中波紫外线诱导的人角质形成细胞凋亡与SSA/Ro抗原表达

目的 为明确紫外线对光敏感型皮肤型红斑狼疮的作用机制,光照后人角质形成细胞表面SSA/Ro抗原的形成机理及其在皮损形成中的作用。方法 用M154培养基体外培养角质形成细胞,经UVB照射后先用相差显微镜观察细胞形态学改变,并用原位末端标记法测定凋亡细胞的断裂DNA片段,同时用间接免疫荧光法测定角质形成细胞表达SSA/Ro抗原情况;然后分别用DNase、RNase、DNase+RNase消化,碘化丙啶复染。用ELISA法测定细胞上清液SSA/Ro抗原;小剂量UVB照射后的角质形成细胞与亲和纯化的SSA/Ro特异血清及正常人血清孵育,台盼蓝染色观察细胞活性。结果 不同剂量UVB(52.8mJ/cm²、105.6mJ/cm²、158.4mJ/cm²、200mJ/cm²、211mJ/cm²)照射后KC凋亡,表面有凋亡小泡,细胞核有断裂DNA缺口并表达SSA/Ro抗原,随UVB剂量的递增,凋亡和表达SSA/Ro抗原的细胞数增多。这种抗原位于凋亡细胞的凋亡小体中,表达于细胞表面的SSA/Ro抗原可在原位诱导补体杀伤,而在细胞上清液中未测到SSA/Ro抗原。结论 UVB照射角质形成细胞通过诱导角质形成细胞凋亡表达自身抗原SSA/Ro,该抗原并不释放到上清液中,在补体和相应抗体存在的情况下可引起细胞损伤。

Correlation between UVB-induced Apoptosis and SSA/Ro Expression in Keratinocytes

Objective To elucidate the mechanism of SSA/Ro formation after UV exposure,and its significance in the pathogenesis of photosensitive LE skin lesion.Methods Human keratinocytes(HKC)were cultured in Medium-154.After ultraviolet-B light(UVB)irradiation,the morphological change of apoptotic HKC was observed under phase contrast microscopy,the DNA fragment of apoptotic cell was detected by in situ nick end-labeling.And the expression of SSA/Ro was detected by indirect immunofluores-cence(IIF).As target cells,UVB irradiated keratinocytes were incubated with affinity-purified anti-SSA/Ro sera,with or without freshly prepared human sera.SSA/Ro antigen from supernatant of irradiated keratinocytes was also detected with an ELISA method.Results The treatment of keratinocytes with serial doses(52.8mJ/cm²、105.6mJ/cm²、158.4mJ/cm²、200mJ/cm²、211mJ/cm²)of UVB induced SSA/Ro expression on cell membrane and apoptosis,leading to apoptotic bleb formation on cell surface,which were proved containing SSA/Ro and ribonucleoprotein.SSA/Ro antigen expressed on UVB irradiated keratinocytes was proved to be bound to affinity purified anti-SSA/Ro sera and lead to complement-depend cytotoxicity.But no SSA/Ro in supernatant was detected.Conclusions UVB irradiation induces SSA/Ro expression on the HKC surface through apoptosis,and no SSA/Ro antigen was found in supernatant.The result suggests that SSA/Ro anti-gen expressed on UVB irradiated keratinocytes could be recognized and presented to immune cells by direct cell-cell contact.