摘要
目的探讨d-α-生育酚对高糖诱导的人腹膜间皮细胞(human peritoneal mesothelium cell,HPMC)己糖激酶(hexokinase,HK)及其同工酶表达的调节作用。方法胰蛋白酶消化法分离培养HPMC,免疫细胞化学染色及扫描电镜对培养细胞进行鉴定。第3代HPMC分为正常对照组(0．1％葡萄糖,相当于5．5 mmol／L)、高糖组(0．5％、1．0％、1．5％、2．5％、4．25％的葡萄糖)及d-α-生育酚干预组。培养24 h后,利用6-磷酸葡萄糖脱氢酶耦联比色法和温度敏感实验检测HK及其同工酶的活性;免疫细胞化学染色检测HK蛋白的细胞内定位;Western印迹及逆转录聚合酶链反应(RT- PCR)法分别检测HK蛋白及mRNA的表达。己糖激酶法检测培养液内葡萄糖的消耗量作为细胞对葡萄糖净利用指标。结果角蛋白和波形蛋白在HPMC的胞质呈阳性表达,扫描电镜可见细胞表面有丰富的微绒毛。1．5％、2．5％及4．25％葡萄糖组,HK总活性上调,与正常对照组(20．6±2．5)U／g蛋白比较,相对活性分别为115．4％、129．1％及155．2％,并以HKⅡ增加为主(P<0．05)。50 mmol／L d-α-生育酚干预组,HK总活性下调,是2．5％葡萄糖组的82．1％(P=0．001),其中HKⅡ受抑为主;d-α-生育酚干预后HPMC对葡萄糖的净利用由(25．3±3．9)mmol／L降至(17．3±2．1)mmol／L(P= 0．018)。正常状态下,HKⅡ蛋白在胞质呈棕色淡染;随葡萄糖浓度的增加(葡萄糖≥1．5％),HKⅡ在核周积聚、浓染,呈珠链样;d-α-生育酚预处理后,HKⅡ在核周的积聚变淡。HKⅡ蛋白及mRNA的表达与HKⅡ活性同步。结论d-α-生育酚抑制了高糖对HKⅡ活性、蛋白及mRNA表达上调的作用,并减少HPMC对葡萄糖的净利用;可能成为增加腹膜透析超滤的手段之一。
Objective To investigate the effect of d-α-tocopherol on the expression of hexokinase （HK） induced by high glucose in cultured human peritoneal mesothelium cell （HPMC） . Methods Specimens of human omentum were obtained from consenting patient undergoing elective abdominal surgery. HPMC were isolated and subcultured by enzymatic disaggregation. Morphology and immunocytochemical method were used for identification. HPMC were divided into normal glucose group （0. 1% glucose, equal to 5.5 mmol/L） , high glucose group （ 0. 5 %, 1.0%, 1.5 %, 2.5 %, 4.25 % glucose ） and d-α-tocopherol group. After 24 h, standard G6PDH-coupled assay, temperature sensitive essay were used to detect the activity of total HK and its isozyme. Immunocytochemical staining was used for observation the intracellular location of HK 11. Western blotting was used to analyze the protein expression of HK 11 and RT-PCR was used to detect the mRNA expression of HK Ⅱ. The net glucose utilization was assayed by glucose disappearance from medium by hexokinase method. Results Primary cultured HPMC reacted positively for cytokeratin and vimentin and had numerous surface microvilli under electron microscope. High glucose induced HK activity in a dose-dependent manner and increased HK Ⅱ isoform selectively. At concentration of 1.5%, 2.5% and 4. 25% glucose for 24 h, the relative activity of total HK were 115.4% , 129. 1% and 155.2% , respectively comparing with normal control （ P 〈 0. 05 ）, and selectively increased HK Ⅱ isoform expression. D-α-tocopherol blocked the activity of total HK and HK Ⅱ induced by glucose. The relative activity of total HK inhibited by d-α-tocopherol was 82. 1% （P = 0.001 ）. After incubated with d-α- toeopherol, the net glucose utilization were decreased from （ 25.3 ± 3.9 ） mmol/L to （ 17.3 ± 2. 1 ） mmol/L （P=0. 018）. HK Ⅱ stained light brown in cytoplasm of HPMC in normal group, accompanying with the increased concentration of glucose, the staining of HK Ⅱ became strong and a
出处
《中华医学杂志》
CAS
CSCD
北大核心
2006年第32期2275-2280,共6页
National Medical Journal of China
基金
辽宁省教育厅资助项目(2004D180)
