CⅡTA反义cDNA抑制Hela细胞表面MHCⅡ类抗原的表达

目的　探讨Ⅱ类主要组织相容性复合物 (MHCⅡ )激活因子 (CⅡTA)的反义cDNA对细胞表面MHCⅡ类分子表达的抑制。方法　克隆 3条CⅡTA反义片段 (arⅡ 1、arⅡ 2、arⅡ 3) ,并稳定转染Hela细胞。免疫组化、流式细胞术检测细胞核内CⅡTA蛋白及其调控的经典MHCⅡ (HLA DR、 DP、 DQ)类抗原表达 ,RT PCR法检测CⅡTA、经典MHCⅡ及恒定链的mRNA水平。结果　三种反义片段中arⅡ 2效果最佳。在重组人IFN γ诱导下 ,arⅡ 2阳性Hela细胞与正义链组比较 ,CⅡTA蛋白抑制率达87.2 3% ,HLA DR、 DP及 DQ抗原诱导型表达分别降低了 79.2 1%、90 .31%及 4 8.30 % ;同时CⅡTA、经典的MHCⅡ及恒定链的mRNA含量明显减少 (P <0 .0 5 )。结论　CⅡTA反义片段抑制了自身mRNA含量 ,从而阻止其调控的MHCⅡ分子的表达 ,为预防移植物抗宿主病提供了一种新思路。

Inhibition effect of CⅡTA anti-sense cDNA on the expression of MHCⅡmolecules of Hela cells

OBJECTIVE: To investigate the inhibiting effect of classII major histocompatibility complex transactivator (CIITA) anti-sense cDNA on MHCII molecules expression. METHODS: CIITA antisense cDNAs (arII1, arII2 and arII3) were obtained from Raji cell by RT-PCR, and then inserted into the pcDNA3.1B plasmid. The stable transfectants of Hela cell were tested for CIITA protein by immunohistochemistry, and classic MHCII molecules (HLA-DR, -DP, -DQ) induction through recombinant human IFN-gamma by flow cytometry. mRNAs of CIITA, invariant chain and classic MHCII molecules were detected by RT-PCR. RESULTS: The effect of arII2 was the best. When induced with IFN-gamma, the expression of CIITA protein was inhibited by 87.23%, and that of HLA-DR, -DP and -DQ was almost totally inhibited on arII2 positive Hela cells. The mRNA contents of CIITA, HLA-DR, -DP, -DQ and invariant chain were decreased significantly (P < 0.05). CONCLUSIONS: arII2 inhibits CIITA and thus the family of MHCIImolecules regulated by CIITA, therefore, it provides a novel approach to graft versus host disease study in hematopoietic stem cell transplantation.