氧化低密度脂蛋白诱导人载脂蛋白AI转基因小鼠血管平滑肌细胞差异表达基因的研究

目的 为研究人载脂蛋白AI(h-ApoA-I)抗氧化低密度脂蛋白(ox-LDL)所导致的平滑肌细胞增生及脂质浸润的分子机制,克隆并分析可明显抑制动脉内膜脂质沉积和粥样斑块形成的h-apoA-I转基因小鼠血管平滑肌细胞(vSMC)抗ox-LDL所致AS过程中表达的相关基因。方法以ox-LDL为刺激物作用于培养的转基因小鼠vSMC,应用抑制性差减杂交方法构建减除文库,筛选、克隆差异表达基因,并进行序列测定及同源性比较。同时用SMART技术构建了ox-LDL诱导的转基因小鼠vSMC全长噬菌体文库。结果从57个差异克隆中随机选取15个克隆测序并分析,得到3个表达序列标签(EST),均为与免疫系统有关的功能基因片段,即：补体C1-抑制物(C1-INH)、植物凝集素及T细胞受体B(TCRB)。有明显抗内膜脂质沉积能力的h-apoA-I转基因小鼠的vSMC在被ox-LDL诱导后,筛选出的3个EST均与免疫系统有关,其中C1-INH及植物凝集素与补体系统密切相关。结论ox-LDL致AS及h-ApoA-I抗AS可能有免疫机制参与,且与补体活化及其调控密切相关。其中C1-INH作为抗AS的潜在靶点值得关注。

Molecular basis of preventive effect of human apolipoprotein-1 on murine vascular smooth muscle cell proliferation and lipid deposition induced by oxidized low density lipoprotein

OBJECTIVE: To study the molecular basis of preventive effect of human apolipoprotein-1 (h-apoA-1) on vascular smooth muscle cell (vSMC) proliferation and lipid deposition induced by oxidized low density lipoprotein (ox-LDL). METHODS: Smooth muscle cells originated from the aortae of h-apo-A-1 transgenic mice were cultured and divided into 2 groups, one group was stimulated by ox-LDL (tester) and the other group was used as control (driver). Subtractive hybridization was used to enrich the genes differentially expressed in the vSMCs induced by ox-LDL. A subtractive library was thus established and confirmed by colony hybridization in situ and dot blot analysis. 15 clones out of the 57 differentially expressed clones were randomly chosen foe sequencing and homology analysis. The whole-length cDNA library of vSMC induced by ox-LDL was established using SMART technique. RESULTS: Three expression sequence tags (EST), all correlated with immune system, were confirmed: C1-inhibitor (C1-INH), lectin, and T cell receptor beta. The whole-length cDNA library contained 1.5 x 10(6) pfu/ml primary recombinants with insertions 0.5 - 3 kb in length. CONCLUSION: The 3 EST may be involved in the mechanism of atherogenesis by ox-LDL and the mechanism of the function of h-ApoA-1 in retarding the progression of atherogenesis induced by ox-LDL.