兔腰椎间盘成分抗原性差异的研究

The study of differences of antigenicity in rabbit intervertebrai disc

目的初步了解兔椎间盘不同成分的免疫源性差别，进一步提示人体内不同椎间盘细胞的抗原性差别。方法根据生物基因相似性原理及在椎间盘不同部位髓核成分含量存在差异的基础上，采用手术切取实验动物不同区域椎间盘组织并埋植于术野中邻近的椎旁肌内，从而建立髓核糖蛋白埋植组（n=20）和纤维环胶原蛋白埋植组（n=20），并且建立埋植自体肌肉的空白对照组（n=20）。分别于第1、2、4、6、8周分批处死动物取标本进行HE染色，观察新生血管化和淋巴细胞浸润；进行免疫组织化学染色，观察CD4、CD8T淋巴细胞阳性率。结果HE染色显示糖蛋白埋植组标本在术后1周可见淋巴细胞浸润，4周可见典型新生血管化，并且持续至第8周；胶原蛋白埋植组标本在术后4周才可见少量淋巴细胞浸润，不典型新生血管化；肌肉埋植组在各个时间点均未见典型淋巴细胞浸润和新生血管化形成。各组标本总新生血管化率差异有统计学意义。各时间点所取标本进行免疫组织化学染色，计数CD4＋和CD8＋T淋巴细胞，发现糖蛋白埋植组CD4＋和CD8＋淋巴细胞计数均高于胶原蛋白埋植组和肌肉埋植组，胶原蛋白埋植组CD4＋和CD8＋淋巴细胞计数高于肌肉埋植组，差异有统计学意义。结论兔腰椎间盘成分中髓核糖蛋白相比纤维环胶原蛋白更易诱导T淋巴细胞分化为CD4＋和CD8＋细胞，进而推论糖蛋白的自身免疫源性强于胶原蛋白，而胶原蛋白强于肌肉组织，具有弱抗原性。

Objective To reveal and compare the immunogenicity of glycoprotein from nucleus pulposus and collagen from anulus fibrosus in rabbit intervertebral disc. Methods According to the similarity principle of genome biology and the amount of nucleus pulposus in different intervertebral disc, Glycoprotein from nucleus pulposns was harvested from the intervertebral disc of 20 rabbits and placed into the psoas by anterolateral approach. Similar implantation of collagen from anulus fibrosus was performed in another 20 rabbits. The implantations of autologous muscle in rabbits serve as a control group. After one week, two weeks, four weeks, six weeks and eight weeks, the rabbits were killed, the tissue was harvested. HE staining was applied to detect the degree of vascularization and infiltration of lymphocytes. Immunohistochemistry were used for analysis of T-helper cells （CD4＋） and cytotoxic T cells （CD8＋） in the tissue. Results HE staining showed the infiltration of lymphocytes was found just one week after implantation and vascularization was seen at the 2nd week in the glycoprotein implanted group. However in collagen implant group, small amounts of lymphocytic and atypical vascularization were seen at the 4th week. Immunohistochemistry revealed that the proportion of activated T cells （CD4＋ and CD8＋） was significantly higher in the glycoproteinimplanted group at all time points, as compared with the collagen-implanted group and sham group. The proportion of activated T cells was higher in collagen-implated group than that of in sham group , Conclusion The glycoprotein in nucleus pulposus is easier to induce the differentiation and mature of T cell. The antigenicity of glycoprotein in nucleus pulposus was stronger than that of collagen in anulus fibrosus in rabbit intervertebral discs.