人乙酰肝素酶基因真核表达载体的构建与表达

目的构建人乙酰肝素酶基因(HPA)的真核表达载体pEGFP-N1/HPA,初步探讨其在人胚肾细胞293T中的表达情况。方法以pOTB7/HPA质粒为模板,PCR扩增HPA基因,经限制性内切酶酶切后,连接到pEGFP-N1载体上,并对重组表达载体pEGFP-N1/HPA进行酶切与测序鉴定。用脂质体lipefectmine2000为介导转染至293T细胞中,观察该基因在293T细胞中的表达情况。结果经限制性内切酶酶切鉴定及测序分析证实pEGFP-N1/HPA基因在771 bp处碱基c突变成t;在919 bp处碱基a突变成g,两处突变均为同义突变。该基因转染293T细胞48 h后,在荧光显微镜下可见转染的293T细胞有绿色荧光蛋白表达。结论成功构建人乙酰肝素酶基因的真核表达载体pEGFP-N1/HPA,并在人293T细胞中成功表达。

乙酰肝素酶基因在子宫内膜异位症患者在位与异位子宫内膜组织中的表达

目的 研究乙酰肝素酶基因在子宫内膜异位症发病中的作用。方法 应用原位杂交技术 ,检测腹腔镜手术中证实为子宫内膜异位症患者 (EM组 ,2 3例 )的在位和异位子宫内膜组织乙酰肝素酶mRNA的表达 ,并与正常妇女 (对照组 ,2 5例 )子宫内膜组织比较。结果 ( 1)EM组在位与异位子宫内膜乙酰肝素酶mRNA阳性表达率一致 ,但异位子宫内膜组织中 ,阳性细胞着色较深。在位子宫内膜腺上皮细胞较间质细胞胞浆染色深 ,异位子宫内膜腺上皮细胞与间质细胞胞浆着色基本一致 ;EM组增生期与分泌期子宫内膜均有乙酰肝素酶mRNA的表达 ,增生期阳性表达率为 83 3%( 10 / 12 ) ;分泌期为 72 7% ( 8/ 11) ,两者比较 ,差异也无显著性 (P >0 0 5 ) ;( 2 )对照组增生期子宫内膜乙酰肝素酶mRNA的阳性表达率为 4 1 7% ( 5 / 12 ) ,分泌期子宫内膜阳性表达率为 7 7% ( 1/ 13) ,两者比较 ,差异有显著性 (P <0 0 5 )。 ( 3)EM组子宫内膜乙酰肝素酶mRNA阳性表达率为 78 3% ( 18/2 3) ;对照组为 2 4 0 % ( 6 / 2 5 ) ,两者比较 ,差异也有显著性 (P <0 0 5 )。结论 乙酰肝素酶基因的表达与子宫内膜异位症的发病相关 ,其可能成为子宫内膜异位症治疗的一个有价值的靶点。

乙酰肝素酶结构、功能及表达调控的研究进展

乙酰肝素酶(heparanase,HPSE)基因有2种基因亚型:HPSE1、HPSE2。HPSE1作为肿瘤细胞浸润转移的关键酶被人们广泛的认识,其主要的生物学功能包括组织修复、再生、免疫监视、胚胎植入、促进肿瘤细胞浸润、转移和血管生成。HPSE2与HPSE1之间有高度的同源性,又分为HPSE2a、HPSE2b、HPSE2c,其在促进肿瘤浸润、转移作用方面与HPSE1类似,其他作用有待进一步研究。

Detection of Syndecan-1 and Heparanase-1 Genes in Esophageal Carcinoma by Quantitative RT-PCR

OBJECTIVE To quantitatively explore the expression of Syndecan-1 and heparanase-1 in esophageal cancer tissue as well as their relationship with the clinicopathological factors, in order to evaluate their roles in tumor invasion and metastasis.METHODS Real-time fluorescence quantitative PCR （Q-PCR） was used to analyze the expression levels of Syndecan-1 and heparanase-1 genes￡？participants included 67 cases with esophageal cancers and 32 healthy volunteers.RESULTS The expression of Heparanase-1 gene in esophageal cancers was higher than that in normal esophageal tissue （P 〈 0.001）, and the expression of Syndecan-1 gene in the normal esophageal tissue was higher compared with esophageal cancers （P 〈 0.001）. The positive rates of Syndecan-1 and Heparanase-1 gene in esophageal cancer were 13.4% （9/67） and 85.1% （57/67）.The expression of Syndecan-1 and Heparanase-1 genes was signifi cantly related to di. erentiation, depth of infi ltration, lymph node metastasis, vessel metastasis, and TNM stages of disease （P 〈 0.05）. In an attempt to measure the association between the 2 agents, this study found that the expression of Syndecan-1 mRNA had a significantly negative correlation with the expression of Heparanase-1 mRNA by using Spearman rank correlation test （OR = -0.572, P 〈 0.001）.CONCLUSION Syndecan-1 and Heparanase-1 play important roles in the invasion and metastasis of esophageal cancer. The reduction of Syndecan-1 and/or the increase of Heparanase-1 may influence the invasion and metastasis of malignant tumors.Thus the combination assay of Syndecan-1 and Heparanase-1 may contribute to the diagnosis and treatment of malignant tumors.