摘要
目的 明确哪一种透明质酸合成酶是人腹膜间皮细胞合成透明质酸及形成细胞外基质/胞衣样结构的主要酶。方法 分离培养人腹膜间皮细胞,以半定量RT-PCR法检测其3种透明质酸合成酶HAS-1、HAS-2、HAS-3 mRNA水平表达情况。明确正常培养人腹膜间皮细胞主要表达HAS-2mRNA和微量HAS-3 mRNA后,设计HAS-2的反义寡核苷酸序列,以脂质体介导法将其转入正常培养的人腹膜间皮细胞。转染前(0 h)、转染后8、24、48 h以RT-PCR法观察HAS-2 mRNA表达情况,并以微粒排除法观察细胞衣样结构面积变化。结果 转染后8和24 h,HAS-2 mRNA表达分别下降58％和89％(P均<0.05);转染后48 h HAS-2 mRNA表达已部分恢复至正常表达的25％水平(P<0.05)。相应地,转染后24 h以微粒排除法观察人腹膜间皮细胞细胞外基质/胞衣样结构面积与细胞体面积比值,亦明显减少至几乎完全消失。作为对照的正义序列和逆转序列则无该作用。结论 HAS-2是正常培养人腹膜间皮细胞合成透明质酸以及细胞外基质/胞衣样结构形成的关键酶。
Objective To define which hyaluronan synthase (HAS), of three hyaluronan synthesizing enzymes HAS-1, HAS-2, and HAS-3, is primarily responsible for hyaluronan synthesis and extracellular matrix/extracellular coat formation in human peritoneal mesothelial cells (HPMCs) . Methods As a prerequisite study, the expression of each HAS mRNA in cultured HPMCs was measured by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Only the expression of HAS-2 and HAS-3 mRNA could be detected. The level of HAS-2 mRNA expression was about 10 fold higher than that of HAS-3. HAS-2 specific antisense oligonucleotide was then transfected into cultured HPMCs by lipofectamine. After 0, 8, 24, and 48 hours, the expression of HAS-2 mRNA was detected by RT-PCR and the extracellular coat was measured by particle exclusion test. Results 8 hours and 24 hours after transfection, the expression of HAS-2 mRNA in HPMCs decreased by 58% and 89% respectively; 48 hours after transfection, the expression of HAS-2 mRNA in HPMCs partially restored to 25% of the normal level. Correspondingly, 24 hours after transfection, the extracellular matrix/extracellular coat in HPMCs almost completely disappeared. However, as control, sense and reverse oligonucleotides showed no effect. Conclusion HAS-2 plays a leading role in HPMCs hyaluronan synthesis and the formation of extracellualr matrix/extracellular coat.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2002年第5期331-336,共6页
Chinese Journal of Nephrology
基金
卫生部临床学科重点项目基金(97040228)
中山医科大学“211”工程基金(98151)
瑞典Karolinska Institute合作基金
