝拒染率测定深低温保存前和深低温保存后胚胎脊髓组织细胞存活率,着色的为死亡细胞、不着色的为存活细胞。算出细胞存活率和核质比例。4周后应用免疫组化法检测受体大鼠移植段脊髓。结果:①经低温保存的胚胎脊髓细胞存活率低于未经低温保存组[(64.78±6.14)%,(50.07±7.58)%,P<0.01];两组的存活细胞形态和核质比无明显改变。②移植后4周,有1只大鼠因感染死亡。镜下观察移植大鼠脊髓组织均可见移植物存活,存活移植物与受体脊髓组织融合良好,移植区边缘均有少量宿主神经细胞空泡变性,2只因移植物与受体之间存在裂隙,只有部分融合,无明显胶质瘢痕,1只移植区外偶见局限淋巴细胞浸润。③免疫组化检测显示,在移植区内可见少量的5羟色胺及神经丝阳性细胞和纤维,在损伤腔边缘部可见大量来自宿主的5-羟色胺阳性纤维进入移植区,移植区周边的纤维较中央部位纤维粗大。结论:经过深低温保存大鼠胚胎脊髓组织保持了原有的细胞形态,并能够在异体脊髓内存活,并与宿主融合良好,保持了原有的活性。更多还原

【Abstract】 AIM: Fetal spinal cord (FSC) transplantation is the most direct evidence to judge cryopreservated cells. To observe the change of shape and survival rate of cryopreservated rat FSC by judging nuclear-cytoplasmic ratio through computer image processing system. METHODS: The experiment was performed in the Clinical Technical Laboratory, Chengde Medical College from December 2004 to March 2005. Fetal rats were harvested from E14 Wistar rats to isolate FSC, which were cut into 1 mm3 block. DMEM cell culture fluid 70 mL, inactivated serum 20 mL and DMSO 10 mL were mixed to make into cryopreservation liquid. 10-20 blocks of FSC tissues were put into the 10 mL liquid in cryopreservation tube, and then the tube was sealed and refrigerated in order to decrease the temperature sequentially. And finally preserved in liquid nitrogen. 15 days later, the tube was put in 37 ℃ thermostatic waterbath for rapid rewarming. Till complete thawing, the tissues were centrifuged at 3 000 r/min for 3 minutes, and then supernatant fluid was removed. DMEM cell culture fluid of the same volume was added and stood for 3 minutes, washed and then implanted in spinal cord of normal rats. Survival rate of FSC cells was tested by trypan blue before and after cryopreservation, and those with staining were dead cells and those without staining were survival cells. Cell survival rate and nuclear-cytoplasmic ratio were calculated. 4 weeks later, spinal cord of receptors was measured by immunohistochemical method. RESULTS: ①The cellular survival rate of the cryopreserved FSC was lower than that of the non-cryopreserved [(64.78±6.14)%,(50.07±7.58)%,P < 0.01]. There was no significant difference in morphology as well as the ratio of nucleus to the cytoplasm between two groups. ②In all 8 rats, one rat died because of the infection 4 week after transplantation. The grafts might survive in allogenic spinal cord and fused with the host tissue under microscope. The fusion of the grafts and the receptor tissue were well in rats. Some vacuolar degeneration appeared in the edge of transplantation region. There were cranny between the grafts and the receptor tissue in 2 rats. Some partial fusion and no glial scar occurred. Partial lymphocyte infiltrated seldom in transplantation region. ③Immunohistochemical features revealed a small quantity of 5-hydroxytryptamine and neurofilament positive fibers in grafts. There was a great deal of 5-hydroxytryptamine positive fibers entering grafts area from the receptor tissue. The fibers around grafts were thickened than those in center. CONCLUSION: Cryopreserved FSC keep intrinsic cellular morphology. The cryopreserved FSC grafts may survive in allogenic spinal cord, well fuse with host and keep original activity.更多还原